Dehydrozingerone (DHZ), 4-(4-hydroxy-3-methoxyphenyl)-3-buten-2-one (1), is a phenolic compound found as a minor component in the rhizomes of ginger (Zingiber officinale Roscoe). Structurally, DHZ is similar to a half of curcumin. Like as curcumin, the compound also exhibits a wide range of biological activities such as anti-inflammatory, antioxidant, antimicrobial, and cytotoxic activity.1-8 Various chemical modification of DHZ has been performed to exploit for diverse biological activities of DHZ derivatives, such as esterification or alkylation of OH phenolic group, replacement of OH with other substituents, substitution of the methoxy group, reduction of double bond, cyclization of α,β-unsaturated carbonyl, and substitution of hydrogen atom bound to the carbonyl α-carbon.2,4,9-11 Some Mannich bases of DHZ have been synthesized and reported to have better anti-inflammatory activity compared to DHZ and fairly good anti-inflammatory activity compared to indomethacin. However, the aminomethyl group was substituted at the carbonyl α-carbon of the compound.2 The substitution in the aromatic ring of DHZ with an aminomethyl group via Mannich reaction has not been reported yet. The Mannich reaction can take place between a phenolic compound containing one active hydrogen atom, secondary amines, and formaldehyde. The hydroxyl group in phenol is an electron-donating group making benzene very reactive to electrophilic substitution.12 In several cases, the Mannich derivatives of phenolic compound exhibit better biological activity than the corresponding parent analogs, such as several Mannich derivatives of diacetylresorcinol, resveratrol, hydroxycoumarin, hydroxybenzopyranone, and chalcone for anti-inflammatory activity, and chalcone, thymol, and flavonone for antioxidant activity.12-15 Herein we report the synthesis, anti-inflammatory, and antioxidant activity of the Mannich bases of DHZ derivatives substituted on the phenyl ring.
MATERIALS AND METHODS
Chemical Material and General Procedures
All of the solvents, chemicals, and reagents were purchased from commercial sources (Sigma-Aldrich, USA; E. Merck, Germany; and Mallinckrodt, USA). Products purifications were performed using a flash column chromatographic method on silica gel 60, 0.063 – 0.200 mm (Merck, Germany). Purity test of the compounds was performed using TLC method on silica gel 60 F254 plates (Merck, Germany) and the spots were detected by UV-Vis lamp (Camag). Melting points were determined in one open-end capillaries method using Analogue Model SMP11 (Stuart Scientific, UK) and are uncorrected. Infrared (IR) spectra were recorded on an FT-IR Spectrophotometer (8400S, Shimadzu, Japan); NMR spectra were recorded on an NMR spectrometer (Agilent, USA) with console system DD2 at 500 MHz for 1H-NMR and 125 MHz for 13C-NMR, using TMS as internal standard and CDCl3 as solvents for all compounds; and the high-resolution mass spectra were recorded on a Water LCT Premier XE ESI-TOF mass spectrometer (Waters Corp., USA).
Synthesis of dehydrozingerone (DHZ) (1)
DHZ (1) was synthesized through condensation between vanillin and acetone (1:10, mol/mol) using a solution of potassium hydroxide as the catalyst as reported previously (Figure 1).3 Vanillin (20 gram; 0.1314 mol) was dissolved in acetone (80 mL; 1089.4 mmol). Potassium hydroxide (11.2 g; 200 mmol) in 80 mL demineralized water was added to the solution then stirred for 24 h at room temperature. The mixture was neutralized with sulfuric acid (50%) to obtain a yellow crystal. The product was filtered, washed with cool demineralized water, dried under vacuum in 75-80°C, and purified by recrystallization from boiling demineralized water to afford pure DHZ (1).
Synthesis of Mannich bases of dehydrozingerone derivatives (2a-e)
The compounds (2a-e) were synthesized by amino-alkylation of DHZ (1) through a Mannich reaction (Figure 1) according to the method of synthesis of Mannich derivatives of vanillin and cyclovalone reported previously.16-17 Synthesis 2a-c: DHZ (1.92 g; 10 mmol) was added to a mixture of formaldehyde solution (1.92 mL; 15 mmol) and secondary amines (15 mmol) in 9 mL of ethanol previously stirred for 30 min, stirred for 30 min, refluxed at 79°C for 30 min, and then stirred until the reaction finished at 25°C for 2-7 h. [Synthesis 2d-e: DHZ (1.92 g; 10 mmol) was added to a mixture of paraformaldehyde (450.45 mg; 15 mmol) and secondary amines (15 mmol) in 50 mL of acetonitrile previously heated at 80°C for 10 min, stirred for 30 min, and refluxed for 3-8 h]. The progress of the reaction was monitored by TLC. After the reaction was completed, about 75% of the solvent was evaporated under reduced pressure and then refrigerated overnight. The product was filtered, washed with cold ethanol, dried under vacuum at 30-40°C and purified by flash column chromatography to afford pure compound 2a-e.
In vitro anti-inflammatory assay
The in vitro anti-inflammatory evaluation was performed using inhibition of heat-induced albumin denaturation method according to the previously reported with slight modification.18-19 The reaction mixture consisted of 1 mL of different concentration (4, 5, 8, 10, 12 mM) of the test compounds or diclofenac sodium standard (purchased from PT Kimia Farma, Indonesia) and 1.4 mL phosphate buffer saline pH 6.3 (purchased from Sigma-Aldrich, USA) were mixed with 25 μL egg albumin obtained from fresh hen’s egg (purchased from local supermarket), incubated at 37°C for 15 min, heated at 70°C in water bath for 5 min, and cooled to reach room temperature. The turbidity was measured as absorbance at 660 nm using UV-Vis spectrophotometer (V-530, Jasco, USA). For negative control was used the mixture of 1 mL of suitable solvent, 1.4 mL phosphate buffer saline pH 6.3, and 25 μL the egg albumin. The experiment was performed in triplicate, and the average of the absorbance was calculated. The percent of inhibition of albumin denaturation was calculated using the following formula:
The concentration of the compound providing 50% inhibition (IC50) was obtained by plotting % inhibition versus concentration. A lower IC50 corresponds to the higher anti-inflammatory activity.
In vitro antioxidant assay
The antioxidant evaluation was performed using a free-radical 1,1’-diphenyl-2-picrylhydrazyl (DPPH) model system according to the methods previously reported20 with slight modification. The reaction mixtures consisted of 0.5 mL of different concentration of the test compounds (10-200 μM) or quercetin standard (6-35 μM) and 0.5 mL DPPH solution in methanol (2.6 mM) were incubated at room temperature in the dark for 30 min. The absorbance was measured at 517 nm using UV-Vis spectrophotometer (V-530, Jasco, USA). The control solution was prepared as the reaction mixture but without any test compounds and methanol was used for baseline correction. The experiment was performed in triplicate, and the average of absorbance was calculated. The percentage (%) inhibition of radical scavenging activity was calculated using the following formula:
The concentration of the compound providing 50% inhibition (IC50) was obtained by plotting % inhibition versus concentration. A lower IC50 corresponds to the higher antioxidant activity.
Physical and spectral data of synthesized compounds
Dehydrozingerone (DHZ) (1)
Yellow crystalline solid; yield 33%; m.p. 126-128°C (according to literature = 129-131°C ). IR (KBr, cm-1) 3376-3225 (broad, OH), 3002 (C-H Ar), 2983, 2948, 2847 (C-H aliphatic), 1677 (α,β-unsaturated C=O), 1582, 1518 (C=C Ar), 1125, 1030 (C-O). 1H-NMR (500 MHz, CDCl3), δ/ppm: 2.37 (s, 3H, CH3), 3.94 (s, 3H, OCH3), 5.92 (s, 1H, OH), 6.59 (d, 1H, J=16.2 Hz, CH=Cethenyl), 6.93 (d, 1H, J=8.2 Hz, HAr), 7.06 (d, 1H, J=1.95 Hz, HAr), 7.09 (dd, 1H, J= 8.2 Hz and 1.9 Hz, HAr), 7.45 (d, 1H, J=16.2 Hz, CH=Cethenyl).
Light yellow crystal; yield 20%; m. p. 122-123°C. HRMS ES- (m/z): found 290.1392, calculated masses of C16H20NO4: 291.1392 (error 3.8 ppm). IR (KBr, cm-1): 3090 (C-H Ar), 2959, 2850 (C-H aliphatic), 1672 (α, β-unsaturated C=O), 1649 (C=C), 1591, 1420 (C=C Ar), 1161, 1117, 1078 (C-O). 1H-NMR (500 MHz, CDCl3, δ/ppm: 2.34 (s, 3H, CH3), 2.59 (m, 4H, -CH2), 3.74 (m, 2H, -CH2), 3.76, 3.75 (s, 4H, -CH2), 3.90 (s, 3H, OCH3), 6.55 and 7.40 (d, 1H, J=16.5 Hz, respectively, -CH=CH-ethenyl), 6.83 (d, 1H, J=0.7 Hz, HAr), 7.00 (d, 1H, J=0.3 Hz, HAr. 13C-NMR (125 MHz, CDCl3), δ/ppm: 27.4 (1C), 52.8 (2C), 55.9 (1C), 61.4 (1C), 66.7 (2C), 110.0 (1C), 120.9 (1C), 122.4 (1C), 124.6 (1C), 125.6 (1C), 143.6 (1C), 148.4 (1C), 149.8 (1C), 198.12 (1C).
Pale yellow crystalline solid; yield 27%; m.p. 153-155°C. HRMS ES- (m/z) found 318.1705, calculated masses of C18H24NO4: 319.1705 (error 2.8 ppm). IR (KBr, cm-1): 3088 (C-H Ar), 2972, 2845, 2821 (C–H aliphatic), 1660 (α,β-unsaturated C=O), 1636 (C=C aliphatic), 1621 (C=C Ar), 1257, 1082 (C–O). 1H-NMR (500 MHz, CDCl3), δ/ppm: 1.15 and 1.16 (two s of 6H, CH3), 1.89 and 2.82 (t, 2H and d 2H of -CH2-N-C), 2.34 (s, 3H, CH3), 3.71 (s, 2H, -N-CH2-Ar overlap with 2H, m, -CH-O-C), 3.90 and 3.91 ( two s of 3H, OCH3), 6.56 and 7.41 (d, 1H, J=16.1 Hz, respectively, -CH=CH-ethenyl), 6.83 (d, 1H, J=1.9 Hz, HAr), 7.01 (d, 1H, J=2.0 Hz, HAr). 13C-NMR (125 MHz, CDCl3), δ/ppm: 19.06 (2C), 27.49 (1C), 56.06 (1C), 58.47 (2C), 61.44 (1C), 71.78 (2C), 109.99 (1C), 121.10 (1C), 122.51 (1C), 124.75 (1C), 125.67 (1C), 143.77 (1C), 148.48 (1C), 150.07 (1C), 198.32 (1C).
Yellow powder; yield 70%; m.p. 165-167°C. HRMS ES- (m/z) found 303.1709, calculated masses of C17H23N2O3: 304.1709 (error 3.3 ppm). IR (KBr, cm-1): 3082 (C-H Ar), 2949, 2823, 2806 (C-H aliphatic), 1656 (α, β-unsaturated C=O), 1595 (C=C), 1255, 1087 (C-O). 1H-NMR (500 MHz, CDCl3), δ/ppm: 2.29 (s, 3H, CH3), 2.34 (s, 3H, CH3), 2.55 (m, 8H, CH2), 2.74 (s, 2H, CH2), 3.90 (s, 3H, CH3), 6.55 and 7.40 (d, 1H, J=16.1 Hz, respectively, -CH=CH-ethenyl), 6.82 (t, 1H, J=2.3 Hz, HAr), 6.99 (d, 1H, J=1.9 Hz, HAr). 13C-NMR (125 MHz, CDCl3), δ/ppm: 27.47 (1C), 46.00 (1C), 52.57 (2C), 54.94 (2C), 56.05 (1C), 61.06 (1C), 109.94 (1C), 121.40 (1C), 122.50 (1C), 124.62 (1C), 143.89 (1C), 148.48 (1C), 150.34 (1C), 198.35 (C).
Brown viscous oil; yield 32%. HRMS ES- (m/z) found 276.1600, calculated masses of C16H22NO3: 277.1600 (error 3.3 ppm). IR (KBr, cm-1): 3092 (C-H Ar), 2960, 2850 (C-H aliphatic), 1670 (α, β-unsaturated C=O), 1580 (C=C), 1260, 1160 (C-O), 1H-NMR (500 MHz, CDCl3), δ/ppm: 1.13 (t, 6H, CH3), 2.34 (s, 3H, CH3), 2.64 (q, 4H, J=7.15, CH2), 3.8 (s, 2H, CH2), 3.9 (s, 3H, CH3), 6.56 and 7.42 (d, 1H, J=16.1 Hz, respectively, -CH=CH-ethenyl), 6.81 (dd, 1H, J=1.9 Hz, HAr), 6.98 (d, 1H, J=1.9 Hz, HAr). 13C-NMR (125 MHz, CDCl3), δ/ppm: 11.30 (2C), 27.44 (1C), 46.52 (2C), 56.04 (1C), 56.82 (1C), 109.74 (1C), 122.18 (1C), 122.32 (1C), 124.33 (1C), 125.01 (1C), 144.19 (1C), 148.53 (1C), 151.34 (1C), 198.44 (1C).
Brownish orange powder; yield 39%; m.p. 95-97°C. HRMS ES- (m/z) found 248.1287, calculated masses of C14H18NO3: 249.1287 (error 3.6 ppm). IR (KBr, cm-1): 3009 (C-H Ar), 2989, 2958, 2830, 2785 (C-H aliphatic), 1667 (α, β-unsaturated C=O), 1616, 1591 (C=C), 1261, 1080 (C-O). 1H-NMR (500 MHz, CDCl3), δ/ppm: 2.35 (d, 8H, J=6.0 Hz, CH3), 3.68 (s, 2H, CH2), 3,90 (s, 3H, OCH3), 6.56 and 7.41 (d, 1H, J=16.0 Hz, respectively, -CH=CH-ethenyl), 6.82 (d, 1H, J=2.0, HAr), 6.99 (d, 1H, J=2.0 Hz, HAr). 13C-NMR (125 MHz, CDCl3), δ/ppm: 27.43 (1C), 44.51 (2C), 56.07 (1C), 62.46 (1C), 110.02 (1C), 122.06 (1C), 122.35 (1C), 124.51 (1C), 144.05 (1C), 149.46 (1C), 150.77 (1C), 198.42 (1C).
Anti-inflammatory and antioxidant activity
The title compounds (2a-e) were screened for their anti-inflammatory and antioxidant activity. The anti-inflammatory evaluation was performed using an inhibition of heat-induced albumin denaturation method, while the antioxidant activity was done using a free-radical DPPH scavenging activity model system. Using the methods, we found that, the inhibition of heat-induced albumin denaturation activity and DPPH radical scavenging activity of the synthesized compounds were concentration-dependent (Figures 2 and 3). The IC50 values of the biological activity of the title compounds calculated from the dose-response data were displayed in Table 1.
|Compounds||R||Charge of OH1)||Anti-inflammatory Activity||Antioxidant Activity|
|IC50 (mM)2)||IC50 (μM)20)|
|DHZ (1)||H||9.51||-||103.35 ± 0.00|
|2a||8.73||12.32 ± 1.06||80.07 ± 0.01|
|2b||8.73||13.04 ± 0.77||147.64 ± 0.02|
|2c||8.73||7.20 ± 0.27||89.91 ± 0.01|
|2d||8.73||33.86 ± 0.27||72.66 ± 0.01|
|2e||8.73||25.31 ± 6.44||50.23 ± 0.01|
|Diclofenac Sodium||-||-||8.03 ± 0.43||n.d.3)|
|Quercetin||-||-||n.d.3)||21.74 ± 0.00|
1) Calculated using MarvinSketch 22.214.171.124
Various chemical modification of dehydrozingerone (DHZ) has been performed to exploit for diverse biological activities of its derivatives.2,4,9-11 However, the substitution in the aromatic ring of the compound with an aminomethyl group via Mannich reaction has not been reported yet. In this study, a series of five novel Mannich bases of DHZ (2a-e) was synthesized. The IR spectra of the synthesized compounds showed the disappearance a broad peak of OH phenolic at 3200-3550 cm-1, and the appeared additional of peaks at between 2700-2970 cm-1 compared to that of compound 1. These indicate the disappearance of intermolecular hydrogen bonding of the OH group and the addition of CH aliphatic chain of the compound 2a-e. The bands at 1078-1261 cm-1 correspond to C-O phenol, C-O ether, and C-N; while the α,β-carbonyl groups of the compound are observed as intense bands at 1660–1670 cm-1. In 1H-NMR spectra, the protons of the aromatic ring remained only two protons appeared at δ 6.8 ppm (1H), and 7.0 ppm (1H) as a doublet with J=1-3 Hz indicated that the Mannich base substituted a proton at the ortho position relative to the OH phenolic group of compound 1. The data were supported by the disappearance of the broad peak of OH phenolic in the FTIR spectra of the compound because of the formation of intramolecular hydrogen bonding between the OH group and N atom of the Mannich base.21-22 The structures were further supported by 13C-NMR spectra which provided the expected number and types carbons of the carbonyl, aromatic, ethylenic and aliphatic moieties of the compounds, and by MS spectra which provided the molecular masses of the compounds. These values are in the complete agreement with the structure assigned.
The synthesized compounds (2a-e) were then screened for their anti-inflammatory using inhibition of heat-induced albumin denaturation method and antioxidant activity using a free-radical DPPH scavenging activity model system.
The inhibitory of heat-induced albumin denaturation activity of the compound ranged in IC50 values from 7.20 mM to 33.86 mM (Table 1, Figure 2). In this series, the compound 2c containing N-methylpiperazine moiety exhibited the highest activity, which was comparable with that of diclofenac sodium (IC50 = 8.03 mM), while the parent compound (DHZ, 1) did not exhibit the heat-induced albumin denaturation. The previous study reported that several anti-inflammatory drugs had shown the ability to inhibit heat-induced albumin denaturation.23-24 Another study indicated that although there was no complete correlation, several compounds of acetamido[(phenyl-4’-yl)-oxymethyl)]-2-(p-substituted phenylamino)-1,2,4-triazoles and -1,3,4-thiadiazoles which showed good inhibition of denaturation also exhibited significant in vivo anti-inflammatory activity by carrageenan-induced edema in the rat paw.25 Therefore the title compounds promising to have potential anti-inflammatory activity.
The free-radical DPPH scavenging activity of the compounds ranged in IC50 values from 50.23 μM to 147.64 μM (Table 1, Figure 3). In this series, the compound 2e containing dimethylamine moiety was found to be the highest and showed moderate activity compared to that of quercetin (IC50 = 21.74 μM). Mostly the antioxidant activity of the Mannich bases derivatives of DHZ was higher than that of the parent compound (DHZ, 1) (IC50 = 103.35 μM). The previous study reported that the hydroxy phenolic group of DHZ is essential for antioxidant activity through hydroxy radical scavengers.3,26 The results indicate that most of the aminomethyl groups (Mannich bases) of the compounds enhance the ability of the hydroxy group to scavenge the radical. At curcumin analogs, the electron-donating groups are improving the antioxidant activity,27 while recent study reported that the Mannich base of cyclovalone derivatives containing dimethylamine and diethylamine moiety had a higher free-radical DPPH scavenging activity than that the parent compound. The higher the basicity of N atom of the Mannich base, the higher the activity.28 However, the relationship is not shown in the Mannich derivatives of DHZ. Therefore further studies need to be done to explain any factors affecting the antioxidant activity of phenolic compounds by the introduction of Mannich bases.
A series of five new Mannich bases of dehydrozingerone (DHZ) derivatives were synthesized successfully and evaluated for their anti-inflammatory activity through inhibiting heat-induced albumin denaturation and antioxidant activity employing free radical DPPH scavenging activity method. Using the method, all the synthesized compounds (2a-e) showed anti-inflammatory and antioxidant activity. Compound 2c containing N-methylpiperazine moiety showed the highest anti-inflammatory activity. The activity was comparable to diclofenac sodium. While compound 2e containing dimethylamine moiety exhibited the highest antioxidant activity. The compound exhibited moderate antioxidant activity compared to quercetin as standard.