Synthesis and Free Radical-scavenging Activities of Di-mannich Bases of Cyclovalone Derivatives

Novel di-Mannich bases of cyclovalone derivatives (1) have been synthesized and evaluated their antioxidant activity using DPPH free radical-scavenger method. The structures of the compounds were confirmed on the basis of FT-IR, 1H-NMR, 13C-NMR and mass spectral data. The result of antioxidant evaluation showed that di-Mannich derivative of cyclovalone with diethylamine ((2E,6E)-2,6-bis({3-[(diethylamino)methyl]-4-hydroxy-5-methoxyphenyl} methylidene) cyclohexan-1-one) (2a) exhibited the highest antioxidant activity with IC50 = 39.0 μM. Structure-activity relationship study showed that the higher pKa of the Mannich base, the higher activity (the lower IC50) of the compound.


INTRODUCTION
Oxidative stress, generating by an imbalance between free radical production and antioxidant defenses, is associated with the damage of various species of molecules including proteins, lipids, and nucleic acids.A role for oxidative stress has been postulated to contribute significantly to the pathogenesis of atherosclerosis, inflammatory conditions, certain cancers, and the process of aging.Besides the complex system of antioxidant metabolites and enzymes that naturally prevent cell damage, the exogenous antioxidant may sometimes be required to keep reactive oxygen species at optimum level [1][2][3] .
A Mannich reaction is a suitable method to introduce aminoalkyl group into a molecule.In several instances, the Mannich derivatives exhibit better biological activity than the corresponding parent analogs.Moreover, the presence of Mannich side chain increases the solubility and hence the bioavailability of the compounds 2,9-13 .Herein we report the synthesis and antioxidant activity of Mannich bases derivatives of cyclovalone.

Chemistry
All used chemicals were purchased from Merck or Aldrich Company and used without further purification.Thin layer chromatography (TLC) was carried out on silica gel 60 F 254 plates (Merck) and spots were detected under an ultraviolet and visible light.Melting points were determined in the capillary tube using electrothermal digital melting point apparatus (Stuart Scientific).The infrared spectra were recorded with FTIR 8400S Spectrometer (Shimadzu).NMR spectra were recorded on NMR spectrometer (Agilent) at 500 MHz for 1 H and 125 MHz for 13 C using TMS as an internal standard.Highresolution mass spectra (HRMS) were measured with a Waters LCT Premier XE (ESI-TOF) system in positive or negative mode.The absorption in the determination of free radical-scavenging activity was measured using UV-Vis Spectrophotometer 1601 (Shimadzu).

General synthesis of di-Manncih bases of cyclovalone derivatives (2a-e)
The di-Mannich bases of cyclovalone were synthesized by Mannich reaction of compound 1 according to the method of synthesis of di-Mannich bases of 1,5-bis(4-hydroxy-3-methoxyphenyl) penta-1,4-dien-3-one reported previously 7 , with little modification: The solution of 2 mmol of compound 1 in acetonitrile (50 ml) was added to mixture of 16 mmol paraformaldehyde and 16 mmol of corresponding secondary amine in acetonitrile (50 ml) which previously was heated at 80 o C for 10 minutes.The reaction mixture then was refluxed until the disappearance of 1.The completion of the reaction was monitored by TLC for 5-27 h.The reaction solvent was removed in vacuo using a rotary evaporator, the crude products were washed with cold acetonitrile and then purified by recrystallization or column chromatography to give compound 2a-e.

Free Radical-Scavenging Activity Evaluation
The antioxidant activities of Mannich bases of cyclovalone derivatives (2a-e) and cyclovalone (1) were evaluated by the free radicalscavenging activity of stable 2,2-diphenyl-1picrylhydrazyl (DPPH) according to the methodology described by Brand-Williams et al. with a little modification [17][18] .Quercetin was used as reference standard.The test or reference compounds were prepared at five different concentrations in methanol.The test or reference solution (0.5 mL) was mixed with 0.5 mL of DPPH radical solution 0.5 mM in methanol and then allowed to stand at room temperature for 30 min. in a dark laboratory condition.The changes in color (from deep violet to light yellow) were measured at 517 nm.The mixture of of methanol (0.5 mL) and of sample solution (0.5 mL) serve as a blank.The control solution was prepared by mixing methanol (0.5 mL) and DPPH radical solution (0.5 mL).The experiment for each test compounds was performed in triplicate.The percent free radical-scavenging activity (% Scavenging) was calculated according to the following equation: The IR spectra of compounds 2a-e appeared CH aliphatic bands at 2,972-2,830 cm -1 and showed the disappearance of OH phenolic peak.The bands at 1,246-1,057 cm -1 and 1,000-1081 cm -1 correspond to C-O phenol, C-O ether, and C-N; while the α,β-carbonyl groups of the cyclovalone are observed as strong bands at 1,639-1,659 cm -1 and 1589-1591 cm -1 .In 1 H-NMR spectra, the protons of two symmetrical aromatic ring remained only four protons appeared at δ 6.9 ppm (2H) and at δ 6.8 ppm (2H) as singlet or doublet with J=1-2 Hz indicated that the Mannich base substituted a proton at the ortho position relative to the hydroxyl group of 1.The data were supported by the disappearance of OH phenolic peak in IR spectra caused by intramolecular hydrogen bond formation between the hydroxyl group and N atom of the Mannich base [14][15] .The structures were further supported by 13 C-NMR and MS spectra of the compounds which showed the complete agreement with the assigned molecular structures.

Antioxidant acitivity
The antioxidant activities of the synthesized compounds were evaluated using DPPH free radical-scavenger method because of the suitability of the antioxidant mechanism with the compounds.Furthermore, the DPPH analysis is a fast and an uncomplicated test ensuring the reliable result.The mechanism of antioxidant activity briefly is that the phenolic compounds act as the hydrogen donor to reduce the radical molecules, and then the radical antioxidant will be stabilized by electron delocalization in the aromatic system or coupled with other or the same radical antioxidant to give non-radical molecules 3,8,[17][18][19] .
The results of DPPH free radicalscavenging activity of the title compounds are listed in Table 1.All the compounds show free radicalscavenging activity.Compound 2a is the most potent, with IC 50 = 39.0 µM or about a half of quercetin's antioxidant activity.The activity of compound 2a and 2b are more potent than that of 1, while compound 2e, 2d and 2c are lower than that of 1. Structure-activity relationship study of the compounds demonstrates the effect of the alkalinity (pKa) of Mannich substituent of the compound on where A sample is the absorption of DPPH with test or reference compounds and A control is the absorption of DPPH without test compounds.Data obtained was then analyzed using a linear regression equation to determine IC 50 of free radical-scavenging activity (FRSA) of the compounds.

Chemistry
The title compounds 2a-e were synthesized in two steps by the method summarized in Scheme 1. Vanillin was reacted with cyclohexanone according to the method previously reported to provide cyclovalone (1) 5 .Treatment of 1 with paraformaldehyde and corresponding secondary amine (a-e) in acetonitrile at reflux temperature for 5-27 h (TLC monitoring) afforded the title compounds 2a-e.the antioxidant activity (Figure 1).The higher pKa of the Mannich base compound, the higher activity (the lower IC 50 ) of the compound.In the previous study was reported that the hydroxyl group of the phenolic antioxidant compound is essential for the activity.The small alkyl substituents (e.g.methyl and ethyl) and electron donating groups (e.g.methoxy) at ortho positions relative to the phenol groups enhanced the activity.On the contrary, bulky alkyl substituents (e.g.isopropyl and t-butyl) retarded the activity 5,8 .

CONCLUSION
A series of di-Mannich bases of cyclovalone derivatives were synthesized and their free radical-scavenging activity evaluated.The data obtained demonstrated the effect of the basicity of Mannich substituent of the compound on the antioxidant activity.The di-Mannich derivative of cyclovalone with diethylamine and dimethylamine (2a and 2b) exhibited higher free radicalscavenging activity than 1.