H. Azuma et al. / Bioorg. Med. Chem. 15 (2007) 2860–2867
2865
4
. Experimental
15.4 Hz); HRMS (FAB, direct) calcd for C H NO ,
1
8
34
2
+
[
MꢀCl] 296.259; found: 296.2586.
All materials obtained commercially (guaranteed re-
agent grade) were used without further purification.
All solvents were freshly distilled under nitrogen before
use; THF and CH Cl were distilled from LiAlH and
4.3. (2S,4E,6E)-2-acetylamido-3-oxo-4,6-octadecadien-
1-ol (B)
2
2
4
CaH , respectively. Column chromatography was per-
2
The reaction was carried out as described for C2-keto-
Cer, using 2 (50 mg, 151 lmol) to give compound B as
a powdery solid (22 mg, 44%). The specific rotation of
B could not be measured accurately, because the com-
pound was decomposed by irradiation with a sodium
formed on silica gel. C2-Cer was prepared according
2
0
to our previous report. Protease inhibitors, Z-VAD-
fmk and Ac-IETD-CHO, were purchased from the Pep-
tide Institute (Osaka, Japan) and Sigma Chemical Co.
(
St. Louis, MO, USA), respectively. Polyclonal antibody
lamp in CHCl ; mp 79 ꢁC; IR (KBr) 3233, 2927, 2851,
3
for caspase-3 and monoclonal antibody for cytochrome
c were purchased from Santa Cruz Biotechnology (San-
ta Cruz, CA, USA). Monoclonal antibodies for PARP
and for b-actin were purchased from Trevigen (Gai-
thersburg, MD, USA) and Abcam (Cambridge, UK),
respectively. Anti-rabbit and anti-mouse IgGs coupled
to alkaline phosphatase were purchased from Sigma
Chemical Co. (St. Louis, MO, USA).
1684, 1630, 1752, 1466, 1379, 1283, 1144, 1069, 995,
ꢀ
1 1
723 cm ; H NMR (400 MHz, CDCl ); d 0.88 (t, 3H,
3
J = 6.8 Hz), 1.26 (br s, 16H), 1.39–1.75 (m, 2H), 2.09
(s, 3H), 2.21 (q, 2H, J = 7.1 Hz), 3.4 (br s, 1H), 3.81–
3.89 (m, 1H), 3.92–4.02 (m, 1H), 4.9 (ddd, 1H, J = 3.2,
4.6, 6.1 Hz), 6.22 (dd, 1H, J = 10.5, 15.1 Hz), 6.24 (d,
1H, J = 15.1 Hz), 6.32 (dt, 1H, J = 15.1, 6.8 Hz), 6.83
(d, 1H, J = 6.1 Hz), 7.44 (dd, 1H, J = 10.5, 15.1 Hz)+;
HRMS (FAB, direct) calcd for C H NO , [M+H]
2
0
36
3
4
.1. (2S,4E)-2-acetylamido-3-oxo-4-octadecen-1-ol (A)
338.2695; found: 338.2691; Anal. Calcd: C, 71.18; H,
0.45; N, 4.15. Found: C, 70.92; H, 10.51; N, 4.15.
1
To a suspension of acetic acid (14 mg, 0.234 mmol),
WSC (45 mg, 0.234 mmol), and HOBt (36 mg,
4.4. (S)-(N-Boc-O-methyl)serine methyl ester (4)
0
.234 mmol) in dry CH Cl (10 mL), a solution of
2 2
1
4
15
(
2S,4E)-2-amino-3-oxo-4-octadecen-1-ol Æ HCl (60 mg,
.18 mmol) and Et N (27 mg, 0.27 mmol) in dry CH Cl
2
(S)-N-Boc-serine methyl ester 3 (2.3 g, 10.5 mmol) was
dissolved in dry acetone (50 mL) containing methyl
0
3
2
(
2 mL) was added dropwise, and the mixture was stirred
iodide (15 mL) and then Ag O (3.77 g, 16.3 mmol) was
2
overnight at room temperature. The resulting mixture
was washed with saturated brine, dried with Na SO ,
and then concentrated. The obtained residue was puri-
fied by column chromatography with hexane/EtOAc
added. The mixture was refluxed and stirred overnight.
The solids were removed by filtration and the solution
was concentrated. The residue was purified by column
chromatography with hexane/EtOAc (4:1) to give 4
2
4
2
0
(
1:3) and recrystallized from hexane to give compound
(1.31 g, 54%) as colorless oil; ½aꢁ +12.2 (c 1.76, CHCl3);
D
IR (NaCl) 3447, 2980, 1749, 1720, 1506, 1456, 1367,
2
0
A (35 mg, 57%) as a colorless powder; mp 64 ꢁC; ½aꢁ
D
9.26 (c 1.05, CHCl ); IR (KBr) 2918, 2851, 2350,
697, 1682, 1568, 1470, 1379, 1302, 1267, 1161, 1067,
ꢀ1
1
+
1167, 1119, 1063, 980, 874, 779 cm
;
H NMR
3
1
9
3
2
3
(400 MHz, CDCl ); d 1.45 (s, 9H), 3.34 (s, 3H), 3.59
3
ꢀ
1 1
70, 719 cm ; H NMR (400 MHz, CDCl ); d 0.88 (t,
(dd, 1H, J = 3.2, 9.3 Hz), 3.77 (s, 3H) 3.8 (dd, 1H,
J = 3.2, 9.3 Hz), 4.38–4.44 (m, 1H), 5.37 (d, 1H,
J = 8 Hz); HRMS (FAB, direct) calcd for C H NO ,
3
H, J = 6.8 Hz), 1.26 (br s, 20H), 1.44–1.52 (m, 2H),
.08 (s, 3H), 2.26 (q, 2H, J = 6.8 Hz), 3.36 (br s, 1H),
.8–3.88 (m, 1H), 3.93–4.01 (m, 1H), 4.91 (ddd, 1H,
1
0
20
5
+
[M+H] 234.1341; found: 234.134.
J = 3.6, 4.8, 6 Hz), 6.27 (d, 1H, J = 15.6 Hz), 6.8 (d,
H, J = 6 Hz), 7.11 (dt, 1H, J = 15.6, 7.2 Hz); HRMS
1
4.5. (3S)-[3-(tert-butoxycarbonyl)amino-4-methoxy-2-
oxo-butyl]phosphonic acid methyl ester (5)
+
FAB, direct) calcd for C H NO : [M+H] 340.2852;
(
found: 340.2852; Anal. Calcd: C, 70.75; H, 10.98; N,
2
0
38
3
4
.13. Found: C, 70.45; H, 11.05; N, 4.13.
To a solution of methylphosphonic acid dimethyl ester
6.14 g, 49.5 mmol) in dry THF (150 mL), n-BuLi
(1.6 M in hexane, 30.9 mL, 49.5 mmol) was added drop-
(
4.2. (2S,4E,6E)-2-amino-3-oxo-4,6-octadecadien-1-ol
hydrochloride (2)
wise at ꢀ78 ꢁC under N . After the mixture was stirred
2
for 30 min at ꢀ78 ꢁC, a solution of 4 (3.85 g, 16.5 mmol)
in dry THF (20 mL) was added dropwise. The mixture
was stirred for 1 h at ꢀ78 ꢁC and then warmed up to
0 ꢁC. The reaction mixture was poured into ice-cooled
1 M HCl and extracted with EtOAc. After drying with
Na SO , the solution was concentrated, and the residue
1
2
To a solution of 1 (210 mg, 0.48 mmol), in MeOH
20 mL), 10% HCl (1.2 mL) was added, and the mixture
(
was stirred for 1 h at reflux. After cooling, the resulting
mixture was washed with hexane and then concentrated.
The crude product was recrystallized from i-PrOH/Et O
2
2
4
to give 2 (80 mg, 50%) as a colorless powder; mp 130 ꢁC;
was purified by column chromatography with CHCl3/
MeOH (20:1) to give 5 (1.18 g, 65%) as colorless oil;
2
5
½
aꢁ +7.35 (c 1, MeOH); IR (KBr) 3420, 2922, 2853,
D
435, 1684, 1636, 1601, 1468, 1213, 1057, 1005,
2
0
2
7
3
2
1
1
½aꢁ +35.3 (c 1.8, CHCl ); IR (NaCl) 3300, 2977, 1711,
D
1520, 1367, 1252, 1169, 1117, 1032, 866, 812 cm ; H
3
ꢀ
1
1
ꢀ1
1
21 cm
;
H NMR (400 MHz, CD OD); d 0.88 (t,
3
H, J = 6.8 Hz), 1.29 (br s, 16H), 1.42–1.52 (m, 2H),
.24 (q, 2H, J = 6.8 Hz), 3.95 (dd, 1H, J = 4.9,
2.2 Hz), 4.02 (dd, 1H, J = 3.7, 12.2 Hz), 4.39–4.43 (m,
H), 6.28–6.46 (m, 3H), 7.44 (dd, 1H, J = 10.2,
NMR (400 MHz, CDCl ); d 1.45 (s, 9H), 3.18 (dd, 1H,
3
J = 14.8, 22 Hz), 3.35 (s, 3H), 3.38 (dd, 1H, J = 14.8,
22 Hz), 3.59 (dd, 1H, J = 4.4, 9.6 Hz), 3.78 (dd, 3H,
J = 1.2, 2.8 Hz), 3.81 (dd, 3H, J = 1.2, 2.8 Hz), 3.85