Antioxidant and neuroprotective effects of synthesized sintenin derivatives

Abstract

Three series of sintenin derivatives (compounds 1–14) were designed and prepared and their antioxidative and neuroprotective effects were evaluated. The in vitro models of scavenging 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals, chelating ferrous ions, inhibiting the rat brain homogenates lipid peroxidation, and protecting neurons damaged by hydrogen peroxide were employed for bioassays. It was found that sintenin derivatives 4 and 13 showed remarkable antioxidative and neuroprotective activities.

Keywords::

• Sintenin derivatives
• lignanoid
• antioxidant
• neuroprotective effects

Introduction

Reactive oxygen species (ROS), generated by the reduction of molecular oxygen, have been established to be involved in various pathological events and the development processes of many diseases 1, 2, 3. Hydrogen peroxide (H₂O₂), one kind of ROS, could be transformed to hydroxyl radical (OH·) at the existence of ferrous ions (Fe^{2 +} ), and it has been implicated in the pathogenesis of brain injuries and neurodegenerative diseases by impairing cells through direct oxidation of lipids, proteins and DNA, or by acting as a signal molecule to trigger the cellular apoptotic pathways [4]. Lipid peroxidation could cause severe damage to cell membrane structure and consequently, alters its fluidity and ability to function properly. Unbalanced oxidative homeostasis and lipid peroxidation are associated with neurodegenerative diseases, e.g. Alzheimer's disease (AD) and Parkinson's disease (PD) [5].

Antioxidants can intervene in the oxidation process by inhibiting the initiation or propagation of oxidizing chain reactions [6]. Therefore, substantially increased attempts are undertaken to discover and develop efficient synthetic or natural antioxidants to prevent or attenuate ROS-induced damages.

The structural and biological diversity of lignanoid has attracted great attentions of related researchers. Sintenin (3-(3,4-dimethoxyphenyl)propyl-3-(3,4-dimethoxy- phenyl) propanoate), a lignanoid isolated from the leaves and stems of Piper sintenense scattered in Taiwan, has been reported to possess selective cytotoxicity against mouse acute lymphocytic leukemia P388 cells [7]. However, to the best of our knowledge, its antioxidative property is not well studied. In continuation of studying the biological activities of sintenin and its derivatives and exploring novel antioxidative agents, three series (A, B and C) of sintenin analogues (1–14) were designed and synthesized for the evaluation of antioxidant properties. Series A (compounds 1–4) and series B (compounds 5–8) were unsaturated esters with either one or two double-bonds between two phenyls, while series C (compounds 9–14) were saturated esters (Figure 1). Different patterns of substituents on the aromatic A and/or B rings were also systematically introduced in the synthetic molecules, e.g., OCH₂OCH₃ to replace OCH₃ groups and demethylation to expose the phenolic OH groups.

Figure 1.  The structures of sintenin and three series of synthetic sintenin derivatives.

The antioxidant activities of synthesized compounds were investigated by scavenging 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) radicals, chelating of Fe^{2 +} , and the inhibition of lipid peroxidation in rat brain homogenates. Moreover, the protective effects of the test compounds on neurons damaged by H₂O₂ were also assayed to preliminarily reveal the diverse bioactivities of synthesized sintenin analogues.

Materials and methods

ESI-MS data were recorded on a Bruker Esquire 3000+ spectrometer. ¹H NMR and ¹³C NMR spectra were measured on a Varian INOVA 400 spectrometer with TMS as internal standard and CDCl₃ as solvent. Preparative TLC was performed on silica gel GF₂₅₄ and RP-18 plates (Merck, Darmstadt, Germany). The silica gel GF₂₅₄ was purchased from Qingdao Marine Chemical Factory, China. DPPH, H₂O₂, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), ferrozine were purchased from Sigma Chemical Co. (St. Louis, USA). Tris bases, DMEM medium were obtained from Gibco (Grand Island, NewYork, USA). Quercetin was prepared in our laboratory (HPLC purity of 99%), and rat pheochromocytoma (PC12) cells were provided by Cell Bank of Chinese Academy of Sciences, Shanghai. All other reagents were of the highest purity commercially available. Sprague-Dawley rats were obtained from the Zhejiang Center of Laboratory Animals, China. The use of animals was in accordance with Guideline for the Care and Use of Laboratory Animals of Zhejiang University.

Chemistry

Compounds 1–4 and 9–14 were synthesized according to the previously reported methods [8]. The structures of the synthetic compounds were identified by MS and NMR measurement, some of which were also directly compared with authentic samples.

The synthetic routes for compounds 5–8 are outlined in Schemes 1 and 2. 4-Methylcinnamic acid was treated with 4-methylphenylpropyl alcohol under catalysis of carbonyldiimidazole (CDI) and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) to afford 5 with a yield of 72% [9]. As illustrated in scheme 2, 4-methoxylmethoxylphenylpropionic acid was reacted with phenylallyl alcohol catalyzed by N,N′-dicyclohexylcarbodiimide (DCC) and 4-dimethylaminopyridine (DMAP) to afford 6 with a yield of 70% [10]. Compound 7 was obtained with a yield of 68% by removing the OCH₂OCH₃ group of 6 by treatment of 10% hydrochloric acid. The preparation of 8 was similar to that of 6, but with a different starting material, and its yield was 69%. The structures of 5 − 8 were elucidated mainly on the basis of ¹H and ¹³C NMR spectra as well as MS spectral data.

Scheme 1 Synthesis of compound 5. Reagents and conditions: (i) CDI, THF, reflux, 0.5 h; DBU, THF, reflux, 12 h.

Scheme 2 Synthesis of compounds 6–8. Reagents and conditions: (i) DCC, CH₂Cl₂, rt, 5 min; DMAP, rt, 12 h (ii) 10% HCl, CH₃OH, reflux.

Synthesis of (E)-4-methylcinnamic acid-4′-methyl phenylpropyl ester (5)

A mixture of 4-methylcinnamic acid (100 mg, 0.62 mM), CDI (313 mg, 1.23 mM), and dry tetrahydrofuran (THF, 10 mL) was refluxed for 30 min. The solution of 4-methylphenylpropanol (100 mg, 0.68 mM) and DBU (103 mg, 0.68 mM) in dry THF (10 mL) was then added for another 12 h refluxing. The solvent was removed by evaporation and the resulted mixture was purified by column chromatography to afford 5 (131 mg, 72%) as a colorless oil; Rf (petroleum ether/EtOAc = 3:1) 0.27; ¹H NMR (400 MHz, CDCl₃): δ 7.64 (1H, d, J = 16.0 Hz, H-1), 7.05 − 7.12 (8H, m, ArH), 6.63 (1H, d, J = 16.0 Hz, H-2), 4.09 (2H, t, J = 6.4 Hz, H-5), 2.70 (2H, t, J = 8.0 Hz, H-7), 2.34 (3H, s, CH₃-4′), 2.33 (3H, s, CH₃-4″), 1.92 (2H, m, H-6); ESI-MS m/z [M + NH₄]⁺374.

Synthesis of 4-methoxylmethoxylphenylpropionic acid-(E)-phenylallyl ester (6)

The mixture of 4-methoxylmethoxylphenylpropionic acid (50 mg, 0.24 mM) and DCC (54 mg, 0.26 mM) in dry dichloromethane (8 mL) was stirred at room temperature for 5 min, and then phenylallyl alcohol (35 mg, 0.26 mM) and 4-DMAP (5.8 mg, 0.048 mM) were added. The mixture was stirred for another 12 h at room temperature. The white insoluble substance was then removed by filtering and the resulted mixture was purified by column chromatography to give 6 (55 mg, 70%) as a colorless oil; Rf (petroleum ether/ EtOAc = 3:1) 0.38; ¹H NMR (400 MHz, CDCl₃): δ 6.95 − 7.40 (9H, m, ArH), 6.72 (1H, d, J = 16.0 Hz, H-7), 6.63 (1H, dt, J = 6.4, 15.6 Hz, H-6), 5.14 (2H, s, CH₃OCH₂O-4′), 4.74 (2H, d, J = 6.4 Hz, H-5), 3.47 (3H, s, CH₃OCH₂O-4′), 2.93 (2H, t, J = 8.0 Hz, H-3), 2.66 (2H, t, J = 8.0 Hz, H-2); ESI-MS m/z [M + NH₄]⁺344.

Synthesis of 4-hydroxylphenylpropionic acid-(E)-phenylallyl ester (7)

Compound 6 (25 mg, 0.078 mM) was dissolved in 6 mL methanol, and then 10% HCl (4 mL) was added to the solution. The mixture was refluxed for 40 min and was cooled to room temperature. Saturated sodium bicarbonate solution was then added to adjust the pH value to 7.0. After removing methanol by evaporation, the mixture was extracted with ethyl acetate for 3 times. The combined ethyl acetate layer was washed by saturated brine and dried over MgSO₄. Compound 7 (15 mg, 68%) was obtained as a colorless oil by column chromatography. Rf (petroleum ether/ EtOAc = 3:1) 0.15; ¹H NMR (400 MHz, CDCl₃): δ 6.74 − 7.40 (9H, m, ArH), 6.62 (1H, d, J = 16.0 Hz, H-7), 6.38 (1H, dt, J = 6.4, 15.6 Hz, H-6), 4.34 (2H, d, J = 6.4 Hz, H-5), 2.88 (2H, t, J = 8.0 Hz, H-3), 2.61 (2H, t, J = 8.0 Hz, H-2); ESI-MS m/z [M + NH₄]⁺300.

Synthesis of phenylpropionic acid-(E)-phenylallyl ester (8)

The preparation of 8 was similar to that of 6. Colorless oil; Rf (petroleum ether/ EtOAc = 3:1) 0.47; ¹H NMR (400 MHz, CDCl₃): δ 7.21 − 7.40 (10H, m, ArH), 6.63 (1H, d, J = 16.0 Hz, H-7), 6.26 (1H, dt, J = 6.4, 15.6 Hz, H-6), 4.74 (2H, d, J = 6.4 Hz, H-5), 2.99 (2H, t, J = 8.0 Hz, H-3), 2.70 (2H, t, J = 8.0 Hz, H-2); ESI-MS m/z [M + NH₄]⁺284.

Biology

DPPH radicals scavenging activity

The quenching of free radicals by the compounds was assayed spectrophotometrically at 517 nm against the absorbance of the stable DPPH radicals [11]. The reaction mixture contained in a total volume of 0.25 mL, 25 μL of various concentrations of the test compounds which were dissolved in dimethyl sulfoxide (DMSO) and 40 μL of DPPH (0.4 mg/mL) dissolved in methanol. The methanolic solution of DPPH served as a control; meanwhile quercetin was applied as a reference. The absorbance was measured at 517 nm after incubating the mixture at 37 °C for 30 min.

Measurement of rat brain homogenates lipid peroxidation

The formation of MDA was used as an indicator of inhibiting lipid peroxidation and was determined by the thiobarbituric acid (TBA) assay on freshly prepared Sprague-Dawley rat brain homogenates using colorimetric analysis [12]. The reaction mixtures which were composed of 200 μL of solution containing an aqueous FeSO₄ (4 μM), vitamin C (50 μM), 50 μL of rat brain homogenate and 5 μL of the test compounds (concentrations from 25 to 100 μg/mL), were incubated at 37 °C in capped tubes for 1 h before 100 μL of trichloroacetic acid (20%, v/v) was added. The mixture reacted at room temperature for 30 min. Finally, 200 μL of HCl (0.1 M) and 100 μL of TBA (1%, w/v) were added into each tube and the mixture was incubated at 100 °C for another 1 h. Centrifugation was then carried out at 5000 rpm for 5 min, and the absorbance of the supernatant was measured at 532 nm. Quercetin and curcumin acted as positive standards in this assay; results are shown as the means ± SD, n = 3.

Ferrous ions chelating assay

The ferrous ions chelating by the test compounds were estimated as reported previously [13]. Briefly, the samples with final concentration at 110 μg/mL were added to a solution of 2 mM FeSO₄ (5 μL) and 80% DMSO (200 μL). The reaction was initiated by adding of 5 mM ferrozine (10 μL), and the mixture was shaken vigorously before left standing at room temperature for 10 min. The absorbance of the solution was measured spectrophotometrically at 562 nm. Quercetin served as a positive standard.

Effect of the tested compounds on protecting PC 12 cells against hydrogen peroxide-induced injury

The protective effects of the compounds on PC 12 cells against hydrogen peroxide-induced injury were assessed with MTT assay as reported previously [14]. PC 12 cells were maintained at 37 °C in a humidified atmosphere containing 5% of CO₂. Cells were seeded into 96 well cell culture plates (Shengyou, China) at a density of 8000 cells per well in DMEM medium which was supplemented with 10% heat-inactivated calf serum, 100 units/mL of penicillin and 100 μg/mL of streptomycin. Following protocols were carried out 48 h after cells were seeded. H₂O₂ was prepared in phosphate buffered saline (PBS) on the day of application to cultures at a final concentration of 600 μM. Samples were dissolved in DMSO and diluted with the medium. The PC 12 cells were preincubated with samples 2 h before the H₂O₂ was added. A fresh solution of MTT (5 mg/mL) prepared in NaCl solution (0.9%) was added to the 96-well cell culture plates 3 h after H₂O₂ was added. The plates were incubated in the CO₂ incubator for 3 h, and the formazan were lysed with DMSO. The plates were analyzed in a multi-well-plate reader at 570 nm; data are shown as means ± SD, n = 3. The cytotoxicity of the test compounds against PC 12 cells was routinely examined with MTT method.

Results and discussion

Chemistry

Totally, 14 sintenin derivatives were synthesized by the procedure described by Hu et al. [8] and their structures were identified by MS and NMR measurement, some of which were also directly compared with the authentic samples. The yields of compounds 1–14 were 74%, 70%, 82%, 33%, 72%, 70%, 68%, 69%, 83%, 90%, 92%, 79%, 69%, and 94%, respectively. The esterification under the catalysis of CDI and DBU is beneficial for the isolation of the target compounds.

DPPH radicals scavenging activity

DPPH is a stable free radical existed in vitro, and bleaching of DPPH absorption is representative of the capacity of the test compounds to scavenge free radicals with aryl and stable characters and independent from any enzyme activity. Among the test compounds, compounds 4 and 13 showed noticeable DPPH scavenging activities (Table I). It should be pointed out that the inhibitory activity of compound 13 (IC₅₀ = 20.0 ± 4.0 μM) is comparable with quercetin (IC₅₀ = 11.9 ± 0.43 μM).

Table I.  DPPH radicals scavenging activity by the test compounds.

Compounds	DPPH inhibition (50 μg/mL)
1	11.20%
2	18.60%
3	16.80%
4	35.8 ± 5.4^a
5	13.40%
6	3.10%
7	14.20%
8	–^b
9	14.00%
10	–^b
11	0.70%
12	1.10%
13	20.0 ± 4.0^a
14	–^b
Quercetin	11.9 ± 0.43^a

^a IC₅₀ ± SD (μM): IC₅₀ values were further generated for those inhibitions greater than 50% at the concentration of 50 μg/mL. Data are expressed means ± SD, n = 3

^b No detectable activity.

Inhibitory effects of the test compounds in rat brain homogenates lipid peroxidation

The inhibition of lipid peroxidation is another practical index for the evaluation of antioxidants. Rat brain homogenates, widely adopted biomaterial for the investigation of lipid peroxidation [15], were carried out to examine the antioxidant properties of the compounds 1–14. The extent of in vitro lipid peroxidation was measured by lipid-derived malondialdehyde (MDA) production. As shown in Figure 2, compounds 4 and 13 exhibited obvious antioxidant activities in rat brain homogenates in dose-dependent manner. Moreover, the IC₅₀ values of these two compounds (86.8 ± 8.1 and 99.0 ± 13.7 μM for compounds 4 and 13, respectively) were lower than that of quercetin (one of the positive controls, IC₅₀ = 157.5 ± 19.5 μM) and were comparable to that of curcumin (the other positive control, IC₅₀ = 69.2 ± 10.6 μM).

Figure 2.  The inhibitory effects of compounds 4, 13, curcumin, and quercetin on iron-induced lipid peroxidation in rat brain homogenates.

Chelating ferrous ions by the test compounds

Fe^{2 +} chelation may lead to important antioxidative effects by retarding metal-catalysed oxidation. The ferrous ions chelation properties of the test compounds were evaluated and the results were illustrated in Table II. It could be found that only compound 4 showed a notable iron affinity with the inhibitory rate of 75.1 ± 4.9% at the concentration of 110 μg/mL among all the synthetic compounds.

Table II.  Ferrous ion chelating ability by the test compound.

Compounds	Ferrous ions chelation (110 μg/mL)
1	10.0%
2	2.4%
3	21.2%
4	75.1 ± 4.9%^a
5	6.4%
6	10.4%
7	7.2%
8	4.8%
9	12.4%
10	11.2%
11	10.4%
12	14.4%
13	40.4%
14	14.8%
Quercetin	46.8%

^a Data are shown as means ± SD, n = 3.

Protective effects of synthetic compounds on H₂O₂ induced insult in PC 12 cells

PC 12 cells, affording homogeneous populations and being easy of manipulation, provide a useful model system for the investigation of neural injury. The protective effects of the test compounds on PC 12 cells damaged with the direct application of hydrogen proxide were investigated. The results of morphological images of the PC 12 cells cultivated together with compounds 4 and 13 are illustrated in Figure 3. It could be observed that most of the cells became round shapes, while the membrane lesions of cells were also observable after 3 h exposure to 600 μM of H₂O₂ in Figure 3B. Though the round shapes of PC 12 cells could be observed in the presence of compounds 4 and 13, the cell viabilities and integralities were assuredly improved (Figure 3C and 3D). This indicated that compounds 4 and 13 possess clear protective effects against H₂O₂ insults. As shown in Figure 4, compounds 4 and 13 demonstrated significant neuroprotective activities in a dose-dependent manner. In addition, the experiments of removing the compounds from the pre-incubated medium of PC 12 cells prior to the injury initiated by H₂O₂ were also carried out. The neuroprotective effects of quercetin declined, while the neuroprotective activities of compounds 4 and 13 still remained visible (Figure 5) after the evacuation of pre-incubated medium containing these compounds. These results suggested that compounds 4 and 13 might work both intracellular and extracellular, whether they accumulate in the cell membrane to attenuate the damage of ROS needs to be identified. Additionally, the cytotoxicities of the active compounds were examined in parallel at the same concentrations. No noticeable cytotoxicities on PC 12 cells were found among these hits. The growth inhibition of PC 12 cells by compounds 4 and 13 was 17.0% and 25.9%, respectively, at 20 μg/mL.

Figure 3.  Effects of compounds 4, 13 and positive control on PC 12 cell injury induced by H₂O₂. (A) PC 12 control cells. (B) PC 12 cells exposed to 600 μM of H₂O₂ for 3 h. Most of the cells become round shapes. The membrane lesion of cells was also observed. (C, D and E) PC 12 cells were pre-incubated with 20 μg/mL of compounds 4, 13 and quercetin, respectively before exposed to 600 μM H₂O₂ for 3 h.

Figure 4.  Protective effects of compounds 4, 13, and quercetin against H₂

induced insult in PC 12 cells. The experiments were repeated three times. *p < .01, compared with the negative control.

Figure 5.  The results of removing test compounds from the pre-incubated medium of PC 12 cells prior to the injury initiated by H₂O₂. (A) PC 12 control cells. (B) PC 12 cells exposed to 600 μM H₂O₂ for 3 h. Most of the cells become round shapes. The membrane lesion of cells was also observed. (C, D and E) PC 12 cells were pre-incubated with 20 μg/mL of compounds 4, 13 and quercetin, respectively, and then the compounds were removed from the medium and the cells were exposed to 600 μM H₂O₂ for 3 h. The cells cultured with compounds 4 and 13 were remarkably improved in cell viabilities and integralities.

Among all of the test compounds, those with ortho-diphenolic hydroxyl groups, i.e., compounds 4 and 13 possessed remarkable antioxidative activities and potential neuroprotective properties. The absence of ortho-diphenolic hydroxyl moieties in series A and C diminished their capabilities as antioxidants (Figure 2, Figure 4, and Table II). Results derived from the DPPH radicals scavenging assays demonstrated that compound 13, which contained free phenolic OH groups on its aromatic A ring of sintenin, is the best DPPH scavenger (Table I). It seems that ortho-diphenolic hydroxyl groups play more important roles in scavenging DPPH radicals than the double bonds existing between two phenyls do.

In addition to the potential of free radical scavenging, compound 4 showed stronger antioxidant activities than compound 13 in the implemented bioassays, especially the capacity of Fe^{2 +} chelating; this might indicate that the unsaturated ester moiety is benefit for its bioactivity via the enhancement of the conjugated system. Furthermore, the mechanisms of ferrous ion-induced lipid peroxidation of brain homogenates were hypothesized as the decomposition of lipid peroxides, the generation of hydroxyl radicals, or the formation of perferryl or ferryl species [15]. According to the abovementioned results, it could be deduced that the Fe^{2 +} -chelating property of compound 4 might be one of the contributors to its anti-peroxidation (Table II).

In conclusion, sintenin derivatives 4 and 13 exhibited remarkable antioxidative and neuroprotective activities. Their DPPH radicals scavenging activity might be due to the diphenolic hydroxyl groups, and compared with the performances of compound 13, the unsaturated ester moiety of 4 played some positively effects on inhibiting lipid peroxidtion, chelating Fe^{2 +} , and neuroprotection. This study might offer some useful information for further development of sintenin analogues as novel antioxidants.

Acknowledgements

This work was partially supported by intramural foundation from Zhejiang University. We thank the Zhejiang Hisun Group for instrumental assistances. One of the authors (Y. Zhao) is grateful to Chinese Ministry of Education and Mr. Ka-Ching Lee for the award of a “Cheung Kong Scholars Professorship”. The authors acknowledge helpful discussions with Prof. H.K. Cheng at Chinese University of Hong Kong.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.