5838 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 19
Leo´n et al.
was purified by column chromatography to give 22 mg of a
colorless solid (53%).
(3H, s, OAc), 2.04 (6H, s, OAc), 2.08 (3H, s, OAc), 2.27 (3H, s,
OAc), 4.04 (1H, dd, J ) 3.1, 11.6 Hz, Ha-1), 4.13 (1H, dd, J )
5.4, 11.7 Hz, Hb-1), 4.45 (1H, m, H-2), 4.80 (1H, dd, J ) 6.7, 9.7
Hz, H-4), 5.05 (1H, dd, J ) 3.4, 7.9 Hz, H-3), 5.23 (1H, d, J ) 3.1
(2S,3S,4R,2′R,3′R)-2-(2′,3′-O-Isopropylideneicosanoylamino)-
1,3,4-docosanetriol (23a). Amorphous solid; [R]25D -11.8 (c 0.022,
CHCl3); IR νmax (film, NaCl) 2917, 2850, 1658, 1529, 1467, 1216,
Hz, H-3′), 5.31 (1H, m, H-2′), 6.62 (1H, d, J ) 9.4 Hz, N-H); 13
C
1
1081 cm-1; H NMR (δ, CDCl3, 400 MHz) 0.87 (6H, m), 1.21-
NMR (δ, CDCl3) 14.1 (C-22 and C-20′), 20.6 (2 × OAc), 20.7
(OAc), 20.9 (2 × OAc), 25.1-31.9 [C-(5-21) and C-(4′-19′)],
47.6 (C-2), 62.7 (C-1), 72.0 (C-3), 73.3 (C-3′), 72.8 (C-4), 74.0
(C-2′), 167.4 (C-1′), 169.3 (CdO), 169.8 (CdO), 170.3 (CdO),
1.35 (62H, m), 1.37 (3H, s), 1.35-1.60 (2H, m), 1.60-1.80 (2H,
m), 1.56 (3H, s), 3.55-3.70 (2H, m), 3.75 (1H, dd, J ) 5.7, 11.5
Hz), 3.88 (1H, dd, J ) 2.4, 11.5 Hz), 4.12 (1H, m), 4.36 (1H, m),
4.46 (1H, d, J ) 7.2 Hz), 7.33 (1H, d, J ) 8.0 Hz); 13C NMR (δ,
CDCl3) 14.1, 22.7, 24.7, 25.7, 26.6, 27.4, 29.4, 29.7, 30.4, 31.9,
33.2, 52.6, 61.4, 72.4, 76.5, 77.6, 77.7, 109.4, 170.7; HR-FABMS
(relative intensity) 740.6797 [M + H]+ (C49H90NO6+, calcd
740.6768) (22), 682 (16), 664 (17), 356 (10), 338 (16).
170.7 (CdO), 171.0 (CdO); HR-EIMS (70 eV) m/z (relative
+
intensity) [M]+ (absent), 790.6172 [M - C5H11O3]+ (C47H84NO8
,
calcd 790.6197) (83), 776 (42), 747 (36), 730 (26), 687 (13), 669
(26); 656 (27), 627 (18), 599 (11), 512.3546 (C28H50NO7+, calcd
512.3587) (86), 484.3729 (C28H52O6+, calcd 484.3764) (75), 452
(44), 428 (24), 424 (19), 411.3085 (C24H43O5+, calcd 411.3110)
(46), 392 (100), 383 (33), 369 (42), 364 (12), 341 (39), 320 (20).
Anal. (C52H95NO11) C, H. N: calcd, 10.52; found, 11.0.
(2S,3S,4R,2′S,3′S)-2-(2′,3′-O-Isopropylideneicosanoylamino)-
1,3,4-docosanetriol (23b). Amorphous solid; [R]25 -5 (c 0.018,
D
CHCl3); IR νmax (film, NaCl) 2917, 2849, 1660, 1530, 1468, 1371,
1
1214, 1066 cm-1; H NMR (δ, CDCl3, 400 MHz) 0.88 (6H, m),
(2S,3S,4R,2′R,3′S)-2-(2′,3′-Diacetoxyeicosanoylamino)-1,3,4-
triacetoxydocosane (27). Amorphous solid (7.1 mg, 0.0074 mmol,
1.15-1.37 (62H, m), 1.38 (3H, s), 1.40-1.80 (4H, m), 1.55 (3H,
s), 3.63 (3H, m), 3.75 (1H, dd, J ) 5.8, 11.1 Hz), 3.93 (1H, b dd,
J ) 2.5, 11.4 Hz), 4.12 (1H, m), 4.33-4.39 (1H, m), 4.47 (1H, d,
J ) 7.4 Hz), 7.35 (1H, d, J ) 7.7 Hz); 13C NMR (δ, CDCl3) 13.8,
22.4, 24.4, 25.4, 26.3, 27.1, 29.1, 29.2, 29.3, 29.42, 29.46, 30.0,
31.6, 33.0, 52.4, 61.6, 72.5, 76.0, 77.4, 77.5, 109.2, 170.4; HR-
FABMS (relative intensity) 740.6732 [M + H]+ (C49H90NO6+, calcd
740.6768) (100), 682 (32), 664 (52), 398 (45), 356 (34), 338 (26).
General Experimental Procedure for the Preparation of
Pentaacetate Ceramides 24 and 25. The acetonide 23a (13 mg,
0.017 mmol) was treated with 3% HCl-MeOH (3 mL) and stirred
at room temperature for 12 h. Then the mixture was neutralized
with Et3N. After removal of the MeOH in vacuo, the obtained white
solid was dissolved in pyridine (2 mL) and an excess of Ac2O was
added. After 8 h, the reaction product was extracted with CH2Cl2
(3 × 10 mL) and the organic layer was washed with brine, dried
(MgSO4), and filtered. The solvent was removed in vacuo to give
a white solid, which was purified by column chromatography to
yield pure 24 (10 mg, 65%).
44%); [R]25 +15.3 (c 0.004, CHCl3); IR νmax (film, NaCl) 3286,
D
1
2917, 2852, 1744, 1699, 1470, 1370, 1220, 1044 cm-1; H NMR
(δ, CDCl3) 0.86 (6H, t, J ) 6.5 Hz, Me-22 and Me-20′), 1.20-
1.40 (62H, br s, 31-CH2), 1.59 (4H, m, CH2-5 and CH2-4′), 2.00
(3H, s, OAc), 2.06 (3H, s, OAc), 2.07 (3H, s, OAc), 2.08 (3H, s,
OAc), 2.27 (3H, s, OAc), 4.01 (1H, dd, J ) 3.6, 11.7 Hz, Ha-1),
4.33 (1H, dd, J ) 7.1, 11.7 Hz, Hb-1), 4.46 (1H, m, H-2), 4.95
(1H, td, J ) 3.4, 7.5 Hz, H-4), 5.01 (1H, dd, J ) 4.3, 6.0 Hz,
H-3), 5.24 (1H, d, J ) 3.6 Hz, H-3′), 5.33 (1H, m, H-2′), 6.68
(1H, d, J ) 9.1 Hz, N-H); 13C NMR (δ, CDCl3) 14.1 (C-22 and
C-20′), 20.6 (2OAc), 20.7 (OAc), 20.8 (OAc), 21.0 (OAc), 25.1-
31.9 [C-(5-21) and C-(4′-19′)], 48.4 (C-2), 62.1 (C-1), 72.3 (C-
3), 72.6 (C-3′), 72.9 (C-4), 73.7 (C-2′), 167.4 (C-1′), 169.3 (Cd
O), 169.7 (CdO), 170.3 (CdO), 170.6 (CdO), 171.1 (CdO); EIMS
(70 eV) m/z (relative intensity) [M]+ (absent), 792 (14), 790 (84),
777 (22), 776 (41), 748 (21), 747 (36), 730 (26), 729 (23), 716 (7),
687 (12), 669 (23); 657 (12), 599 (12), 424 (19), 411 (46), 392
(100), 383 (33), 369 (43). Anal. (C52H95NO11) C, H, N.
Cell Culture. Human SK-MEL-1 melanoma cells (DSMZ No.
ACC 303, DSMZ, German Collection of Microorganisms and Cell
Cultures, Braunschweig, Germany) were grown in RPMI 1640
(Sigma) supplemented with 10% (v/v) heat-inactivated fetal bovine
serum (Sigma) and 100 units/mL penicillin and 100 µg/mL
streptomycin at 37 °C in a humidified atmosphere containing 5%
CO2. The cell numbers were counted by a hematocytometer, and
the viability was always greater than 95% in all experiments as
assayed by the 0.025% trypan blue exclusion method. Stock
solutions of 10 mM ceramides were made in ethanol/dodecane (49:
1), and aliquots were frozen at -20 °C. Further dilutions were made
in culture media just before use. In all experiments, the final
concentration of ethanol/dodecane (49:1) did not exceed 0.5% (v/
v), a concentration that is nontoxic to the cells. Cells were
resuspended in fresh medium 24 h before each treatment.
Assay for Growth Inhibition and Cell Viability. The cytotoxi-
city of ceramides was assessed using the 3-[4,5-dimethylthiazol-
2-yl]-2,5-diphenyltetrazolium bromide assay.23 Briefly, 1 × 104
exponentially growing cells were seeded in 96-well microculture
plates with various ceramide concentrations in a volume of 100 µL
for 72 h at 37 °C. Controls were always treated with the
same amount of ethanol-dodecane (49:1) as used in the corre-
sponding experiments. Surviving cells were detected on the basis
of their ability to metabolize 3-[4,5-dimethylthiazol-2-yl]-2,5-
diphenyltetrazolium bromide (MTT) into formazan crystals. Optical
density at 570 nm was used as a measure of cell viability. Cell
survival was calculated as the fraction of cells alive relative to
control for each point:
(2S,3S,4R,2′R,3′R)-2-(2′,3′-Diacetoxyeicosanoylamino)-1,3,4-
triacetoxydocosane (24). Amorphous solid; [R]D -7.5 (c 0.02, CH-
Cl3). Anal. (C52H95NO11) C, H. N: calcd, 10.52; found, 10.07. The
1
IR, MS, and H and 13C NMR data were identical to those of 2a.
(2S,3S,4R,2′S,3′S)-2-(2′,3′-Diacetoxyeicosanoylamino)-1,3,4-
triacetoxydocosane (25). Amorphous solid; [R]25D -2.0 (c 0.035,
CHCl3); IR νmax (film, NaCl) 2918, 2850, 1746, 1698, 1523, 1467,
1371, 1225, 1047 cm-1; 1H NMR (δ, CDCl3) 0.87 (6H, m), 1.15-
1.35 (62H, m), 1.55-1.75 (4H, m), 2.04 (9H, br s), 2.08 (3H, s),
2.22 (3H, s), 4.07 (1H, dd, J ) 3.2, 11.7 Hz), 4.20 (1H, dd, J )
5.6, 11.6 Hz), 4.44-4.50 (1H, m), 4.81-4.85 (1H, m), 5.04 (1H,
dd, J ) 3.6, 7.5 Hz), 5.19-5.26 (1H, m), 5.33 (1H, d J ) 3.0 Hz),
6.76 (1H, d J ) 9.2 Hz); 13C NMR (δ, CDCl3) 13.9, 20.5, 20.6,
20.8, 22.4, 25.2, 25.2, 28.4, 29.1, 29.2, 29.3, 2935, 31.7, 47.3, 62.3,
72.1, 72.5, 72.8, 73.8, 166.4, 169.2, 170.0, 170.1, 170.5, 170.8;
HR-FABMS m/z (relative intensity) 932 (6), 910.6927 [M + H]+
(C52H96NO11+, calcd 910.6983) (12), 850 (20), 790 (22), 320 (22).
Anal. (C52H95NO11) C, H. N: calcd, 10.52; found, 9.97.
General Experimental Procedure for the Preparation of
Pentaacetate Ceramides 26-27. To a mixture of acid 17a (8 mg,
0.0128 mmol), amine 4 (5.5 mg, 0.0128 mmol), and 1-hydroxy-
benzotriazole (HOBt) (5 mg, 0.037 mmol) in dry CH2Cl2 (2 mL)
was added EDCI (3.68 mg, 0.0192 mmol). The reaction mixture
was stirred for 48 h, and then it was extracted with AcOEt. The
organic layers were washed using a saturated solution of NaCl and
dried over MgSO4. This residue was dissolved in pyridine. Acetic
anhydride was added, and the solution was stirred for 24 h at room
temperature. The product was dried in vacuo to furnish 26.
(2S,3S,4R,2′S,3′R)-2-(2′,3′-Diacetoxyeicosanoylamino)-1,3,4-
triacetoxydocosane (26). Amorphous solid (6.8 mg, 0.0074 mmol,
mean absorbance in treated cells
mean absorbance in control wells
cell survival (%) )
× 100
58%); [R]25 +8.5 (c 0.02, CHCl3); IR νmax (film, NaCl) 3285,
D
1
2918, 2850, 1746, 1698, 1469, 1372, 1220, 1044 cm-1; H NMR
(δ, CDCl3) 0.87 (6H, t, J ) 6.5 Hz, Me-22 and Me-20′), 1.20-
1.40 (62H, br s, 31-CH2), 1.60 (4H, m, CH2-5 and CH2-4′), 2.03
Concentrations inducing a 50% inhibition of cell growth (IC50) were
determined graphically for each experiment using the curve-fitting