590
K. Seno et al. / Bioorg. Med. Chem. Lett. 11 (2001) 587±590
5. The inversion of stereochemistry at the 4-position of pyr-
rolidine was achieved by the Mitsunobu reaction. The reaction
conditions are as follows: (1) SOCl2, MeOH, 0À40 ꢀC, 3 h;(2)
Boc2O, Et3N, EtOAc, 0 ꢀC±rt, 15 h;(3) HCOOH, DIAD, PPh 3,
THF, 0 ꢀC±rt, 1 h;(4) 1N NaOH (1.0 equiv), MeOH, 0 ꢀC, 1 h.
Overall yield from trans-4-hydroxy-l-proline (1) was 82%.
6. 1H NMR (CDCl3) d: 1.35±1.42 (9H, m), 1.75±1.90 (1H, m),
2.82±3.35 (3H, m), 3.57±3.62 (4H, m), 4.08±4.217 (1H, m),
7.18±7.31 (9H, m), 7.42±7.45 (6H, m). MS: m/z 504 (M+ for
C30H33NO4S).
7. 1H NMR (CDCl3) d: 1.42 (9H, s), 1.50±1.64 (1H, m), 1.71±
1.81 (1H, m), 2.72±3.39 (5H, m), 3.70±3.89 (1H, m), 7.20±
7.32±7.31 (9H, m), 7.46 (6H, d, J=7.8 Hz). IR nmax (CHCl3):
2105, 1686 cmÀ1. Anal. calcd for C29H32N4O2S: C, 69.57;H,
6.44;N, 11.19;S, 6.40%. Found: C, 69.30;H, 6.56;N, 11.23;
S, 6.31%.
8. 1H NMR (CDCl3) d: 1.88±1.97 (2H, m), 2.58 (1H, dd,
J=7.2, 10.8 Hz), 2.69 (1H, dd, J=7.2, 10.8 Hz), 3.07 (1H,
quintet, J=7.5 Hz), 3.35 (1H, dd, J=3.3, 6.3 Hz), 3.49 (1H,
dd, J=5.7, 12.3 Hz), 4.20±4.25 (1H, m), 6.80±7.00 (2H, m),
7.15±7.64 (20H, m). IR nmax (CHCl3): 2103, 1669, 1640,
1609 cmÀ1. Anal. calcd for C38H30N4O2SF2: C, 71.63;H, 4.86;
N, 8.33;S, 4.77;F, 5.65%. Found: C, 71.80;H, 4.99;N, 8.29;
S, 4.81;F, 5.52%.
sn-glycero-3-phosphocholine (50 mCi/nmol) and 1.25 mM sn-1,2-
dioleoylglycerol according to the method described previously.1c
11. Cellular assay. Human THP-1 cells were grown in RPMI
1640 containing 10% fetal calf serum and preincubated with
1.3% dimethyl sulfoxide for 2 days. After washing with phos-
phate-buered saline, the cells were suspended in Hanks'
balanced salt saline containing 0.1% bovine serum albumin.
The cell suspension was preincubated with inhibitor at 37 ꢀC
for 15 min and then incubated with 3 mM A23187 at 37 ꢀC for
20 min. Arachidonic acid, PGE2, and LTC4 were quanti®ed
according to methods described previously.4,15
12. Yoshimoto, T.;Yokoyama, C.;Ochi, K.;Yamamoto, S.;
Biochim. Bio-
Maki, Y.;Ashida, Y.;Terao, S.;Shiraishi, M.
phys. Acta 1982, 713, 470.
13. Whole blood assay. Freshly drawn heparinized venous
blood was obtained from healthy volunteers. After pre-
incubation with the drug at 37 ꢀC for 15 min, 30 mM A23187
was added and incubated at 37 ꢀC for 60 min. The plasma was
separated by centrifugation at 1600Âg for 20 min at 4 ꢀC.
Arachidonic acid was extracted with Dole's extraction proce-
dure,16 labeled with 9-anthryldiazomethane, and quanti®ed by
high-performance liquid chromatography as described pre-
viously.4,15 PGE2, TXB2, and LTB4 were extracted with ace-
tone and evaporated in vacuo, then quanti®ed by radio
immunoassay and enzyme immunoassay.
14. (a) Street, I. P.;Lin, H.-K.;Laliberte , F.;Ghomashchi, F.;
Wang, Z.;Perrier, H.;Tremblay, N. M.;Huang, Z.;Weech,
P. K.;Gelb, M. H. Biochemistry 1993, 32, 5935. (b) Riendeau,
D.;Guay, J.;Weech, P. K.;Laliberte , F.;Yergey, J.;Li, C.;
Desmarais, S.;Perrier, H.;Liu, S.;Nicoll-Grith, D.;Street,
I. P. J. Biol. Chem. 1994, 269, 15619. (c) Bartoli, F.;Lin, H.-
K.;Ghomashchi, F.;Gelb, M. H.;Jain, M. K.;Apitz-Castro,
R. J. Biol. Chem. 1994, 269, 15625.
9. Mp: 158±160 ꢀC. NMR (CDCl3) d: 2.03±2.21 (2H, m),
2.26±2.34 (1H, m), 2.51 (1H, dd, J=7.8, 11.1 Hz), 2.90±3.01
(1H, m), 3.77±3.93 (2H, m), 4.19±4.46 (1H, m), 6.93±7.10
(10H, m), 7.22±7.29 (7H, m), 7.39 (2H, d, J=8.1 Hz), 7.51±
7.65 (4H, m), 7.79 (1H, s), 7.88 (2H, d, J=8.1 Hz), 8.12±8.16
(1H, m), 9.35±9.50 (1H, br. s), 0.65 mol of EtOH peaks
were found accompanying these peaks. IR nmax (KBr):
3411, 1751, 1709, 1662, 1610, 1537, 1498, 1284 cmÀ1
.
[a]D=À190.4Æ2.3ꢀ (c 1.002, CHCl3, t=23 ꢀC). Anal. calcd for
.
.
C49H37F2N3O5S2 H2O 0.65EtOH: C, 67.28;H, 4.59;N, 4.68;
S, 7.14;F, 4.23;H 2O, 2.01%. Found: C, 67.48;H, 4.61;N,
4.86;S, 7.10;F, 4.13;H 2O, 1.76%.
15. (a) Tojo, H.;Ono, T.;Okamoto, T. J. Lipid Res. 1993, 34,
837. (b) Hanasaki, K.;Ono, T.;Saiga, A.;Morioka, Y.;Ikeda,
M.;Kawamoto, K.;Higashino, K.;Nakano, K.;Yamada, K.;
Ishizaki, J.;Arita, H. J. Biol. Chem. 1999, 274, 34203.
16. Dole, V. P.;Meinertz, H. J. Biol. Chem. 1960, 235, 2595.
10. Enzyme assay. The PLA2 activity was measured using the
liposome containing 2.5 mM 1-palmitoyl-2-[14C]arachidonoyl-