SUPPLEMENTARY MATERIAL
The semisynthetic spin-labeled derivatives of 3-hydroxybutanolide as potential
oxidative stress inhibitors
Qing Liua, Zhen-Ling Liub*, Jing Tianc, Wei Shib and Yin-qian Liuc
a
Department of Chemical Engineering and Pharmacy, College of Chemical
Engineering, Huaqiao University, Xiamen, Fujian 362011, P.R. China; bState Key
Laboratory of Applied Organic Chemistry, College of Chemistry and
Engineering, Lanzhou University, Lanzhou 730000, P. R. China;
Chemical
c
School of
Pharmacy, Lanzhou University, Lanzhou, 730000, P.R. China
Abstract To obtain more accessible oxidative stress inhibitors, a series of novel
spin-labeled derivatives of 3-hydroxybutanolide (2a–d, 3a–d) with the natural active
compound (kinsenoside) as the lead compound was designed, synthesized from the
nitroxide free radical piperidine (pyrroline) and the main structural unit of kinsenoside:
3-hydroxybutanolide. Antioxidant activity screening of these derivatives was
performed using MTT method on rat pheochromocytoma PC12 cells. The
antioxidative stress effect was further investigated on the changes of the important
antioxidant enzyme activities and intracellular ROS production. Among the
derivatives, 2b-d, 3a and 3c showed comparable or superior antioxidative stress
activity to kinsenoside. Also, most of tested derivatives displayed obvious
antioxidative ability in concentrations. Cytotoxic assay simultaneously indicated that
all compounds had very low toxicity to normal cells. Based on the observed results,
the structure–activity relationship (SAR) of these derivatives was discussed.
Keywords: 3-hydroxybutanolide, spin-labeled, antioxidant activity, synthesis.
Experimental
Materials and general methods
Materials
Rat pheochromocytoma PC12 cells were purchased from Shanghai Fu Xiang
Biological Technology Co., Ltd (Shanghai, China). Cell culture products were
purchased from Gibco (GranIsland, NY, USA). All chemicals were from Sigma (St
Louis, MO, USA). Reagent kits of superoxide dismutase (SOD) and catalase (CAT)
*
Corresponding author. Email: liuzhl@lzu.edu.cn
1
came from the Nanjing jiancheng Biological Engineering Institute (Nanjing, China).
Vitamin E was from Xiamen xingsha Pharmaceutical Co (Xiamen, China). Silica gel
200–300 mesh for column chromatography and silica GF254 for TLC were supplied
by the Qingdao Marine Chemical Inc., China. A. roxburghii was purchased from
Fuzhou Yuefeng Chinese herb medicine Co. Ltd., China, batch NO. 060501 and
identified by Prof. Chen-Zi Yang of Chinese medicine identification section of Fujian
university of traditional Chinese medicine. Kinsenoside was obtained according to the
reference (Liu et al. 2013).
General methods
Melting points were determined with a Kofler apparatus which was uncorrected.
IR spectra were measured on a Nicolet 5DX-FT-IR spectrometer on neat samples
placed between KBr plates.
1
H NMR spectra were measured on a Varian
Mecury-400BB spectrometer with TMS as an internal standard. Optical rotations
were measured on a Perkin Elmer 341 spectrometer. Mass spectra were recorded on a
Bruker Daltonics APEX II 49e and VGZAB-HS (70 ev) spectrometer with ESI and
FAB source as ionization, respectively. The electron spin resonance (ESR) spectra
were obtained from 10-5 M methyl alcohol solution, using a Bruker ER200D-SRC
spectrometer.
Physical and spectroscopy characters of kinsenoside and compounds 2a–d and
3a–d.
Kinsenoside (Fig. 1) was identified since its 1D NMR (1HNMR and
13
CNMR),
ESI-MS and optical rotation were consistent with that of kinsenoside (Liu et al. 2013;
Suzuki et al. 2005).
2
Table S1. The cytotoxicity of compounds on PC12 cells.
test groups with different dosage
compound
kin
2a
3a
2b
3b
2c
3c
2d
3d
control
100±0.00
10 (μg/mL)
30 (μg/mL)
50 (μg/mL)
70 (μg/mL)
90 (μg/mL)
99.03±8.95
99.05±8.43
99.62±19.35
100.58±4.90
106.78±12.59
115.88±5.75
96.99±14.61
99.90±9.83
100.17±11.15
94.76±0.14
99.91±11.97
98.41±16.64
97.66±1.80
102.11±11.80
97.01±11.95
97.74±10.77
99.05±17.32
103.09±7.73
100.35±7.40
108.25±14.25
96.15±13.96
104.95±7.53
105.56±14.58
114.14±5.70
100.07±13.10
108.40±11.26
107.47±15.75
97.12±9.76
90.81±9.10
96.87±16.35
102.79±10.19
103.52±14.84
102.66±10.90
96.06±16.29
103.54±16.73
100.69±19.15
98.78±10.65
99.29±10.60
93.33±12.65
99.38±3.76
93.13±7.16
97.70±8.71
101.66±4.93
106.25±16.06
108.10±6.98
Data are expressed as a percentage of control and are the mean ± SD from 6 experiments. A value of P
< 0.05 was considered to be statistically difference.
References
Liu ZL, Liu Q, Xiao B, Zhou J, Zhang JG, Li Y. 2013. The vascular protective
properties of kinsenoside isolated from Anoectochilus roxburghii under high
glucose condition. Fitoterapia. 86: 163-170.
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K,
Suzuki N,
Yainaura
M,
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3-O-β-D-glucopyranosyl-(3R)-hydroxybutanolide
3-O-β-D-glucopyranosyl-(3S)-hydroxybutanolide
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3
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(Kinsenoside)
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