Organic & Biomolecular Chemistry
Paper
Synthesis of (E)-3-(4-((dimethylcarbamoyl)thio)-3-methoxy- Spectrophotometric assays
phenyl)acrylic acid (4). Aldehyde 3 (0.61 g, 2.54 mmol) was dis-
•
Sodium persulfate, DPPH , 2,2′-azino-bis(3-ethylbenzothiazo-
solved in anhydrous pyridine (3 mL, 37.1 mmol). Then,
malonic acid (0.41 g, 3.90 mmol) and piperidine (88 µL,
•
line-6-sulfonic acid) diammonium salt (ABTS) and GO were
acquired from Sigma Aldrich. In these methods, the scaven-
ging of the colored radical by an antioxidant is followed by
spectrophotometry. The absorbance decrease was monitored
in a multiplate reader (BioTek Synergy HT from BioTek
Instruments, Winooski, VT, USA). The results were expressed
in IC50 as mean ± standard deviation (n = 3). Trolox (Sigma
Aldrich), a water-soluble vitamin E derivative, was used as a
standard reference. Kinetic curves were prepared using
GraphPad PRISM version 6 for Windows (GraphPad Software®,
San Diego, CA, USA).
0
1
.89 mmol) were added. The mixture was stirred at 60 °C over
h. Once the reaction was complete, the mixture was poured
onto water (15 mL), cooled on an ice bath and acidified with
HCl 6 M. The product was collected by filtration under
reduced pressure and washed with water. After suspension in
methanol, the solid was centrifuged at 3000g for 20 min. The
supernatant was discarded, yielding the purified product. TLC
analysis was performed in dichloromethane/methanol/formic
acid (9 : 1 : 0.01). The general procedure was adapted from
2
9
1
Teixeira et al. with some modifications. η = 91%. H NMR
DMSO-d ): δ (ppm) = 2.91 (bs, 3H, NCH ), 3.05 (bs, 3H,
NCH ), 3.83 (s, 3H, OCH ), 6.65 (d, J = 16.0 Hz, 1H, Hα), 7.26
dd, J = 1.5 Hz, 8.0 Hz, 1H, H6), 7.41 (m, 2H, H2, H5), 7.60 (d,
(
6
3
•
DPPH radical assay
3
3
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DPPH radical scavenging activity was performed as described
(
2
9
1
3
by Teixeira et al. Briefly, solutions of the test compounds
J = 16.0, 1H, Hβ), 12.46 (bs, 1H, COOH). C NMR (DMSO-d
δ (ppm) = 37.0 (2 × NCH ), 56.6 (OCH ), 111.4 (C2), 119.2 (C4),
21.1 (Cα), 121.2 (C6), 137.8 (C1), 138.2 (C5), 143.7 (Cβ), 160.3
6
):
with increasing concentrations (ranging between 10 μM and
3
3
•
1
mM) were prepared in ethanol. A DPPH ethanolic solution
1
•
+
(6.85 μM) was also prepared and then diluted to reach the
(C3), 164.7 (COS), 167.9 (COOH). EI/MS m/z (%): 281.2 (M ,
absorbance of 0.72 ± 0.02 at 515 nm. After the addition of
4
5), 192.1 (29), 72.1 (100).
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DPPH solution (180 μL) to the compound solution (20 μL) in
Synthesis of (E)-3-(4-sulfanyl-3-methoxyphenyl)acrylic acid
triplicate, the absorbance at 515 nm was recorded every
minute over 45 min. The percent inhibition of the radical was
based on the comparison between the blank (20 μL of ethanol
(
TFA). To a solution of compound 5 (0.44 g, 1.57 mmol) in
methanol (2 mL), aqueous solution of NaOH 2 M (4.0 mL,
.00 mmol) was added dropwise. The reaction mixture was
8
•
and 180 μL of DPPH solution), which corresponds to 100% of
refluxed for 1.5 h. Upon completion, the mixture was allowed
to cool at room temperature, then poured onto cooled aqueous
the radical, and the test compound solutions. The dose–
response curves allowed the determination of IC50 values.
saturated solution of NH Cl (15 mL) and acidified with
4
aqueous solution of acetic acid 5 M. The solid was filtered
under reduced pressure and washed with water. TLC analysis
was performed in ethyl acetate/dichloromethane/formic acid
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ABTS radical cation assay
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+
ABTS scavenging activity was evaluated as described by
2
9
1
8
Teixeira et al. Briefly, ethanolic solutions of the test com-
pounds with increasing concentrations (ranging between
(3 : 7 : 0.01). The procedure was adapted from Garcia et al.
with some modifications. η = 80% (0.26 g; 1.25 mmol); white
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+
1
50 μM and 500 μM) were prepared. ABTS radical cation solu-
tion was obtained by the addition of aqueous solution of pot-
assium persulfate (150 mM, 163 μL) to the aqueous solution of
ABTS (7 mM, 10 mL) and the storage in the dark at room temp-
erature for 16 h (2.45 mM final concentration). The solution
was then diluted in ethanol to reach the absorbance of 0.72 ±
0.02 at 734 nm. After the addition of ABTS solution (180 μL)
to the compound solution (20 μL) in triplicate, the spectropho-
tometric measurement was carried out every minute over
solid. H NMR (MeOD-d ): δ (ppm) = 3.92 (s, 3H, OCH ), 6.44
4
3
(d, J = 15.9 Hz, 1H, Hα), 7.07 (dd, J = 1.5 Hz, 8.0 Hz, 1H, H6),
7
.15 (d, J = 1.6 Hz, 1H, H2), 7.26 (d, J = 7.9 Hz, 1H, H5), 7.61
1
3
(
(
(
d, J = 16.0 Hz, 1H, Hβ). C NMR (MeOD-d ): δ (ppm) = 55.1
OCH
C5), 135.6 (C1), 144.7 (Cβ), 155.0 (C3), 169.1 (COOH). EI/MS
4
3
), 109.2 (C2), 117.0 (Cα), 121.1 (C6), 124.9 (C4), 128.5
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•
+
m/z (%): 210.0 (M , 100), 195.0 (19), 177.0 (9), 165.0 (23), 150.0
13), 121.0 (22), 89.0 (14), 77.0 (17). 72.0 (19).
(
1
5 min at 734 nm. The percent inhibition of the radical was
NMR stability studies
based on the comparison between the blank (20 μL of ethanol
and 180 μL of ABTS solution), which corresponds to 100% of
radical, and the test compound solutions. The dose–response
curves allowed the determination of IC50 values.
TFA solutions (0.7 M; 650 µL) were prepared in EtOD-d
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or
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1
DMSO-d . H NMR spectra were acquired at various timepoints
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for up to 24 h (15 min, 30 min, 1 h, 2 h, 4 h, 6 h and 24 h).
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Radical scavenging activity
GO radical assay
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To measure the radical scavenging activity of FA and TFA, spec- GO radical scavenging protocol was adapted from the
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0–32
trophotometric methods (1,1-diphenyl-2-picrylhydrazyl radical, literature.
Solutions of test compounds with increasing
•
(
DPPH ) 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) concentrations (ranging between 5 µM to 1 mM) were prepared
radical cation (ABTS ) and galvinoxyl (GO ) assays) and fluoro- in ethanol. An ethanolic solution of GO 5 mM was prepared
metric methods (oxygen radical absorbance capacity (ORAC) and diluted to reach the absorbance of 1.00 ± 0.02 at 428 nm.
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+
•
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assay) were adopted.
After the addition in triplicate of compound solution (20 µL)
This journal is © The Royal Society of Chemistry 2019
Org. Biomol. Chem., 2019, 17, 9646–9654 | 9651