Synthesis and biological evaluation of 1,2,4-triazole derivatives as potential neuroprotectant against ischemic brain injury

Article abstract of DOI:10.1016/j.ejmech.2020.112114

A series of 1,2,4-triazole derivatives 1–14 was synthesized to investigate their neuroprotective effects and mechanisms of action. Compounds 5–11 noticeably protected PC12 cells from the cytotoxicity of H₂O₂ or sodium nitroprusside (SNP). Compound 11 was the most effective derivative. Compound 11 chelated Fe (II) iron, scavenged reactive oxygen species (ROS), and restored the mitochondrial membrane potential (MMP). Moreover, it enhanced the activity of the antioxidant defense system by increasing the serum level of superoxide dismutase (SOD) and promoting the nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2). Compound 11 caused certain improvements in behavior, the cerebral infarction area, and serum levels of biochemical indicators (TNF-α, IL-1β, SOD and MDA) in a rat MCAO model. The lethal dose (LD₅₀) of compound 11 in mice receiving intraperitoneal injections was greater than 400 mg/kg. Meanwhile, pharmacokinetic experiments revealed high bioavailability of this compound after both oral and intravenous administration (F = 60.76%, CL = 0.014 mg/kg/h) and a longer half-life (4.26 and 5.11 h after oral and intravenous administration, respectively). Based on these findings, compound 11 may be a promising neuroprotectant for the treatment of ischemic stroke.

Full text of DOI:10.1016/j.ejmech.2020.112114

Journal Pre-proof
Synthesis and biological evaluation of 1,2,4-triazole derivatives as potential
neuroprotectant against ischemic brain injury
Liping Liao, Caibao Jiang, Jianwen Chen, Jinguo Shi, Xinhua Li, Yang Wang, Jin
Wen, Shujia Zhou, Jie Liang, Yaoqiang Lao, Jingxia Zhang
PII:
S0223-5234(20)30081-7
DOI:
https://doi.org/10.1016/j.ejmech.2020.112114
EJMECH 112114
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 12 December 2019
Revised Date: 31 January 2020
Accepted Date: 1 February 2020
Please cite this article as: L. Liao, C. Jiang, J. Chen, J. Shi, X. Li, Y. Wang, J. Wen, S. Zhou, J.
Liang, Y. Lao, J. Zhang, Synthesis and biological evaluation of 1,2,4-triazole derivatives as potential
neuroprotectant against ischemic brain injury, European Journal of Medicinal Chemistry (2020), doi:
https://doi.org/10.1016/j.ejmech.2020.112114.
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition
of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of
record. This version will undergo additional copyediting, typesetting and review before it is published
in its final form, but we are providing this version to give early visibility of the article. Please note that,
during the production process, errors may be discovered which could affect the content, and all legal
disclaimers that apply to the journal pertain.
© 2020 Published by Elsevier Masson SAS.

Graphical abstract:
Scheme summarizing the neuroprotection of compound 11 in vivo and in vitro through enhancing the
antioxidant system via Nrf2 pathway and enhancing certain anti-inflammatory action. Compound 11 could
activate the Nrf2 pathway enhancing the endogenous antioxidant system, and maintain MMP level, and effectively
avoid cell necrosis caused by oxidative damage. Furthermore, it ameliorated the behavior in rats with cerebral
ischemia, reduced the release of inflammatory factors (TNF-α and IL-β) and increased the level of antioxidants
(SOD and MDA). Eventually, compound 11 exhibited neuroprotective effects in vitro and in vivo.

Synthesis and biological evaluation of 1,2,4-triazole derivatives as potential neuroprotectant against
ischemic brain injury
Liping Liao,1 Caibao Jiang,1 Jianwen Chen1, Jinguo Shi, Xinhua Li, Yang Wang, Jin Wen, Shujia Zhou, Jie Liang,
Yaoqiang Lao, Jingxia Zhang*
Department of Medicinal chemistry, School of Pharmaceutical Science, Sun Yat-sen University, Guangzhou,
510006, P. R. China.
E-mail: zhjingx@mail.sysu.edu.cn
Highlights:
A series of 1,2,4-triazole derivatives 1-14 were synthesized to investigate their neuroprotective effects.
Compound 11 scavenged reactive oxygen species (ROS), and restores mitochondrial membrane potential (MMP).
Compound 11 ameliorated rat behavior, cerebral infarct size, and cytokine levels in the MCAO model.
Compound 11 had high bioavailability and longer half-life, LD50<400mg/kg.
Compound 11 might enhance antioxidant defense system via activation of Nrf2 signal.
Abstract: A series of 1,2,4-triazole derivatives 1-14 was synthesized to investigate their neuroprotective effects
and mechanisms of action. Compounds 5-11 noticeably protected PC12 cells from the cytotoxicity of H2O2 or
sodium nitroprusside (SNP). Compound 11 was the most effective derivative. Compound 11 chelated Fe (II) iron,
scavenged reactive oxygen species (ROS), and restored the mitochondrial membrane potential (MMP). Moreover,
it enhanced the activity of the antioxidant defense system by increasing the serum level of superoxide dismutase
(SOD) and promoting the nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2). Compound 11
caused certain improvements in behavior, the cerebral infarction area, and serum levels of biochemical indicators
(TNF-α, IL-1β, SOD and MDA) in a rat MCAO model. The lethal dose (LD50) of compound 11 in mice receiving
intraperitoneal injections was greater than 400 mg/kg. Meanwhile, pharmacokinetic experiments revealed high
bioavailability of this compound after both oral and intravenous administration (F=60.76%, CL=0.014mg/kg/h)
and a longer half-life (4.26 and 5.11 h after oral and intravenous administration, respectively). Based on these
findings, compound 11 may be a promising neuroprotectant for the treatment of ischemic stroke.
Keywords 1,2,4-triazole derivatives, neuroprotection, oxidative stress, Nrf2 signaling pathway
Abbreviations
SNP, sodium nitroprusside; SAR, structure-activity relationship; ROS, reactive oxygen species; TNF-α, tumor
necrosis factor-α; IL-1β, Interleukin – 1; MDA, malondialdehyde; MCAO, middle cerebral artery occlusion; KM,
kunming; SOD, superoxide dismutase; tPA, tissue plasminogen activator; ATP, adenosine triphosphate; DNA,
deoxyribonucleic acid; RNA, ribonucleic acid; CNS, central nervous system; MTT, 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide; LDH, lactate dehydrogenase; FBS, fetal bovine serum; HS, horse serum;
DMEM, duibecco’s modified eagie’s medium; DCFH-DA, 2,7-dichlorodi-hydrofluorescein diacetate; MMP,
mitochondrial membrane potential; JC-1, Tetrechloro-tetraethylbenzimidazol carbocyanine iodide; TTC, 2,3,5-
triphenyltetrazolium chloride; GSH, glutathione; TLC, thin layer chromatography; UV, ultraviolet; HRMS, high-
resolution mass spectrometry; DMSO, dimethyl sulfoxide;Me4Si, tetramethylsilane; SPF, specific pathogen free;
SD, sprague-dawley; SRM, selected reaction monitoring mode; PK, Pharmacokinetics; t1/2, half-life; Tmax, time of
maximum concentration; Cmax, maximum concentration; AUC0-∞, area under the plasma concentration timecurve;
CL, clearance; and F, oral bioavailability.

1. Introduction
Ischemic stroke is a leading cause of death and adult disability worldwide, substantially altering patients’
quality of life. Apart from tissue plasminogen activator (tPA), no effective clinical treatment strategy has been
reported to date[1,2,3]. During cerebral ischemia, insufficient local blood flow causes oxygen and glucose
deprivation, leading to energy metabolism disorders, mitochondrial adenosine triphosphate (ATP) synthase
disorders, oxidative stress caused by reactive oxygen species (ROS), and inflammatory reactions[4,5,6]
.
Neuroprotective agents with anti-oxidative stress and anti-inflammatory properties represent potential therapeutic
strategies for ischemic stroke[7].
Iron is an essential element involved in fundamental biological processes as a component of DNA, RNA, and
protein synthesis[8], oxygen transport, electron transfer[9], etc. However, iron-overload is cytotoxic due to the
generation of hydroxyl radicals and hydroxyl anions by the Fenton reaction[9,10], and evidence supports that
iron-overload induces oxidative stress in the pathogenesis of ischemic stroke[11]. Iron chelators have important
therapeutic potential and clinical applications[12]. Deferasirox, a potential oral iron chelator, is undergoing clinic
trials and has produced very promising results for neurodegenerative disorders[13], its triazole ring and two phenol
structures are the active groups.
Inflammatory reactions may trigger and amplify nerve injury, leading to an increase in the severity of stroke.
Some anti-inflammatory drugs possess an appropriate free-radical scavenging capacity[14], potent antioxidant
activities[15], and anti-inflammatory properties and have been developed as novel neuroprotective agents[16,17]
.
Celecoxib, a cyclooxygenase-2 specific inhibitor, increase neuroprotection in different cellular and animal models
of neurodegenerative disorders[18,19], and its pyrazole ring and benzene sulfonamide structure are the active groups.
Nuclear factor erythroid 2 related factor 2 (Nrf2) plays a vital role in the amelioration of various diseases
induced by inflammation and oxidative stress [20]. Upon exposure to stimuli and inducers, Nrf2 is translocated from
the cytoplasm to the nucleus resulting in enhanced cytoprotection, particularly the antioxidant defense system[20,21]
,
by increasing the levels of superoxide dismutase (SOD), glutathione peroxidase (GSH-px) and reduced glutathione
(GSH)[22]. Activation of the Nrf2 signaling pathway may contribute to antioxidant and anti-inflammatory effects
and reduce ischemic stroke.
In our search, we aimed to synthesize a series of compounds with antioxidant and anti-inflammatory
properties to identify potential neuroprotective agents for the treatment of ischemic brain injury. Deferasirox, an
iron-chelating agent reduces the level of hydroxyl radicals by inhibiting the Fenton reaction induced by iron
overload [9,10]. Considering the crucial iron-chelating role of the phenolic hydroxyl and 1,2,4-triazole groups in
deferasirox[23], we maintained that active scaffold to retain the antioxidant effect. In addition, extensive SAR
studies of celecoxib indicated that the phenyl sulfonamide moiety was a major determinant of the
anti-inflammatory effects and in vivo efficacy[24].Thus, based on the information described above, target
compounds containing both active sites (antioxidant and anti-inflammatory units) were prepared to obtain more
powerful neuroprotectants (Fig.1). The most potent analog was further evaluated to determine its mechanism, and
neuroprotective effects on a rat cerebral ischemia model.
2. Materials and methods
2.1. Reagents and chemical
Substituted benzoic acid and o-hydroxy benzamide, 4-hydrazinobenzene-1-sulfonamide hydrochloride,
edaravone and curcumin were purchased from Aladdin (Shanghai, China). FeSO4·7H2O was purchased from
Macklin. Hydrogen peroxide (H2O2), Sodium nitroprusside (SNP), and dimethyl sulfoxide (DMSO) were
purchased from Sigma-Aldrich Co (St. Louis, MO, USA). Fetal bovine serum (FBS), horse serum (HS) and
Dulbecco's modified Eagle's medium (DMEM) were obtained from Gibco (Maryland, USA). Antibodies against

Nrf2 (catalog number: ab137550) was obtained from Abcam Inc (Cambridge, MA, USA). β-Actin (catalog
number:30101ES10), peroxidase AffiniPure Goat Anti-Mouse IgG (H+L)(catalog number:33201ES60), peroxidase
AffiniPure Goat Anti-Rabbit IgG (H+L) (catalog number:33101ES50), Thiazolyl blue tetrazolium bromide (MTT),
phosphate buffer saline (PBS), penicillin-streptomycin (PS), mitochondrial membrane potential assay kit with JC-1,
and Lactate dehydrogenase (LDH) detection kit were purchased from Yeasen (Shanghai, China). Hoechst 33258
staining kit, LY294002, BCA assay kit, 2, 7-Dichlorodi-hydrofluorescein diacetate (DCFH-DA), total superoxide
dismutase (SOD) activity detection kit and glutathione peroxidase (GSH-px) detection kit were obtained from
Beyotime Institute of Biotechnology (Nanjing, Jiangsu, China). Reduced glutathione (GSH) detection kit was
obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, Jiangsu, China).
2.2. General procedure for synthesis of 3,5-Diphenyl-1,2,4-triazole
Substituted benzoic acid (1.2-2.0 g, 10 mmol) and o-hydroxy benzamide (1.5-2.3 g, 11 mmol) were added
into 20 mL of xylene, then anhydrous pyridine (catalytic amount) was added, and thionyl chloride (1.5 mL, 20
mmol) in xylene (8 mL) was added dropwise over 30 mins at room temperature. After that, stirring was undertaken
at 130-140 . The mixture was stirred for 2 h (TLC show acid was consumed) and the solvent was distilled off
under reduced pressure to obtain intermediate. The intermediate, 4-hydrazinobenzene-1-sulfonamide
hydrochloride (1.1 g, 5 mmol) and triethylamine (1.0 g, 10 mmol), were added to 15 mL of absolute ethanol, and
the mixture was stirred at 80 for 18 h. When the reaction was complete, the solution was filtered, and saturated
sodium bicarbonate was added to adjust pH to 7. The solution was extracted with ethyl acetate, and the organic
layer was washed with 20 mL of saturated brine and dried over anhydrous sodium. The solvent was evaporated
under reduced pressure to obtain the target compounds. The target compounds were recrystallized from methanol.
Reactions were monitored using TLC which was performed on GF254 plates and then detected under
ultraviolet (UV) light at 254 nm. The structural characteristics of target compounds were identified by NMR and
high-resolution mass spectrometry (HRMS). 1H and 13C NMR spectra were recorded in DMSO using
AvanceIII-400 and AvanceIII-500 spectrometer (Bruker, Germany) where the tetramethylsilane (Me4Si) was used
as an internal standard and chemical shifts were expressed in δ (ppm). Mass spectrometry was carried out by
Thermo Scientific TSQ Fortis Triple Quadrupole Mass Spectrometer (Thermo Fisher Scientific, Waltham, MA,
USA). The target compounds showed purity higher than 98% by high-performance liquid chromatography (HPLC)
using an Agilent 1260 infinity HPLC system (Agilent Technologies Inc., Santa Clara, CA, USA).
2.2.1.4-[5-(2-Hydroxyphenyl)-3-phenyl-1H-1,2,4-triazol-1-yl]benzenesulfonamide (1)
Compound 1 was prepared according to the general procedure, using benzoic acid (1.2 g, 10 mmol),
2-hydroxybenzamide (1.5 g, 11 mmol) and 4-hydrazinobenzene-1-sulfonamide hydrochloride (1.1 g, 5 mmol) to
obtain pure compound 1 as an off-white powder (1.3 g, yield: 68.0 %). m. p. 282~284.1H NMR (400 MHz,
DMSO-d6) δ 10.11 (s, 1H), 8.21 (d, J = 7.8 Hz, 1H), 7.94 (d, J = 9.0 Hz, 2H), 7.68 (d, J = 8.7 Hz, 1H), 7.65-7.61
(m, 2H), 7.59 (dd, J = 3.9, 2.2 Hz, 2H), 7.57 (s, 2H), 7.46 (td, J = 8.0, 2.0 Hz, 1H), 7.06 (t, J = 7.4 Hz, 1H), 6.95 (d,
J = 8.2 Hz, 1H).13C NMR (126 MHz, DMSO-d6) δ 161.4(s), 155.6(s), 153.8 (s), 143.8(s), 141.1(s), 132.6(s), 131.5
(s), 130.8 (s), 130.2 (s), 129.4 (s), 127.2 (s), 126.5(s), 123.9(s), 119.8(s), 116.5(s), 115.8(s). HRMS (m/z):
calculated for C20H17O3N4S [M+H]+ 393.1016, found 393.1020.
2.2.2.4-[5-(2-Hydroxyphenyl)-3-(4-(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl]benzenesulfonamide(2)
Compound 2 was prepared according to the general procedure, using 4-trifluoromethybenzoic acid (1.9 g, 10
mmol), 2-hydroxybenzamide (1.5 g, 11 mmol) and 4-hydrazinobenzene-1-sulfonamide hydrochloride (1.1 g, 5
1
mmol) to obtain pure compound 2 as an off-white powder (1.5 g, yield: 65.2 %). m. p. 264~286. H NMR (500
MHz, DMSO-d6) δ 10.04 (s, 1H), 8.33 (d, J = 8.0 Hz, 2H), 7.91 (d, J = 8.1 Hz, 2H), 7.87 (d, J = 7.6 Hz, 2H), 7.62
(dd, J = 8.6, 2.0 Hz, 2H), 7.55 (d, J = 7.7 Hz, 1H), 7.50 (s, 2H), 7.39 (t, J = 7.8 Hz, 1H), 6.99 (t, J = 7.6 Hz, 1H),
6.87 (d, J = 8.3 Hz, 1H).13C NMR (126 MHz, DMSO) δ 160.3 (s), 155.6 (s), 154.3 (s), 144.0 (s), 141.0 (s), 134.6

(s), 132.8 (s), 131.6 (s), 130.1 (q, J = 31.5 Hz),127.2 (s), 127.2 (s),126.4 (q, J = 3.8 Hz),124.6 (q, J = 273.4 Hz),
124.0 (s), 119.8 (s), 116.5 (s), 115.6 (s). HRMS(m/z): calculated forC21H16O3N4F3S[M+H]+ 461.0890, found
461.0892.
2.2.3.4-[3-(4-Fluorophenyl)-5-(2-hydroxyphenyl)-1H-1,2,4-triazol-1-yl]benzenesulfonamide (3)
Compound 3 was prepared according to the general procedure, using 4-fluorobenzoic acid (1.4 g, 10 mmol)
and 2-hydroxybenzamide (1.5 g, 11 mmol) and 4-hydrazinobenzene-1-sulfonamide hydrochloride (1.1 g, 5 mmol)
to obtain pure compound 3 as an off-white powder (1.5 g, yield: 72.4 %). m. p.270~272.1H NMR (500 MHz,
DMSO-d6) δ 10.02 (s, 1H), 8.21-8.09 (m, 2H), 7.86 (dd, J = 8.1, 2.3 Hz, 2H), 7.60 (d, J = 8.2 Hz, 2H), 7.52 (dd, J
= 7.5, 1.3 Hz, 1H), 7.48 (s, 1H), 7.43-7.29 (m, 3H), 6.97 (td, J = 7.5, 2.3 Hz, 1H), 6.86 (dd, J = 8.2, 2.2 Hz, 1H).
13C NMR (126 MHz, DMSO) δ 163.4 (d, J = 246.9 Hz), 160.7 (s), 155.6 (s), 153.9 (s), 143.9 (s), 141.1 (s), 132.7
(s), 131.5 (s), 128.7 (d, J = 7.6 Hz), 127.3 (d, J = 2.5 Hz), 127.2 (s), 123.9 (s), 119.8 (s), 116.5 (s), 116.3 (s), 115.7
(s). HRMS (m/z): calculated for C20H16O3N4FS [M+H]+ 411.0922, found 411.0923.
2.2.4.4-[5-(2-Hydroxyphenyl)-3-(4-nitrophenyl)-1H-1,2,4-triazol-1-yl]benzenesulfonamide(4)
Compound 4 was prepared according to the general procedure, using 4-nitrobenzoic acid (1.7 g, 10 mmol)
and 2-hydroxybenzamide (1.5 g, 11 mmol) and 4-hydrazinobenzene-1-sulfonamide hydrochloride (1.1 g, 5 mmol)
to obtain pure compound 4 as a white powder (1.3 g, yield: 57.8 %). m. p. 291~293. 1H NMR (500 MHz,
DMSO-d6) δ 10.05 (s, 1H), 8.53-8.21 (m, 4H), 7.88 (d, J = 8.2 Hz, 2H), 7.63 (d, J = 8.0 Hz, 2H), 7.56 (dd, J = 7.7,
2.0 Hz, 1H), 7.51 (s,2H), 7.40 (td, J = 7.4, 2.4 Hz, 1H), 6.99 (td, J = 7.5, 2.3 Hz, 1H), 6.87 (dd, J = 8.2, 2.3 Hz,
1H).13C NMR (126 MHz, DMSO-d6) δ 159.8 (s), 155.6 (s), 154.5 (s), 148.5 (s), 144.2 (s), 140.9 (s), 136.7 (s),
132.9 (s), 131.6 (s), 127.6 (s), 127.2 (s), 124.8 (s), 124.1 (s), 119.9 (s), 116.5 (s), 115.4 (s). HRMS (m/z):
calculated for C20H16O5N5S [M+H]+ 438.0867, found 438.0873.
2.2.5.4-[3,5-Bis(2-hydroxyphenyl)-1H-1,2,4-triazol-1-yl]benzenesulfonamide (5)
Compound 5 was prepared according to the general procedure, using 2-hydroxylbenzoic acid (1.4 g, 10
mmol) and 2-hydroxybenzamide (1.5 g, 11 mmol) and 4-hydrazinobenzene-1-sulfonamide hydrochloride (1.1 g, 5
1
mmol) to obtain pure compound 5 as a white powder (1.4 g, yield: 70.3 %). m. p.272~274. H NMR (500 MHz,
DMSO-d6) δ 10.77 (s, 1H), 10.12 (s, 1H), 8.05 (dd, J = 7.8, 2.0 Hz, 1H), 7.87 (d, J = 8.0 Hz, 2H), 7.64 (d, J = 8.6
Hz, 2H), 7.57 (dd, J = 7.5, 2.1 Hz, 1H), 7.50 (s, 2H), 7.44-7.35 (m, 2H), 7.06-6.96 (m, 3H), 6.88 (dd, J = 8.4, 2.4
Hz, 1H).13C NMR (126 MHz, DMSO-d6) δ 160.4 (s), 156.8 (s), 155.6 (s), 152.6 (s), 144.2 (s), 140.7 (s), 133.1 (s),
132.0 (s), 131.6 (s), 127.3 (s), 127.2 (s), 124.3 (s), 120.2 (s), 120.0 (s), 117.6 (s), 116.6 (s), 114.8 (s), 114.1 (s).
HRMS (m/z): calculated for C20H17O4N4S [M+H]+ 409.0965, found 409.0969.
2.2.6.4-[3-(2-Hydroxy-4-(trifluoromethyl)phenyl)-5-(2-hydroxyphenyl)-1H-1,2,4-triazol-1-yl]benzenesulfonamide(
6)
Compound 6 was prepared according to the general procedure, using 2-hydroxy-4-trifluoromethylbenzoic
acid (2.0 g, 10 mmol) and 2-hydroxybenzamide (1.5 g, 11 mmol) and 4-hydrazinobenzene-1-sulfonamide
hydrochloride (1.1 g, 5 mmol) to obtain pure compound 6 as a white powder (1.0 g, yield: 43.8 %). m. p.
240~242.1H NMR (500 MHz, DMSO-d6) δ 11.14 (s, 1H), 10.14 (s, 1H), 8.24 (d, J = 8.1 Hz, 1H), 7.88 (d, J = 8.5
Hz, 2H), 7.66 (d, J = 8.7 Hz, 2H), 7.58 (d, J = 7.7 Hz, 1H), 7.51 (s, 2H), 7.45-7.31 (m, 3H), 7.00 (td, J = 7.2, 2.5
Hz, 1H), 6.87 (dd, J = 8.2, 2.6 Hz, 1H). 13C NMR (126 MHz, DMSO) δ 159.2 (s), 156.9 (s), 155.6 (s), 153.0 (s),
144.4 (s), 140.6 (s), 133.2 (s), 131.8 (q, J = 32.8 Hz), 131.6 (s), 128.7 (s), 127.2 (s), 124.4 (s), 123.2 (q, J = 273.4
Hz), 120.0 (s), 118.1 (s), 116.6 (s), 116.5 (s),114.6 (s),114.3 (q, J = 3.8 Hz). HRMS (m/z): calculated for
C21H16O4N4F3S [M+H]+ 477.0839, found 477.0841.
2.2.7.4-[3-(4-Fluoro-2-hydroxyphenyl)-5-(2-hydroxyphenyl)-1H-1,2,4-triazol-1-yl]benzenesulfonamide (7)
Compound 7 was prepared according to the general procedure, using 4-fluoro-2-hydroxybenzoic acid (1.5 g,
10 mmol) and 2-hydroxybenzamide (1.5 g, 11 mmol) and 4-hydrazinobenzene-1-sulfonamide hydrochloride (1.1 g,

5 mmol) to obtain pure compound 7 as a white powder (0.3 g, yield: 14 %). m. p. 280~282.1H NMR (500 MHz,
DMSO-d6) δ 11.11 (s, 1H), 10.12 (s, 1H), 8.06 (td, J = 7.3, 2.6 Hz, 1H), 7.89 (d, J = 8.7 Hz, 2H), 7.64 (d, J = 7.8
Hz, 2H), 7.56 (d, J = 7.8 Hz, 1H), 7.50 (s, 1H), 7.40 (t, J = 8.1 Hz, 1H), 6.99 (td, J = 7.5, 2.3 Hz, 1H), 6.95-6.81
(m, 3H). 13C NMR (126 MHz, DMSO) δ 164.2 (d, J = 246.9 Hz), 159.7 (s), 158.5 (d, J = 13.0 Hz), 155.6 (s), 152.6
(s), 144.3 (s), 140.6 (s), 133.1 (s), 131.6 (s), 129.1 (d, J = 10.6 Hz), 127.2 (s), 124.3 (s), 120.0 (s), 116.6 (s), 114.7
(s), 111.2 (s), 107.7 (d, J = 22.4 Hz), 104.6 (d, J = 24.4 Hz). HRMS (m/z): calculated for C20H16O4N4FS [M+H]+
427.0871, found 427.0872.
2.2.8.4-[3-(2-Hydroxy-4-nitrophenyl)-5-(2-hydroxyphenyl)-1H-1,2,4-triazol-1-yl]benzenesulfonamide(8)
Compound 8 was prepared according to the general procedure, using 2-hydroxy-4-nitrobenzoic acid (1.8 g,
10 mmol) and 2-hydroxybenzamide (1.5 g, 11 mmol) and 4-hydrazinobenzene-1-sulfonamide hydrochloride (1.1 g,
5 mmol) to obtain pure compound 8 as a yellow powder (0.8 g, yield: 36.7 %). m. p. 280~282.1H NMR (500 MHz,
DMSO-d6) δ 11.29 (s, 1H), 10.15 (s, 1H), 8.28 (dd, J = 8.9, 2.6 Hz, 1H), 7.97-7.79 (m, 4H), 7.66 (d, J = 8.0 Hz,
2H), 7.58 (d, J = 7.5 Hz, 1H), 7.52 (s, 2H), 7.41 (t, J = 8.0 Hz, 1H), 7.00 (td, J = 7.6, 2.5 Hz, 1H), 6.88 (dd, J = 8.3,
2.5 Hz, 1H).13C NMR (126 MHz, DMSO-d6) δ 158.8(s), 157.0(s), 155.6(s), 153.1(s), 149.4 (s), 144.5 (s), 140.6 (s),
133.2(s), 131.6(s), 129.0(s), 127.3 (s), 124.4(s), 120.8(s), 120.0 (s), 116.6(s), 115.0 (s), 114.5(s), 112.3(s). HRMS
(m/z): calculated for C20H16O6N5S [M+H]+ 454.0816, found 454.0817.
2.2.9.4-[3-(4-Chloro-2-hydroxyphenyl)-5-(2-hydroxyphenyl)-1H-1,2,4-triazol-1-yl]benzenesulfonamide (9)
Compound 9 was prepared according to the general procedure, using 4-chloro-2-hydroxybenzoic acid (1.7 g,
10 mmol) and 2-hydroxybenzamide (1.5 g, 11 mmol) and 4-hydrazinobenzene-1-sulfonamide hydrochloride (1.1 g,
5 mmol) to obtain pure compound 9 as a white powder (1.5 g, yield: 66.8 %). m. p.267~269.1H NMR (500 MHz,
DMSO-d6) δ 11.03 (s, 1H), 10.12 (s, 1H), 8.04 (d, J = 8.2 Hz, 1H), 7.88 (d, J = 8.4 Hz, 2H), 7.64 (d, J = 8.4 Hz,
2H), 7.56 (dd, J = 7.8, 2.0 Hz, 1H), 7.50 (s, 2H), 7.45-7.33 (m, 1H), 7.13 (s, 1H), 7.11-7.04 (m, 1H), 6.99 (td, J =
7.7, 2.5 Hz, 1H), 6.87 (dd, J = 8.3, 2.5 Hz, 1H).13C NMR (126 MHz, DMSO-d6) δ 159.6 (s), 157.6 (s), 155.6(s),
152.7 (s), 144.3(s), 140.6(s), 135.8 (s), 133.1(s), 131.6(s), 128.8(s), 127.2(s), 124.3(s), 120.5 (s), 120.0 (s), 117.4
(s), 116.6(s), 114.7 (s), 113.5 (s). HRMS (m/z): calculated for C20H16O4N4ClS [M+H]+ 443.0575, found 443.0577.
2.2.10.4-[3-(2-Hydroxy-4-methylphenyl)-5-(2-hydroxyphenyl)-1H-1,2,4-triazol-1-yl]benzenesulfonamide(10)
Compound 10 was prepared according to the general procedure, using 2-hydroxy-4-methylbenzoic acid (1.5
g, 10 mmol) and 2-hydroxybenzamide (1.5 g, 11 mmol) and 4-hydrazinobenzene-1-sulfonamide hydrochloride
(1.1 g, 5 mmol) to obtain pure compound 10 as an off-white powder (1.6 g, yield: 76.7 %).m. p.252~254.1H NMR
1
(400 MHz, DMSO-d6) H NMR (400 MHz, DMSO-d6) δ 10.68 (s, 1H), 10.09 (s, 1H), 7.93 (d, J = 7.9 Hz, 1H),
7.91-7.85 (m, 2H), 7.66-7.60 (m, 2H), 7.56 (dd, J = 7.7, 1.7 Hz, 1H), 7.49 (s, 2H), 7.44-7.38 (m, 1H), 7.00 (td, J =
7.5, 1.1 Hz, 1H), 6.90-6.81 (m, 3H), 2.33 (s, 3H). 13C NMR (126 MHz, DMSO-d6) δ 160.5(s), 156.7(s), 155.6(s),
152.5(s), 144.2 (s), 142.0(s), 140.7(s), 133.0(s), 131.6 (s), 127.2(s), 127.0(s), 124.2(s), 121.2(s), 119.9(s), 117.8(s),
116.6 (s), 114.9 (s), 111.4(s), 21.6 (s). HRMS (m/z): calculated for C21H19O4N4S [M+H]+ 423.1122, found
423.1123.
2.2.11.4-[3-(2-Hydroxy-4-methoxyphenyl)-5-(2-hydroxyphenyl)-1H-1,2,4-triazol-1-yl]benzenesulfonamide (11)
Compound 11 was prepared according to the general procedure, using 2-hydroxy-4-methoxylbenzoic acid
(1.7 g, 10 mmol) and 2-hydroxybenzamide (1.5 g, 11 mmol) and 4-hydrazinobenzene-1-sulfonamide
hydrochloride (1.1 g, 5 mmol) to obtain pure compound 11 as a yellow powder (1.7 g, yield: 78.0 %). m.
p.254~256.1H NMR (500 MHz, DMSO-d6) δ 10.73 (s, 1H), 9.59 (s, 1H), 8.05 (d, J = 7.7 Hz, 1H), 7.89 (d, J = 8.1
Hz, 2H), 7.65 (d, J = 8.0 Hz, 2H), 7.50 (s, 2H), 7.38 (t, J = 7.9 Hz, 1H), 7.13 (s, 1H), 7.05-6.97 (m, 3H), 6.79 (dd,
J = 8.9, 2.4 Hz, 1H), 3.73 (s, 2H).13C NMR (126 MHz, DMSO-d6) δ 160.4 (s), 156.8 (s), 152.5 (s), 152.4 (s),
149.4(s), 144.2(s), 140.7 (s), 132.0 (s), 127.3(s), 127.2(s), 124.3(s), 120.2 (s), 119.2 (s), 117.6 (s), 117.6 (s),
115.7(s), 114.9 (s), 114.1(s), 56.0(s). HRMS(m/z): calculated for C21H19O5N4S [M+H]+ 439.1071, found 439.1074.

2.2.12.4-[3,5-Bis(4-fluoro-2-hydroxyphenyl)-1H-1,2,4-triazol-1-yl]benzenesulfonamide(12)
Compound 12 was prepared according to the general procedure, using4-fluoro-2-hydroxybenzoic acid (1.6 g,
10 mmol) and 4-fluoro-2-hydroxybenzamide (1.7 g, 11 mmol) and 4-hydrazinobenzene-1-sulfonamide
hydrochloride (1.1 g, 5 mmol) to obtain pure compound 12 as a white powder (1.7 g, yield: 79.2 %). m. p.264~266.
1H NMR (400 MHz, DMSO-d6) δ 11.04 (s, 1H), 10.67 (s, 1H), 8.07 (dd, J = 8.6, 6.8 Hz, 1H), 7.97-7.83 (m, 2H),
7.74-7.55 (m, 3H), 7.50 (s, 2H), 7.00-6.75 (m, 3H), 6.65 (dd, J = 10.7, 2.5 Hz, 1H). 13C NMR (126 MHz, DMSO)
δ 164.9 (d, J = 248.2 Hz), 164.3 (d, J = 246.9 Hz), 159.7 (s), 158.5 (d, J = 12.8 Hz), 157.4 (d, J = 11.9 Hz), 151.8
(s), 144.3 (s), 140.5 (s), 133.5 (d, J = 11.1 Hz), 129.2 (d, J = 10.8 Hz), 127.3 (s), 124.3 (s), 111.5 (d, J = 2.7 Hz),
111.2 (d, J = 2.7 Hz), 107.7 (d, J = 22.1 Hz), 107.3 (d, J = 22.1 Hz), 104.5 (d, J = 24.4 Hz), 103.6 (d, J = 24.3 Hz).
HRMS (m/z): calculated for C20H15O4N4 F2S [M+H]+ 445.0777, found 445.0778.
2.2.13.4-[3,5-Bis(2-hydroxy-4-(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl]benzenesulfonamide(13)
Compound 13 was prepared according to the general procedure, using 2-hydroxy-4-trifluoromethylbenzoic
acid (2.0 g, 10 mmol) and 2-hydroxy-4-trifluoromethylbenzamide (2.3 g, 11 mmol) and 4-hydrazinobenzene-1-
sulfonamide hydrochloride (1.1 g, 5 mmol) to obtain pure compound 13 as a white powder (2.1 g, yield: 77.5 %).
m. p.271~273. 1H NMR (500 MHz, DMSO-d6) δ 11.05 (s, 1H), 10.69 (s, 1H), 8.06 (dd, J = 7.1, 2.3 Hz, 1H), 7.90
(d, J = 8.3 Hz, 2H), 7.64 (d, J = 7.9 Hz, 3H), 7.51 (s, 2H), 6.95-6.81 (m, 3H), 6.65 (dd, J = 10.6, 2.6 Hz, 1H). 13
C
NMR (126 MHz, DMSO) δ 159.5 (s), 156.8 (s), 156.1 (s), 151.6 (s), 144.6 (s), 140.4 (s), 133.3 (s),133.1 (d, J =
31.5Hz), 131.9 (d, J = 31.5Hz), 128.9 (s), 127.4 (s), 124.4 (s), 124.2 (d, J = 273.4Hz),124.0 (d, J = 273.4
Hz),118.8 (s), 118.2 (s),116.6 (d, J = 3.78Hz),116.4 (d, J = 3.78 Hz),114,4 (d, J = 3.78 Hz), 112.9 (d, J = 3.78 Hz).
HRMS (m/z): calculated for C22H15O4N4 F6S [M+H]+ 545.0713, found 545.0717.
2.2.14.4-[3,5-Bis(2-hydroxy-4-methylphenyl)-1H-1,2,4-triazol-1-yl]benzenesulfonamide(14)
Compound 14 was prepared according to the general procedure, using 2-hydroxy-4-methylbenzoic acid (1.5g,
10 mmol) and 2-hydroxy-4-methylbenzamide (1.6 g, 11 mmol) and 4-hydrazinobenzene-1-sulfonamide
hydrochloride (1.1 g, 5 mmol) to obtain pure compound 14 as a white powder (1.7 g, yield: 78.9 %). m. p.268~270.
1H NMR (500 MHz, DMSO-d6) δ 10.74 (s, 1H), 10.00 (s, 1H), 7.92 (d, J = 7.9 Hz, 1H), 7.88 (d, J = 7.9 Hz, 2H),
7.63 (d, J = 8.0 Hz, 2H), 7.50 (s, 2H), 7.43 (d, J = 8.2 Hz, 1H), 6.90-6.76 (m, 3H), 6.68 (s, 1H), 2.32 (s, 3H), 2.29
(s, 3H).13C NMR (126 MHz, DMSO-d6) δ 160.4(s), 156.8 (s), 155.5 (s), 152.6 (s), 144.1(s), 143.1 (s), 142.0 (s),
140.9 (s), 131.3(s), 127.2(s), 127.0(s), 124.3 (s), 121.2 (s), 120.9 (s), 117.8(s), 117.0(s), 112.0(s), 111.4(s), 21.6(s),
21.6 (s). HRMS (m/z): calculated for C22H21O4N4S [M+H]+ 437.1278, found 437.1280.
2.3. Cell culture
The PC12 cell line was purchased from the Experimental Animal Center of Sun Yat-sen University
(Guangzhou, China). Cells were maintained in DMEM containing 10% (v/v) HS, 5% (v/v) FBS, and 1% (v/v) PS,
and incubated in a humidified atmosphere of 5% CO2 at 37 °C (Thermo, BB15, USA).
2.4. Cell viability assay
Cell viability was measured using the MTT assay. Briefly, PC12 cells were seeded in 96-well plates (5×103
cells/well) and cultured for 24 h. Cells were then pre-treated with the indicated concentrations of target compounds
for 1 h, and incubated with 500 µM H2O2 for another 5 h or 300 µM SNP for 24 h. 10 µL of 5 mg/mL MTT were
added to pre-treated cells for 3 h at 37 °C in the presence of 5% CO2. The supernatant was discarded, 100 µL of
DMSO were added to the wells of the 96-well plates, and the solutions were mixed thoroughly. The absorbance
was recorded at 570 nm using a microplate reader (BioTek, Epoch, USA). The relative viability of treated cells
was calculated by comparing the values with the OD value of control group. In inhibitor experiments, cells were
pretreated with LY294002 (10 µM) for 1 h prior to the addition of the target compounds. All data presented in this
phase were normalized to the control group. The experiments were repeated at least 3 times.

2.5. Measurement of lactate dehydrogenase (LDH) release
The amount of LDH released from treated-cells cultured in 96-well plates was determined using an LDH
assay kit according to the manufacturer’s instructions. Briefly, PC12 cells were seeded in 96-well plates (1×104
cells/well) and cultured for24 h. Cells were then pre-treated with active compounds 5-11 (2.5, 5 or 10µM) for 1 h,
and then incubated with 500 µM H2O2 for another 5 h or 300 µM SNP for 24 h. The INT solution (1X) working
and LDH detection solution were prepared in advance. 60 µL of the supernatant were removed from each well of
the 96-well plate and added to a new 96-well plate. Then, 30 µL of the working LDH detection solution were
added, and mixed with supernatant. The mixture was incubated in the dark at room temperature for 30 minutes and
then the absorbance was measured at 490 nm using a microplate reader (BioTek, Epoch, USA). All data presented
in this phase were normalized to the H2O2 or SNP group. The experiments were repeated at least 3 times.
2.6. Hoechst 33258 staining assay
PC12 cells were seeded in 6-well plates, cultured for 24 h, pre-treated with compound 11 (2.5, 5, or 10 µM)
for 1 h, and incubated with 500 µM H2O2 for another 5 h or 300 µM SNP for 24 h. Next, the cells were stained
with Hoechst 33258 (10µg/mL) for 30 min in the dark in an incubator. The medium was removed and cells were
washed twice with phosphate-buffered saline (PBS). The relative fluorescence intensities of Hoechst 33258 were
detected using a fluorescence microscope (Life Technologies, EVOS FL, USA).
2.7. Detecting of ROS levels
PC12 cells were seeded into glass-bottomed cell culture dishes and cultured for 24 h. Cells were then
pre-treated with compound 11 (2.5, 5, or 10 µM) for 1 h and incubated with 500 µM H2O2 for another 5 h or 300
µM SNP for 24 h. Next, the medium was changed to serum-free medium and 10 µM DCFH-DA was co-incubated
with the cells for 30 min in the dark in an incubator. The medium was removed and cells were washed twice with
PBS. The relative fluorescence intensities of ROS were detected using fluorescence microscope (Olympus,
FV3000, Japan) at excitation and emission wavelengths of 488 nm and 525 nm, respectively.
2.8. Changes in the mitochondrial membrane potential (MMP)
PC12 cells were seeded into glass-bottomed cell culture dishes and cultured for 24 h. The cells were then
pre-treated with compound 11 (2.5, 5, or 10 µM) for 1 h and incubated with 500 µM H2O2 for another 5 h or 300
µM SNP for 24 h. The working JC-1 detection solution was prepared in advance and co-incubated with cells for 20
mins in the dark in an incubator. The medium was removed and cells were washed twice with the JC-1 solution
buffer. The relative fluorescence intensities were detected with a fluorescence microscope (Olympus, FV3000,
Japan) at excitation and emission wavelengths of 490 nm and 530 nm, respectively, to detect the JC-1 monomer,
and 525 nm and 590 nm, respectively, to detect JC-1 aggregates.
2.9. Evaluation of the binding to iron ions and determination of stoichiometries of the complexes
Evaluation of the binding to iron ions. The binding of ferrous iron ions to the series of triazole compounds
was determined using a previously described method[25]. Briefly, FeSO4 (dissolved in 200 µL of water to a final
concentration of 15 µM) was mixed with the target compounds (dissolved in 200 µL of ethanol to a final sample
concentration of 0−50 µM), followed by ferrozine (dissolved in 600 µL of ethanol to a final concentration of 30
µM). The mixture was incubated for 10 mins at room temperature. The absorbance of the purple colored Fe
(II)-ferrozine complex in the presence or absence of target compounds was recorded at 562 nm using UV-visible
spectrophotometer (Shimadzu, 2600, Japan). The experiments were repeated at least 3 times.
Spectrophotometric studies. Solutions of compound 11 and FeSO4·7H2O were prepared with concentrations
of 5×10 4 mol/L in methanol and aqueous solution, respectively. Absorption spectra of compound 11 (1×10 4
−
−
mol/L) were recorded between 275 and 500 nm. Then, 1mL 5×10−4 mol/L compound 11 and 1mL 5×10−4 mol/L

FeSO4·7H2O solution were mixed and diluted to 5 mL. Absorption spectrum of compound 11-Fe (II)complex were
recorded between 275 and 500 nm using a UV-2600 ultraviolet-visible spectrophotometer.
Determination of stoichiometries of the complexes. Stoichiometries of the complexes were determined using
mole ration method[26,27]. 10 mL volumetric flasks were prepared and numbered in advance.1 mL of 5 × 10-4 mol /
L FeSO4·7H2O solution was added to each numbered volumetric flask, and followed by 0, 1, 2, 3, 4, 5, 6 mL of 5 ×
10-4 mol / L compound 11, and then each volumetric flask was diluted to 10 mL, respectively. The absorbance of
each complex was measured at an appropriate wavelength, at which the complex solution showed intense
absorption but compound 11 did not. The absorbance (OD value) was taken as the Y axis, and the molar ratio of
compound 11 to Fe (II) was plotted on the X axis.
2.10. Western blot analysis
PC12 cells were seeded in 6-well plates and treated with different concentrations of compound 11 (1.25, 2.5,
or 5 µM) for 24 h. Cells were lysed in using cell lysis buffer (containing 1 mM phenylmethylsulfonyl fluoride
(PMSF)) to obtain the total proteins. Nuclear and cytoplasmic proteins were extracted using a Nuclear and
Cytoplasmic Protein Extraction Kit according to the manufacturer's protocol (Biotechnology, Nanjing, Jiangsu,
China). For the western blot analysis, protein extracts were quantified using a BCA assay kit, and equal amounts
(40 mg/lane) of proteins were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (10%
SDS-PAGE), and then transferred onto polyvinylidene difluoride (PVDF) membranes (Bio-Rad, Philadelphia, PA,
USA). The membranes were blocked with 5% skim milk for 2 h at room temperature, washed, and then incubated
overnight at 4°C with a Nrf2 primary antibody (dilution, 1:1000; Abcam, Cambridge, UK). Next, membranes were
washed three times and incubated with the secondary antibody (1:5000; Yeasen, Shanghai, China) for 2 h. After
washing the membranes, the protein bands were detected with a chemiluminescence (ECL) detection kit using an
ImageQuant chemiluminescence system and Image J. software was used to quantify the bands intensities. In the
inhibitor experiment, the cells were pretreated with LY294002 (10 µM) for 1 h before the addition of the target
compounds.
2.11. Inhibitor treatment
PC12 cells were pretreated with or without compound 11 for 1 h, and co-treated with or without 10 µM
LY294002 (PI3K/AKT pathway inhibitor) for 24 h. Cells were then lysed and protein levels were determined prior
to the Western blot analysis.
Cells were pre-treated with compound 11 for 1 h, co-treated with or without LY294002 (10 µM), and then
incubated with 500 µM H2O2 or 300 µM SNP for 5 h or 24 h, respectively, to analyze the roles of signaling
pathways in the neuroprotective effects of compound 11 on PC12 cells. Cell viability was measured using the MTT
assay.
2.12. Detecting the levels of GSH, GSH-px, and SOD
PC12 cells were seeded in 6-well plates, cultured for 24 h, pre-treated with compound 11 (2.5, 5 or 10 µM)
for 1 h, and then incubated with 500 µM H2O2 or 300 µM SNP for another 5 h or 24 h, respectively. Cells were
lysed on ice in cell lysis buffer (containing 1 mM PMSF) to prepare total protein extracts. The proteins present in
cell lysates were centrifuged at 12000 g for 10 mins at 4 °C. The supernatant was collected, quantified BCA kit,
and then subjected to the measurement of SOD, GSH-px and GSH levels using SOD, GSH-px, and GSH detection
kits, respectively, according to the manufacturer’s instructions.
2.13. Establishment of a rat model of ischemic stroke
Animals. In this study, 72 male specific-pathogen-free (SPF) Sprague-Dawley (SD) rats (body weight,
180-220 g) were provided by the Experimental Animal Center of Sun Yat-sen University (Guangzhou, China;

License No. SYXK (Guangdong) 2018-0002)
Middle Cerebral Artery Occlusion[28]. The rats were anesthetized by administering an intraperitoneal
injection of 7% chloral hydrate (5 mL/kg). The left common carotid artery and external carotid artery were
separated and ligated through a median cervical incision, and the pterygofrontal artery was separated as well. An
arterial clamp was placed in the proximal and distal carotid arteries, and no. 4-0 nylon thread was inserted through
the incision, to a depth of 17-20 mm. The wound was disinfected with iodophor and the skin was sutured. In the
sham-operated group, the other steps were the same except that no thread was inserted. The animals were
randomly divided into six groups: sham-operated group, model-based control group, positive control group
(edaravone injection, 10mg/kg), low- (3mg/kg), medium- (10mg/kg) and high-dose (30mg/kg) groups (n=12 rats
in each group). The treatments were administered via intraperitoneal injection at the onset of reperfusion period by
an investigator blinded to the groups. All procedures were approved by the local Institutional Animal Care
Committee, and were conducted in compliance with the Chinese legislation.
Behavioral testing[29]. Behavioral tests were performed 24 h after the operation according to the 4-point
scoring system proposed by Zea Longa. The specific criteria were 0 points, no nerve injury symptoms; 1 point,
unable to completely extend the opposite front paw; 2 points, turning to the outside; 3 points, tilting to the opposite
side; and 4 points, unable to walk spontaneously and loss of consciousness.
Cerebral Infarction Area[28]. After completing the behavioral tests, the animals were euthanized and brain
sections were prepared for TTC staining to observe the cerebral infarction area. The whole brain was quickly
removed from the skull, the olfactory bulb, cerebellum and lower brainstem were removed, and the tissue was
frozen for 25 mins. Then, tissues were coronally cut into 5-7 pieces, quickly placed in 1% TTC for 20 mins, and
incubated at 37°C for 20 mins. Tissues were flipped every 7-8 mins and fixed with 4% paraformaldehyde after
staining. The normal tissues were stained red and the injured infarcted tissues were stained white. After capturing
pictures, the percentage of the cerebral infarction area was calculated using an image analysis method.
Determination of Biochemical Indicators. After completing the behavioral testing, the animals were
euthanized, blood was collected from the abdominal vein, and the serum was stored at -20 °C. The serum levels
of inflammatory markers, such as IL-1β, TNF-α, SOD, and MDA, were detected using enzyme-linked
immunosorbent assay (ELISA) kits.
2.14. Acute toxicity test
Animals. Twenty SPF KM mice (half male and half female, 18-22 g) were provided by Experimental Animal
Center of Sun Yat-sen University, License No. SYXK (Guangdong) 2018-0002.
Acute toxicity[30]. Twenty KM mice (18-22 g) were divided into two groups: a compound 11 treated group
(400 mg/kg) and solvent control group (0.4 mL/10 g). The animals in each group were administered intraperitoneal
injections. The reactions were observed immediately after administration for 12 h, and then observed once on the
1st, 3rd, 5th, and 7th days. The general state and body weights were recorded for 7 days. The observations
included changes in the animals’ weights, diets, appearance, behaviors, secretion, and excretion. The contents of
observations of toxic symptoms were based on Appendix II of Technical Guiding Principles for the Study of Acute
Toxicity of Chemical Drugs. The mortality rate, poisoning symptoms and the onset time, severity, and duration of
poisoning reactions were recorded as well. The animals were weighed at each time point. According to the
mortality rate recorded within 7 days, lethal dose, the 50% lethal dose (LD50) was calculated using the weighted
regression method. The treatments were administered by intraperitoneal injection at the onset of the reperfusion

period by an investigator who was blinded to the groups.
2.15. Pharmacokinetic studies in SD rats
Compound 11 was dissolved in solvent and administered to 12 female SD rats (weight ranging from 220 g to
250 g) for i.v. and p.o. administration. The dosing volume was 2 mg/kg (i.v.) or 20 mL/kg (p.o.). After
administration, blood samples were collected at various time points 1/12, 1/6, 3/10, 0.5,0.75, 1, 2, 3, 4, 6, 8, 12 and
24 h (i.v.) or 0.25, 0.5, 0.75, 1, 2, 3, 4, 6, 8, 12and 24 h (p.o.) for analyses. The collected blood samples were
centrifuged at 4000 rpm for 10 mins at 4 °C, and then analyzed after protein precipitation. An LC/MS/MS analysis
of compound 11 was performed under optimized conditions to obtain the best sensitivity and selectivity of the
analyte in selected reaction monitoring mode (SRM) in samples containing an internal standard[31]. Plasma
concentration-time data were measured with a noncompartmental approach using the Excel program on a
computer to calculate pharmacokinetic parameters.
2.16. Statistical analysis
Biomedical experimental data were processed using GraphPad Prism 8.0 software, and all measurements
were performed in triplicate. All results are reported as means ± standard deviations (SD). The statistical
significance of differences between the two groups were assessed using GraphPad Prism8.0 software, and P < 0.05
was considered statistically significant. Animal experimental data were statistically processed using GraphPad
Prism8.0 software, and measured data are reported as means ± standard errors of the means (SEM) and analyzed
using single-factor analysis of variance (ANOVA) combined with Dunnett's test, and count data were analyzed
using the Kruskal-Wallis test.
3. Results
3.1. Chemical findings
Three series of triazole compounds were synthesized (shown in Scheme 1), the first contained R1= H, R2 = H,
CF3, F, NO2, and R3 = H, the second contained R1 = OH, R2 = H, CF3, F, NO2, Cl, CH3, OCH3, and R3 = H, and the
third contained R1=OH, R2, R3=F, CF3,CH3. The substituents were derived from substituted o-hydroxyl benzamide
and benzoic acid. These compounds were synthesized to compare whether the activity of dihydroxy compounds
was better than mono hydroxyl compounds, and to compare the potency of disubstituted and monosubstituted
compounds at the meta-position, as relevant researches support meta-substituted analogs are with good activity.
(The results of the NMR analysis of compounds 1-14 are presented in the supporting information, purity＞98%)
3.2. Evaluating the ability of the compounds to protect cells from H2O2 or SNP-induced death
In ischemic stroke, oxidative stress produces excessive ROS levels, resulting in cytotoxicity and eventually
more severe nerve injury. PC12 cells, which are derived from a pheochromocytoma of the rat adrenal medulla, are
generally used as a screening model to study nerve injuries. H2O2 produces hydroxyl radical and is commonly used
to generate oxidative stress-based models. SNP is a medication used to lower blood pressure, that is also widely
utilized to induce cerebral ischemia-based models in nerve cells[32]. Treatment of PC12 cells with H2O2 (500 µM)
for 5 h or SNP (300 µM) for 24 h causes approximately 60% of cells to die (Fig. 2A and 2C). An increase in cell
viability attributed to the potent protective effects of 10 µM treatments with the target compounds on H2O2- or
SNP- induced death was first assessed using the MTT assay, as shown in Fig. 2B and 2D.
Compounds 5-11 containing dihydroxy and monosubstituted groups, actively protected PC12 cells from
H2O2- or SNP- induced cell death, and showed higher activities than curcumin and edaravone. Nevertheless,
compounds 1-4 containing the monohydroxy group showed a poor potency, indicating a significant role of the
hydroxyl group. Additionally, compounds 12-14 containing dihydroxyl and disubstituted groups did not show
stronger potency, indicating that the disubstituted compounds exhibited a weaker protective effect.

3.3. Protective effects of compounds 5-11 on prominent activities
As shown in Fig. 3, compounds 5-11 were more active than the remaining compounds. The protective
efficacy of different concentrations of compounds 5-11 was assessed using MTT and LDH assays to further
investigate and compare their potency. LDH is often used as an indicator of cytotoxicity. Pretreatment with
compounds 5-11 dose-dependently (2.5, 5, and 10 µM) increased cell viability (Fig. 3A and 3C), and significantly
reduced LDH levels (Fig. 3B and 3D) in H2O2- or SNP treated PC12 cells. The results of the MTT assay were
consistent with the results of the LDH assay. Based on our findings, compound 11 was the most active compound,
and the hydroxyl and 4-methoxy groups of the benzene ring were considered effective pharmaceutically active
groups contributing to the neuroprotective effects.
3.4. Compound 11 dose-dependently alleviates H2O2 or SNP- induced apoptosis
The appearance of a condensed nucleus is a typical characteristic of apoptosis. Consistent with previous
reports, H2O2 or SNP caused cytotoxicity to PC12 cells mainly by inducing apoptosis. Quantitatively, the number
of living cells in the H2O2 group was substantially decreased, and the pretreatment with compound 11 (2.5, 5 and
10 µM) effectively increased the number of living cells. In terms of morphology, the cells in the injured group
exhibited nuclear condensation (brightening), and the cells were not adherent, became rounded, and displayed an
unclear shape. However, the morphology of cells in the compound 11 group often appeared normal (Fig. 4A).
Notably, SNP produced the same result as H2O2 (Fig. 4B). Thus, compound 11 effectively prevented H2O2 or SNP-
induced apoptosis by reducing the number of condensed nuclei.
3.5. Compound 11 dose-dependently inhibits ROS accumulation
ROS levels were detected with 2,7-dichlorodi-hydrofluorescein diacetate (DCFH-DA). DCFH-DA freely
crosses the cell membrane and is hydrolyzed by an esterase to generate 2,7-dichlorodi-hydrofluorescein (DCFH).
DCFH is oxidized by intracellular ROS to the green fluorescent compound dichlorofluorescein (DCF). The green
fluorescence intensity is proportional to the ROS level. As shown in Fig. 5, compared with the control group, the
green fluorescence of the H2O2 (Fig. 5A) or SNP group (Fig. 5B) was significantly increased, while three different
concentrations (2.5, 5, and 10 µM) compound 11 remarkably reduced the ROS levels in cells in the H2O2 or SNP
group. Oxidative stress potentially disrupts the homeostasis between pro-oxidant and antioxidant systems by
abnormally increasing ROS levels, indicating that compound 11 effectively antagonizes H2O2- or SNP- induced
increases in ROS levels, and may protect cells by reducing ROS levels.
3.6. Compound 11 restores the mitochondrial membrane potential (MMP)
A number of scholars have described the mitochondria as a target for the treatment of neurodegenerative
diseases[33]. Since mitochondria are the energy supply center for electron transport and oxidation reactions[34]
,
mitochondria are the main intracellular source of ROS production and are also particularly susceptible to oxidative
damage[35]. The MMP is the most direct and simplest measurement that reflects the function of mitochondria.
5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine (JC-1) is a dye that is commonly used to rapidly and
sensitively detect changes in the MMP of cells. In normal mitochondria, JC-1 aggregates to form a polymer,
emitting intense red fluorescence, while in unhealthy mitochondria, JC-1 exists as a monomer to produce green
fluorescence due to a decrease or loss of the MMP. Since green fluorescence indicates unhealthy mitochondria, a
shift in fluorescence from red to green indicates a reduction in the MMP. As illustrated in Fig. 6, red fluorescence
was more intense in the normal group compared with green fluorescence, suggesting that the mitochondria were in
a healthy state. After treatment with H2O2 (Fig. 6A) and SNP (Fig. 6B), cells with brighter green fluorescence were
observed, reflecting a decrease in the MMP. The conversion of the fluorescence from green to red in the compound
11 (2.5, 5, and 10 µM) group indicates that the MMP was restored.

3.7. Ability of compounds 1-14 to bind iron ions and Stoichiometries of the compound 11-Fe (II)complex
Iron is the core component of the mitochondrial enzyme complexes I-IV in the electron transport chain[10]
.
Notably, mitochondria are the major site of iron utilization forming iron sulfur clusters, that are essential for the
normal function of brain and biological processes. Excess iron ions cause mitochondrial dysfunction, which is
closely associated with neurodegenerative diseases[36]. In the present study, the ability of the target compounds to
chelate Fe (II) ions was assessed to explore the neuroprotective mechanisms of the compounds. We compared the
relative binding of Fe (II) by the compounds with ferrozine, as shown in Fig. 7. Ferrozine is often used as a
positive control due to its capability to interact with Fe (II) to form a purple complex that produces an absorption
signal at 562 nm[37]. The absorbance of the purple-colored Fe (II)-ferrozine complex was measured in the presence
or absence of targeted compounds. Compounds 5-14 inhibited the binding of ferrozine (30 µM) to iron, suggesting
that these compounds were more effective than ferrozine (Fig. 7). However, compounds 1-4 were not more active
than ferrozine, suggesting the importance of dihydroxy groups in iron chelation. IC50 values for compounds 5-11
(neuroprotection in vitro) ranged from 22-45 µM (Table 1). The above-mentioned outcomes supported the ability
of dihydroxy compounds to bind iron ions.
The mole ration method was used to evaluate the stoichiometric ratio of complex. The absorption spectra of
compound 11 and compound 11–Fe (II) complex were represented in Fig. 8A and 8B. It can be seen from the
spectrum that the maximum absorbance of compound 11 and compound 11–Fe (II) complex were both determined
at 305 nm. But at 380 nm, compound 11-Fe (II) complex showed intense absorption while compound 11 did not.
Therefore, 380 nm was selected to evaluate the stoichiometric ratio of the complex, thereby avoiding the
interference of compound 11 by mole ration method. In Fig. 8C, as the mole ratio of compound 11 to Fe (II)
increased, the absorbance first increased and then tended to be constant. When the mole ratio of compound 11 to
Fe (II) was equal to or more than 2:1, the curve became stable. Thence, it can be concluded that the stoichiometric
ratio of compound 11–Fe (II) complex was 2: 1.
3.8. Compound 11 alleviates cerebral ischemic injury
A rat model of middle cerebral artery occlusion (MCAO) was exposed to compound 11, and then the cerebral
infarction volume was evaluated to confirm the protective effect of compound 11 on ischemic disease. We
performed 2,3,5-triphenyltetrazolium chloride (TTC) staining, one of the most commonly used methods for
reliable macroscopic identification of an infarcted tissue[38]. After 24 h, brain sections were prepared and TTC
staining was performed. Compared with the cerebral infarction area of the sham-operated group and the
model-based control group (sham-operated: 0.00 ± 0.00%, model-based control group: 28.67 ± 2.90%), the
cerebral infarction areas in the positive control, low-dose, middle-dose, and high-dose groups were significantly
decreased (positive control: 15.76 ± 3.21%, low-dose: 20.52 ± 2.05%, middle-dose: 12.59 ± 1.83%, and high-dose:
18.06±2.24%,)(Fig. 9A and 9B). The effect of the middle-dose on alleviating the cerebral infarction area was
greater than the positive control.
3.9. Compound 11 improves behavior after ischemic stroke
After 24 h, the animals’ behaviors were scored using the 4-point scoring system described by Zea Long [33]
.
Compared with the behavioral scores of the sham-operated group and the model-based control group
(sham-operated: 0.00 ± 0.00, model-based control group: 2.54 ± 0.27), the scores of the positive control, low-dose,
middle-dose, and high-dose groups were noticeably lower (positive control: 1.64 ± 0.28, low-dose: 2.00 ± 0.33,
medium-dose: 1.54 ± 0.28, and high-dose: 1.73 ± 0.30), as shown in Fig.10. The medium-dose of compound 11
remarkably improved the behavior of animals after ischemic stroke, and its effect was superior to edaravone. The
results were consistent with previous findings, confirming that compound 11 attenuated cerebral ischemic injury.
As a result, the effect of medium-dose was better than that of high-dose, which may be due to the relatively narrow

therapeutic window of compound 11 or low animal sensitivity. Further repeated and in-depth mechanism of action
will be studied in the future work.
3.10. Compound 11 improves the serum levels of biochemical markers in a rat model of MCAO
We examined levels of superoxide dismutase (SOD), and malondialdehyde (MDA), and inflammatory
cytokines (interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α)) in experimental animal models to elucidate
the mechanism underlying the physiological improvement induced by compound 11 after cerebral
ischemia-reperfusion injury, and the results are shown in Fig. 11. The group treated with a medium dose of
compound 11 displayed significantly increased levels of SOD and MDA, but decreased levels of IL-1β and TNF-α,
and its effect was superior to edaravone.
3.11. Preliminary assessment of the acute toxicity of compound 11 by following an intraperitoneal injection into
Kunming mice
Solvent control group: After the intraperitoneal injection, the animals’ activity was slightly decreased, and
returned to a normal level after 12 h. The body weight increased normally within 7 days, and no obvious abnormal
reaction was noted.
Compound 11 group (400 mg/kg): After the intraperitoneal injection, the animal’ activity was slightly
decreased and returned to a normal level after 12 h. The body weight increased slowly within 7 days, and no
remarkable abnormal reaction was observed.
In summary, the LD50 of KM mice injected intraperitoneally with compound 11 was greater than 400 mg/kg
(Table 2).
3.12. Compound 11 induces Nrf2 nuclear translocation
Any disruption of redox homeostasis may lead to excess ROS production, causing oxidative damage. Nrf2 is
a stress-responsive transcription factor that is important for protecting cells from oxidative damage and particularly
neurodegenerative diseases[21]. The translocation of Nrf2 from the cytoplasm to the nucleus is the key factor to
induce the expression of antioxidants[39], including GSH, SOD, and others, which are up-regulated to remove
excess free radicals and protect nerve cells from oxidative stress[22]
.
We further explored whether compound 11 induced Nrf2 translocation from the cytoplasm to the nucleus.
After treating cells with compound 11 for 24 hours, we obtained total protein, nuclear protein, and cytoplasmic
protein extracts. A change in the level of total Nrf2 protein was not observed, and the level of the nuclear Nrf2
protein increased, while the cytosolic Nrf2 level decreased after 24 h of treatment with compound 11 (Fig. 12A).
The quantitative analysis revealed (Fig. 12B-D) that compound 11 promotes the translocation of Nrf2 to the
nucleus, potentially allowing cells to resist oxidative damage by regulating the translocation of the Nrf2 protein.
Antioxidant defense systems are essential for scavenging free radicals and protecting proteins and DNA from
peroxidation[40,41]. GSH is the main non-enzymatic antioxidant in cells, and GSH-px and SOD are antioxidant
enzymes[42]. Additionally, GSH, GSH-px, and SOD together increase the ability of cells to remove ROS through
endogenous antioxidant reactions [43]. In the present study, we measured the intracellular contents of these three
antioxidants. As shown in Table 3, treatment with H2O2 or SNP reduced the levels of GSH-px, SOD, and GSH in
PC12 cells. However, compound 11 increased their levels to some extent. The antioxidant activity of compound 11
was dose-dependent, particularly at 10 µM, and the levels of antioxidants in the cells were distinctly improved by
compound 11. Based on these results, compound 11 increased the cellular antioxidant activity by modulating the
levels of endogenous antioxidants.
Here, the PI3K inhibitor LY294002 was used to decrease the expression of Nrf2 and determine whether Nrf2
is involved in the neuroprotective effect of compound 11. As displayed in Fig. 13A and 13B, LY294002 reduced
the total level of Nrf2. Western blot analyses revealed a reversal of the Nrf2 by LY294002- induced decrease in

Nrf2 levels in cells treated with compound 11. Based on the results of the MTT assay, a treatment with 10 µM
compound 11 produced approximately 30% higher neuroprotective activity than the H2O2 group (Fig. 13C) the
SNP group (Fig. 13D), consistent with the results shown in Fig. 2. However, the addition of LY294002 reduced the
neuroprotective capacity of compound 11 to prevent H2O2- or SNP- induced cell death compared with co-treatment
with compound 11 and H2O2 or SNP. Thus, the Nrf2 pathway is at least partially involved in the neuroprotective
effects of compound 11 on PC12 cells.
3.13. Pharmacokinetics studies
In vivo PK properties of compound 11 were determined in rats. As shown in Table 4, the in vivo half-life (t1/2
)
of compound 11 for after oral administration and intravenous administration was 4.26 and 5.11 h, respectively, and
the peak time (Tmax) of plasma concentration after oral administration was 2.00 h. The area under the curve
(AUC0-∞) at the time of administration was 14160.53 and 2331.53 ng/mL*h, respectively. The drug retention time
were 6.32 and 2.37 h, respectively, and the relative bioavailability (F) of the drug was 60.76%. Based on these
characteristics, compound 11 possesses great advantages of drug-likeness characteristics.
4. Discussion
Excess oxidative stress and/or inflammation are postulated to play critical roles in the pathogenesis of
cerebral ischemia. Oxidative stress stimulates the expression of pro-inflammatory genes, and inflammatory factors
also trigger the excess production of reactive oxygen species (ROS), causing a vicious cycle[20]. Deferasirox, an
effective iron chelator, is considered resistant to oxidative stress[44]. Celecoxib is a commonly used
anti-inflammatory agent in the clinic. In this study, we used deferasirox and celecoxib to design and synthesize
new compounds with triazole and two phenol moieties to explore the antioxidant and anti-inflammatory effects in
vitro and in vivo.
Oxidative stress is a negative consequence of the imbalance between excess free radicals and the antioxidant
system[45]. Free radicals are divided into oxygen radicals and nitrogen radicals. Hydrogen peroxide is a substance
that induces the production of hydroxyl radicals, which are oxygen radicals. Sodium nitroprusside induces the
formation of nitric oxide radicals, which are nitrogen radicals[46]. At the same time, iron ions have been reported to
generate hydroxyl radicals through the Fenton reaction in the body[9,10]. The presence of a large number of free
radicals potentially causes a rapid increase in the levels of reactive oxygen species and produces neurotoxicity.
According to the results of the MTT assays, compounds 5-11 allowed cells to resist necrosis caused by oxidative
stress (both hydroxyl radicals and nitric oxide radicals), and compound 11 was the most active derivative.
Simultaneously, the finding supported the good antioxidant effect of iron chelators. Furthermore, compound 11
significantly inhibited the increased production of reactive oxygen species (ROS).
In recent years, mitochondria have been considered a target for neurodegeneration[33]. Mitochondria are
factories that provide energy. In addition, mitochondria are the center of electron production and transfer, rendering
them very vulnerable to oxidative damage[35]. The mitochondrial membrane potential (MMP) is the most direct
method for assessing mitochondrial function. Compound 11 effectively restored the decrease in the mitochondrial
membrane potential caused by oxidative stress. Thus, compound 11 may maintain stable mitochondrial function
when cells are exposed to an adverse environment.
Furthermore, in vivo experiments were performed to verify the findings from the cell-based experiments
described above. Cerebral ischemia is more common in the pathological process of various neurological diseases
and is also known as "small stroke"[47]. The rat model of middle cerebral artery occlusion (MCAO) is a model that
simulates human cerebral ischemia. In the MCAO model, neurological function was impaired resulting in
behavioral abnormalities, and the cerebral infarction volume increased with cytokine abnormalities. Based on our
data, compound 11 ameliorated the changes in rat behavior and reduced the cerebral infarct size. In addition, it
reduced the levels of inflammatory factors, including tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β).

Additionally, it increased the antioxidant capacity by increasing superoxide dismutase (SOD) levels and reducing
malondialdehyde (MDA) levels. Moreover, the 50% lethal dose (LD50) calculated in Kunming (KM) mice injected
intraperitoneally with compound 11 was greater than 400 mg/kg. Oral administration of compound 11 resulted in a
longer half-life, slower elimination, and higher bioavailability (relative to the intravenous injection, the
bioavailability is approximately 60.76%). Collectively, these data confirmed the anti-ischemic effect of compound
11 and supported its potential use as a promising neuroprotective agent for the treatment of ischemic stroke.
Additionally, the Nrf2 signaling pathway is essential for neuroprotection, particularly protection against
oxidative stress[21]. Generally, the nuclear transfer of Nrf2 is the key step to activate the antioxidant defense
system[39]. The intracellular antioxidant system includes antioxidant enzymes (such as SOD and GSH-px) and
non-enzymatic antioxidants (GSH)[22]. Strengthening the endogenous antioxidant defense system will allow the
cell resist exogenous oxidative damage. Here, compound 11 obviously increased the levels of SOD, GSH-px and
GSH in PC12 cells. LY294002, an inhibitor of the PI3K/AKT pathway, reduced Nrf2 levels in cells. Western
blots-showed that compound 11 promoted the translocation of Nrf2 from the cytoplasm to the nucleus, reversed the
changes in the Nrf2 content, and increased cell viability which was reduced by LY294002. Based on the data, Nrf2
pathway is at least partially involved in the neuroprotective effects of compound 11on PC12 cells.
In conclusion, in this study, we designed and synthesized a series of triazole compounds containing
substitutions with different groups. These compounds (5-11) were effective neuroprotectants that attenuated H2O2
or SNP- induced neurotoxicity. The compounds were effective Fe (II)-chelating agents, and significantly reduced
ROS production and maintained the change in the MMP in PC12 cells. Compound 11 outperformed curcumin or
edaravone. Compound 11 improved the behavioral score of rats after acute ischemic stroke, reduced the infarct
size in rats, and ameliorated the changes in the serum levels biochemical indicators (TNF-α, IL-1β, SOD, and
MDA) in a rat model of MCAO. This compound was also less toxic in vivo. Moreover, compound 11 promoted the
nuclear translocation of Nrf2 to activate the downstream signaling pathway and increased the activity of the
antioxidant system. Compound 11 exerts its neuroprotective effect by regulating antioxidants to reduce oxidative
stress and inflammation; thus, it represents as a potential neuroprotective agent for the treatment of ischemic stroke
in the future.
Scheme 1. Synthesis of triazole derivatives.
Reagents and conditions: i) SOCl2, xylene, Pyridine; ii) 4-hydrazinobenzene-1-sulfonamide hydrochloride,
Ethanol.

Figure 1. Design of triazole derivatives
Figure 2. Target compounds conferred protection against H2O2- or SNP-induced cell damage in PC12 cells.
(A) Cytotoxicity of H2O2 on PC12 cells. PC12 cells were treated with different concentration of H2O2 for 5 h.
(B) PC12 cells were pretreated with 10 µM curcumin and target compounds for 1 h followed by treatment with
500 µM H2O2 for another 5 h. (C) Cytotoxicity of SNP on PC12 cells. PC12 cells were treated with different doses
of SNP for 24 h. (D) PC12 cells were pretreated with 10 µM edaravone and target compounds for 1h followed by
treatment with 300 µM SNP for another 24 h. (Data are expressed as the mean ± SD of three replicates
***p<0.001,**p<0.01 and *p<0.5 compared to H2O2 or SNP group).

Figure 3. Neuroprotective effect of active compounds 5-11 on H2O2 or SNP induced PC12 damage. PC12 cells
were pretreated with compounds 5-11 and curcumin for 1 h followed by treatment with 500 µM H2O2 for another 5
h and measured by MTT (A) and LDH (B) assay. PC12 cells were pretreated with compounds 5-11 and edaravone
for 1 h followed by treatment with 300 µM SNP (C) or 400 µM SNP (D) for another 24 h. (Data are expressed as
the mean ± SD of three independent assays)
Figure 4. Compound 11 attenuates H2O2- or SNP-induced apoptosis of PC12 cells. Cell were pre-treated with
Compound 11 (2.5, 5 and 10 µM) or 10% FBS, 5% HS DMEM for 1 h and then incubated with or without. (A)
500 µM H2O2 for another 5 h. or (B) 300 µM SNP for another 24 h at 37℃. 10 µg/mL Hoechst were co-incubated
for 30 mins, then detected by fluorescent microscope. Scale bar=200 µm.
Figure 5. Compound 11 alleviated ROS production in PC12 cells. Pretreated with compound 11 (2.5, 5 and 10 µM)
for 1 h, 500 µM H2O2 (A) or 300 µM SNP (B) was added for another 5 or 24 h. Then 10 µM DCFH-DA was
incubated for 30 mins. Excitation and emission wavelengths were 488 nm and 525 nm, respectively, and ROS
level was detected by laser confocal. Scale Bar=50 µm.

Figure 6. Compound 11 restored the mitochondrial membrane potential of PC12 cells induced by H2O2 or SNP.
After pretreated with compound 11 (2.5, 5 and 10 µM) for 1 h, 500 µM H2O2 (A) or 300 µM SNP (B) was added
for another 5 or 24 h. Mitochondrial membrane potential was determined using JC-1 dye. Scale Bar=50 µm.
Figure 7. Fe (II)-coupling ability of compounds by phenanthrene assay. Chelation of ferrous ions by 15 µM FeSO4
was incubated with the tested compounds and with 30 µM ferrozine for 10 minS at room temperature. The
absorbance of the formed Fe (II)−ferrozine complex in the presence (Acompound) or absence of the tested compounds
(Acontrol) was measured at 562 nm.
Figure 8. Determination of stoichiometries of the complexes. (A) Absorption spectra of compound 11 (1×10 4
−
mol/L) were recorded between 275 and 500 nm. (B) 1mL 5×10−4 mol/L compound 11 and 1mL 5×10−4 mol/L
FeSO4·7H2O solution were mixed and diluted to 5 mL. Absorption spectra of compound 11-Fe (II) complex were
recorded between 275 and 500 nm. (C) 1 ml of 5×10-4 mol/L FeSO4·7H2O solution was added to each volumetric
flask, and followed by 0, 1, 2, 3, 4, 5, 6 ml of 5×10-4 mol / L compound 11, and then each volumetric flask was
diluted to 10 mL, respectively. The absorbance of each complex was measured at 380 nm. All absorption spectra
were recorded using a UV-2600 ultraviolet-visible spectrophotometer (Shimadzu, Japan)

A
B
Fig. 9 Cerebral infarction of rats in each group after 24 h of cerebral ischemia. (A) Effects of compound 11 on
acute cerebral ischemia in rats 24 h after MCAO. Rats were intraperitoneally injected with low dose (3mg/kg),
medium dose (10mg/kg) and high dose (30mg/kg) of compound 11 or edaravone (10mg/kg) at 2 h, 4 h and 6 h
after ischemia onset. TTC staining was used to obtain brain sections from each group of cerebral ischemia animals.
(B) The proportion of cerebral infarction area in each group of cerebral ischemia animals after 24 h. n=72, *P <
0.05 vs model control group; **P < 0.01 vs model control group
Fig. 10 Behavioral scoring results of cerebral ischemia in each group with reference to the 4-point scoring standard
of Zea Longa after 24 hours. n=72, *P < 0.05 vs model control group; **P < 0.01 vs model control group.

Fig. 11 Serum biochemical indicators in rats 24 hours after sham surgery or permanent MCAO. (A) Serum IL-1β;
(B) Serum TNF-α; (C) Serum SOD; (D) Serum MDA. n=72, * p<0.05 vs model control group; **p<0.01 vs model
control group.
Figure 12. Compound 11 promotes Nrf2 nuclear translocation. PC12 cells were treated with compound 11 (1.25,
2.5, 5 µM) for 24 h. Nuclear Nrf2, cytosolic Nrf2, and total Nrf2 were analyzed by Western blots. (A) Blot bands.
(B) Quantification of the nuclear Nrf2. (C) cytosolic Nrf2. (D) total Nrf2 blots (110 kDa). Results from three
independent experiments are expressed as means ± SD. ***p<0.001, **p<0.01 and *p<0.5 vs the control group

Figure 13. Nrf2 is involved in the cytoprotection of compound 11. (A) Validation of Nrf2 levels of LY294002 in
PC12 cells. (B) Quantification of the blot in A. (C) PI3K inhibitor LY294002 attenuated compound 11
neuroprotection in H2O2- induced PC12 cells model. (D) PI3K inhibitor LY294002 attenuated compound 11
neuroprotection in SNP- induced PC12 cells model.
Table 1. IC50 of compounds 5-11 Inhibition of Ferrozine-Fe2+
Compound
IC50 (µM) a
42.94±0.11
22.61±0.54
46.17±3.88
27.53±0.43
Compound
IC50 (µM)a
35.99±0.49
35.12±0.67
36.27±1.74
5
6
7
8
9
10
11
a Values represent the mean ± SD of three experiments. Binding rate%=(Acontrol-Acompound)/Acontrol*100%.
Table 2. Weight and Mortality of Male Mice compound 11 pre-test (n=4, g,
X
+SD)
Groups
Zeroth days
First days
Third
days
Fifth days Seventh days
Number of
deaths/total
0/10
Control
24.4±1.3
24.6±1.3
25.1±1.6
24.3±1.0
26.1±1.9
24.5±0.7
27.1±1.3
24.9±0.6
28.4±0.9
25.7±0.5
0/10
11
Table 3. Effect of compound 11 on GSH, GSH-px and SOD on H2O2 or SNP- Treated PC12 cells (n=3, Mean ±
S.D.)
GSH
GSH-px
SOD
GSH
GSH-px
SOD
(nM/mg)
(mU/mg)
(U/mg)
(nM/mg)
(mU/mg)
(U/mg)
88.85±1.47
65.52±1.44***
70.42±2.05
80.89±5.68
45.92±3.64**
51.03±3.82
42.59±1.86
29.69±1.65**
36.30±2.35
94.76±2.23
75.78±1.74**
79.07±1.99
55.70±3.38
37.70±2.88*
42.45±7.76
44.38±6.56
52.78±3.46#
42.87±1.82
23.74±2.02**
34.63±1.88
34.85±2.74#
39.07±3.83#
Control
Control
H2O2(500µM)
H2O2+11(2.5µM)
H2O2+11(5µM)
H2O2+11(10µM)
SNP (300µM)
SNP+11(2.5µM)
SNP+11(5µM)
SNP+11(10µM)
72.51±1.15#
80.57±1.16##
65.15±2.54#
77.94±1.62##
38.85±1.90#
41.19±1.40##
86.74±1.86#
89.16±1.72##
***p<0.001, **p<0.01 and *p<0.5 vs the control group; ##p<0.01 and #p<0.5 vs the H2O2 or SNP group.

Table 4. In vivo PK profiles of compound 11 in rat a, b
N.O.
t1/2 (h)
20 mg/kg (p.o.)
2mg/kg (i.v.)
5.11
4.26
2.00
Tmax (h)
0.083
Cmax (ng/ml)
AUC0-∞ (ng/ml*h)
MRT (h)
2027.47
14160.53
6.32
2746.08
2330.53
2.37
CL [(mg/kg)/(ng/ml)/h]
F (%)
0.0014
60.76
0.0009
-
a
t1/2, half-life; Tmax, time of maximum concentration; Cmax, maximum concentration; AUC0-∞, area under the
plasma concentration time curve; CL, clearance; and F, oral bioavailability.
b Data reported as the average of three animals.
Acknowledges
This research was financially supported by the Science and Technology Planning Program of Guangzhou (Grant
No. 201704020146) and Science and Technology Planning Program of Guangdong Province (Grant No.
509164523031).
Conflict of interest
The authors declare that they have no conflict of interest.
References
[1] Li, S. Y.; Lu, X. S.; Shao, Q.; Chen, Z., Huang, Q.; Jiao, Z.;Huang, X. D.; Yue, M. S.; Peng, J. W.;Zhou, X.;
Chao, D. C.; Zhao, H.; Ji, J. L.; Ji, Y. H.; Ji, Q. H. Early Histone Deacetylase Inhibition Mitigates
Ischemia/Reperfusion Brain Injury by Reducing Microglia Activation and Modulating Their Phenotype. Front
Neurol. 2019, 10, 893.
[2] Hu, J. N.; Huang, S. W.; Zhu, L.; Huang, W. J.; Zhao, Y. P.; Jin, K. L.; ZhuGe, Q. C. Tissue Plasminogen
Activator-Porous Magnetic Microrods forTargeted Thrombolytic Therapy after Ischemic Stroke. ACS Appl Mater
Inter. 2018, 10, 32988-32997.
[3] Benjamin, E. J.; Blaha, M. J.; Chiuve, S. E.; Cushman, M.; Das, S. R.; Deo, R.; et al. Heart disease and stroke
statistics-2017 update: a report from the American Heart Association. Circulation. 2017, 135, e146–e603.
[4] Jing, H. Y.; Liu, L. Y.; Jia, Y.; Yao, H. Y.; Ma, F. Z. Overexpression of the long non-coding RNA Oprm1
alleviates apoptosis from cerebral ischemia-reperfusion injury through the Oprm1/miR-155/GATA3 axis. Artif
Cells Nanomed Biotechnol. 2019, 47, 2431-2439.
[5] Ya, B. L.; Liu, Q.; Li, H. F.; Cheng, H. J.; Yu, T.; Chen, L.; Wang, Y.; Yuan, L. L.; Li, W. J.; Liu, W. Y.; Bai, B.
Uric Acid Protects against Focal CerebralIschemia/Reperfusion-Induced Oxidative Stress via ActivatingNrf2 and
Regulating Neurotrophic Factor Expression. Oxid Med Cell Longev. 2018, 2018, 6069150.
[6] Matsuda, T.; Zhai, P. Y.; Sciarretta, S.; Zhang, Y.; Jeong, J. I.; Ikeda, S.; Park, J.; Hsu, C.; Tian, B.; Pan, D. J.;
Sadoshima, J.; Del Re, D. P. NF2 Activates Hippo Signaling and Promotes Ischemia/Reperfusion Injury in the
Heart. Circ Res. 2016, 119, 596-606.
[7] Peyravian, N.; Dikici, E.; Deo, S.; Toborek, M.; Daunert, S. Opioid antagonists as potential therapeutics for
ischemic stroke. Prog Neurobiol. 2019, 101679.
[8] Kerins, M. J.; Ooi, A. The Roles of NRF2 in Modulating Cellular Iron Homeostasis. Antioxid Redox Sign.
2018, 29, 1756-1773.
[9] Illes, E.; Mizrahi, A.; Marks, V.; Meyerstein, D. Carbonate-radical-anions, and not hydroxyl radicals, are the
products of the Fenton reaction in neutral solutions containing bicarbonate. Free Radic Biol Med. 2019, 131, 1-6.

[10] Szulczyk, D.; Dobrowolski, M. A.; Roszkowski, P.; Bielenica, A.; Stefanska, J.; Kolinski, M.; Kmiecik, S.;
Jozwiak, M.; Wrzosek, M.; Olejarz, W.; Struga, M. Design and synthesis of novel 1H-tetrazol-5-amine based
potent antimicrobial agents: DNA topoisomerase IV and gyrase affinity evaluation supported by molecular
docking studies. Eur J Med Chem. 2018, 156, 631-640.
[11] Abdel-Aziz, A. A.; Abou-Zeid, L. A.; ElTahir, K. E. H.; Ayyad, R. R.; El-Sayed, M. A.; & El-Azab, A. S.
Synthesis, anti-inflammatory, analgesic, COX-1/2 inhibitory activities and molecular docking studies of
substituted 2-mercapto-4(3H)-quinazolinones. Eur J Med Chem. 2016, 121, 410-421.
[12] Bertamino, A.; Iraci, N.; Ostacolo, C.; Ambrosino, P.; Musella, S.; Di Sarno, V.; Ciaglia, T.; Pepe, G.; Sala, M.;
Soldovieri, M. V.; Mosca, I.; Gonzalez-Rodriguez, S.; Fernandez-Carvajal, A.; Ferrer-Montiel, A.; Novellino, E.;
Taglialatela, M.; Campiglia, P.; Gomez-Monterrey, I. Identification of a Potent Tryptophan-Based TRPM8
Antagonist With in Vivo Analgesic Activity. J Med Chem. 2018, 61, 6140-6152.
[13] Gu, Z. S.; Zhou, A. N.; Xiao, Y.; Zhang, Q. W.; Li, J. Q. Synthesis and antidepressant-like activity of novel
aralkyl piperazine derivatives targeting SSRI/5-HT1A/5-HT7. Eur J Med Chem. 2018, 144, 701-715.
[14] Lewandowski, M.; Gwozdzinski, K. Nitroxides as Antioxidants and Anticancer Drugs. Int J Mol Sci. 2017, 18,
2490.
[15] Iraji, A.; Firuzi, O.; Khoshneviszadeh, M.; Tavakkoli, M.; Mahdavi, M.; Nadri, H.; Edraki, N.; Miri, R.
Multifunctional iminochromene-2H-carboxamide derivatives containing different aminomethylene triazole with
BACE1 inhibitory, neuroprotective and metal chelating properties targeting Alzheimer's disease. Eur J Med Chem.
2017, 141, 690-702.
[16] El-Sherief, H. A. M.; Youssif, B. G. M.; Abbas Bukhari, S. N.; Abdelazeem, A. H.; Abdel-Aziz, M.;
Abdel-Rahman, H. M. Synthesis, anticancer activity and molecular modeling studies of 1,2,4-triazole derivatives
as EGFR inhibitors. Eur J Med Chem. 2018, 156, 774-789.
[17] Singh, R.; Thota, S.; & Bansal, R. Studies on 16,17-Pyrazoline Substituted Heterosteroids as Anti-Alzheimer
and Anti-Parkinsonian Agents Using LPS Induced Neuroinflammation Models of Mice and Rats. ACS Chem
Neurosci. 2018, 9 , 272-283.
[18] Guo, J. W.; Guan, P. P.; Ding, W. Y.; Wang, S. L.; Huang, X. S.; Wang, Z. Y.; Wang, P. Erythrocyte
membrane-encapsulated celecoxib improves thecognitive decline of Alzheimer's disease by concurrently
inducingneurogenesis and reducing apoptosis in APP/PS1 transgenic mice. Biomaterials. 2017, 145, 106-127.
[19] Sayed, A. S.; El Sayed, N. S. Co-administration of 3-Acetyl-11-Keto-Beta-Boswellic Acid Potentiates the
Protective Effect of Celecoxib in Lipopolysaccharide-Induced Cognitive Impairment in Mice: Possible Implication
of Anti-inflammatory and Antiglutamatergic Pathways. J Mol Neurosci. 2016, 59, 58-67.
[20] Lv, H. M.; Liu, Q. M.; Wen, Z. M.; Feng, H. H.; Deng, X. M.; Ci, X. X. Xanthohumol ameliorates
lipopolysaccharide (LPS)-induced acute lunginjury via induction of AMPK/GSK3β-Nrf2 signal axis. Redox Biol.
2017, 12, 311-324.
[21] Dinkova-Kostova, A. T.; Kostov, R. V.; Kazantsev, A. G. The role of Nrf2 signaling in counteracting
neurodegenerative diseases. FEBS J. 2018, 285, 3576-3590.
[22] Kavian, N.; Mehlal, S.; Jeljeli, M.; Saidu, N. E. B.; Nicco, C.; Cerles, O.; Chouzenoux, S.; Cauvet, A.; Camus,
C.; Ait-Djoudi, M.; Chéreau, C.; Kerdine-Römer, S.; Allanore, Y.; Batteux, F. The Nrf2-Antioxidant Response
Element Signaling Pathway Controls Fibrosis and Autoimmunity in Scleroderma. Front Immunol. 2018, 9, 1896.
[23] Salehi, S.; Saljooghi, A. S.; Izadyar, M. A theoretical study on the electronic structures and equilibrium
constants evaluation of deferasirox iron complexes. Comput Biol Chem. 2016, 64, 99-106.
[24] Hassan, G. S.; Abou-Seri, S. M.; Kamel, G.; Ali, M. M. Celecoxib analogs bearing benzofuran moiety as
cyclooxygenase-2 inhibitors: Design, synthesis and evaluation as potential anti-nflammatory agents. Eur J Med
Chem. 2014, 76, 482-493.

[25] Azran, S.; Danino, O.; Förster, D.; Kenigsberg, S.; Reiser, G.; Dixit, M.; Singh, V.; Major, D.T.; Fischer, B.
Identification of highly promising anti-oxidants/neuroprotectants based on nucleoside 5'-phosphorothioate scaffold.
Synthesis, activity, and mechanisms of action. J Med Chem. 2015, 58, 8427-8443.
[26] Fu, Z. F.; Chen, R. Study of Complexes of tannic acid with Fe(III) and Fe(II). J Anal Methods Chem. 2019,
2019, 3894571.
[27] Sungur, S.; Uzar, A. Investigation of complexes tannic acid and myricetin with Fe(III). Spectrochim Acta A.
2008, 69, 225-229.
[28] Trotman-Lucas, M.; Kelly, M. E.; Janus, J.; Gibson, C. L.Middle Cerebral Artery Occlusion Allowing
Reperfusion via Common Carotid Artery Repair in Mice. J Vis Exp. 2019, 143, e58191.
[29] Zhao, Q. P.; Wang, X. B.; Chen, A, L.; Cheng, X. L.; Zhang, G. X.; Sun, J. M.; Zhao, Y. S.; Huang, Y.; Zhu, Y.
F. Rhein protects against cerebral ischemic-/reperfusion-induced oxidative stress and apoptosis in rats. Int J Mol
Med. 2018, 41, 2802-2812.
[30] Yuan, J. N.; Zeng, C.; Cao, W.; Zhou, X. X.; Pan, Y.; Xie, Y. H.;Zhang, Y. H.; Yang, Q.; Wang, S. W.
Bufalin-Loaded PEGylated Liposomes: Antitumor Efficacy, Acute Toxicity, and Tissue Distribution. Nanoscale
Res Lett. 2019, 14, 223.
[31] Wang, M. S.; Zhuo, L. S.; Yang, F. P.; Wang, W. J.; Huang, W.; Yang, G. F. Synthesis and biological
evaluation of new MET inhibitors with 1,6-naphthyridinone scaffold. Eur J Med Chem. 2020, 185, 111803.
[32] Zhao, C. Y.; Zhang, J. L.; Hu, H. Y.; Qiao, M. X.; Chen, D. W.; Zhao, X. L.; Yang, C. R. Design of lactoferrin
modified lipid nano-carriers for efficient brain-targeteddelivery of nimodipine. Mat Sci Eng C-Mater. 2018, 92,
1031-1040.
[33] Salinaro, A. T.; Pennisi, M.; Paola, R. D.; Scuto, M.; Crupi, R.; Cambria, M. T.; Ontario. M. L.; Tomasello, M.;
Uva, M.; Maiolino, L.; Calabrese, E. J.; Cuzzocrea, S.;Calabrese, V. Neuroinflammation and neurohormesis in the
pathogenesis of Alzheimer's disease and Alzheimer-linked pathologies: modulation by nutritional mushrooms.
Immun Ageing. 2018, 15, 8.
[34] Liao, L. P.; Shi, J. G.; Jiang, C. B.; Zhang, L. T.; Feng, L. S.; Liu, J. Y.; Zhang, J. X. Activation of anti-oxidant
of curcumin pyrazole derivatives throughpreservation of mitochondria function and Nrf2 signaling pathway.
Neurochem Int. 2019, 125, 82-90.
[35] Buglak, N. E.; Batrakova, E. V.; Mota, R.; Bahnson, E. S. M. Insights on Localized and Systemic Delivery of
Redox-Based Therapeutics. Oxid Med Cell Longev.2018, 2468457.
[36] Amit, T.; Bar-Am, O.; Mechlovich, D.; Kupershmidt, L.; Youdim, M. B. H.; Weinreb, O. The novel
multitarget iron chelating and propargylamine drug M30 affects APP regulation and processing activities in
Alzheimer's disease models. Neuropharmacology. 2017, 123, 359-367.
[37] Azran, S.; Danino, O.; Forster, D.; Kenigsberg, S.; Reiser, G.; Dixit, M.; Singh, V.; Major, D. T.; Fischer, B.
Identification of Highly Promising Antioxidants/Neuroprotectants Based on Nucleoside 5'-Phosphorothioate
Scaffold. Synthesis, Activity, and Mechanisms of Action. J Med Chem. 2015, 58, 8427-8443.
[38] Kim, R.; Hur, D.; Kim, H. K.; Han, J.; Mishchenko, N. P.; Fedoreyev, S. A.;Stonik, V. A.; Chang, W.
Echinochrome A Attenuates Cerebral Ischemic Injury through Regulation of Cell Survival after Middle Cerebral
Artery Occlusion in Rat. Mar Drugs. 2019, 17, 501.
[39] Xu, L. L.; Zhang, X.; Jiang, Z. Y.; You, Q. D. Molecular similarity guided optimization of novel Nrf2
activators with 1,2,4-oxadiazole core. Bioorg Med Chem. 2016, 24, 3540-3547.
[40] Fang, C.; Gu, L.; Smerin, D.; Mao, S.; Xiong, X. The Interrelation between Reactive Oxygen Species and
Autophagy in Neurological Disorders. Oxid Med Cell Longev. 2017, 2017, 8495160/1-8495160/16.
[41] Panahi, Y.; Rajaee, S. M.; Johnston, T. P.; Sahebkar, A. Neuroprotective effects of antioxidants in the
management of neurodegenerative disorders: A literature review. J Cell Biochem. 2019, 120 , 2742-2748.

[42] Liguori, I.; Russo, G.; Curcio, F.; Bulli, G.; Aran, L.; Della-Morte, D.;Gargiulo, G.; Testa, G.; Cacciatore, F.;
Bonaduce, D.; Abete, P. Oxidative stress, aging, and diseases. Clin Interv Aging. 2018, 13, 757-772.
[43] Pohl, F.; Kong Thoo Lin, P. The Potential Use of Plant Natural Products and Plant Extracts with Antioxidant
Properties for the Prevention/Treatment of Neurodegenerative Diseases: In Vitro, In Vivo and Clinical Trials.
Molecules. 2018, 23, 3283.
[44] Jimenez-Solas, T.; Lopez-Cadenas, F.;Aires-Mejia, I.; Caballero-Berrocal, J. C.; Ortega, R.; Redondo, A. M.;
Sanchez-Guijo, F.; Muntion, S.; Garcia-Martin, L.; Albarran, B.; AlonsoJ. M.; Canizo C. D.;
Hernandez-HernandezA.; Dıez-Campelo M.Deferasirox reduces oxidative DNA damage in bone marrowcells from
myelodysplastic patients andimproves their differentiationcapacity. Brit J Haematol. 2019, 187, 93-104.
[45] Tonnies, E.; Trushina, E. Oxidative Stress, Synaptic Dysfunction, and Alzheimer’s Disease. J Alzheimers Dis.
2017, 57, 1105-1121.
[46] Zheng, W. H.; Chong, C. M.; Wang, H. T.; Zhou, X. H.; Zhang, L.; Wang, R. K.; Meng, Q.; Lazarovici, P.;
Fang, J. K. Artemisinin conferred ERK mediated neuroprotection to PC12 cells and cortical neurons exposed to
sodium nitroprusside-induced oxidative insult. Free Radical Bio Med. 2016, 97, 158-167.
[47] Kim, Y. O.; Kim, H. J.; Abu-Taweel, G. M.; Oh. J. S.; Sung, G. H. Neuroprotective and therapeutic effect of
Cordyceps militaris onischemia-induced neuronal death and cognitive impairments. Saudi J Biol Sci. 2019, 26,
1352-1357.