The semisynthetic spin-labelled derivatives of 3-hydroxybutanolide as potential oxidative stress inhibitors

Article abstract of DOI:10.1080/14786419.2014.903477

To obtain more accessible oxidative stress inhibitors, a series of novel spin-labelled derivatives of 3-hydroxybutanolide (2a-d, 3a-d) with the natural active compound (kinsenoside) as the lead compound were designed, synthesised 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 reactive oxygen species production. Among the derivatives, 2b-d, 3a and 3c showed comparable or superior antioxidative stress activity to kinsenoside. Also, most of the 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 of these derivatives was discussed.

Full text of DOI:10.1080/14786419.2014.903477

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Natural Product Research: Formerly
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The semisynthetic spin-labelled
derivatives of 3-hydroxybutanolide as
potential oxidative stress inhibitors
Qing Liua, Zhen-Ling Liub, Jing Tianc, Wei Shib & Yin-Qian Liuc
a Department of Chemical Engineering and Pharmacy, College of
Chemical Engineering, Huaqiao University, Xiamen, Fujian 362011,
P.R. China
b State Key Laboratory of Applied Organic Chemistry, College of
Chemistry and Chemical Engineering, Lanzhou University, Lanzhou
730000, P.R. China
c School of Pharmacy, Lanzhou University, Lanzhou 730000, P.R.
China
Published online: 04 Apr 2014.
To cite this article: Qing Liu, Zhen-Ling Liu, Jing Tian, Wei Shi & Yin-Qian Liu (2014) The
semisynthetic spin-labelled derivatives of 3-hydroxybutanolide as potential oxidative stress
inhibitors, Natural Product Research: Formerly Natural Product Letters, 28:14, 1037-1044, DOI:
10.1080/14786419.2014.903477
To link to this article: http://dx.doi.org/10.1080/14786419.2014.903477
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Natural Product Research, 2014
Vol. 28, No. 14, 1037–1044, http://dx.doi.org/10.1080/14786419.2014.903477
The semisynthetic spin-labelled derivatives of 3-hydroxybutanolide as
potential oxidative stress inhibitors
Qing Liua, Zhen-Ling Liub*, Jing Tianc, Wei Shib and Yin-Qian Liuc
aDepartment of Chemical Engineering and Pharmacy, College of Chemical Engineering, Huaqiao
University, Xiamen, Fujian 362011, P.R. China; State Key Laboratory of Applied Organic Chemistry,
b
College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou 730000, P.R. China;
cSchool of Pharmacy, Lanzhou University, Lanzhou 730000, P.R. China
(Received 6 November 2013; final version received 9 March 2014)
To obtain more accessible oxidative stress inhibitors, a series of novel spin-labelled
derivatives of 3-hydroxybutanolide (2a–d, 3a–d) with the natural active compound
(kinsenoside) as the lead compound were designed, synthesised 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 reactive oxygen species production. Among the
derivatives, 2b–d, 3a and 3c showed comparable or superior antioxidative stress
activity to kinsenoside. Also, most of the 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 of these derivatives was discussed.
Keywords: 3-hydroxybutanolide; spin-labelled; antioxidant activity; synthesis
1. Introduction
There are many derivatives of 3-hydroxybutanolide existing in nature. Kinsenoside is a classic
one. It is 3-(R)-3-b-D-glucopyranosyloxybutanolide (Figure 1) and a major ingredient of
Anoectochilus roxburghii which is one rare species belonging to the genus Anoectochilus
(Orchidaceae). A. roxburghii is a Chinese folk medicine used to treat diabetes (Zhang et al.
2007), cancers (Tseng et al. 2006), liver diseases (Shih et al. 2004), cardiovascular diseases
(Liu et al. 2013), etc. A. roxburghii is also made into popular nutraceutical healthcare products in
China and other Asian countries because of its healthcare effects. Recently, kinsenoside
exhibited significantly antihyperglycaemic activity and possessed antioxidant, anti-inflamma-
tory, immunostimulating and hepatoprotective activities (Wu et al. 2007; Du et al. 2008; Hsiao
et al. 2013). Zhang et al. (2007) reported that the antihyperglycaemic effect of kinsenoside might
be attributed to modulating the activity of enzymatic antioxidants, scavenging free radicals and
reducing the content of factor NO in diabetic rats. In our previous study, kinsenoside showed
significant vascular protective effects in in vitro and in vivo experiments. And this effect of
kinsenoside was speculated due to the oxidative stress inhibition and the reduction of nuclear
factor kappa B (NF-kB) mRNA expression levels in high glucose conditions (Liu et al. 2013).
Researches have revealed that oxidative stress was caused by an imbalance between the
production of reactive oxygen species (ROS) and a biological system’s ability to readily detoxify
*Corresponding author. Email: liuzhl@lzu.edu.cn
q 2014 Taylor & Francis

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Q. Liu et al.
Figure 1. The structure of kinsenoside (1) and the synthesis of compounds 2a–d and 3a–d.
the reactive intermediates or to repair the resulting damage. It is thought that oxidative stress was
involved in the progression of pathogenesis of diabetes, atheromatous plaque, stroke,
Alzheimer’s disease, Parkinson’s disease, etc. (Dewapriya et al. 2013).
Oxidative stress resistance strategy is used to treat the diseases related to oxidative stress and
proved to be a practicable treatment based on the root cause of diseases. Antioxidant stress drugs
are the key to oxidative stress resistance strategy. Many known antioxidants, such as ferulic acid
and quercetin (Hsieh et al. 2010), cannot be ideal antioxidant stress drugs because of their
prooxidant side effects on normal cells or being synergies with other agentia, such as vitamin C
or glutathione. The discovery of a new antioxidant and development of excellent antioxidant
stress drugs have still attracted the interest of researchers. Kinsenoside is a good potential
oxidative stress inhibitor (Du et al. 2008). Our results also showed that kinsenoside was a good
antioxidant lead. In the study, the protective effect of kinsenoside against H2O2-induced
oxidative damage in PC12 cells was obvious (Table 2) and the toxicity of kinsenoside to normal
PC12 cells was quite low (Table S1). But kinsenoside is not easily available because of scarce
natural resources and artificially synthesised approach which is a multi-step process or difficult
in separating (Zhang et al. 2005). A series of derivatives with kinsenoside as the lead compound
were therefore designed to obtain more accessible antioxidants (Figure 1 and Table 1).
(R)-3-Hydroxybutyrolactone is the main structural unit of kinsenoside and might play an
important role for biological function of kinsenoside. The stable nitroxides are the antioxidants
which can provide cytoprotection in mammalian cells against diverse types of oxidative insult and
identifystructuraldeterminantsoptimalforprotectionagainst individualtypesofdamage. Synthesis
of stable nitroxyl radicals spin-labelled bioactive molecules was proved to be an efficient method to
obtain the effective therapeutic agents with high activities and less toxicity to normal cells
(Hendricks et al. 2012). Herein, we continue the study of our group on the discovery and
development of bioactive spin-labelled molecules derived from natural products (Liu et al. 2012).
Stable nitroxyl radicals spin-labelled derivatives of 3-hydroxybutanolide (2a–d) and the
corresponding epimers (3a–d) were synthesised and biologically evaluated because the
configuration at 3-position was simultaneously considered as another important factor affecting
bioactivities.
2. Results and discussion
The new compounds (2a–d, 3a–d) synthesised (Figure 1, Table 1) were screened for
antioxidant activity through the suppression test on the H2O2-induced oxidative stress damage in

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PC12 cells by the MTT method. As shown in Table 2, all compounds exhibited in vitro
antioxidant activity within the experimental dose range (10–90 mg/mL). Particularly, like
kinsenoside, compounds 2b–d, 3a and 3c remarkably protected PC12 cells against H2O2-
induced cytotoxicity on different doses and simultaneously had very low toxicity to normal cells
in the cytotoxicity assay (Table S1). The results showed that the replacement of 3-glycosyl group
in the kinsenoside structure with nitroxide radical could bring about comparable or superior
antioxidants to kinsenoside.
The antioxidative stress ability of the molecules was further studied. The effects of 2a–d,
3a–d on up-regulating the activities of the two important antioxidant enzymes (superoxide
dismutase (SOD) and catalase (CAT)) and down-regulating intracellular ROS production were
detected in injured PC12 cells treated with H2O2 (Figure 2). As seen in Figure 2(A)–(C), SOD
seemed sensitive to all compounds although the activity of CAT substantially had consistent
change trend with that of SOD. Meanwhile, the intracellular ROS production showed a contrary
tendency with the changes of the antioxidant enzyme activity after the cells were pretreated with
the compounds. Among the compounds, 2b–d, 3a and 3c increased the antioxidant enzyme
activity and inhibited intracellular ROS generation significantly.
Based on these results, some preliminary structure–activity relationships (SAR) on these
derivatives werethen investigated. Forthe derivatives(2a–d) with 3R-configuration, they generally
emerges higher antioxidative ability than the derivatives (3a–d) with 3S-configuration in no matter
screening test or the enzyme activity assay, or even in the intracellular ROS detection. This
phenomenon is analogical to the phenomenon that kinsenoside has higher anti-hyperliposis effect
than its epimer: (3S)-3-b-D-glucopyranosyloxy (butanolide, goodyeroside A) (Du et al. 2008).
A possible explanation for this phenomenon is that configuration affects the affinity between drug
molecules and receptor. This phenomenon also provides an evidence for (R)-3-hydroxybutyr-
olactone as the main active structural unit of kinsenoside. In addition, in the experiments of this
study, the activities of derivatives of (R)-3-hydroxybutanolide with saturated pyrroline (piperidine)
(2b and 2d) were stronger than those of derivatives with unsaturated pyrroline (piperidine) (2a and
2c). But the reverse happened in the derivatives of (S)-3-hydroxybutanolide with piperidine and
piperidine (3a . 3b, 3c . 3d). The law and the mechanism are a subject of further investigations.
In summary, a series of novel spin-labelled derivatives of 3-hydroxybutanolide with
kinsenoside as the lead compound were designed, synthesised and evaluated to develop more
potential oxidative stress inhibitors. Some derivatives (2b–d, 3a and 3c) showed very
impressive antioxidative stress activity and the 3-nitroxide radical derivative was demonstrated
an effective method to obtain potential oxidative stress inhibitors. The different antioxidative
activity of these compounds indicated that the 3-configuration of kinsenoside structure markedly
affected the activity profiles of this compound class, and the important SAR information has
been revealed. It will pave the way for the design and development of derivatives of
3-hydroxybutanolide as potential oxidative stress inhibitors in the future.
3. Experimental
3.1. Materials and general methods (See supplementary material)
3.2. Synthesis
The nitroxide free radical piperidine (pyrroline) was synthesised according to the literature
(Elmer et al. 1976). As described in Figure 1, under argon atmosphere, (R)-3-
hydroxybutyrolactone or (S)-3-hydroxybutyrolactone (102 mg, 1.0 mmol) and nitroxide free
radical piperidine (pyrroline) (1.0 mmol) were dissolved in dry dichloromethane (DCM)
(20 mL). Then 4-dimethylaminopyridine (24 mg, 0.2 mmol) was added into the above solution.
After the mixture was stirred for 5 min at room temperature, dicyclohexyl carbodiimine (206 mg,

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Figure 2. Effects of compounds on the activities of SOD, CAT and ROS production on H2O2-injured PC12
cells. Data are mean ^ SD from three experiments. A value of P , 0.05 was considered to be statistically
*
difference. P , 0.05, **P , 0.01 versus control; #P , 0.05, ##P , 0.01 versus model group.
1.0 mmol) was successively added. The reaction mixture was stirred for another 2 h until TLC
revealed the absence of the starting material. The mixture was filtered and the filtrate was
concentrated in vacuum. The residue was purified by column chromatography on silica gel using
DCM/acetone (15/1) as eluent to give compounds 2a–d, 3a–d.
3.3. Physical and spectroscopy characters of kinsenoside and compounds 2a–d
and 3a–d (See supplementary material and Table 1)
3.4. In vitro antioxidant activity assay
3.4.1. Cell culture
PC12 cells were seeded onto 96-well plates at a density of 1.0 £ 105 cells/mL in Dulbecco’s
Modified Eagle’s Medium, supplemented with 10% new calf serum, 100 U/mL penicillin, 100 mg/
mL streptomycin and 2 mM L-glutamine at 378C under a 5% CO2 humidified atmosphere.

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3.4.2. Effect of the derivatives on cells viability
To assess the cytotoxicity of the derivatives of 3-hydroxybutanolide (10–90 mg/mL), the cells
were incubated in MTT solution (0.5 mg/mL) in phosphate-buffered saline (PBS) for 4 h. The
formation of purple formazan was quantified by measuring the absorbance at a wavelength of
570 nm with background subtraction using a wavelength of 630 nm on a microplate reader
(Thermo Fisher Scientific, Inc., Waltham, MA, USA)
3.4.3. The cell protection assay
To test the protective effects of kinsenoside and the derivatives in PC12 cells, the proliferative
activity was evaluated (Li et al. 2003). Hydrogen peroxide (H2O2) was stored at 48C, and
100 mM stock solution was freshly prepared in PBS before being applied to the cultures with a
final concentration of 200 mM. The PC12 cells were pretreated with different derivatives for 1 h,
and then the cells were incubated with H2O2 for 24 h. Meanwhile, the test groups were divided
into the control (standard growth medium), the model (high H2O2), vitamin E (800 mM)
treatment and the derivative (10, 30 and 50 mg/mL) treatment groups. Cell survival was
evaluated by the colorimetric assay of MTT to test the protection.
3.4.4. Antioxidant assay
To evaluate the antioxidant effects of the derivatives, the activities of SOD and CAT and the
ROS production were examined.
Intracellular ROS production was measured by using a fluorescent dye, 20,70-
dichlorofluorescin diacetate (DCFH-DA; Zhao et al. 2011). Briefly, cells were seeded into 24-
well plates and pretreated with derivatives for 1 h, and then the cells were incubated with H2O2
for 24 h. The treated cells were loaded with 10 mM DCFH-DA in the medium for 30 min. After
washing three times with PBS, the cells were digested with 0.25% trypsin. The digestive cells
were collected and transferred into EP tube. Then, the single cell suspension was made. The
fluorescence was analysed by flow cytometry through the FL1 channel. ROS levels are
expressed as mean fluorescence intensity.
3.4.5. Statistical analysis
All data are presented as means ^ SD for three or six independent experiments. Statistical
analysis was performed by one-way ANOVA followed by LSD test. A P-value of less than 0.05
was considered to be statistically significant.
4. Conclusion
Our results have not only enriched the family of derivatives of 3-hydroxybutanolide, but have
also encouraged the synthesis of spin-labelled derivatives for improving antioxidant activity.
Supplementary material
Experimental details relating to this article are available online, alongside Table S1.
Acknowledgements
This work was supported by the grants from the National Natural Science Foundation of China (No.
20972061), the National Fundamental Fund for Personnel Training (No. J1103307) and Fujian Province
Natural Science Foundation of China (No. 2013J01337).

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