1448 J ournal of Natural Products, 2004, Vol. 67, No. 9
Shoeib et al.
0.25 M phosphate buffer (pH 8, 3 mL). The mixture was stirred
for 10 min and carefully acidified to pH 5 using dilute HCl.
All reaction steps were performed in an ice bath to minimize
side reactions. 3 (0.19 g, yield 64%) was extracted with CHCl3
and precipitated upon concentration as a white powder: mp
132-134 °C; 1H NMR (acetone-d6) δ 4.58 (2H, s, ArCH2), 7.25
(1H, s, 6-H), 8.45 (1H, br s, OH); 13C NMR (acetone-d6) δ 63.0
(ArCH2), 108.5, 108.7, 121.8 (C-6), 134.0 (C-1), 142.2, 143.7;
EIMS m/z 300 (23), 298 (48), 296 (25) [M]+, 219 (38), 217 (45)
[M - Br]+, 190 (25), 188 (27) [M - Br - HCO]+, 138 (12) [M
- 2Br]+, 110 (100) [M - 2Br - CO]+, 53 (15); GLC-MS, tR 27.17
min; EIMS of silylated derivative m/z 516 (5), 514 (9), 512 (4)
[M]+, 499 (3) [M - CH3]+, 435 (8), 433 (7) [M - Br]+, 411 (7)
[M - OSi(CH3)3 - CH2]+, 339 (24), 337 (47), 335 (24) [M -
OSi(CH3)3 - Si(CH3)3 - CH3]+, 147 (11) [(CH3)2SidO-
Si(CH3)3]+, 73 (100) [Si(CH3)3]+; anal. C 28.52%, H 1.61%, calcd
for C7H6Br2O3, C 28.20%, H 2.00%.
2,5-Dibr om o-3,4-d ih yd r oxyben zyl Meth yl Eth er (3a ).
A solution of 3 (50 mg, 0.17 mmol) in 95% MeOH (13 mL) was
heated under reflux for 20 h when reaction was complete, as
judged by monitoring with TLC (silica gel GF254, n-hexane/
EtOAc/MeOH, 6.5:2.5:1). After evaporation of the solvent, the
residue (39 mg, yield 74%) was recrystallized from CCl4 to
afford yellowish white crystals: mp 106-107 °C; 1H NMR
(acetone-d6) δ 3.35 (3H, s, CH3), 4.39 (2H, s, ArCH2), 7.14 (1H,
s, 6-H); 13C NMR (acetone-d6) δ 57.6 (CH3), 73.0 (ArCH2), 108.5,
109.7, 123.0 (C-6), 130.6 (C-1), 142.8, 143.9; EIMS m/z 314 (29),
312 (63), 310 (31) [M]+, 283 (49), 281 (100), 279 (51) [M -
OCH3]+, 233 (80), 231 (81) [M - Br]+, 53 (23), 31 (23); GLC-
MS tR 25.17 min; EIMS of silylated derivative m/z 458 (9), 456
(18), 454 (8) [M]+, 425 (3) [M - OCH3]+, 339 (54), 337 (100),
335 (55) [M - OCH3 - Si(CH3)3 - CH3]+, 289 (7) [M - Br -
Si(CH3)3 - CH3]+, 73 (78) [Si(CH3)3]+; anal. C 30.60%, H 2.20%,
calcd for C8H8Br2O3, C 30.77%, H 2.56%.
no significant effect. These results suggest inhibition of
early DNA synthesis. The mechanism of action of 3c cannot
be completely understood from the above experiments, and
other investigations are necessary such as multiparametric
flow cytometry using bromodeoxyuridine,20 Western analy-
ses, and kinase assays.21
Exp er im en ta l Section
Gen er a l Exp er im en ta l P r oced u r es. GLC-MS analysis
was carried out on a Hewlett-Packard 5890 series II gas
chromatograph equipped with a 5972 A series mass selective
detector and a 30 m capillary column, 0.25 mm internal
diameter and 25 µm thickness, of stationary phase (Alltech-
AT-5MS). Helium was used as the carrier gas at a linear
velocity of 20 cm s-1. The injector and detector temperatures
were held constant at 280 and 310 °C, respectively. A solvent
delay of 3.5 min and a temperature program from 100 to 300
°C at 4 °C min-1 were applied. EIMS were recorded on an
Associated Electrical Industries Ltd. (AEI) MS 902 spectrom-
eter. NMR spectra were acquired on a J EOL Delta GX270
1
(observing H at 270.17 MHz and 13C at 67.80 MHz) or J EOL
ECA600 spectrometer (observing 1H at 600.17 MHz and 13C
at 150.91 MHz). Melting points were carried out on an
Electrothermal IA9000 series digital melting point apparatus.
CHN analyses were determined using a Carlo Erba 1108
elemental analyzer at the University of Newcastle, U.K.
Alga Ma ter ia l. P. lanosa was collected from Kimmeridge
Bay, Dorset, on the south coast of England in J uly 1999 and
authenticated by one of us (G.B.). A voucher sample is
deposited in the herbarium of the Hampshire County Council
Museums Service, Winchester, Hampshire, U.K. (Index Her-
bariorum code HCMS; accession number Bi 2000.16.271). The
alga (seaweed) was quickly dried at 50 °C and powdered.
Extr a ction a n d F r a ction a tion . The dried powdered sea-
weed (5 g) was extracted three times with MeOH (100 mL) at
room temperature, and the extracts were concentrated under
reduced pressure, dried first under nitrogen and then under
vacuum at room temperature. The dried extract was kept at
4 °C for cytotoxicity assay. The available amount (75 g) of P.
lanosa powder of the same collection was successively ex-
tracted with n-hexane, followed by MeOH. The MeOH extract
was then concentrated and partitioned between CHCl3 and
H2O to afford 0.62 g of CHCl3 residue.
2,5-Dibr om o-3,4-d ih yd r oxyben zyl Eth yl Eth er (3b). A
solution of 3 (50 mg, 0.17 mmol) in 95% EtOH (10 mL) was
heated under reflux for 12 h (TLC monitored). Similar to 3a ,
the recrystallized residue (46 mg, yield 83%) gave yellowish
white crystals: mp 82-84 °C; 1H NMR (acetone-d6) δ 1.18 (3H,
t, J ) 7.05 Hz, CH3), 3.55 (2H, q, J ) 7.05 Hz, CH2CH3), 4.41
(2H, s, ArCH2), 7.15 (1H, s, 6-H), 8.45 (1H, s, OH); 13C NMR
(acetone-d6) δ 14.7 (CH3), 65.7 (CH2CH3), 71.1(ArCH2), 108.4,
109.54, 122.8 (C-6), 131.0 (C-1), 142.7, 143.7; EIMS m/z 328
(19), 326 (40), 324 (20) [M]+, 283 (44), 281 (91), 279 (46) [M -
OCH2CH3]+, 203 (89), 201 (100) [M - OCH2CH3 - Br + H]+,
110 (66), 29 (57); GLC-MS tR 26.06 min; EIMS of silylated
derivative m/z 472 (8), 470 (15), 468 (7) [M]+, 426 (4) [M -
The CHCl3 fraction (0.5 g) was loaded onto a silica gel (45
g) column and eluted with n-hexane/EtOAc of increasing
polarities to obtain nine fractions, each of 100 mL (F1-F9),
followed by a gradient of EtOAc and MeOH to afford 11
fractions each of 100 mL (F10-F20). By performing TLC (silica
gel GF254, n-hexane/EtOAc/MeOH, 6.5:2.5:1) and spraying with
5% FeCl3, no phenolic compounds could be detected in fractions
F1-F5. Fractions F6 (n-hexane/EtOAc, 3:2) and F7 (n-hexane/
EtOAc, 1:1) contained a single phenolic compound, which was
further purified by recrystallization from CCl4 to afford a
yellowish white powder (3 mg). By spectroscopic analysis and
GLC-MS, the compound was found to be the methyl ether (1a )
of lanosol. 7 All other fractions were mixtures. Similar fractions
were combined and tested for cytotoxic activity. Only three
fractions {F9, 6 mg (100% EtOAc), F10, 25 mg, and F11, 28 mg
(EtOAc/MeOH, 9:1)} were found to have cytotoxic activity
higher than that of the parent CHCl3 fraction (Table 1).
CHOCH3]+, 339 (47), 337 (88), 335 (45) [M - OCH2CH3
-
Si(CH3)3 - CH3]+, 259, 257 (8) [M+ - OCH2CH3 - Br - Si-
(CH3)3 - CH3 + H], 73 (100) [Si(CH3)3]+; anal. C 32.58%, H
2.63% calcd for C9H10Br2O3 0.08 CCl4, C 32.24%, H 2.98%.
2,5-Dibr om o-3,4-d ih yd r oxyben zyl n -P r op yl Eth er (3c).
Similar to above, a solution of 3 (70 mg, 0.23 mmol) in 95%
n-propanol (11 mL) was heated under reflux for 6 h, and the
recrystallized residue (67 mg, yield 86%), similarly, afforded
pale brown crystals: mp 81-82 °C; 1H NMR (acetone-d6) δ
0.91 (3H, t, J ) 7.39 Hz, CH3), 1.58 (2H, sext, J ) 7.39, 6.53
Hz, CH2CH2CH3), 3.45 (2H, t, J ) 6.53 Hz, OCH2CH2), 4.41
(2H, s, ArCH2), 7.13 (1H, s, 6-H), 8.4 (1H, s, OH); 13C NMR
(acetone-d6) δ 10.2 (CH3), 22.8 (CH2CH3), 71.3 (OCH2), 72.05
(ArCH2), 108.5, 109.6, 122.8 (C-6), 131.0 (C-1), 142.7, 143.8;
EIMS m/z 342 (14), 340 (28), 338 (14) [M]+, 283 (54), 281 (100),
279 (51) [M - OCH2CH2CH3]+, 219 (26), 217 (27) [M -
CH2CH2CH3 - Br + H]+, 203 (40), 201 (50) [M - OCH2CH2-
CH3 - Br +H]+, 59 (20) [OCH2CH2CH3]+, 31 (73); GLC-MS tR
28.07 min; EIMS of silylated derivative m/z 486 (7), 484 (12),
482 (6) [M]+, 425 (6) [M - OCH2CH2CH3]+, 339 (45), 337 (87),
335 (44) [M - OCH2CH2CH3 - Si(CH3)3 - CH3]+, 259, 257 (9)
[M - OCH2CH2CH3 - Br - Si(CH3)3 - CH3 + H]+, 73 (100)
[Si(CH3)3]+; anal. C 34.35%, H 3.19%, calcd for C10H12Br2O3
0.1 CCl4, C 34.13%, H 3.40%.
GLC-MS. The most active fraction (F10, IC50: 2.35 µg mL-1
)
was subjected to GLC-MS analysis. Identification of the
bromophenols was made from the mass spectra of the trim-
ethylsilyl derivatives after GLC.8 The fraction (3 mg) was
silylated by adding 40 µL of pyridine and 40 µL of BSTFA
(N,O-bis-TMS-trifluoroacetamide containing 1% TMCS as
catalyst) at 40 °C for 15 min.
Syn th esis of P h en olic Com p ou n d s. Novel Syn th etic
Isom er s. Note that signals for OH groups did not always
appear in the 1H NMR spectra.
2,5-Dibr om o-3,4-d ih yd r oxyben zyl Alcoh ol (3). KBH4
(0.054 g, 1 mmol) dissolved in H2O (1.2 mL) was added
dropwise to a suspension of aldehyde 6 (0.296 g, 1 mmol) in
In -Vitr o Cytotoxicity Assa y. Human colon cell lines DLD-
122 and HCT-116,23 representatives of a common malignancy,
were used, which are available at the Tom Connors Cancer
Research Centre, Bradford University, U.K. These cell lines