Materials and methods
Unless otherwise noted, the chemicals required for synthesis and antioxidant activity were purchased from Hi-media Laboratories. The biological hMAO activity evaluation of the test drugs was examined by quantifying their action on the generation of H2O2 by p-tyramine (general substrate for hMAO-B and hMAO-A), utilizing the Amplex Red MAO assay kit (Sigma USA) and MAO isoforms (microsomal) obtained from insect cells (BTI-TN-5B14) expressed as recombinant baculovirus consisting cDNA probes for hMAO-A or hMAO-B. Reactions were monitored by thin layer chromatography TLC executed over silica gel precoated plates (0.25 mm) purchased from Merck, envisioned of single spots was carried in iodine and UV chambers, in mobile media TLC- Benzene:Chloroform (7:3). Melting points were recorded on Sonar melting point apparatus in open capillary tubes. The nuclear magnetic resonance (NMR) spectra 1H NMR and 13C NMR spectra were confirmed in DMSO and deuterated CDCl3 respectively on Bruker Avance II 400 NMR spectrometer at a frequency of 400 MHz downfield to tetramethylsilane standard. Coupling constants (J) were reported in Hertz (Hz) and chemical shifts were depicted as d (parts per million). Infrared (IR) spectra were recorded on Perkin Elmer FTIR spectrophotometer by using KBr pellets technique. Waters Micromass Q-ToF Micro instrument was used for Mass spectra recording.
General procedure for the synthesis of caffeic acid chloride
In a round bottom flask (250 mL) thionyl chloride (10 mL) was added to caffeic acid (20 mmol, 2.46 g) in the presence of diethyl ether as a solvent and few drops of pyridine as the catalyst. The above reaction mixture was refluxed with stirring at 80 °C for 1–4 h. The progress of the reaction was scrutinized by TLC. The surplus amount of thionyl chloride was removed under low-pressure distillation. Lastly, the purity of the product was detected by single spot TLC under UV lamp and IR: peak shifted 1640 (carboxylic) to 1768 (acid chloride); Yield: 78%; MP: 155–157 °C.
General procedure for the synthesis of natural hybrid caffeic acid esters
Natural hybrid esters derived from caffeic acid were prepared by refluxing aromatic alcohols with a solution of caffeic acid chloride (0.05 mol) with ether (50 mL) at 70–80 °C for 8–10 h (Scheme 1). The mixture was refluxed on the water bath until the evolution of hydrogen chloride gas was stopped consequently the reaction completion was confirmed by single spot TLC. Finally, this mixture was placed at room temperature for solvent evaporation which yielded the crude product. This was extracted with ether (50 mL) to obtain ester, which was subjected to final recrystallization by acetone.
General procedure for the synthesis of amides
The solutions of equivalent amine/aniline (0.1 mol) in ether (50 mL) dropped to a caffeic acid chloride solution (0.1 mol) in ether (50 mL) kept at 0–10 °C temperature (Scheme 1). This reaction mixture was stirred up to 40 min and yielded precipitates were separated by filtration. Amides formed as crude precipitates were recrystallized with ethanol and further acidified with 5% hydrochloric acid and then treated with 4% sodium carbonate to remove water and residual aniline finally the extracted anilides were recrystallized with methanol.
Spectral data
(E)-N-(2-chloro-4-nitrophenyl)-3-(3,4-dihydroxyphenyl)acrylamide 1)
Rf TLC mobile phase: Benzene:Chloroform (7:3) = 0.67; (%) Yield = 67.3; M.P = 230–231 °C; IR (KBR pellets) cm−1; 3475 (O–H str., Ar), 3354 (N–H str., amide), 1627 (C=O str., amide), 1504 (NO2 str., nitro), 648 (Cl str., chloro); 1H NMR (400 MHz, DMSO-d6) δ = 8.27 (d, J = 1.5 Hz, 2H), 8.06 (dd, J = 7.5, 1.5 Hz, 2H), 7.90 (d, J = 7.5 Hz, 2H), 7.43 (dd, J = 14.9, 0.8 Hz, 2H), 7.06 (d, J = 1.5 Hz, 2H), 7.02 (s, 1H), 6.99–6.93 (m, 3H), 6.77 (d, J = 7.5 Hz, 2H); 13C NMR (400 MHz, CDCl3) δ = 168.5, 152.5, 145.9, 142.8, 139.8, 136.3, 128.2, 123.8, 121.6, 120.5, 119.8, 118.8, 117.3, 115.3, 112.8; MS ES + (ToF): m/z 333.34 [M++2]; CHN: Calc. C15H11ClN2O5: C, 53.83; H, 3.31; N, 8.37; Found: C, 53.79; H, 3.27; N, 8.20.
(E)-N-(4-chloro-2-nitrophenyl)-3-(3,4-dihydroxyphenyl)acrylamide 2)
Rf TLC mobile phase: Benzene:Chloroform (7:3) = 0.73; (%) Yield = 73.2; M.P = 200–202 °C; IR (KBR pellets) cm−1; 3437 (O–H str., Ar), 3313 (N–H str., amide), 1629 (C=O str., amide), 1536 (NO2 str., nitro), 721 (Cl str., chloro); 1H NMR (400 MHz, DMSO-d6) δ = 8.15 (d, J = 1.5 Hz, 2H), 7.86 (d, J = 7.5 Hz, 2H), 7.61 (dd, J = 7.5, 1.5 Hz, 2H), 7.41 (dd, J = 14.9, 0.8 Hz, 2H), 7.26 (d, J = 1.4 Hz, 2H), 7.12 (s, 1H), 6.98–6.95 (m, 3H), 5.03 (d, J = 7.5 Hz, 2H); 13C NMR (400 MHz, CDCl3) δ = 167.9, 150.5, 149.9, 138.8, 137.7, 135.5, 132.5, 130.1, 127.2, 124.4, 123.3, 120.8, 118.3, 115.3, 114.8; MS ES + (ToF): m/z 333.09 [M++2]; CHN: Calc. C15H11ClN2O5: C, 53.83; H, 3.31; N, 8.37; Found: C, 53.80; H, 3.27; N, 8.33.
(E)-N-(4-bromophenyl)-3-(3,4-dihydroxyphenyl)acrylamide 3)
Rf TLC mobile phase: Benzene:Chloroform (7:3) = 0.8; (%) Yield = 59.9; M.P = 167–167.8 °C; IR (KBR pellets) cm−1; 3536 (O–H str., Ar), 3174 (N–H str., amide), 1637 (C=O str., amide), 572 (Br str., bromo); 1H NMR (400 MHz, DMSO-d6) δ = 8.61–8.54 (m, 8H), 7.45–7.37 (m, 2H), 7.09 (d, J = 1.4 Hz, 2H), 7.07 (s, 1H), 7.02–6.94 (m, 3H), 5.14 (d, J = 7.5 Hz, 2H); 13C NMR (400 MHz, CDCl3) δ = 168.7, 151.5, 144.9, 143.8, 140.4, 135.8, 127.2, 121.7, 120.8, 118.5, 115.8, 111.3, 110.8; MS ES + (ToF): m/z 333.00 [M++2]; CHN: Calc. C15H12BrNO3: C, 53.91; H, 3.62; N, 4.19; Found: C, 53.89; H, 3.60; N, 4.16.
(E)-3-(3,4-dihydroxyphenyl)-N-(3-fluorophenyl)acrylamide 4)
Rf TLC mobile phase: Benzene:Chloroform (7:3) = 0.57; (%) Yield = 71.6; M.P = 189–186 °C; IR (KBR pellets) cm−1; 3374 (O–H str., Ar), 3044 (N–H str., amide), 1632 (C=O str., amide), 1192 (F str., fluro); 1H NMR (400 MHz, DMSO-d6) δ = 7.60 (dt, J = 7.5, 1.5 Hz, 2H), 7.13–7.03 (m, 6H), 6.95 (d, J = 1.4 Hz, 2H), 6.92 (s, 1H), 6.91–6.87 (m, 3H), 6.77 (ddt, J = 8.0, 7.5, 1.5 Hz, 2H), 6.13 (d, J = 7.5 Hz, 2H); 13C NMR (400 MHz, CDCl3) δ = 165.1, 163.8, 161.3, 150.5, 147.9, 144.8, 138.3, 131.3, 127.2, 124.8, 120.5, 119.5, 116.3, 114.8, 112.1, 110.9, 108.22, 108.2; MS ES + (ToF): m/z 272.23 [M++2]; CHN: Calc. C15H12FNO3: C, 74.74; H, 4.95; N, 4.59; Found: C, 74.71; H, 4.92; N, 4.55.
(E)-3-(3,4-dihydroxyphenyl)-N-(naphthalen-1-yl)acrylamide 5)
Rf TLC mobile phase: Benzene:Chloroform (7:3) = 0.71; (%) Yield = 82.1; M.P = 176–176.3 °C; IR (KBR pellets) cm−1; 3498 (O–H str., Ar), 3258 (N–H str., amide), 1627 (C=O str., amide), 1595 (C=C skeletal str., naphthyl); 1H NMR (400 MHz, DMSO-d6) δ = 7.88–7.81 (m, 2H), 7.72–7.65 (m, 2H), 7.64–7.57 (m, 2H), 7.54–7.51 (m, 3H), 7.52–7.42 (m, 3H), 7.39 (d, J = 0.7 Hz, 1H), 7.20 (td, J = 7.5, 0.5 Hz, 2H), 7.10 (d, J = 1.5 Hz, 2H), 7.01 (s, 1H), 7.00–6.93 (m, 3H), 6.78 (d, J = 7.5 Hz, 2H); 13C NMR = (400 MHz, CDCl3) δ 167.1, 149.5, 147.9, 141.8, 136.5, 135.1, 127.8, 126.4–126.1 (m), 125.6, 125.4, 124.2, 123.8, 119.3, 117.2, 114.8; MS ES + (ToF): m/z 272.23 [M++1]; CHN: Calc. C19H15NO3: C, 65.93; H, 4.43; N, 5.13; Found: C, 65.89; H, 4.41; N, 5.11.
(E)-4-formyl-2-methoxyphenyl 3-(3,4-dihydroxyphenyl)acrylate 6)
Rf TLC mobile phase: Benzene:Chloroform (7:3) = 0.49; (%) Yield = 75.5; M.P = 150–151 °C; IR (KBR pellets) cm−1; 3432 (O–H str., Ar), 1725 (C=O str., ester), 1644 (C=C skeletal str., alkene), 2839, 2734 (C–H str., aldehydes), 1234 (C–O str., ester), 1150 (C–O–C assym. str., Ar–O–CH3); 1H NMR (400 MHz, DMSO-d6) δ = 9.83 (t, J = 0.5 Hz, 1H), 7.69 (dd, J = 15.0, 0.8 Hz, 1H), 7.49–7.45 (m, 2H), 7.36 (d, J = 7.4 Hz, 1H), 7.06 (d, J = 1.6 Hz, 1H), 6.96 (ddd, J = 7.5, 1.5, 0.6 Hz, 1H), 6.89 (d, J = 15.1 Hz, 1H), 6.78 (d, J = 7.5 Hz, 1H), 3.87 (s, 3H); 13C NMR (400 MHz, CDCl3) δ = 194.3, 163.1, 152.6, 149.5, 148.2, 146.5, 141.6, 135.6, 127.8, 123.6, 122.6, 120.5, 117.3, 115.4, 115.8, 110.1 57.1; MS ES + (ToF): m/z 313.05 [M++1]; CHN: Calc. C17H14O6: C, 64.97; H, 4.49; Found: C, 64.95; H, 4.46.
(E)-4-allyl-2-methoxyphenyl 3-(3,4-dihydroxyphenyl)acrylate 7)
Rf TLC mobile phase: Benzene:Chloroform (7:3) = 0.67; (%) Yield = 73.6; M.P = 161–161.7 °C; IR (KBR pellets) cm−1; 3436 (O–H str., Ar), 1732 (C=O str., ester), 1268 (C–O str., ester), 1124 (C–O–C assym. str., Ar–O–CH3); 1H NMR (400 MHz, DMSO-d6) δ 8.16 (dd, J = 14.9, 0.8 Hz, 2H), 7.10–7.05 (m, 4H), 6.91 (ddd, J = 7.5, 1.5, 0.6 Hz, 2H), 6.84 (d, J = 15.1 Hz, 2H), 6.79–6.75 (m, 6H), 5.94 (ddt, J = 16.8, 10.1, 6.2 Hz, 2H), 5.19–5.11 (m, 3H), 5.08 (dt, J = 2.1, 1.0 Hz, 1H), 3.84 (s, 6H), 3.32 (dp, J = 6.2, 1.0 Hz, 4H); 13C NMR (400 MHz, CDCl3) δ 165.1, 153.3, 149.5, 145.2, 142.6, 140.2, 139.8, 138.2, 127.8, 123.6, 121.6, 119.8, 117.3, 117.3, 114.48, 114.8, 113.2, 55.9, 40.4; MS ES + (ToF): m/z 325.05 [M++1]; CHN: Calc. C19H18O5: C, 69.93; H, 5.56; Found: C, 69.90; H, 5.54.
(E)-5-isopropyl-2-methylphenyl 3-(3,4-dihydroxyphenyl)acrylate 8)
Rf TLC mobile phase: Benzene:Chloroform (7:3) = 0.79; (%) Yield = 69.5; M.P = 182–182.6 °C; IR (KBR pellets) cm−1; 3394 (O–H str., Ar), 1875 (C=O str., ester), 1616 (C=C skeletal str., alkene), 1255 (C–O str., ester); 1H NMR (400 MHz, DMSO-d6) δ = 8.52 (dd, J = 14.9, 0.8 Hz, 1H), 7.49 (d, J = 1.6 Hz, 1H), 7.01 (dq, J = 7.8, 0.9 Hz, 1H), 6.83–6.74 (m, 2H), 6.64–6.23 (m, 2H), 6.17 (d, J = 7.5 Hz, 1H), 2.92 (dtt, J = 13.5, 6.7, 0.8 Hz, 1H), 2.18 (d, J = 1.0 Hz, 3H), 1.28 (d, J = 6.8 Hz, 6H); 13C NMR (400 MHz, CDCl3) δ = 168.2, 150.4, 148.5, 145.2, 145.02, 144.8, 129.7, 128.4, 126.8, 122.7, 118.5, 116.3, 114.5, 112.0, 33.2, 23.9, 15.9; MS ES + (ToF): m/z 311.05 [M++1]; CHN: Calc. C19H20O4: C, 73.06; H, 6.45; Found: C, 73.02; H, 6.41.
(E)-3-hydroxyphenyl 3-(3,4-dihydroxyphenyl)acrylate 9)
Rf TLC mobile phase: Benzene:Chloroform (7:3) = 0.65; (%) Yield = 63.9; M.P = 198–198.4 °C; IR (KBR pellets) cm−1; 3375 (O–H str., Ar), 1810 (C=O str., ester), 1595 (C=C skeletal str., alkene), 1255 (C–O str., ester); 1H NMR (400 MHz, DMSO-d6) δ = 7.71–7.62 (m, 2H), 7.16 (tt, J = 7.6, 0.9 Hz, 2H), 7.06 (d, J = 1.5 Hz, 2H), 6.99–6.89 (m, 4H), 6.86 (dd, J = 1.5, 0.7 Hz, 3H), 6.86–6.77 (m, 4H), 6.77 (s, 1H), 13C NMR (400 MHz, CDCl3) δ = 169.9, 156.5, 152.3, 150.5, 146.2, 143.2, 129.9, 125.8, 123.6, 118.3, 118.1, 114.8, 112.3, 110.7, 107.1; MS ES + (ToF): m/z 271.02 [M++1]; CHN: Calc. C15H12O5: C, 66.17; H, 4.44; Found: C, 66.14; H, 4.41.
(E)-3-(3,4-dihydroxyphenyl)-N-ethylacrylamide 10)
Rf TLC mobile phase: Benzene:Chloroform (7:3) = 0.52; (%) Yield = 72.6; M.P = 194–194.2 °C; IR (KBR pellets) cm−1; 3464 (O–H str., Ar), 3278 (N–H str., amide), 1647 (C=O str., amine); 1H NMR (400 MHz, DMSO-d6) δ = 7.40–7.31 (m, 1H), 7.08–7.04 (m, 1H), 6.96 (ddd, J = 7.5, 1.5, 0.6 Hz, 1H), 6.78 (d, J = 7.5 Hz, 1H), 6.51 (d, J = 15.1 Hz, 1H), 3.24 (q, J = 8.0 Hz, 2H), 1.24 (t, J = 8.0 Hz, 3H); 13C NMR (100 MHz, Chloroform-d) δ = 167.9, 148.9, 148.5, 147.2, 145.2, 133.9, 131.5, 129.3, 127.5, 126.8, 126.4, 122.6, 121.2, 116.3, 116.1, 115.7, 115.8; MS ES + (ToF): m/z 206.03 [M++1]; CHN: Calc. C11H13NO3: C, 63.76; H, 6.32; N, 6.76; Found: C, 63.74; H, 6.29; N, 6.73.
(E)-naphthalen-2-yl 3-(3,4-dihydroxyphenyl)acrylate 11)
Rf TLC mobile phase: Benzene:Chloroform (7:3) = 0.8; (%) Yield = 75.4; M.P = 176–176.9 °C; IR (KBR pellets) cm−1; 3275 (O–H str., Ar), 1780 (C=O str., ester), 1601 (C=C skeletal str., naphthyl), 1216 (C–O str., ester); 1H NMR (400 MHz, DMSO-d6) δ = 7.88 (ddd, J = 7.5, 1.4, 0.6 Hz, 2H), 7.75 (dtd, J = 7.4, 1.4, 0.5 Hz, 2H), 7.71–7.62 (m, 4H), 7.58–7.48 (m, 4H), 7.45 (tdd, J = 7.4, 1.5, 0.5 Hz, 2H), 7.23 (ddd, J = 7.5, 1.5, 0.5 Hz, 2H), 7.07 (d, J = 1.5 Hz, 2H), 6.97 (ddd, J = 7.5, 1.5, 0.6 Hz, 2H), 6.86–6.77 (m, 3H), 6.77 (s, 1H); 13C NMR (400 MHz, CDCl3) δ = 166.3, 150.4, 150.5, 148.2, 144.2, 134.9, 130.5, 127.3, 126.5, 124.8, 124.4, 123.6, 120.2, 116.36, 115.1, 114.7, 114.8; MS ES + (ToF): m/z 305.05 [M++1]; CHN: Calc. C19H14O4: C, 74.50; H, 4.61; Found: C, 74.47; H, 4.59.
(E)-2-isopropyl-5-methylcyclohexyl 3-(3,4-dihydroxyphenyl)acrylate 12)
Rf TLC mobile phase: Benzene:Chloroform (7:3) = 0.74; (%) Yield = 78.3; M.P = 185–186 °C; IR (KBR pellets) cm−1; 3445 (O–H str., Ar), 1715 (C=O str., ester), 1612 (C=C skeletal str., Ar), 1198 (C–O str., ester), 1H NMR (400 MHz, DMSO-d6) δ = 7.47 (dd, J = 15.3, 0.8 Hz, 2H), 7.09 (d, J = 1.5 Hz, 2H), 6.95 (ddd, J = 7.5, 1.5, 0.6 Hz, 2H), 6.78 (d, J = 7.5 Hz, 2H), 6.15 (d, J = 15.1 Hz, 2H), 5.08 (q, J = 7.0 Hz, 2H), 2.04–1.87 (m, 4H), 1.87–1.82 (m, 4H), 1.80 (ddt, J = 8.5, 5.4, 1.5 Hz, 1H), 1.72–1.58 (m, 4H), 1.58–1.50 (m, 1H), 1.46–1.33 (m, 2H), 0.93–0.81 (m, 16H), 13C NMR (400 MHz, CDCl3) δ = 165.9, 149.5, 147.2, 146.9, 129.5, 122.7, 119.4, 117.1, 75.6, 46.4, 37.6, 34.1, 30.8, 27.8, 23.5, 21.6, 19.6; MS ES + (ToF): m/z 317.14 [M++1]; CHN: Calc. C19H26O4: C, 71.67; H, 8.23; Found: C, 71.64; H, 8.19.
Monoamine oxidase-A and MAO-B assays
All synthesized compounds were evaluated for their capability to inhibit the MAO-A and MAO-B isoforms of human MAO by a fluorometric method. The MAO inhibitory property of the test compounds was examined with the recombinant human enzymes using an Amplex Red Monoamine Oxidase Assay Kit (Sigma USA). The incessant peroxidase-linked photometric assay was performed on the fluorometer. Due to their limited aqueous solubility, a few compounds were solubilized in DMSO whose final concentration of 3.3% (v/v) does not affect MAO activity. Distilled water was used as a negative control. In brief, 0.1 mL of sodium phosphate buffer (0.05 M, pH 7.4) with the test new compounds/reference inhibitors in different concentrations and sufficient quantity of recombinant hMAO-A or hMAO-B requisite and in step to complete our investigational protocol with the equal reaction velocity, (hMAO-A: 1.1 mg protein; explicit activity: 150 nmol of p-tyramine oxidized to p-hydroxyphenylacetaldehyde/min/mg protein; hMAO-B: 7.5 mg protein; explicit activity: 22 nmol of p-tyramine changed/min/mg protein) were incubated for 15 min at 37 °C, placed in the dark fluorometric compartment. After incubation, the effect was attained through the addition of 200 mM Amplex Red reagent (final concentrations), 1 mM p-tyramine, and 1 U/mL horseradish peroxidase. Finally, the generation of hydrogen peroxide and, subsequently, of resorufin was measured at 37 °C in fluorescence reader (λemission, 585 nm, λexcitation, 530 nm) after 15 min, by the time fluorescence amplified linearly. All the control experiments were performed concurrently by changing the reference inhibitors and test compounds with suitable dilutions in the solvents. Moreover, the activity of abovementioned evaluated compounds to alter the fluorescence produced in the combination reaction by (e.g. for reacting with Amplex Red reagent directly) non-enzymatic inhibition was identified by addition of these compounds to solutions having just the Amplex Red reagent in a sodium phosphate buffer. Clorgyline and pargyline were taken as the standard inhibitor at the same concentrations with the compounds. The fluorescence reading was computed by subtraction of the background activity and was evaluated from wells with all components not including the hMAO isoforms that was substituted by a sodium phosphate buffer solution. All data was processed in Microsoft Excel, to calculate IC50 values.
Kinetic parameters of MAO activity
Kinetic constants of steady-state (Vmax, maximum rate, and Km, Michaelis constant,) were measured by studying the effects of substrate concentration on the primary rate of reaction of MAO-A or MAO-B in absence and presence of compounds at diverse concentrations. To examine the mode of action by synthesized derivatives all of them were solubilized in dimethyl sulfoxide, with the concentration of 1%, and utilized in a wide concentration range. Construction of Lineweaver–Burk plots illustrated the mode of MAO inhibition. Selectivity index SI (Ki (MAO-A)/Ki (MAO-B)) was also computed. Enzyme kinetics for the interaction of the compounds with the MAO enzymes was computed on the GraphPad Prism 7 software.
Statistical analysis
Results in the studies are depicted as mean ± SEM (standard error mean). Experimental data were evaluated statistically by utilizing one-way analysis of variance (ANOVA). However, ANOVA revealed considerable difference as revealed by ANOVA/Dunnett’s test. P < 0.05 was regarded as to be statistically significant. The statistical evaluation was carried out by Graph Pad Prism 5.0 Version for Windows (San Diego, CA, USA).