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Radical-scavenging activities of marine-derived xyloketals and related chromanes
Acta Pharmaceutica Sinica B 2013;3(5):322–327 Institute of Materia Medica, Chinese Academy of Medical Sciences Chinese Pharmaceutical Association
Acta Pharmaceutica Sinica B www.elsevier.com/locate/apsb www.sciencedirect.com
ORIGINAL ARTICLE
Radical-scavenging activities of marine-derived xyloketals and related chromanes Zhongliang Xua, Bingtai Lua, Qi Xiangb, Yiying Lia, Shichang Lia, Yongcheng Lina, Jiyan Panga,n a
School of Chemistry & Chemical Engineering, Sun Yat-Sen University, Guangzhou 510275, China Biopharmaceutical R&D Center, Jinan University, Guangzhou 510632, China
b
Received 28 April 2013; revised 27 May 2013; accepted 21 June 2013
KEY WORDS Xyloketals; Radical scavenging activities; DFT; SPLET
Abstract Xyloketals, a new type of antioxidants from a marine mangrove fungus, have potential pharmacological properties. In this paper, the radical-scavenging activities of a series of synthetic xyloketals and related chromanes toward 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-azino-bis(3ethylbenzothiazoline-6-sulfonic acid) (ABTS) were evaluated by absorption spectrometry. One of the compounds (compound 10) displayed significant antioxidative action against DPPH and ABTS. A structure-activity analysis showed that the reactive sites on these compounds correlated with a hydroxygroup and also with ketal or aromatic H substituents. Based in part on a density functional theory (DFT) calculation of compound 10, the antioxidant mechanism of this chromane was deduced as a possible radical-scavenging mechanism by a sequential proton loss electron-transfer (SPLET) process. & 2013 Institute of Materia Medica, Chinese Academy of Medical Sciences and Chinese Pharmaceutical Association. Production and hosting by Elsevier B.V. All rights reserved.
n
Corresponding author. E-mail address: [email protected] (Jiyan Pang). Peer review under responsibility of Institute of Materia Medica, Chinese Academy of Medical Sciences and Chinese Pharmaceutical Association.
2211-3835 & 2013 Institute of Materia Medica, Chinese Academy of Medical Sciences and Chinese Pharmaceutical Association. Production and hosting by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.apsb.2013.06.008
Radical-scavenging activities of marine-derived xyloketals and related chromanes 1.
Introduction
Free radicals and other reactive oxygen species (ROS) are inevitable by-products of biological redox reactions, and play important roles in disease and cellular damage1–4. Consequently, selecting efficient antioxidants to scavenge free radicals has aroused great interest. In recent years numerous studies have shown that phenols exhibit potent antioxidant activities5–7. In particular, benzopyran-type phenols such as vitamin E and quercetin show high radical scavenging activities, since they would be readily converted to the corresponding O-quinones and further products8–10. However, to completely understand the antioxidant mechanisms of phenols and explain why they show high radical scavenging activities, and whether the formation of O-quinones or H-atom abstraction is the key event, further studies are required. Xyloketals, a series of novel benzopyran compounds derived from natural products isolated from a marine mangrove endophytic fungus11–13, possess multiple strong pharmacological properties, such as blood vessel relaxation, promoting endothelial cell NO production, inhibiting NADPH oxidase activity and attenuating oxLDL-induced oxidative stress14,15. Furthermore, (7)-xyloketal B was able to protect PC12 cells against oxygen glucose deprivation (OGD)-induced cell injury and to directly scavenge DPPH free radicals and protect mitochondria against oxidative insult16. The antioxidative property and protective action on mitochondria may account for its neuroprotective action. It was speculated that the pharmacological effects of xyloketals may arise from their antioxidative or radical-scavenging activities. Exploring the antioxida-
323
tive and radical-scavenging mechanisms of these compounds is therefore quite attractive. In this paper, the antioxidative ability of xyloketals and benzopyran derivatives 1–10 (Fig. 1) against DPPH and ABTS were assessed by utilizing absorption spectrometry, and density functional theory (DFT) calculations were used to dissect the antioxidative mechanism of this series of compounds. Herein we report our observations and discuss mechanistic interpretations. 2. 2.1.
Results and discussion Chemistry
Compounds 1–10 (Fig. 1) were prepared from commercially available orcinol with methyl vinyl ketone in methanol via intramolecular Michael addition and followed by a ketal cyclization in the presence of p-TsOH with 72–95% yield (Scheme 1)17. Compound 9 is a novel compound. The structure of chromane rac-9 was further confirmed by X-ray diffraction crystallography (Fig. 2). 2.2.
Crystallographic data
Single-crystal growth was carried on in hexane at room temperature. Crystal data for 9: C17H24O4, crystal dimension 0.41 mm 0.41 mm 0.26 mm, space group Monoclinic, C2/c; unit cell dimensions a ¼11.521 (2) Å, b ¼13.206(2) Å, c ¼11.2193(19) Å, volume¼ 1546.5(5) Å3, Z¼ 4, Dcalcd ¼1.256 Mg/m3, m¼0.088 mm1,
Figure 1 The structures of xyloketals, related benzopyran compounds, phloroglucinol, vitamin C, quercetin, and ()-epigallocatechin.
324
Z. Xu et al. O
R
+ OH
p- TsOH, MgSO4 Et2O or MeOH
HO or
72-95%
O OH +
HO
OH
or R
R
O
O
O
O
1-9 OH
p-TsOH, Et2O
HO
O
50 °C, 8 h, 87 % OH 10
Scheme 1 Synthesis of the target compounds.
and 9, showed low activities. Therefore, the hydroxyl group is an important functional group for antioxidative action. The activity of compound 6 substituted in H-2 is two-fold weaker than 10 with no substituent in H-2, and it may mean that a hydrogen at the C-2 or C-6 position is important in the radical-scavenging activities of these compounds. With compounds 2, 3a–c and 4, ABTSscavenging activities were not consistent with their DPPHscavenging properties, suggesting that the reactive site of these compounds with free radicals correlated not only with the OH group but also with a ketal or aromatic H. The results suggested that these derivatives are unique compounds with potential implication in the treatment of certain diseases with lipid oxidation induced by free-radical formation and it provided valuable information for further structural modification. Fig. 3a exemplifies the absorption spectrum of 10 after reaction with DPPH. In addition, the DPPH-scavenging activities of compounds 2, 8, and 10 showed good time-dependence (Fig. 3b). The relative rates of DPPH scavenging are in the order of 104842 based on the analyses of the relationship between the absorbance intensity and time.
2.4. Figure 2
X-ray structure of chromane 9.
F000¼632. All single-crystal data were collected using the hemisphere technique on a Bruker SMART 1000 CCD system diffractometer with graphite-monochromated Mo Kα radiation λ¼0.7l073 at 173(2) K. The structure was solved by the direct method. The final value of R was 0.0395, wR2¼0.1076 [I42s (I)]. 2.3. Antioxidant activities of xyloketals and related chromanes against DPPH and ABTS Various antioxidant activity assays have been used to evaluate the actions of antioxidant compounds18–21. Among them, the radical scavenging activity testing method such as DPPH, ABTS scavenging activity screenings, is described as a simple, rapid and convenient method independent of sample polarities for radical scavenging effects screening. It can be used for solid or liquid samples and is not specific to any particular antioxidant component, but applies to the overall antioxidant capacity of the samples22,23. These advantages aroused many interests for testing radicals scavenging abilities of various natural products to find out promising candidates for antioxidants. DPPH and ABTS radical-scavenging activities of xyloketals and benzopyran derivatives (1–10), which possess electron-withdrawing/donating substituents on the aromatic ring and different benzopyran rings, were evaluated in methanol by absorption spectroscopy, and phloroglucinol, vitamin C and quercetin were used as positive controls. Due to slight different experimental conditions, the results showed some drift. For example, the IC50 of vitamin C was reported from 28 to 460 μM in scavenging activities against DPPH24–27. In our research, the IC50 of vitamin C is 154.5 μM, which proved the reliability of the operation. The results showed that most of the examined compounds had moderate to strong radical scavenging activities, and among them, compound 10 displayed significant antioxidative activity both against DPPH and ABTS. It can be seen from Table 1 that the compounds which lack a hydroxyl group, such as compounds 1, 5,
SPLET mechanism and BDE calculations
The role of OH groups in the antioxidant activity of benzopyrans was further deduced by semi-empirica quantum chemistry calculation. We studied the free-radical scavenging mechanism of compound 10, which has the best antioxidant activity of these compounds. Electron delocalization in the parent molecule (ArOH) and in the phenoxy radical (ArOd) is one major electronic feature correlated with a high antioxidant capacity28. Generally, the redox reactivity mode of phenolic compounds (ArOH) can follow three different chemical pathways: One-step H-atom transfer (HAT): DPPHd+ArOH-DPPHH+ArOd
(1)
Stepwise electron-transfer/proton-transfer (ET/EP): ArOH+DPPHd-ArOHd++DPPH-ArOd+DPPHH
(2)
sequential proton loss electron-transfer (SPLET): ArOH-ArO+H+, ArO+DPPHdArOd+DPPH, DPPH+H+-DPPHH
(3)
As shown in Table 2, the O-H BDE at position 11 and 12 of compound 10 is 84.76 and 85.63 kcal/mol, respectively. However, the BDE of DPPHH is low (around 82.6 kcal/mol), indicating that this compound does not utilize the HAT mechanism. Judging from the IP value of compound 10, it is strongly localized on the O atom from where the H atom is removed (170.90 kcal/mol); therefore, the ET/EP mechanism is not likely in this process. The dissociation energy of the 11-phenolic anion and 12-phenolic anion are 53.67 and 51.47 kcal/mol, respectively. Comparably, the IP value of DPPH free radical is 93.9 kcal/mol29, higher than the IP of anions of 10. On the other hand, electron transfer between DPPH and 11- or 12-phenolic anion occurred spontaneously. Thus, a SPLET mechanism is favored in the DPPH-scavenging process. It should be noted that the IP of 12-OH is lower than that
Radical-scavenging activities of marine-derived xyloketals and related chromanes
Table 1 Compd.
1 2 3a 3b 3c
325
The IC50 of xyloketals and benzopyran derivatives for DPPH and ABTS free radicals. IC50 (μM)
Compd.
DPPH
ABTS
4200 102 20.6 4200 4200
4200 11.7 7.1 15.4 21.8
4 5 6 7 8
IC50 (μM)
Compd.
DPPH
ABTS
4200 4200 15.3 4200 31.5
21.8 4200 14.7 4200 21.8
IC50 (μM)
9 10 Phloroglucinol Vitamin C Quercetin
DPPH
ABTS
4200 7.2 47.5 154.5 5.5
4200 8.2 6.93 36.0 25.2
Figure 3 (a) Consecutive spectra of the visible absorbance (517 nm) of DPPH (0.04 mmol) solution in MeOH at 298 K with addition of 10; (b) DPPH scavenging as a function of reaction time for compound 2(■), 8(●), 10(▲). Table 2
(RO)B3LYP/6-311+G(2d, 2p)//(RO)B3LYP/6-31+G(d)-calculated BDEs and IPs of 10 or derived anions or radicals.
Compound or anions or radicals
SPE (hartree)
ZPVE (hartree)
TCE (hartree)
a
653.49149868
0.242655
0.243599
b
653.21872847
0.242232
–
c
652.84427051
0.229537
0.230482
d
652.92544487
0.228687
–
e
652.84457356
0.229382
0.230326
f
652.92917019
0.228452
–
BDE (kcal/mol)
IP (kcal/mol)
170.90
85.63
51.47
84.76
53.67
SPE, Single point energy; ZPVE, Zero point vibrational energy; TCE, Thermal correction to enthalpy; BDE, Bond dissociation energy; IP, Ionization potential.
326
Z. Xu et al.
of 11-OH by 2.2 kcal/mol, demonstrating that the 12-OH group more easily generates a radical cation and is likely more active in DPPH-scavenging than the 11-OH group. This calculated result is also supported by the structure-activity relationship analysis and the kinetics of oxidation of this type of compounds as demonstrated by the absorption spectrometry analysis. 3. 3.1.
Materials and method Chemicals, reagents and materials
All the solvents were of the highest commercially available purity and were used as received. Melting points were uncorrected. The NMR spectra were taken on 400 MHz spectrometers. Chemical shifts are given in ppm (δ-scale), coupling constants (J) in Hz. Complete assignment of all NMR signals was performed using a combination of H, H-COZY, H, C-HSQC and H, C-HMBC experiments. Mass spectra (EI) were measured on a dual sector mass spectrometer using direct inlet and the lowest temperature enabling evaporation. Compounds 1–9 were prepared according to reported procedures17. Column chromatography was performed using flash silica gel. Yields are given for isolated products showing one spot on a TLC plate and no impurities detectable in the NMR spectrum. The DPPH and ABTS (from AldrichSigma) radicals were purchased with the highest purity available and used as received. Chromane 9: To a suspension of orcin (1.00 g, 7.94 mmol), methyl vinyl ketone (1.30 g, 18.26 mmol), anhydrous magnesium sulfate (1.50 g) and methanol (30 mL) at 0 1C was added p-TsOH (1.43 g, 7.94 mmol). The mixture was allowed to warm to room temperature and was stirred for 8 h. The mixture was then diluted with ethyl acetate. The resultant solution was washed with a saturated aqueous solution of ammonium chloride, brine, dried over anhydrous magnesium sulfate and concentrated in vacuo. Purification by flash chromatography using petroleum ether and ethyl acetate as the eluant afforded compound 9 (1.68 g, 94%) as a white solid. Mp. 166–167 1C, 1H NMR (CDCl3, 400 MHz) δ: 1.52 (s, 6H), 1.80 (dd, J ¼6.5, 13.5, 1H), 1.82 (dd, J ¼ 6.5, 13.5, 1H), 2.09 (s, 3H), 2.15 (dd, J ¼6.5, 13.5, 1H), 2.16 (dd, J ¼ 6.5, 13.5, 1H), 2.55 (dd, J ¼6.5, 16.0, 1H), 2.56 (dd, J ¼ 6.5, 16.0, 1H), 2.70 (dd, J ¼6.5, 16.0, 1H), 2.73 (dd, J ¼ 6.5, 16.0, 1H), 3.24 (s, 6H), 6.29 (s, 1H); 13C NMR (CDCl3, 101 MHz): 14.7, 19.9, 23.4, 32.5, 49.3, 97.7, 103.0, 114.1, 135.1, 151.3. HR-EI-MS calcd. for C17H24O4, 292.1674, found 292.1672. 3.2.
Antioxidant assays
Quantification of radical scavenging activity was based on a procedure described previously with some modifications30,31. The solutions of the test compounds (AH) in methanol (0.1 mL) were added to the respective solution of the stable free radicals DPPH and ABTS (2.4 mL, 100 μM) at different molar ratios. After 30 min of incubation at 3770.5 1C maintained by an outer watercirculating bath, the absorbance at 517 nm and 735 nm was measured. Vitamin C, phloroglucinol and quercetin were used as positive controls. Results are expressed as the sample concentration that quenches 50% of the DPPH radical (IC50, μM), calculated by linear regression. The inhibition ratio (%) was calculated using the following formula: inhibition ratio (%) ¼(AAl)/A 100, where A is the absorbance of the control and Al is the absorbance of the test sample.
3.3.
Calculations
In this paper, BDEs were calculated according to the following procedures. The molecular geometries were optimized first by the molecular mechanics method MMX, and then, by the semiempirical quantum chemical method AM1. Finally, according to the proposal of Wright et al.32, (RO) B3LYP functional basis set of 6–311+G (2d, 2p) was used for the full geometry optimization. The zero point vibrational energy (ZPVE) and the vibrational contribution to the energy were scaled by a factor of 0.9805 according to the definition of BDE, BDE ¼Hr+HhHp, in which, Hr is the enthalpy for radical generated after H-abstraction reaction, Hh is the enthalpy for hydrogen atom, 0.49792 hartree, and Hp is the enthalpy for the parent molecule. Adiabatic IPs were calculated by means of a combined DFT method, which affords accuracy and economy. (RO) B3LYP functional basis set of 6–31 +G (d) was used to calculate single point energy (SPE) on the basis of the PM3-optimized structure. Thus, the molecular energy (e) consists of (RO) B3LYP/6-31+G (d) calculated SPE and PM3 calculated ZPVE (scaled by a factor of 0.947). In these calculations IP¼ ecep, in which ec is the energy for cation radical, while ep is the energy for parent molecule. All of the quantum chemical calculations were accomplished with the Gaussian 03 program. Considering the lipophilicity of edaravone, the calculations were achieved in gas phase. Acknowledgments This work was supported by the National Natural Science Foundation of China (21172271), Natural Science Foundation of Guangdong Province, China (Grant No. S2011020001231), and the Fundamental Research Funds for the Central Universities (11lgpy75). References 1. Kadenbach B, Ramzan R, Vogt S. Degenerative diseases, oxidative stress and cytochrome c oxidase function. Trends Mol Med 2009;15:139–47. 2. Farooqui T, Farooqui AA. Aging: an important factor for the pathogenesis of neurodegenerative diseases. Mech Ageing Dev 2009;130:203–15. 3. Mladenka P, Zatloukalova L, Filipsky T, Hrdina R. Cardiovascular effects of flavonoids are not caused only by direct antioxidant activity. Free Radical Biol Med 2010;49:963–75. 4. Rudkowska I. Functional foods for health: focus on diabetes. Maturitas 2009;62:263–9. 5. Rossi L, Mazzitelli S, Arciello M, Capo CR, Rotilio G. Benefits from dietary polyphenols for brain aging and Alzheimer's disease. Neurochem Res 2008;33:2390–400. 6. Mandel SA, Amit T, Kalfon L, Reznichenko L, Youdim MB. Targeting multiple neurodegenerative diseases etiologies with multimodal-acting green tea catechins. J Nutr 2008;138:S1578–83. 7. Hong JT, Yen JH, Wang L, Lo YH, Chen ZT, Wu MJ. Regulation of heme oxygenase-1 expression and MAPK pathways in response to kaempferol and rhamnocitrin in PC12 cells. Toxicol Appl Pharmacol 2009;237:59–68. 8. Ikuo N, Kentaro M, Tomokazu S, Yuko I, Keiko I, Masataka M, et al. Kinetic study of the electron-transfer oxidation of the phenolate anion of a vitamin E model by molecular oxygen generating superoxide anion in an aprotic medium. Org Biomol Chem 2003;1:4085–8. 9. Burton GW, Ingold KU. Vitamin E: application of the principles of physical organic chemistry to the exploration of its structure and function. Acc Chem Res 1986;19:194–201.
Radical-scavenging activities of marine-derived xyloketals and related chromanes 10. Burkitt MJ. A critical overview of the chemistry of copper-dependent low density lipoprotein oxidation: roles of lipid hydroperoxides, alphatocopherol, thiols, and ceruloplasmin. Arch Biochem Biophys 2001;394:117–35. 11. Lin Y, Wu X, Feng S, Jiang G, Luo J, Zhou S, et al. Five unique compounds: xyloketals from mangrove fungus Xylaria sp. from the South China Sea coast. J Org Chem 2001;66:6252–6. 12. Wu X, Liu X, Lin Y, Luo J, She Z, Li H, et al. Xyloketal F: a strong L-calcium channel blocker from the mangrove fungus Xylaria sp (♯2508) from the South China Sea coast. Eur J Org Chem 2005;19:4061–4. 13. Wu X, Liu X, Jiang G, Lin Y, Chan W, Vrijmoed LLP. Xyloketal G: a novel metabolite from the mangrove fungus Xylaria sp. 2508. Chem Nat Compd 2005;41:27–9. 14. Chen W, Qian Y, Meng W, Pang J, Lin Y, Guan Y, et al. A novel marine compound xyloketal B protects against oxidized LDL-induced cell injury in vitro. Biochem Pharmacol 2009;78:941–50. 15. Xu Z, Li Y, Xiang Q, Pei Z, Lu X, Chen W, et al. Design and synthesis of novel xyloketal derivatives and their vasorelaxing activities in rat thoracic aorta and angiogenic activities in zebrafish angiogenesis screen. J Med Chem 2010;53:4642–53. 16. Zhao J, Li L, Chen L, Li J, Pang J, Lin Y, et al. Marine compound xyloketal B protects PC12 cells against OGD-induced cell damage. Brain Res 2009;1302:240–7. 17. Xu Z, Li Y, Lu B, Pang J, Lin Y. An expedient approach to the benzopyran core: application to synthesis of the natural products (7)xyloketals and (7)-alboatrin. Chin J Chem 2010;28:2441–6. 18. Alam MN, Bristi NJ, Rafiquzzaman M. Review on in vivo and in vitro methods evaluation of antioxidant activity. Saudi Pharm J 2013;21:143–52. 19. Chanda S, Dave R. In vitro models for antioxidant activity evaluation and some medicinal plants possessing antioxidant properties: an overview. Afr J Microbiol Res 2009;3:981–96. 20. Badarinath AV, Rao KM, Chetty CMS, Ramkanth V, Rajan TVS, Gnanaprakash K. A review on in vitro antioxidant methods: comparisons, correlations and considerations. Int J PharmTech Res 2010;2:1276–85.
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21. Camilo L, Ana D. Evaluating the antioxidant capacity of natural products: a review on chemical and cellular-based assays. Anal Chim Acta 2013;763:1–10. 22. Miller NJ, Rice-Evans C, Davies MJ, Gopinathan V, Milner A. A novel method for measuring antioxidant capacity and its application to monitoring the antioxidant status in premature neonates. Clin Sci 1993;84:407–12. 23. Brand-Williams W, Cuvelier ME, Berset C. Use of a free radical method to evaluate antioxidant activity. LWT-Food Sci Technol 1995;28:25–30. 24. Wang XQ. Evaluation on anti-oxidant activity of Curcuma longa extracts based on DPPH free radical scavenging capability. Drug Eval Res 2011;34:360–3. 25. Toumy SA, Souda SS, Mohamed TK, Brouard I, Bermejo J. Anthraquinone glycosides from Cassia roxburghii and evaluation of its free radical scavenging activity. Carbohydr Res 2012;1:47–51. 26. Tang YZ, Liu ZQ. Free-radical-scavenging effect of carbazole derivatives on DPPH and ABTS radicals. J Am Oil Chem Soc 2007;84:1095–100. 27. Khan RA. Evaluation of flavonoids and diverse antioxidant activities of Sonchus arvensis. Chem Cent J 2012;29:126. 28. Wright JS, Johnson ER, DiLabio GA. Predicting the activity of phenolic antioxidants: theoretical method, analysis of substituent effects, and application to major families of antioxidants. J Am Chem Soc 2001;123:1173–83. 29. Shen L, Zhang H, Ji H. A thermodynamic investigation of DPPH radical-scavenging mechanisms of folates. Theochem 2008;856: 119–23. 30. Manna P, Sinha M, Sil PC. Aqueous extract of Terminalia arjuna prevents carbon tetrachloride induced hepatic and renal disorders. BMC Complementary Altern Med 2006;6:33. 31. Re R, Pellegrini N, Proteggente A, Pannala A, Yang M, Rice-Evans C. Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radical Biol Med 1999;26:1231–7. 32. Wright JS, Carpenter DJ, McKay DJ, Ingold KU. Theoretical calculation of substituent effects on the OH bond strength of phenolic antioxidants related to vitamin E. J Am Chem Soc 1997;119:4245–52.
Acta Pharmaceutica Sinica B www.elsevier.com/locate/apsb www.sciencedirect.com
ORIGINAL ARTICLE
Radical-scavenging activities of marine-derived xyloketals and related chromanes Zhongliang Xua, Bingtai Lua, Qi Xiangb, Yiying Lia, Shichang Lia, Yongcheng Lina, Jiyan Panga,n a
School of Chemistry & Chemical Engineering, Sun Yat-Sen University, Guangzhou 510275, China Biopharmaceutical R&D Center, Jinan University, Guangzhou 510632, China
b
Received 28 April 2013; revised 27 May 2013; accepted 21 June 2013
KEY WORDS Xyloketals; Radical scavenging activities; DFT; SPLET
Abstract Xyloketals, a new type of antioxidants from a marine mangrove fungus, have potential pharmacological properties. In this paper, the radical-scavenging activities of a series of synthetic xyloketals and related chromanes toward 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-azino-bis(3ethylbenzothiazoline-6-sulfonic acid) (ABTS) were evaluated by absorption spectrometry. One of the compounds (compound 10) displayed significant antioxidative action against DPPH and ABTS. A structure-activity analysis showed that the reactive sites on these compounds correlated with a hydroxygroup and also with ketal or aromatic H substituents. Based in part on a density functional theory (DFT) calculation of compound 10, the antioxidant mechanism of this chromane was deduced as a possible radical-scavenging mechanism by a sequential proton loss electron-transfer (SPLET) process. & 2013 Institute of Materia Medica, Chinese Academy of Medical Sciences and Chinese Pharmaceutical Association. Production and hosting by Elsevier B.V. All rights reserved.
n
Corresponding author. E-mail address: [email protected] (Jiyan Pang). Peer review under responsibility of Institute of Materia Medica, Chinese Academy of Medical Sciences and Chinese Pharmaceutical Association.
2211-3835 & 2013 Institute of Materia Medica, Chinese Academy of Medical Sciences and Chinese Pharmaceutical Association. Production and hosting by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.apsb.2013.06.008
Radical-scavenging activities of marine-derived xyloketals and related chromanes 1.
Introduction
Free radicals and other reactive oxygen species (ROS) are inevitable by-products of biological redox reactions, and play important roles in disease and cellular damage1–4. Consequently, selecting efficient antioxidants to scavenge free radicals has aroused great interest. In recent years numerous studies have shown that phenols exhibit potent antioxidant activities5–7. In particular, benzopyran-type phenols such as vitamin E and quercetin show high radical scavenging activities, since they would be readily converted to the corresponding O-quinones and further products8–10. However, to completely understand the antioxidant mechanisms of phenols and explain why they show high radical scavenging activities, and whether the formation of O-quinones or H-atom abstraction is the key event, further studies are required. Xyloketals, a series of novel benzopyran compounds derived from natural products isolated from a marine mangrove endophytic fungus11–13, possess multiple strong pharmacological properties, such as blood vessel relaxation, promoting endothelial cell NO production, inhibiting NADPH oxidase activity and attenuating oxLDL-induced oxidative stress14,15. Furthermore, (7)-xyloketal B was able to protect PC12 cells against oxygen glucose deprivation (OGD)-induced cell injury and to directly scavenge DPPH free radicals and protect mitochondria against oxidative insult16. The antioxidative property and protective action on mitochondria may account for its neuroprotective action. It was speculated that the pharmacological effects of xyloketals may arise from their antioxidative or radical-scavenging activities. Exploring the antioxida-
323
tive and radical-scavenging mechanisms of these compounds is therefore quite attractive. In this paper, the antioxidative ability of xyloketals and benzopyran derivatives 1–10 (Fig. 1) against DPPH and ABTS were assessed by utilizing absorption spectrometry, and density functional theory (DFT) calculations were used to dissect the antioxidative mechanism of this series of compounds. Herein we report our observations and discuss mechanistic interpretations. 2. 2.1.
Results and discussion Chemistry
Compounds 1–10 (Fig. 1) were prepared from commercially available orcinol with methyl vinyl ketone in methanol via intramolecular Michael addition and followed by a ketal cyclization in the presence of p-TsOH with 72–95% yield (Scheme 1)17. Compound 9 is a novel compound. The structure of chromane rac-9 was further confirmed by X-ray diffraction crystallography (Fig. 2). 2.2.
Crystallographic data
Single-crystal growth was carried on in hexane at room temperature. Crystal data for 9: C17H24O4, crystal dimension 0.41 mm 0.41 mm 0.26 mm, space group Monoclinic, C2/c; unit cell dimensions a ¼11.521 (2) Å, b ¼13.206(2) Å, c ¼11.2193(19) Å, volume¼ 1546.5(5) Å3, Z¼ 4, Dcalcd ¼1.256 Mg/m3, m¼0.088 mm1,
Figure 1 The structures of xyloketals, related benzopyran compounds, phloroglucinol, vitamin C, quercetin, and ()-epigallocatechin.
324
Z. Xu et al. O
R
+ OH
p- TsOH, MgSO4 Et2O or MeOH
HO or
72-95%
O OH +
HO
OH
or R
R
O
O
O
O
1-9 OH
p-TsOH, Et2O
HO
O
50 °C, 8 h, 87 % OH 10
Scheme 1 Synthesis of the target compounds.
and 9, showed low activities. Therefore, the hydroxyl group is an important functional group for antioxidative action. The activity of compound 6 substituted in H-2 is two-fold weaker than 10 with no substituent in H-2, and it may mean that a hydrogen at the C-2 or C-6 position is important in the radical-scavenging activities of these compounds. With compounds 2, 3a–c and 4, ABTSscavenging activities were not consistent with their DPPHscavenging properties, suggesting that the reactive site of these compounds with free radicals correlated not only with the OH group but also with a ketal or aromatic H. The results suggested that these derivatives are unique compounds with potential implication in the treatment of certain diseases with lipid oxidation induced by free-radical formation and it provided valuable information for further structural modification. Fig. 3a exemplifies the absorption spectrum of 10 after reaction with DPPH. In addition, the DPPH-scavenging activities of compounds 2, 8, and 10 showed good time-dependence (Fig. 3b). The relative rates of DPPH scavenging are in the order of 104842 based on the analyses of the relationship between the absorbance intensity and time.
2.4. Figure 2
X-ray structure of chromane 9.
F000¼632. All single-crystal data were collected using the hemisphere technique on a Bruker SMART 1000 CCD system diffractometer with graphite-monochromated Mo Kα radiation λ¼0.7l073 at 173(2) K. The structure was solved by the direct method. The final value of R was 0.0395, wR2¼0.1076 [I42s (I)]. 2.3. Antioxidant activities of xyloketals and related chromanes against DPPH and ABTS Various antioxidant activity assays have been used to evaluate the actions of antioxidant compounds18–21. Among them, the radical scavenging activity testing method such as DPPH, ABTS scavenging activity screenings, is described as a simple, rapid and convenient method independent of sample polarities for radical scavenging effects screening. It can be used for solid or liquid samples and is not specific to any particular antioxidant component, but applies to the overall antioxidant capacity of the samples22,23. These advantages aroused many interests for testing radicals scavenging abilities of various natural products to find out promising candidates for antioxidants. DPPH and ABTS radical-scavenging activities of xyloketals and benzopyran derivatives (1–10), which possess electron-withdrawing/donating substituents on the aromatic ring and different benzopyran rings, were evaluated in methanol by absorption spectroscopy, and phloroglucinol, vitamin C and quercetin were used as positive controls. Due to slight different experimental conditions, the results showed some drift. For example, the IC50 of vitamin C was reported from 28 to 460 μM in scavenging activities against DPPH24–27. In our research, the IC50 of vitamin C is 154.5 μM, which proved the reliability of the operation. The results showed that most of the examined compounds had moderate to strong radical scavenging activities, and among them, compound 10 displayed significant antioxidative activity both against DPPH and ABTS. It can be seen from Table 1 that the compounds which lack a hydroxyl group, such as compounds 1, 5,
SPLET mechanism and BDE calculations
The role of OH groups in the antioxidant activity of benzopyrans was further deduced by semi-empirica quantum chemistry calculation. We studied the free-radical scavenging mechanism of compound 10, which has the best antioxidant activity of these compounds. Electron delocalization in the parent molecule (ArOH) and in the phenoxy radical (ArOd) is one major electronic feature correlated with a high antioxidant capacity28. Generally, the redox reactivity mode of phenolic compounds (ArOH) can follow three different chemical pathways: One-step H-atom transfer (HAT): DPPHd+ArOH-DPPHH+ArOd
(1)
Stepwise electron-transfer/proton-transfer (ET/EP): ArOH+DPPHd-ArOHd++DPPH-ArOd+DPPHH
(2)
sequential proton loss electron-transfer (SPLET): ArOH-ArO+H+, ArO+DPPHdArOd+DPPH, DPPH+H+-DPPHH
(3)
As shown in Table 2, the O-H BDE at position 11 and 12 of compound 10 is 84.76 and 85.63 kcal/mol, respectively. However, the BDE of DPPHH is low (around 82.6 kcal/mol), indicating that this compound does not utilize the HAT mechanism. Judging from the IP value of compound 10, it is strongly localized on the O atom from where the H atom is removed (170.90 kcal/mol); therefore, the ET/EP mechanism is not likely in this process. The dissociation energy of the 11-phenolic anion and 12-phenolic anion are 53.67 and 51.47 kcal/mol, respectively. Comparably, the IP value of DPPH free radical is 93.9 kcal/mol29, higher than the IP of anions of 10. On the other hand, electron transfer between DPPH and 11- or 12-phenolic anion occurred spontaneously. Thus, a SPLET mechanism is favored in the DPPH-scavenging process. It should be noted that the IP of 12-OH is lower than that
Radical-scavenging activities of marine-derived xyloketals and related chromanes
Table 1 Compd.
1 2 3a 3b 3c
325
The IC50 of xyloketals and benzopyran derivatives for DPPH and ABTS free radicals. IC50 (μM)
Compd.
DPPH
ABTS
4200 102 20.6 4200 4200
4200 11.7 7.1 15.4 21.8
4 5 6 7 8
IC50 (μM)
Compd.
DPPH
ABTS
4200 4200 15.3 4200 31.5
21.8 4200 14.7 4200 21.8
IC50 (μM)
9 10 Phloroglucinol Vitamin C Quercetin
DPPH
ABTS
4200 7.2 47.5 154.5 5.5
4200 8.2 6.93 36.0 25.2
Figure 3 (a) Consecutive spectra of the visible absorbance (517 nm) of DPPH (0.04 mmol) solution in MeOH at 298 K with addition of 10; (b) DPPH scavenging as a function of reaction time for compound 2(■), 8(●), 10(▲). Table 2
(RO)B3LYP/6-311+G(2d, 2p)//(RO)B3LYP/6-31+G(d)-calculated BDEs and IPs of 10 or derived anions or radicals.
Compound or anions or radicals
SPE (hartree)
ZPVE (hartree)
TCE (hartree)
a
653.49149868
0.242655
0.243599
b
653.21872847
0.242232
–
c
652.84427051
0.229537
0.230482
d
652.92544487
0.228687
–
e
652.84457356
0.229382
0.230326
f
652.92917019
0.228452
–
BDE (kcal/mol)
IP (kcal/mol)
170.90
85.63
51.47
84.76
53.67
SPE, Single point energy; ZPVE, Zero point vibrational energy; TCE, Thermal correction to enthalpy; BDE, Bond dissociation energy; IP, Ionization potential.
326
Z. Xu et al.
of 11-OH by 2.2 kcal/mol, demonstrating that the 12-OH group more easily generates a radical cation and is likely more active in DPPH-scavenging than the 11-OH group. This calculated result is also supported by the structure-activity relationship analysis and the kinetics of oxidation of this type of compounds as demonstrated by the absorption spectrometry analysis. 3. 3.1.
Materials and method Chemicals, reagents and materials
All the solvents were of the highest commercially available purity and were used as received. Melting points were uncorrected. The NMR spectra were taken on 400 MHz spectrometers. Chemical shifts are given in ppm (δ-scale), coupling constants (J) in Hz. Complete assignment of all NMR signals was performed using a combination of H, H-COZY, H, C-HSQC and H, C-HMBC experiments. Mass spectra (EI) were measured on a dual sector mass spectrometer using direct inlet and the lowest temperature enabling evaporation. Compounds 1–9 were prepared according to reported procedures17. Column chromatography was performed using flash silica gel. Yields are given for isolated products showing one spot on a TLC plate and no impurities detectable in the NMR spectrum. The DPPH and ABTS (from AldrichSigma) radicals were purchased with the highest purity available and used as received. Chromane 9: To a suspension of orcin (1.00 g, 7.94 mmol), methyl vinyl ketone (1.30 g, 18.26 mmol), anhydrous magnesium sulfate (1.50 g) and methanol (30 mL) at 0 1C was added p-TsOH (1.43 g, 7.94 mmol). The mixture was allowed to warm to room temperature and was stirred for 8 h. The mixture was then diluted with ethyl acetate. The resultant solution was washed with a saturated aqueous solution of ammonium chloride, brine, dried over anhydrous magnesium sulfate and concentrated in vacuo. Purification by flash chromatography using petroleum ether and ethyl acetate as the eluant afforded compound 9 (1.68 g, 94%) as a white solid. Mp. 166–167 1C, 1H NMR (CDCl3, 400 MHz) δ: 1.52 (s, 6H), 1.80 (dd, J ¼6.5, 13.5, 1H), 1.82 (dd, J ¼ 6.5, 13.5, 1H), 2.09 (s, 3H), 2.15 (dd, J ¼6.5, 13.5, 1H), 2.16 (dd, J ¼ 6.5, 13.5, 1H), 2.55 (dd, J ¼6.5, 16.0, 1H), 2.56 (dd, J ¼ 6.5, 16.0, 1H), 2.70 (dd, J ¼6.5, 16.0, 1H), 2.73 (dd, J ¼ 6.5, 16.0, 1H), 3.24 (s, 6H), 6.29 (s, 1H); 13C NMR (CDCl3, 101 MHz): 14.7, 19.9, 23.4, 32.5, 49.3, 97.7, 103.0, 114.1, 135.1, 151.3. HR-EI-MS calcd. for C17H24O4, 292.1674, found 292.1672. 3.2.
Antioxidant assays
Quantification of radical scavenging activity was based on a procedure described previously with some modifications30,31. The solutions of the test compounds (AH) in methanol (0.1 mL) were added to the respective solution of the stable free radicals DPPH and ABTS (2.4 mL, 100 μM) at different molar ratios. After 30 min of incubation at 3770.5 1C maintained by an outer watercirculating bath, the absorbance at 517 nm and 735 nm was measured. Vitamin C, phloroglucinol and quercetin were used as positive controls. Results are expressed as the sample concentration that quenches 50% of the DPPH radical (IC50, μM), calculated by linear regression. The inhibition ratio (%) was calculated using the following formula: inhibition ratio (%) ¼(AAl)/A 100, where A is the absorbance of the control and Al is the absorbance of the test sample.
3.3.
Calculations
In this paper, BDEs were calculated according to the following procedures. The molecular geometries were optimized first by the molecular mechanics method MMX, and then, by the semiempirical quantum chemical method AM1. Finally, according to the proposal of Wright et al.32, (RO) B3LYP functional basis set of 6–311+G (2d, 2p) was used for the full geometry optimization. The zero point vibrational energy (ZPVE) and the vibrational contribution to the energy were scaled by a factor of 0.9805 according to the definition of BDE, BDE ¼Hr+HhHp, in which, Hr is the enthalpy for radical generated after H-abstraction reaction, Hh is the enthalpy for hydrogen atom, 0.49792 hartree, and Hp is the enthalpy for the parent molecule. Adiabatic IPs were calculated by means of a combined DFT method, which affords accuracy and economy. (RO) B3LYP functional basis set of 6–31 +G (d) was used to calculate single point energy (SPE) on the basis of the PM3-optimized structure. Thus, the molecular energy (e) consists of (RO) B3LYP/6-31+G (d) calculated SPE and PM3 calculated ZPVE (scaled by a factor of 0.947). In these calculations IP¼ ecep, in which ec is the energy for cation radical, while ep is the energy for parent molecule. All of the quantum chemical calculations were accomplished with the Gaussian 03 program. Considering the lipophilicity of edaravone, the calculations were achieved in gas phase. Acknowledgments This work was supported by the National Natural Science Foundation of China (21172271), Natural Science Foundation of Guangdong Province, China (Grant No. S2011020001231), and the Fundamental Research Funds for the Central Universities (11lgpy75). References 1. Kadenbach B, Ramzan R, Vogt S. Degenerative diseases, oxidative stress and cytochrome c oxidase function. Trends Mol Med 2009;15:139–47. 2. Farooqui T, Farooqui AA. Aging: an important factor for the pathogenesis of neurodegenerative diseases. Mech Ageing Dev 2009;130:203–15. 3. Mladenka P, Zatloukalova L, Filipsky T, Hrdina R. Cardiovascular effects of flavonoids are not caused only by direct antioxidant activity. Free Radical Biol Med 2010;49:963–75. 4. Rudkowska I. Functional foods for health: focus on diabetes. Maturitas 2009;62:263–9. 5. Rossi L, Mazzitelli S, Arciello M, Capo CR, Rotilio G. Benefits from dietary polyphenols for brain aging and Alzheimer's disease. Neurochem Res 2008;33:2390–400. 6. Mandel SA, Amit T, Kalfon L, Reznichenko L, Youdim MB. Targeting multiple neurodegenerative diseases etiologies with multimodal-acting green tea catechins. J Nutr 2008;138:S1578–83. 7. Hong JT, Yen JH, Wang L, Lo YH, Chen ZT, Wu MJ. Regulation of heme oxygenase-1 expression and MAPK pathways in response to kaempferol and rhamnocitrin in PC12 cells. Toxicol Appl Pharmacol 2009;237:59–68. 8. Ikuo N, Kentaro M, Tomokazu S, Yuko I, Keiko I, Masataka M, et al. Kinetic study of the electron-transfer oxidation of the phenolate anion of a vitamin E model by molecular oxygen generating superoxide anion in an aprotic medium. Org Biomol Chem 2003;1:4085–8. 9. Burton GW, Ingold KU. Vitamin E: application of the principles of physical organic chemistry to the exploration of its structure and function. Acc Chem Res 1986;19:194–201.
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