3026
A.-N. Abulrob et al. / Phytochemistry 65 (2004) 3021–3027
0
0
m, H-4 or H-5 ), 1.78 (3H, s, vinyl CH ), 1.71 (2H, m,
3
dichloromethane – 2 mL), readily prepared by the
reaction of umbelliferone and geranyl bromide in
anhydrous acetone in the presence of potassium car-
bonate as the base, was added meta-chloro-peroxyben-
zoic acid (31 mg, 0.18 mmol) and the reaction stirred
at room temperature under nitrogen for 2 h. The reac-
tion mixture was diluted with dichloromethane (3
mL), washed with aqueous NaHCO3 (3 mL) then
0
0
H-4 or H-5 ), 1.36 (3H, s, CH ), 1.33 (3H, s, CH );
3
3
1
3
C NMR (CDCl ) d 161.68 (C@O, C-2), 158.53 (C,
3
C-8a), 156.25 (C, C-9a), 145.38 (CH, C-7), 142.52 (C,
0
C-5), 141.86 (C, C-3 ), 139.97 (CH, C-4), 119.86 (CH,
0
C-2 ), 113.00 (CH, C-3), 112.90 (C, C-5a), 107.85 (C,
C-4a), 105.45 (CH, C-6), 94.64 (CH, C-9), 69.99 (CH ,
2
0
0
0
C-1 ), 64.20 (CH, C-6 ), 58.79 (C, C-7 ), 36.70 (CH ,
2
0
0
0
0
C-4 or C-5 ), 27.53 (CH , C-4 or C-5 ), 25.26 and
2
the dichloromethane solution dried (MgSO ) and con-
4
1
CIMS m/z 372 (M + NH ) , 355 (M + H) , 153 (side
9.19 (2· CH , C(CH ) ), 17.13 (CH , vinyl CH );
centrated under reduced pressure to give a pale yellow
waxy solid, crude yield: 53 mg. Purification was
undertaken by column chromatography (petroleum
ether–ethyl acetate 4:1 v/v), and collection and evapo-
ration of the fractions containing pure material, gave
the product (3) as a pale yellow solid. Yield: 28 mg
(53%).
3
3 2
3
3
+
+
4
+
chain) (see Fig. 2(b)).
7
-{[(E)-5-(3,3-Dimethyl-2-oxiranyl)-3-methyl-2-pen-
1
tenyl]oxy}-2H-2-chromenone (3): H NMR (CDCl ) d
3
7.68 (1H, d, J4,3 = 9.5 Hz, H-4), 7.41 (1H, d, J5,6 = 8.5
Hz, H-5), 6.88 (1H, dd, J6,8 = 2.4 Hz, J6,5 = 8.5 Hz, H-
6
J3,4 = 9.5 Hz, H-3), 5.56 (1H, t, J
), 6.85 (1H, d, J8,6 = 2.3 Hz, H-8), 6.28 (1H, d,
0
0
0
= 6.5 Hz, H-2 ),
= 6.5 Hz, H-1 ), 2.76 (1H, t,
2 ,1
3
.4. Bacterial strains
0
4
.65 (2H, d, J1
0
0
0
,2
0
0
J6 = 6.2 Hz, H-6 ), 2.29 (2H, m, H-5 ), 1.82 (3H, s, vi-
nyl CH ), 1.74 (2H, q, J
CH ), 1.31 (3H, s, CH ); C NMR (CDCl ) d 162.46 (C,
0
,5
The bacterial strains consisted of S. aureus (Oxford
NCTC 6571) and ATCC 83254, both of which were
0
0
= 7.3 Hz, H-4 ), 1.34 (3H, s,
4 ,5
0
1
3
–
3
3
3
3
antibiotic-sensitive. MRSA clinical isolates strains 5, 7,
543, 50325 and 16565 were obtained from appropri-
C-7), 161.70 (C@O, C-2), 156.26 (C, C-8a), 143.89 (CH,
C-4), 141.87 (C, C-3 ), 129.16 (CH, C-5), 119.45 (CH, C-
9
0
ate sources. Their antibiotic susceptibility and resist-
ance were described earlier (Suller and Russell, 1999,
2
0
2
4
), 113.59 (CH, C-6), 113.42 (CH, C-3), 112.90 (C, C-
0
a), 101.96 (CH, C-8), 65.75 (CH , C-1 ), 64.28 (CH,
2
000).
0
0
0
C-6 ), 58.83 (C, C-7 ), 36.65 (CH , C- C-5 ), 27.49
2
0
(
(
(
(
CH , C-4 ), 25.25 and 19.18 (2· CH , C(CH ) ), 17.20
2
3
3 2
+
CH , vinyl CH ); CIMS m/z 332 (M + NH ) , 315
3.5. MIC determination
3
3
4
+
+
+
+
M + H) , 314 (M) , 180 (coumarin) , 153 (side chain)
see Fig. 2(b)).
MIC determinations were undertaken for the test
compound alone, antimicrobial agent alone and a
combination of the two to determine intrinsic antibac-
terial activity. The agar plates were autoclaved and al-
lowed to cool under sterile conditions. Following this
the chemical agents were aseptically incorporated into
molten agar at about 40 ꢁC and plates allowed to dry.
The MIC for the combination of ethidium bromide
and each of the agents was determined and compared
with the combination of CCCP (0.75 mg/L) and ethi-
dium bromide. The range of ethidium bromide con-
centration used was 0–100 mg/L. CCCP was
dissolved in absolute ethanol with a final experimental
concentration of 1% v/v. This final concentration
of ethanol had no deleterious effect on bacterial
growth.
3
.3. Synthesis of active components from GFO
The chemical scheme for the synthesis of the individ-
ual major components (components 1, 2 and 3) isolated
from GFO is shown in Fig. 3.
Preparation of 4-{[(E)-5-(3,3-dimethyl-2-oxiranyl)-
3-methyl-2-pentenyl]oxy}-7H-furo[3,2-g]chromen-7-one
(component 2): To a solution of bergamottin (15 mg,
0.044 mmol) in dry dichloromethane (1 mL) was added
meta-chloro-peroxybenzoic acid (8.2 mg, 0.047 mmol)
and the reaction stirred at room temperature under
nitrogen overnight. The reaction mixture was diluted
with dichloromethane (2 mL), washed with aqueous
NaHCO (2 mL), then the dichloromethane solution
3
dried (MgSO ) and concentrated under reduced pres-
4
Bacterial strains were grown overnight in Tryptone
Soya Broth in a shaking water bath operating at 100
oscillations/min at 37 ꢁC. One microliter of an
overnight culture (diluted 10-fold), equivalent to ca.
5 · 10 cfu/spot was used to inoculate the surface of
a Nutrient agar (Oxoid) plate using a Denley
sure. Purification was undertaken by column chroma-
tography (petroleum ether–ethyl acetate 4:1 v/v),
collection and evaporation of the fractions containing
pure material, gave the product (2) as a yellow oil. Yield:
4
1
1.8 mg (75%).
Preparation of 7-{[(E)-5-(3,3-dimethyl-2-oxiranyl)-
-methyl-2-pentenyl]oxy}-2H-2-chromenone (3): To a
multipoint inoculator (Denley, Billinghurst, UK).
Plates were incubated at 37 ꢁC for 24 h and the
MIC taken as the lowest concentration that inhibited
growth.
3
solution of 7-{[(2E)-3,7-dimethyl-2,6-octadienyl]oxy}-
H-2-chromenone (1) (50 mg, 0.17 mmol in dry
2