Natural and synthetic 2,2-dimethylpyranocoumarins with antibacterial activity

Article abstract of DOI:10.1021/np0497447

A new efficient synthetic approach to the natural coumarins 5-hydroxyseselin (5), 5-methoxyseselin (3), and (±) cis-grandmarin (9) is described as well as the synthesis of some new derivatives in the 5-methoxyseselin series (10-15). The natural coumarins 7-hydroxyalloxanthyletin (6), alloxanthoxyletin (8), and dipetalolactone (7) have also been obtained as secondary products. The type of fusion of the pyrano ring in all cases has been established by 2D NMR spectroscopy. The compounds have been studied for their in vitro antibacterial activity, which has been compared with that of some previously synthesized seselin derivatives. The most active compounds were 3, 7, 8, 11, and 14. Some structure-activity relationships are discussed.

Full text of DOI:10.1021/np0497447

78
J. Nat. Prod. 2005, 68, 78-82
Natural and Synthetic 2,2-Dimethylpyranocoumarins with Antibacterial
Activity
Eleni Melliou,† Prokopios Magiatis,*,† Sofia Mitaku,† Alexios-Leandros Skaltsounis,† Efrosini Chinou,‡ and
Ioanna Chinou†
Division of Pharmacognosy and Natural Products Chemistry, Department of Pharmacy, University of Athens,
Panepistimiopolis, Zografou, GR-15771 Athens, Greece, and Laboratory of Microbiology,
Anticancer Hospital “Ag. Savvas”, Alexandras Av., Greece
Received August 3, 2004
A new efficient synthetic approach to the natural coumarins 5-hydroxyseselin (5), 5-methoxyseselin (3),
and (() cis-grandmarin (9) is described as well as the synthesis of some new derivatives in the
5-methoxyseselin series (10-15). The natural coumarins 7-hydroxyalloxanthyletin (6), alloxanthoxyletin
(8), and dipetalolactone (7) have also been obtained as secondary products. The type of fusion of the
pyrano ring in all cases has been established by 2D NMR spectroscopy. The compounds have been studied
for their in vitro antibacterial activity, which has been compared with that of some previously synthesized
seselin derivatives. The most active compounds were 3, 7, 8, 11, and 14. Some structure-activity
relationships are discussed.
Coumarins are widely distributed natural products
exhibiting a broad pharmacological profile.1 A few years
ago, we described the synthesis of some seselin (1) and
xanthyletin (2) derivatives with interesting cytotoxic activi-
ties.2 Some seselin derivatives, including derivatives of
5-methoxyseselin (3), were found to be potent anti-HIV
agents.3 Besides the cytotoxic and antiviral activities,
coumarins also possess antibacterial activities.4
As part of our research program on bioactive compounds
with a pyranocoumarin nucleus, the synthesis of 5-meth-
oxyseselin (3) as well as the synthesis of some natural and
semisynthetic derivatives was envisaged. Synthesis of
aromatic oxygenated pyranocoumarins is difficult, and
thus, the development of new synthetic approaches is of
interest. The antibacterial activity of the synthesized
compounds is presented herein. Additionally, from com-
parison with the previously synthesized non-oxygenated
2,2-dimethylpyranocoumarins,2 some structure-activity
relationships are discussed.
because these conditions led to higher yields of dipetalolac-
tone (7). Subsequent methylation of 5-hydroxyseselin (5)
and 7-hydroxyalloxanthyletin (6) with methyl iodide in
acetone led to 5-methoxyseselin (3) and alloxanthoxyletin
or 7-methoxyalloxanthyletin (8), respectively. Acetylation
of 5 and 6 with acetic anhydride in pyridine led to the
corresponding acetate esters 3a and 8a.
Compounds 3, 5, 6, 7, and 8 obtained in the above-
described synthetic approach occur in nature. 5-Methoxy-
seselin (3) has been isolated from Citrus grandis8 and
Citrus paradisi x Citrus tangerine,9 5-hydroxyseselin (5)
from Citrus paradisi x Citrus tangerina9 and from Citrus
natsudaidai,10 alloxanthoxyletin (8) from Zanthoxylum
americanum,11 7-hydroxyalloxanthyletin (6) from Pilocar-
pus goudotianus,12 and dipetalolactone (7) from Zanthoxy-
lum dipetalum13 and Hortia arborea.14
Interestingly, compounds 3, 5, 6, and 7 were identified
1
by H NMR spectroscopy, and detailed 2D NMR evidence
for the type of fusion of the pyrano ring was never provided.
Alloxanthoxyletin (8) was recently synthesized, but 13C
NMR data15 were incomplete.
Results and Discussion
HMBC and NOESY experiments were executed on 3 and
8, because the carbon bearing the methoxy group could be
unequivocally identified from the HMBC spectrum. For
5-methoxyseselin (3) C-5 was correlated with H-4 and not
with H-4′, while for alloxanthoxyletin (8) C-7 was correlated
with H-4′ and not with H-4. The above observations were
confirmed by a NOESY spectrum, which was mainly used
to exclude the linear isomer. Both 3 and 8 showed a
correlation between the OMe protons and the aromatic
proton, revealing that both isomers were angular.
The overall yield from phloroglucinol to 5-methoxyseselin
was 35% in only three steps, while previous synthetic
approaches3,16,17 suffered from low yields over several
reaction steps.3
5-Methoxyseselin (3) derivatives were also synthesized.
(()-cis-Grandmarin (9) was obtained by catalytic osmium
oxidation of 3, using 4-methyl-morpholine-N-oxide to re-
generate the oxidizing agent (Scheme 1). Treatment of cis
diol 9 with excess acetic anhydride in pyridine afforded the
corresponding diester 10. (()-trans-3′-Bromo-4′-hydroxy-
5-methoxy-3′,4′-dihydroseselin (11) was obtained by treat-
Our synthetic approach toward the aforementioned
pyranocoumarins was based on the successful use of
3-methyl-2-butenal in the synthesis of pyranoacridones5
and pyranoquinones.6 As starting material we used 5,7-
dihydroxycoumarin (4), which was easily obtained in
almost quantitative yield from phloroglucinol and ethyl
propiolate.7 5,7-Diacetoxycoumarin (4a) and 5,7-dimethoxy-
coumarin (4b) were also prepared, for structure-activity
studies. Direct cyclization of 4 with 3-methyl-2-butenal in
pyridine for 4 h led to a separable mixture of 5-hydroxy-
seselin (5) (26%) and 7-hydroxyalloxanthyletin (6) (8%).
Dipetalolactone (7) was obtained as a byproduct (4%). No
trace of the linear isomer 5-hydroxyxanthyletin could be
detected. The yield of 5-hydroxyseselin (5) could be in-
creased to 42% if the residual starting material (62%) was
reacted again. As expected, the yield could not be increased
using excess 3-methyl-2-butenal or by prolonged reaction
* To whom correspondence should be addressed. Tel: (++30210)7274052.
Fax: (++30210)7274594. E-mail: magiatis@pharm.uoa.gr.
† University of Athens.
‡ Anticancer Hospital “Ag. Savvas”.
10.1021/np0497447 CCC: $30.25
© 2005 American Chemical Society and American Society of Pharmacognosy
Published on Web 01/12/2005

2,2-Dimethylpyranocoumarins
Journal of Natural Products, 2005, Vol. 68, No. 1 79
Scheme 1a
a Conditions: (i) ethyl propiolate, ZnCl2, 100 °C; (ii) 3-methyl-2-butenal, Py, 115 °C; (iii) MeI, K2CO3, Me2CO, 56 °C; (iv) OsO4, N-methylmorpholine-N-
oxide, t-BuOH, THF, H2O, rt; (v) NBS, THF, H2O, 0 °C; (vi) Ac2O, Py, rt; (vii) (PhCO)2O, Py, rt; (viii) Bu3SnH, AIBN, toluene, 110 °C.
ment of 3 with N-bromosuccinimide in aqueous tetra-
hydrofuran solution. Bromohydrin 11 was smoothly debro-
minated with tributyltin hydride to afford 12. Treatment
of 11 with excess acetic anhydride or benzoic anhydride in
pyridine gave the corresponding esters 13 and 14, respec-
tively. Similarly, alcohol 12 afforded the corresponding
acetate 15.
The antibacterial activity of compounds 3-15 and of the
previously synthesized2 non-oxygenated pyranocoumarins
1, 2, and 16-22 was evaluated in vitro using the diffusion
technique of Bauer-Kirby (disk method) as previously
described.18 The results are reported in Table 1. The most
active pyranocoumarins were 3, 7, 8, 11, and 14.
The compounds showed a broad diversity regarding
growth-inhibitory activity (Table 1). Five compounds (5-
methoxyseselin (3) and its brominated derivatives (11, 14),
alloxanthoxyletine (8), the acetylated derivatives 3a, 8a,
and dipetalolactone (7)) were active against seven tested
bacteria. A seselin derivative, 3-bromo-4-benzoyloxyseselin
(20), showed moderate activity, while three other cou-
marins (13, 15, 19) expressed a specific activity only
against the two Gram-positive bacteria. Interestingly,
seselin (1), xanthyletin (2), 5-hydroxyseselin (5), and 7-hy-
droxyalloxanthyletin (6) were found to be inactive.
Comparing the methoxy or acetoxy derivatives of seselin,
alloxanthyletin, or simple coumarins with the respective
non-oxygenated or hydroxy derivatives, it could be consid-
ered that the presence of additional oxygenated substitu-
ents in the ether or ester form generally enhances the
antibacterial activity, while the presence of free hydroxyl
reduces or abolishes the activity. This fact could be at least
partially attributed to the reduced lipophilicity of the
hydroxyl derivatives, which hinders the penetration through
the bacterial cell wall.19 Further comparison showed that
the brominated derivatives are generally more active than
the unsubstituted compounds. Results for 13, 15, and 19
showed that the presence of an acetoxy group on the pyrano
ring enhances the selectivity against Gram-positive bac-
teria. Additionally, the activity found for dipyranocoumarin
7 indicates that, besides the oxygenation pattern and the
functionalization of the pyrano ring, the polycyclic nature
may also be important and should be further studied.

80 Journal of Natural Products, 2005, Vol. 68, No. 1
Table 1. Antibacterial Activitya
Melliou et al.
compound
S. aureus
S. epidermidis
P. aeruginosa
E. cloacae
K. pneumoniae
E. coli
Proteus mirabilis
3
+
+
++
+
+
+
++
+
++
+
+
+
+
+
+
+
+
-
+
-
-
+
3a
+
+
+
+
4
+
+
+
+
+
+
4a
+
++
++
+
+
+
+
+
4b
+
++
++
++
+
+
+
++
++
++
+
7
8
++
+
+
+
+
+
+
8a
+
+
+
+
11
+
+
++
-
++
-
-
+
+
+
++
-
++
-
-
+
13
+
++
+
-
-
14
+
+
+
15
++
++
+
++
++
+
-
-
-
-
19
20
+
+
netilmicin
amoxycillin
clavulanic acid
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
a
Zone of inhibition around each disk (in mm) <7 mm (-), 7-10 mm (+), 11-16 mm (++), >16 mm (+++). Compounds 1, 2, 5, 6, 9,
10, 12, 16, 17, 18, 21, and 22 were tested and found to be inactive.
Experimental Section
7-Hydroxyalloxanthyletin (6): mp 220 °C (EtOAc); 1H
NMR (CDCl3, 400 MHz) δ 7.97 (1H, d, J ) 9.8 Hz, H-4), 6.62
(1H, d, J ) 9.8 Hz, H-4′), 6.53 (1H, s, H-8), 6.12 (1H, d, J )
General Experimental Procedures. Spectra were re-
corded on the following apparatus. MS: Nermag R10-10C in
desorption-chemical ionization, using NH3 as reagent gas.
NMR: Bruker AC200, 13C NMR (50 MHz); Bruker DRX400,
1H NMR (400 MHz). Chemical shifts are given in δ values with
TMS as an internal standard. Coupling constants (J) are given
in Hz. The signals of 1H and 13C spectra were unambiguously
assigned using 2D NMR techniques: COSY, NOESY, HMQC,
and HMBC. These 2D experiments were performed using
standard Bruker microprograms. Column chromatography was
conducted using Merck flash silica gel 60 (40-63 µm), with
an overpressure of 300 mbar.
5,7-Dihydroxycoumarin (4). A mixture of phloroglucinol
(2.00 g, 15.8 mmol), ZnCl2 (1.67 g, 12.3 mmol), and ethyl
propiolate (1.9 mL) was stirred for 2 h at 100 °C. Then the
mixture was cooled, and 5% hydrochloric acid (40 mL) was
added. The precipitate was filtered off and washed with boiling
H2O to give 4 (2.53 g, 90%): 1H NMR (DMSO, 400 MHz) δ
10.65 (1H, br s, OH), 10.40 (1H, br s, OH), 7.94 (1H, d, J )
9.7 Hz, H-4), 6.25 (1H, d, J ) 2.2 Hz, H-8), 6.17 (1H, d, J )
2.2 Hz, H-6), 6.02 (1H, d, J ) 9.7, H-3).
9.8, H-3), 5.55 (1H, d, J ) 9.8, H-3′), 1.44 (6H, s, 2×CH3); 13
C
NMR (CDCl3, 50 MHz) δ 162.6 (C-2), 155.9 (C-9), 151.1 (C-7),
150.6 (C-5), 139.5 (C-4), 127.5 (C-3′), 116.0 (C-4′), 110.1 (C-3),
106.4 (C-6), 103.5 (C-10), 95.5 (C-8), 77.9 (C-2′), 28.0 (2×CH3);
MS-DCI m/z 245 (M + H)+.
Dipetalolactone (7): 1H NMR (CDCl3, 400 MHz) δ 7.94
(1H, d, J ) 9.8 Hz, H-4), 6.78 (1H, d, J ) 10 Hz, H-4′), 6.62
(1H, d, J ) 10 Hz, H-4′′), 6.12 (1H, d, J ) 9.8, H-3), 5.58 (1H,
d, J ) 10 Hz, H-3′), 5.54 (1H, d, J ) 10, H-3′′), 1.44 (12H, s,
4×CH3); 13C NMR (CDCl3, 50 MHz) δ 161.2 (C-2), 151.8 (C-7),
150.0 (C-5,9), 138.8 (C-4), 127.6 (C-3′*), 127.5 (C-3′′*), 115.8
(C-4′*), 115.0 (C-4′′*), 110.4 (C-3), 105.9 (C-8*), 103.1 (C-10),
102.1 (C-6*), 77.9 (C-2′,2′′), 28.0 (2×CH3), 27.9 (2×CH3); MS-
DCI m/z 311 (M + H)+; *assignments can be interchanged.
5-Methoxyseselin (3). Treatment of 5-hydroxyseselin (5)
(250 mg, 1.02 mmol) in conditions similar to those described
for the preparation of 4b afforded 3 (245 mg, 93%); mp 162 °C
(diethyl ether); 1H NMR as described in ref 3; 13C NMR (CDCl3,
50 MHz) δ 161.1 (C-2), 157.3 (C-7), 156.4 (C-5), 150.9 (C-9),
138.9 (C-4), 127.4 (C-3′), 114.8 (C-4′), 110.2 (C-3), 103.5 (C-
10), 102.4 (C-8), 95.2 (C-6), 77.8 (C-2′), 55.8 (OCH3), 28.0
(2×CH3); MS-DCI m/z 259 (M + H)+.
5,7-Diacetoxycoumarin (4a). To a solution of 4 (24 mg,
0.13 mmol) in dry pyridine (1.5 mL) was added Ac2O (1 mL,
15 mmol). The reaction was stirred for 24 h at room temper-
ature, and the reagents were removed under reduced pressure.
The residue was purified by flash chromatography on silica
gel with cyclohexane-EtOAc (1:1) to give compound 4a (28
5-Acetoxyseselin (3a). Treatment of 5 (25 mg, 0.10 mmol)
in conditions similar to those described for the preparation of
1
4a afforded 3a (29 mg, 96%). H NMR as described in ref 17.
Alloxanthoxyletin (8). Treatment of 7-hydroxyalloxanth-
yletin (6) (70 mg, 0.29 mmol) in conditions similar to those
described for the preparation of 4b afforded 8 (69 mg, 93%):
mp 115 °C (EtOAc); 1H NMR as described in ref 12; 13C NMR
(CDCl3, 50 MHz) δ 161.2 (C-2), 158.1 (C-7), 155.7 (C-9), 150.0
(C-5), 138.4 (C-4), 127.4 (C-3′), 115.9 (C-4′), 111.0 (C-3), 106.3
(C-6), 102.4 (C-10), 91.3 (C-8), 77.8 (C-2′), 55.8 (OCH3), 27.7
(2×CH3); MS-DCI m/z 259 (M + H)+.
1
mg, 92%). H NMR as described in ref 20.
5,7-Dimethoxycoumarin (4b). To a solution of 4 (250 mg,
1.40 mmol) in dry acetone (15 mL) was added anhydrous
K2CO3 (1.1 g) and MeI (3 mL). The reaction mixture was
stirred for 4 h at 60 °C. Then, the mixture was filtered and
the reagents were removed under reduced pressure. The solid
residue was submitted to flash chromatography with CH2Cl2-
MeOH (99.9:0.1) to give 4b (260 mg, 90%). 1H NMR as
described in ref 21.
Reaction of 5,7-Dihydroxycoumarin (4) with 3-Methyl-
2-butenal. To a solution of 5,7-dihydroxycoumarin (4) (1.000
g, 5.62 mmol) in dry pyridine (3.0 mL) was added 3-methyl-
2-butenal (1.1 mL), and the reaction mixture was stirred for
4 h at 115 °C. Then, the reagents were removed under reduced
pressure. The solid residue was submitted to flash chroma-
tography with CH2Cl2-MeOH (99.9:0.1 to 94:6) to afford
5-hydroxyseselin (5) (357 mg, 26%), 7-hydroxyalloxanthyletin
(6) (110 mg, 8%), and dipetalolactone (7) (65 mg, 4%).
5-Hydroxyseselin (5): mp 212 °C (EtOAc); 1H NMR as
described in ref 10; 13C NMR (CDCl3, 50 MHz) δ 161.8 (C-2),
157.3 (C-7), 153.2 (C-5), 151.0 (C-9), 139.6 (C-4), 127.9 (C-3′),
115.4 (C-4′), 110.3 (C-3), 103.0 (C-8, 10), 99.3 (C-6), 78.1 (C-
2′), 28.7 (2×CH3); MS-DCI m/z 245 (M + H)+.
7-Acetoxyalloxanthyletin (8a). Treatment of 6 (25 mg,
0.10 mmol) in conditions similar to those described for the
preparation of 4a afforded 8a (29 mg, 96%). 1H NMR as
described in ref 12.
(()-cis-3′,4′-Dihydroxy-3′,4′-dihydro-5-methoxysese-
lin (9). To a solution of 3 (50 mg, 0.19 mmol) in 7 mL of
t-BuOH-THF-H2O (10:3:1 v/v/v) was added a solution of OsO4
2.5% (w/v) in t-BuOH (0.25 mL) and 50 mg (0.33 mmol) of
4-methylmorpholine-N-oxide. The reaction mixture was stirred
for 48 h at room temperature. NaHSO3 (sat.) was added, and
the mixture was stirred for 1 h. The reaction mixture was
extracted with CH2Cl2-H2O, and the organic layer was col-
lected. The solvent was removed under reduced pressure, and
compound 9 was purified by flash chromatography on silica
gel with cyclohexane-EtOAc (60:40 to 90:10) (32 mg, 58%):
1
mp 230 °C (hexane-diethyl ether); H NMR as described in

2,2-Dimethylpyranocoumarins
Journal of Natural Products, 2005, Vol. 68, No. 1 81
ref 10; 13C NMR (CDCl3, 50 MHz) δ 161.9 (C-2), 157.7 (C-7),
157.2 (C-5), 156.0 (C-9), 140.0 (C-4), 110.5 (C-3), 104.3 (C-8,-
10), 96.0 (C-6), 79.5 (C-2′), 71.7 (C-3′), 61.4 (C-4′), 56.4 (OCH3),
25.7 (CH3), 21.9 (CH3); MS-DCI m/z 293 (M + H)+.
m/z 461, 459 (M + H)+; anal. C 57.65%, H 4.21%, Br 17.55%
calcd for C22H19BrO6, C 57.53%, H 4.17%, Br 17.40%.
(()-4′-Hydroxy-3′,4′-dihydro-5-methoxyseselin (12). Com-
pound 11 (78 mg, 0.22 mmol) was dissolved in anhydrous
toluene (10 mL), and the solution was refluxed for 15 min
under argon. Then AIBN (azo-bis-2,2′-(methyl-2-propionitrile))
(10 mg) was added, and after 5 min, a solution of tributyltin
hydride (0.5 mL in 4 mL of toluene) was added over a period
of 40 min. The reaction mixture was refluxed for 1 h. The
solvent was evaporated and the residue was purified by flash
chromatography on silica gel with cyclohexane-EtOAc (80:
20) to give compound 12 (35 mg, 59%): UV (CHCl3) λmax (log
ꢀ) 330 (3.90), 261 (3.66), 251 (3.60) nm; IR (CHCl3) νmax 3350,
1630, 1604, 1220 cm-1; 1H NMR (CDCl3, 400 MHz) δ 8.02 (1H,
d, J ) 9.8 Hz, H-4), 6.24 (1H, s, H-6), 6.18 (1H, d, J ) 9.8 Hz,
H-3), 5.20 (1H, m, H-4′), 3.89 (3H, s, OCH3), 3.27 (1H, d, J )
3.0 Hz, OH), 2.12 (1H, d, J ) 4.9, H-3′), 1.51 (3H, s, CH3),
1.43 (1H, s, CH3); 13C NMR (CDCl3, 50 MHz) δ 161.4 (C-2),
157.8 (C-7), 156.7 (C-5), 139.4 (C-4), 109.9 (C-3), 104.2 (C-8),
104.1 (C-10), 95.7 (C-6), 77.0 (C-2′), 59.0 (C-4′), 55.9 (OCH3),
40.2 (C-3′), 28.0 (CH3), 26.5 (CH3); MS-DCI m/z: 277 (M +
H)+; anal. C 65.33%, H 5.76%, calcd for C15H16O5, C 65.21%,
H 5.84%.
(()-cis-3′,4′-Diacetoxy-3′,4′-dihydro-5-methoxyseselin
(10). Treatment of 9 (24 mg, 0.08 mmol) in conditions similar
to those described for the preparation of 4a afforded 10 (28
mg, 92%): UV (CHCl3) λmax (log ꢀ) 344 (sh), 326 (4.15), 259
(3.95), 249 (3.92) nm; IR (CHCl3) νmax 1747, 1729 (sh),1629,
1
1603, 1236 cm-1; H NMR (CDCl3, 200 MHz) δ 7.92 (1H, d, J
) 9.5 Hz, H-4), 6.44 (1H, d, J ) 5.0 Hz, H-4′), 6.22 (1H, s,
H-6), 6.13 (1H, d, J ) 9.5 Hz, H-3), 5.25 (1H, d, J ) 5.0 Hz,
H-3′), 3.86 (3H, s, OCH3), 2.20 (3H, s, COCH3), 2.09 (6H, s,
COCH3), 1.42 (3H, s, 2×CH3); 13C NMR (CDCl3, 50 MHz) δ
171.0 (COCH3), 161.9 (C-2), 157.7 (C-5), 157.2 (C-7), 156.0 (C-
9), 139.7 (C-4), 112.8 (C-3), 104.3 (C-10), 100.0 (C-8), 96.7 (C-
6), 78.5 (C-2′), 71.5 (C-3′), 62.3 (C-4′), 57.3 (OCH3), 26.4 (CH3),
25.4 (CH3), 21.8 (COCH3); MS-DCI m/z 377 (M + H)+; anal. C
60.75%, H 5.39%, calcd for C19H20O8, C 60.64%, H 5.36%.
(()-trans-3′-Bromo-4′-hydroxy-3′,4′-dihydro-5-methoxy-
seselin (11). To a solution of 3 (140 mg, 0.54 mmol) in THF
(5 mL) and H2O (5 mL) was added N-bromosuccinimide (100
mg, 0.56 mmol). The reaction mixture was stirred for 1 h at 0
°C, and then the reaction mixture was extracted with NaCl
(sat.)-Et2O and the organic layer was collected. The solvent
was removed under reduced pressure, and compound 11 was
purified by crystallization with Et2O (110 mg, 57%): UV
(CHCl3) λmax (log ꢀ) 331 (3.99), 261 (3.75), 251 (3.71) nm; IR
(()-4′-Acetoxy-3′,4′-dihydro-5-methoxyseselin (15). Treat-
ment of compound 12 (23 mg, 0.08 mmol) in conditions
essentially similar to those described for the preparation of
4a afforded 15 (24 mg, 92%): UV (CHCl3) λmax (log ꢀ) 345 (sh),
329 (4.02), 261 (3.77), 251 (3.74) nm; IR (CHCl3) νmax 1732,
1630, 1604, 1220 cm-1; 1H NMR (CDCl3, 400 MHz) δ 7.98 (1H,
d, J ) 9.7 Hz, H-4), 6.24 (1H, s, H-6), 6.23 (1H, t, J ) 4.9,
H-4′), 6.17 (1H, d, J ) 9.7, H-3), 3.90 (3H, s, OCH3), 2.12 (2H,
1
(CHCl3) νmax 3350, 1630, 1605, 1225 cm-1; H NMR (DMSO,
200 MHz) δ 8.02 (1H, d, J ) 9.7 Hz, H-4), 6.28 (1H, s, H-6),
6.20 (1H, d, J ) 9.7 Hz, H-3), 5.35 (1H, d, J ) 5.1 H-4′), 4.25
(1H, s, br, OH), 4.29 (1H, d, J ) 5.1, H-3′), 3.90 (3H, s, OCH3),
1.65 (3H, s, CH3), 1.55 (3H, s, CH3); 13C NMR (DMSO, 50 MHz)
δ 161.6 (C-2), 157.3 (C-5), 156.9 (C-7), 155.2 (C-9), 139.9 (C-
4), 110.9 (C-3), 104.9 (C-10), 104.4 (C-8), 96.0 (C-6), 79.2 (C-
2′), 67.2 (C-4′), 58.2 (C-3′), 56.4 (OCH3), 26.5 (CH3), 25.3 (CH3);
MS-DCI m/z 357, 355 (M + H)+; anal. C 50.61%, H 4.19%, Br
22.45% calcd for C15H15BrO5, C 50.72%, H 4.26%, Br 22.50%.
(()-trans-3′-Bromo-4′-acetoxy-3′,4′-dihydro-5-methoxy-
seselin (13). Treatment of 11 (35 mg, 0.10 mmol) in conditions
essentially similar to those described for the preparation of
4a afforded 13 (36 mg, 92%): UV (CHCl3) λmax (log ꢀ) 344 (sh),
327 (4.11), 260 (3.90), 251 (3.82) nm; IR (CHCl3) νmax 1733,
1631, 1604, 1220 cm-1; 1H NMR (CDCl3, 400 MHz) δ 7.95 (1H,
d, J ) 9.7 Hz, H-4), 6.44 (1H, d, J ) 4.2 Hz, H-4′), 6.28 (1H,
s, H-6), 6.18 (1H, d, J ) 9.7, H-3), 4.33 (1H, d, J ) 4.2 Hz,
H-3′), 3.90 (3H, s, OCH3), 2.15 (3H, s, COCH3), 1.59 (6H, s,
2×CH3); 13C NMR (CDCl3, 50 MHz) δ 170.4 (COCH3), 160.6
(C-2), 157.7 (C-5), 157.6 (C-7), 155.4 (C-9), 138.9 (C-4), 111.8
(C-3), 104.9 (C-10), 98.5 (C-8), 96.1 (C-6), 78.3 (C-2′), 67.4 (C-
4′), 56.4 (OCH3), 54.3 (C-3′), 26.4 (2×CH3), 21.3 (COCH3); MS-
DCI m/z 397, 395 (M + H)+; anal. C 51.41%, H 4.27%, Br
20.22% calcd for C17H17BrO6, C 51.40%, H 4.31%, Br 20.12%.
(()-trans-3′-Bromo-4′-benzoyloxy-3′,4′-dihydro-5-meth-
oxyseselin (14). To a solution of 11 (28 mg, 0.08 mmol) in
dry pyridine (1.5 mL) was added benzoic anhydride (62 mg,
0.29 mmol). The reaction mixture was stirred for 48 h at room
temperature, and the reagents were removed under reduced
pressure. The residue mixture was extracted with EtOAc-
NaHCO3 (sat.), and the organic layer was collected. The solvent
was removed under reduced pressure, and the remaining
residue was purified by flash chromatography on silica gel with
cyclohexane-CH2Cl2 (1:1) to give compound 14 (23 mg, 63%):
UV (CHCl3) λmax (log ꢀ) 346 (sh), 329 (4.10), 297 (sh), 284 (sh),
258 (3.69), 242 (3.98) nm; IR (CHCl3) νmax 1736, 1629 (sh),
1608, 1604, 1220 cm-1; 1H NMR (CDCl3, 200 MHz) δ 8.02 (2H,
d, J ) 7.5, H-2′′,6′′), 7.98 (1H, d, J ) 9.5, H-4), 7,57 (1H, t, J
) 7.5 Hz, H-4′′), 7.43 (2H, t, J ) 7.5 Hz, H-3′′,5′′), 6.70 (1H, d,
J ) 2.9, H-4′), 6.36 (1H, s, H-6), 6.16 (1H, d, J ) 9.5, H-3),
4.53 (1H, d, J ) 2.9, H-3′), 3.94 (3H, s, OCH3), 1.67 (3H, s,
CH3), 1.66 (3H, s, CH3); 13C NMR (CDCl3, 50 MHz) δ 165.6
(COPh-9), 160.7 (C-2), 158.0 (C-5), 157.8 (C-7), 155.6 (C-9),
133.7 (C-4′′), 130.3 (C-2′′,6′′), 129.8 (C-1′), 129.5 (C-3′′,5′′), 111.5
(C-3), 104.5 (C-10), 97.8 (C-8), 95.5 (C-6), 77.6 (C-2′), 67.3 (C-
4′), 56.1 (OCH3), 53.5 (C-3′), 28.4 (CH3), 24.7 (CH3); MS-DCI
d, J ) 4.9, H-3′), 2.12 (3H, s, OCOCH3), 1.46 (1H, s, CH3); 13
C
NMR (CDCl3, 50 MHz) δ 170.8 (COCH3), 161.3 (C-2), 158.0
(C-7), 157.6 (C-5), 155.3 (C-9), 138.6 (C-4), 110.8 (C-3), 104.1
(C-10), 100.6 (C-8), 95.5 (C-6), 76.6 (C-2′), 61.3 (C-4′), 55.9
(OCH3), 38.4 (C-3′), 28.7 (CH3), 25.8 (CH3), 21.1 (COCH3); MS-
DCI m/z 319 (M + H)+; anal. C 64.09%, H 5.66%, calcd for
C17H18O6, C 64.14%, H 5.70%.
Antibacterial Activity. Antibacterial activities of com-
pounds were determined using the diffusion technique of
Bauer-Kirby (disk method) by measuring the zone of inhibi-
tion against two Gram-positive bacteria, Staphylococcus au-
reus (ATCC 25923) and Staphylococcus epidermidis (ATCC
12228), and five Gram-negative bacteria, Pseudomonas aerugi-
nosa (ATCC 27853), Escherichia coli (ATCC 25922), Entero-
bacter cloacae (ATCC 13047), Klebsiella pneumoniae (ATCC
13883), and Proteus mirabilis. Netilmicin, amoxycillin, and
clavulanic acid were used as control antibiotics. The results
were reported as the diameter of the zone of inhibition around
each disk (in mm), and evaluation of inhibition corresponds
to <7 mm (-), 7-10 mm (+), 11-16 mm (++), >16 mm (+++).
The compounds were dissolved in DMSO. For each experiment
control disks with solvent were used as negative control. All
paper disks had a diameter of 6 mm and were deposited on
the surface of the seeded trypticase Muller-Hinton agar. Petri
dishes were previously inoculated with the organisms to give
a final cell concentration of 107 cell/mL. Volumes of 10 µL of
the above solutions were required to wet the test paper disks.
The incubation conditions used were 24 h at 37 °C. The
experiments were repeated three times, and the results were
expressed as average values. All strains were standard refer-
ence strains (American Type Culture Collection).
References and Notes
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(6) Melliou, E.; Magiatis, P.; Mitaku, S.; Skaltsounis, A. L.; Pierre´, A.;
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