T.-S. Jeong et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1525–1527
1527
brine, dried over anhydrous MgSO4, filtered, and evapo-
rated to give the crude product. Purification by flash
column chromatography on silica gel n-hexane–EtOAc
(1:1) gave the pure (E)-2a (3.0 g, 50%) and (Z)-2a
(0.32 g, 5%). To a solution of (E)-2a (0.22 g, 1.8 mmol)
in CH2Cl2 (10 mL) was added benzoyl chloride (0.3 mL,
2.3 mmol) in the presence of Et3N (0.32 mL, 2.3 mmol) at
0 °C. After being stirred for 1 h, the reaction mixture was
quenched with 1 N HCl and extracted with CH2Cl2 to give
the residue, which was washed with saturated NaHCO3
aqueous solution and brine and dried over anhydrous
MgSO4. Purification by flash column chromatography on
silica gel [n-hexane–EtOAc (1:1)] gave the pure (E)-3a
(0.26, 65%) as colorless prisms, mp 98–105 °C (n-hexane–
CH2Cl2); 1H NMR (300 MHz, CDCl3) d 7.48 (m, 5H),
7.62 (m, 1H), 7.82 (dd, J = 1.8, 7.8 Hz, 2H), 8.14 (dd,
J = 2.1, 8.4 Hz, 2H), 8.57 (s, 1H).
11. Lp-PLA2 the enzyme is also known as platelet-activating
factor acetylhydrolase (PAF-AH), activity was measured
using [3H] PAF as a substrate.13,16 Briefly, a micelle
substrate was prepared with unlabeled PAF and [3H] PAF
(100 lCi/mL, 21.5 Ci/mmol, NET 910) in 10 mM
phosphate-buffered saline (PBS), pH 7.4, containing
2.7 mM EDTA (PBS–EDTA). The reaction mixture,
containing 20 lL of diluted human LDL (4–5 lg protein),
120 lL of PBS–EDTA, and 20 lL of test sample, was
preincubated at 37 °C for 15 min. The reaction was
initiated by the addition of 40 lL micelle substrate
(0.05 lCi, final concn 80 lM PAF) to measure initial rates
of PAF-AH activity. The reaction was stopped by
vortexing with 600 lL of CHCl3/MeOH (2:1) and the
CHCl3 and aqueous layers were separated by centrifuga-
tion. The aqueous layer was removed (250 lL) and
vortexed with 250 lL of CHCl3. The aqueous layer was
again removed and the [3H] acetate determined by
scintillation counting (1450 Microbeta Trilux, Qallac Oy,
Turku, Finland). The raw counts were corrected for
background using a nonenzyme-containing blank and
were expressed as nanomoles of PAF degraded per hour
per milligram of protein.
8. Vermillion, G.; Hauser, C. R. J. Org. Chem. 1940, 62,
2939.
9. Caslake, M. J.; Packard, C. J.; Suckling, K. E.; Holmes, S.
D.; Chamberlain, P.; Macphee, C. H. Atherosclerosis 2000,
150, 413.
10. Ahn, B. T.; Lee, S.; Lee, S. B.; Lee, E. S.; Kim, J. G.; Bok,
S. H.; Jeong, T. S. J. Nat. Prod. 2001, 64, 1562, Procedure
for isolation of LDL: Blood was collected from normal-
ipidemic volunteers. EDTA was used as anticoagulant
(1.5 mg/mL of blood). After low-speed centrifugation of
the whole blood to obtain plasma and to prevent
lipoprotein modification, EDTA (0.1%), NaN3 (0.05%),
and PMSF (0.015%) were added. LDL was isolated from
the plasma by discontinuous density gradient ultracentri-
fugation.14 Briefly, the plasma was centrifuged at 100,000g
at 4 °C for 20 h. After the top layers containing chylomi-
cron and very low-density lipoprotein (VLDL) were
removed, the density of remaining plasma fractions was
adjusted to 1.063 with NaBr solution and they were
recentrifuged at 100,000g for an additional 24 h. The LDL
fraction in the top of the tube was collected and dialyzed
overnight against three changes of phosphate buffer
(pH 7.4), containing NaCl (150 mM) in the dark at 4 °C
to remove NaBr and EDTA. The LDL in PBS was stored
at 4 °C and used within 4 weeks. The purity of the fraction
was confirmed by agarose gel electrophoresis and SDS-
PAGE.15 Concentration of LDL protein was determined
using bovine serum albumin (BSA) as a standard.
12. Bloomer, J. C.; Boyd, H. F.; Hickey, D. M. B.; Ife, R. J.;
Leach, C. A.; Macphee, C. H.; Milliner, K. J.; Pinto, I. L.;
Rawlings, D. A.; Smith, S. A.; Stansfield, I. G.; Stanway,
S. J.; Taylor, M. A.; Theobald, C. J.; Whittaker, C. M.
Bioorg. Med. Chem. Lett. 2001, 11, 1925, and compound
33 (SB-381320) in this literature.
13. Boyd, H. F.; Fell, S. C. M.; Flynn, S. T.; Hickey, D. M. B.;
Ife, R. J.; Leach, C. A.; Macphee, C. H.; Milliner, K. J.;
Moores, K. E.; Pinto, I. L.; Porter, R. A.; Rawlings, D.
A.; Smith, S. A.; Stansfield, I. G.; Tew, D. G.; Theobald,
C. J.; Whittaker, C. M. Bioorg. Med. Chem. Lett. 2000, 10,
2557.
14. Havel, R. J.; Edger, H. A.; Bradgon, J. H. J. Clin. Invest.
1955, 34, 1345.
15. Chapman, M. J.; Goldstein, S.; Lagrange, D.; Laplaud, P.
M. J. Lipid Res. 1981, 22, 399.
16. (a) Tew, D. G.; Southan, C.; Rice, S. Q. J.; Lawrence, G.
M. P.; Li, H.; Boyd, H. F.; Moores, K.; Gloger, I. S.;
Macphee, C. H. Arterioscler. Throm. Vasc. Biol. 1996, 16,
591; (b) McCall, M. R.; Belle, M. L.; Forte, T. M.; Krauss,
R. M.; Takanami, Y.; Tribble, D. L. Biochem. Biophys.
Acta 1999, 1437, 23.