468 J ournal of Natural Products, 2003, Vol. 66, No. 4
Kwon et al.
column (CH2Cl2-MeOH, 1:1) and purified using a Lobar A RP-
18 column (90% MeOH) to afford 1 (3 mg), 2 (5 mg), 3 (15 mg),
and 4 (17 mg).
br m, H-3), 5.60 (1H, dd, J ) 15.0, 6.0 Hz, H-4), 5.73 (1H, dt,
J ) 15.0, 7.8 Hz, H-7), 6.03 (1H, dd, J ) 15.0, 10.5 Hz, H-6),
6.28 (1H, br d, J ) 7.2 Hz, NH), 6.29 (1H, dd, J ) 15.0, 10.5
Hz, H-5); 13C NMR (CDCl3, 125 MHz) δ 14.8 (C-14, C-22′), 23.4
(C-13, C-21′), 26.5 (C-3′), 29.9, 30.0, 30.1, 30,2, 30.3, 30.4 (C-
9-C-11, C-4′-C-19′), 32.5, 32.6 (C-12, C-20′), 33.4 (C-8), 37.5
(C-2′), 55.2 (C-2), 63.2 (C-1), 75.2 (C-3), 129.6, 129.7 (C-5, C-6),
133.5 (C-4), 137.4 (C-7), 174.7 (C-1′); EIMS m/z 563 [M]+ (2),
545 (12), 515 (17), 416 (12), 382 (27), 365 (97), 340 (35), 224
(32), 206 (20), 194 (15), 60 (100); FABCIDMS m/z 586 (100),
565 (1.9), 556 (0.9), 542 (0.7), 528 (0.7), 514 (0.7), 500 (0.7),
486 (1.0), 472 (0.9), 458 (1.1), 444 (0.8), 430 (1.0), 416 (0.8),
402 (0.7), 388 (0.8), 374 (1.0), 358 (1.2), 332 (1.0), 318 (2.0),
206 (0.6); HRFABMS m/z 564.5344 (calcd for C36H70NO3,
564.5277).
Meth a n olysis of 1-4. Compound 1 (1 mg) was heated with
5% HCl in MeOH for 24 h at 74 °C. After adding H2O, the
reaction mixture was extracted with hexane and dried with
anhydrous magnesium sulfate. The resulting hexane-soluble
fraction was concentrated to yield a fatty acid methyl ester
1b, which was analyzed by GC-MS. Compounds 2, 3, and 4
were cleaved by the same method to afford 2b, 3b, and 4b,
respectively.
(4E ,2S ,3R )-2-N -Oct a d e ca n oyl-4-t e t r a d e ca sp h in ge -
n in e (1): white powder; [R]20D -3.5° (c 0.012, CHCl3); IR (neat)
1
νmax 3330, 2920, 2850, 1650, 1617, 1550, 1465, 1050 cm-1; H
NMR (CDCl3, 500 MHz) δ 0.88 (6H, t, J ) 7.0 Hz, H-14 and
H-18′), 1.20-1.39 (42H, m, H-7-H-13, H-4′-H-17′), 1.62 (2H,
br sep, J ≈ 7.5 Hz, H-3′), 2.05 (2H, br q, J ) 7.0 Hz, H-6), 2.23
(2H, t, J ) 7.5 Hz, H-2′), 2.65 (2H, br s, OH), 3.71 (1H, br d,
J ≈ 11.5 Hz, H-1), 3.91 (1H, ddd, J ) 11.5, 4.0, 3.0 Hz, H-1),
3.96 (1H, d br q, J ) 7.2, 3.5 Hz, H-2), 4.32 (1H, br dd, J ≈
6.5, 3.5 Hz, H-3), 5.53 (1H, dd, J ) 15.7, 6.5 Hz, H-4), 5.79
(1H, dt, J ) 15.7, 6.5 Hz, H-5), 6.26 (1H, br d, J ) 7.2 Hz,
NH); 13C NMR (CDCl3, 125 MHz) δ 14.8 (C-14, C-18′), 23.4
(C-13, C-17′), 26.5 (C-3′), 29.8, 30.1, 30.4, 30.4 (C-7-C-11,
C-4′-C-15′), 32.6 (C-12, C-16′), 33.0 (C-6), 37.6 (C-2′), 55.2 (C-
2), 63.2 (C-1), 75.3 (C-3), 129.5 (C-4), 135.1 (C-5), 174.7 (C-1′);
EIMS m/z 509 [M]+ (0.5), 491 (6), 461 (6), 360 (4), 326 (35),
309 (100), 278 (23), 267 (16), 226 (11), 208 (18), 60 (97).
(4E,6E,2S,3R)-2-N-E icosa n oyl-4,6-t et r a d eca sp h in ga -
d ien in e (2): white powder; mp 73.6 °C; [R]20 -3.2° (c 0.05,
D
CHCl3); UV (EtOH) λmax (log ꢀ) 233 (4.23) nm; IR (neat) νmax
3340, 2930, 2850, 1648, 1625, 1535, 1470, 1070, 1050 cm-1
;
Octa d eca n oic a cid m eth yl ester (1b): EIMS m/z (rel int)
298 (18), 267 (8), 255 (17), 241 (4), 213 (5), 199 (15), 185 (5),
171 (2), 157 (5), 143 (28), 129 (10), 97 (14), 87 (74), 74 (100),
55 (22), 43 (28).
Eicosa n oic a cid m eth yl ester (2b a n d 3b): EIMS m/z
326 (30), 295 (10), 283 (20), 269 (4), 255 (5), 241 (7), 227 (8),
213 (2), 199 (8), 185 (6), 171 (4), 157 (4), 143 (30), 129 (10), 97
(10), 87 (75), 74 (100), 55 (24), 43 (30).
1H NMR (CDCl3, 500 MHz) δ 0.88 (6H, t, J ) 7.0 Hz, H-14
and H-20′), 1.20-1.39 (42H, m, H-9-H-13, H-4′-H-19′), 1.61
(2H, br sep, J ≈ 7.5 Hz, H-3′), 2.06 (2H, m, H-8), 2.22 (2H, t,
J ) 7.5 Hz, H-2′), 2.74 (1H, br s, OH), 2.88 (1H, br s, OH),
3.70 (1H, br d, J ) 10.5 Hz, H-1), 3.89-3.96 (2H, m, H-1 and
H-2), 4.39 (1H, br m, H-3), 5.61 (1H, dd, J ) 15.0, 6.0 Hz, H-4),
5.73 (1H, dt, J ) 15.0, 7.5 Hz, H-7), 6.03 (1H, dd, J ) 15.0,
10.5 Hz, H-6), 6.26 (1H, br d, J ) 7.0 Hz, NH), 6.29 (1H, dd,
J ) 15.0, 10.5 Hz, H-5); 13C NMR (CDCl3,125 MHz) δ 14.8
(C-14, C-20′), 23.4 (C-13, C-19′), 26.5 (C-3′), 29.9, 30.0, 30.1,
30.2, 30.3, 30.4 (C-9-C-13, C-4′-C-17′), 32.5, 32.6 (C-12,
C-18′), 33.4 (C-8), 37.6 (C-2′), 55.2 (C-2), 63.2 (C-1), 75.3 (C-
3), 129.6 (C-5, C-6), 133.5 (C-4), 137.5 (C-7), 174.6 (C-1′); EIMS
m/z 535 [M]+ (1), 517 (10), 487 (19), 388 (15), 354 (17), 337
(90), 312 (24), 306 (20), 224 (38), 206 (22), 193 (21), 57 (100);
FABCIDMS m/z 558 (100), 537 (1.9), 528 (0.9), 514 (0.7), 500
(0.7), 486 (1.0), 472 (0.9), 458 (1.1), 444 (0.8), 430 (1.0), 416
(0.8), 402 (0.7), 388 (0.8), 374 (1.0), 358 (1.2), 332 (1.0), 318
(2.0), 206 (0.6); HRFABMS m/z 558.4874 (calcd for C34H65NO3-
Na, 558.4862).
Docosa n oic a cid m eth yl ester (4b): EIMS m/z 354 (40),
323 (8), 311 (18), 297 (4), 283 (2), 269 (5), 255 (10), 241 (4),
227 (1), 213 (2), 199 (10), 185 (6), 171 (2), 157 (3), 143 (30),
129 (12), 115 (4), 101 (8), 87 (80), 74 (100), 55 (30), 43 (45).
Mea su r em en t of Neu r ite Ou tgr ow th . Measurement of
NGF-potentiating activity and morphological observations of
PC12 cells were performed as previously reported.16 PC12 cells
were maintained in a humidified atmosphere of 5% CO and
95% air in RPMI1640 supplemented with 1% fetal bovine
serum, 2% horse serum, and 2 mM glutamine. Cells were
seeded in six-well culture plates at 3 × 104 cells/well in poly-
L-lysine-coated wells. PC12 cells in six-well plates were treated
with each sphingolipid (1-4) (10 µM) and NGF (50 ng/mL).
The culture medium was changed every day. Neurite out-
growth was measured under a microscope after 48 h of
treatment,17 when each culture was fixed with 2% glutaral-
dehyde in PBS and stored in PBS solution. Randomly selected
fields were photographed using a camera attached to the light
microscope (model CK-2; ×100 magnification). Neurite exten-
sion was evaluated in terms of lengths equivalent to one
diameter of the cell body. All data are expressed as means (
SD. Statistical significance was tested by one-way ANOVA.
(4E ,2S ,3R )-2-N -E icosa n oyl-4-t e t r a d e ca sp h in ge n in e
(3): white powder; mp 81.7 °C; [R]20 -4.0° (c 0.082, CHCl3);
D
IR (neat) νmax 3332, 2920, 2851, 1650, 1615, 1550, 1465, 1045
1
cm-1; H NMR (CDCl3, 500 MHz) δ 0.88 (6H, t, J ) 7.0 Hz,
H-14 and H-20′), 1.20-1.39 (46H, m, H-7-H-13, H-4′-H-19′),
1.64 (2H, br sep, J ≈ 7.5 Hz, H-3′), 2.05 (2H, br q, J ) 7.5 Hz
H-6), 2.22 (2H, t, J ) 7.5 Hz, H-2′), 2.84 (1H, br s, OH), 2.85
(1H, br s, OH), 3.70 (1H, m, H-1), 3.90 (1H, ddd, J ) 11.3, 4.0,
3.5 Hz, H-1), 3.94 (1H, d br q, J ) 7.2, 3.5 Hz, H-2), 4.31 (1H,
br dd, J ≈ 6.3, 3.5 Hz, H-3), 5.52 (1H, ddt, J ) 15.5, 6.3, 1.2
Hz, H-4), 5.79 (1H, dtd, J ) 15.5, 6.3, 1.2 Hz, H-5), 6.26 (1H,
br d, J ) 7.2 Hz, NH); 13C NMR (CDCl3, 125 MHz) δ 14.8 (C-
14, C-20′), 23.40 (C-13, C-19′), 26.5 (C-3′), 29.8, 29.9, 30.0, 30.1,
30.2, 30.3, 30.4 (C-7-C-11, C-4′-C-17′), 32.6 (C-16, C-18′), 33.0
(C-6), 37.6 (C-2′), 55.2 (C-2), 63.2 (C-1), 75.3 (C-3), 129.5 (C-
4), 135.0 (C-5), 174.7 (C-1′); EIMS m/z 537 [M]+ (0.6), 519 (10),
489 (12), 388 (5), 354 (18), 337 (100), 306 (22), 280 (10), 195
(15), 60 (66); FABCIDMS m/z 560 (100), 539 (1.9), 530 (0.9),
516 (0.7), 502 (0.7), 488 (1.0), 474 (0.9), 460 (1.1), 446 (0.8),
432 (1.0), 418 (0.8), 404 (0.7), 390 (0.8), 376 (1.0), 360 (1.2),
334 (1.0), 320 (2.0), 208 (0.6); HRFABMS m/z 538.5195 (calcd
for C34H68NO3, 558.5121).
Ack n ow led gm en t. The authors would like to thank Dr.
E. J . Bang and Dr. J . J . Seo at Korea Basic Science Institute
for measuring the NMR and MS spectra.
Su p p or tin g In for m a tion Ava ila ble: This material is available
Refer en ces a n d Notes
(1) Shanghai Science and Technologic Publisher and Shougakukan. The
Dictionary of Chinese Drugs; Shougakukan: Tokyo, 1995; pp 2238-
2240.
(2) Pemberton, R. W. J . Ethnopharmacol. 1999, 65, 207-216.
(3) Cheng, K. P.; Nagano, H.; Bang, L.; Ourisson, G.; Beck, J . P. J . Chem.
Res. (S) 1977, 217.
(4E,6E,2S,3R)-2-N-Docosa n oyl-4,6-t et r a d eca sp h in ga -
d ien in e (4): white powder; mp 71.8 °C; [R]20 -3.6° (c 0.176,
(4) Kwon, H. C.; Moon, H. I.; Choi, S. H.; Lee, J . O.; Cho, S. Y.; J ung, I.
Y.; Kim, S. Y.; Lee, K. R. Yakhak Hoeji 1999, 43, 169-172.
(5) Kwon, H. C.; Bang, E. J .; Choi, S. U.; Lee, W. C.; Cho, S. Y.; J ung, I.
Y.; Kim, S. Y.; Lee, K. R. Yakhak Hoeji 2000, 44, 115-118.
(6) Connor, B.; Dragunow, M. Brain. Res. Rev. 1998, 27, 1-39.
(7) Yuen, E. C.; Mobby, W. C. Annu. Neurol. 1996, 40, 346-354.
(8) Greene, L. A.; Aletta, J . M.; Rukenstein, A.; Green, S. H. Methods
Enzymol. 1987, 147, 207-216.
D
neat
CHCl3); UV (EtOH) λmax (log ꢀ) 233 (4.10) nm; IR νmax
3340,
2930, 2850, 1648, 1623, 1535, 1470, 1050 cm-1
;
1H NMR
(CDCl3, 500 MHz) δ 0.88 (6H, t, J ) 7.0 Hz, H-14 and H-22′),
1.20-1.39 (46H, m, H-9-H-13, H-4′-H-21′), 1.63 (2H, br sep,
J ≈ 7.5 Hz, H-3′), 2.06 (2H, m, H-8), 2.22 (2H, t, J ) 7.5 Hz,
H-2′), 2.85 (1H, br s, OH), 2.99 (1H, br s, OH), 3.70 (1H, br d,
J ) 10.5 Hz, H-1), 3.89-3.96 (2H, m, H-1 and H-2), 4.38 (1H,
(9) Zhang, H.; Buckley, N. E.; Gibson, K.; Spiegel, S. J . Biol. Chem. 1990
265, 76-81.