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F. Bailly et al. / Bioorg. Med. Chem. 12 (2004) 5611–5618
(s, 1H), 6.91–7.01 (m, 2H), 7.23(m, 1H), 8.20 (dd, 1H,
3J = 7.75Hz, 4J = 1.50Hz). Compound 3b: 23% yield;
black powder; mp 135–137ꢁC (dec); 1H NMR
(DMSO-d6, 200MHz) 3.70 (s, 3H), 3.79 (s, 3H), 6.70
Compound 4d: 11% yield; reddish brown powder; mp
229–231ꢁC; 1H NMR (DMSO-d6, 200MHz) 6.78
(dd, 1H, 3J = 8.80Hz, 4J = 2.60Hz, H-7), 6.84 (d,
4
1H, J = 2.60Hz, H-5), 7.02 (s, 1H, H-4), 7.15 (d, 1H,
3
4
(s, 1H), 6.90 (dd, 1H, J = 8.05Hz, J = 1.60Hz), 7.05
(dd, 1H, 3J = 8.05Hz, 3J = 7.75Hz), 7.78 (dd, 1H,
3J = 7.75Hz, 4J = 1.60Hz). Compound 3c: 29% yield;
brown powder; mp 156–158ꢁC (dec); 1H NMR
(DMSO-d6, 200MHz) 3.79 (s, 3H), 3.82 (s, 3H),
3J = 8.80Hz, H-8), 9.55 (b s, 1H, OH), 10.18 (b s, 1H,
OH); 13C NMR (DMSO-d6, 50MHz) 112.6 (C-5),
115.8 (C-4), 117.3(C-8), 120.1 (C-10), 120.4 (C-7),
142.4 (C-3), 147.2 (C-9), 154.1 (C-6), 160.4 (C-2); Anal.
Calcd for C9H6O4: C, 60.68; H, 3.39. Found: C, 60.79;
H, 3.36.
4
6.44 (d, 1H, J = 2.25Hz), 6.53(dd, 1H, 3J = 8.90Hz,
3
4J = 2.25Hz), 7.07 (s, 1H), 8.10 (d, 1H, J = 8.90Hz).
Compound 3d: 40% yield; black powder; mp 157–
1
Compound 4e: 8% yield; black powder; mp 273ꢁC; H
1
159ꢁC (dec); H NMR (DMSO-d6, 200MHz) 3.70 (s,
3H), 3.76 (s, 3H), 6.76 (s, 1H), 6.79–6.94 (m, 2H), 7.79
NMR (DMSO-d6, 200MHz) 6.70 (s, 1H, H-8), 6.81 (s,
1H, H-5), 6.99 (s, 1H, H-4), 9.19 (b s, 1H, OH), 9.67
(b s, 1H, OH), 10.21 (b s, 1H, OH); 13C NMR
(DMSO-d6, 50MHz) 102.7 (C-8), 110.9 (C-10), 111.8
(C-5), 117.4 (C-4), 142.6 (C-3), 143.4 (C-6), 148.3 (C-
9), 150.5 (C-7), 161.7 (C-2); Anal. Calcd for C9H6O5:
C, 55.68; H, 3.12. Found: C, 55.44; H, 3.30.
4
(d, 1H, J = 2.10Hz). Compound 3e: 55% yield; black
powder; mp 174–176ꢁC (dec); 1H NMR (DMSO-d6,
200MHz) 3.68 (s, 3H), 3.81 (2s, 6H), 6.68 (s, 1H), 6.75
(s, 1H), 7.86 (s, 1H).
4.1.3. Synthesis of 3-hydroxycoumarins 4; general proce-
dure. A solution of (Z)-3-aryl-2-hydroxypropenoic acid
3 (xmol) in CH2Cl2 (25mL) was treated by a 1.0M solu-
tion of BBr3 in CH2Cl2 ([n + 4] equivalents, n being the
number of methoxy groups of 3). The solution was stir-
red at room temperature for 1h and then hydrolysed by
water (30mL). After 30min stirring, the formed precip-
itate was filtered. The organic layer was separated, dried
over Na2SO4 and the organic solvent was evaporated in
vacuo to give a residue. Column chromatography on
grouped precipitate and residue (typical solvent system:
hexane/ethyl acetate, 50/50) gave benzofuran-2-carb-
oxylic acids and the desired 3-hydroxycoumarins.
4.1.4. 3-Acetamido-6,7-diacetoxycoumarin. 2,4,5-Trihydr-
oxybenzaldehyde (2.31g, 15mmol), 2.10g of acetylgly-
cine (23mmol) and 1.59g of sodium acetate (20mmol)
in 7.5g of acetic anhydride were refluxed for 4h. The
solution was cooled to room temperature and iced-water
(10mL) was added. The precipitate was filtered, washed
with ethanol/water 50/50 (4 · 10mL) and dried to give
3.26g (68%) of 3-acetamido-6,7-diacetoxycoumarin;
mp 235–236ꢁC (EtOH); 1H NMR (CDCl3): 2.23(s,
3H), 2.313 (s, 3H), 2.316 (s, 3H), 7.22 (s, 1H), 7.33 (s,
1H), 8.09 (b s, 1H), 8.62 (s, 1H).
4.1.5. 3,6,7-Trihydroxycoumarin 4e. A solution of 1.59g
of 3-acetamido-6,7-diacetoxycoumarin (5mmol) in
50mL of HCl 3M and 2mL of acetic acid was refluxed
for 1h. The solution was cooled to room temperature
and the precipitate was filtered, washed with water and
dried to give 0.65g (67%) of 4e.
Compound 4a: 15% yield; brown powder; mp 150–
152ꢁC; 1H NMR (DMSO-d6, 200MHz) 7.11 (s, 1H,
H-4), 7.25 (td, 1H, 3J = 7.05Hz, 4J = 1.45Hz, H-6),
3
4
7.27 (dd, 1H, J = 7.05Hz, J = 1.45Hz, H-8), 7.38 (td,
1H, 3J = 7.05Hz, 4J = 1.45Hz, H-7), 7.42 (dd, 1H,
4
3J = 7.05Hz, J = 1.45Hz, H-5), 10.15 (b s, 1H, OH);
13C NMR (DMSO-d6, 50MHz) 115.0 (C-4), 115.6 (C-
8), 120.7 (C-10), 124.5 (C-6), 126.3(C-5), 127.5 (C-
7), 141.8 (C-3), 149.2 (C-9), 158.5 (C-2); Anal.
Calcd for C9H6O3: C, 66.67; H, 3.73. Found: C, 66.49;
H, 3.84.
4.2. Superoxide anion assay
Reaction mixtures contained xanthine (100lM), bovine
heart cytochrome
c (25lM) and test compound
(100lM) in air-saturated sodium-phosphate buffer
(50mM, pH7.4) supplemented with 0.1mM EDTA.
The reaction was started by the addition at 37ꢁC of xan-
thine oxidase (0.07U/mL) and reduced cytochrome c
was spectrophotometrically monitored at 550nm after
8min using an Uvikon 932 spectrophotometer.
Compound 4b: 12% yield; black powder; mp 214–
1
216ꢁC; H NMR (DMSO-d6, 200MHz) 6.90 (m, 2H),
7.04 (m, 1H), 7.05 (s, 1H, H-4), 10.05 (b s, 1H, OH),
10.29 (b s, 1H, OH); 13C NMR (DMSO-d6, 50 MHz)
115.2 (C-4), 118.4 (C-7), 118.5 (C-5), 119.9 (C-10),
124.5 (C-6), 141.5 (C-3), 142.6 (C-9), 144.8 (C-8),
159.9 (C-2); Anal. Calcd for C9H6O4: C, 60.68; H,
3.39. Found: C, 60.39; H, 3.46.
4.3. Hydroxyl radical assay
Buffer solutions (3mL, borate 50mM, pH9.2) contain-
ing Cu+ (500lM CuCl) were freshly prepared and vort-
exed in a test tube with 2.4lL of the test compound
stock solution (0.01M in DMF, 8.0lM final concentra-
tion) and 150lL of the luminol stock solution (5.0mM
in 0.05M NaOH; 250lM final concentration). Then
the reaction was initiated by the addition of 13.1lL of
H2O2 aqueous solution (500lM final concentration)
and chemiluminescence was recorded continuously for
1min at room temperature on a luminometer analyser
(Packard Pico-lite).
Compound 4c: 10% yield; black powder; mp 284–
286ꢁC; 1H NMR (DMSO-d6, 200MHz) 6.75 (d, 1H,
4J = 1.50Hz, H-8), 6.80 (dd, 1H, 3J = 9.50Hz,
4J = 1.50Hz, H-6), 7.15 (s, 1H, H-4), 7.40 (d, 1H,
3J = 9.50Hz, H-5), 10.0 (b s, 2H, 2 OH); 13C NMR
(DMSO-d6, 50MHz) 102.5 (C-8), 111.7 (C-10), 113.7
(C-6), 115.9 (C-4), 129.8 (C-5), 142.2 (C-3), 154.9 (C-
9), 160.8 (C-2), 161.3(C-7); Anal. Calcd for C H6O4:
9
C, 60.68; H, 3.39. Found: C, 60.81; H, 3.51.