Synthesis and growth inhibitory activity of chiral 5-hydroxy-2-N-acyl-(3E)-sphingenines: Ceramides with an unusual sphingoid backbone

Article abstract of DOI:10.1021/jo026242u

The unusual sphingoid base 5-hydroxy-3-sphingenine was identified in the hydrolysate of brain sphingolipids more than 40 years ago. We present here the first synthesis of the 5R and 5S diastereoisomers of the N-acyl derivatives of 5-hydroxy-3-sphingenine, 2 and 3, respectively, which represent regioisomers of (2S,3R)-ceramide (1). The key steps include the synthesis of a,β-unsaturated ketone intermediates 4 and 5 from N-Cbz- and N-Boc -L-serine and diastereoselective reduction of the enones. The configuration at the new carbinol center was deduced by proton NMR analysis of (R)- and (S)-Mosher [methoxy(trifluoromethyl)phenylacetate] ester derivatives. Ceramide analogues 2 and 3 showed a markedly higher antiproliferative activity than 1 on MCF-7 cells.

Full text of DOI:10.1021/jo026242u

Syn th esis a n d Gr ow th In h ibitor y Activity of Ch ir a l
5-Hyd r oxy-2-N-Acyl-(3E)-Sp h in gen in es: Cer a m id es w ith a n
Un u su a l Sp h in goid Ba ck bon e
J iong Chun,† Hoe-Sup Byun,† Gilbert Arthur,‡ and Robert Bittman*,†
Department of Chemistry and Biochemistry, Queens College of The City University of New York,
Flushing, New York 11367-1597, and Department of Biochemistry and Medical Genetics, University of
Manitoba, Winnipeg, Manitoba, Canada R3E 0W3
robert_bittman@qc.edu
Received J uly 25, 2002
The unusual sphingoid base 5-hydroxy-3-sphingenine was identified in the hydrolysate of brain
sphingolipids more than 40 years ago. We present here the first synthesis of the 5R and 5S
diastereoisomers of the N-acyl derivatives of 5-hydroxy-3-sphingenine, 2 and 3, respectively, which
represent regioisomers of (2S,3R)-ceramide (1). The key steps include the synthesis of R,â-
unsaturated ketone intermediates 4 and 5 from N-Cbz- and N-Boc-L-serine and diastereoselective
reduction of the enones. The configuration at the new carbinol center was deduced by proton NMR
analysis of (R)- and (S)-Mosher [methoxy(trifluoromethyl)phenylacetate] ester derivatives. Ceramide
analogues 2 and 3 showed a markedly higher antiproliferative activity than 1 on MCF-7 cells.
CHART 1
In tr od u ction
Sphingolipids are widely distributed in mammalian
membranes, where they play a structural role and also
participate in a plethora of cellular events. They all have,
by definition, a “sphingoid base” backbone, the most
common of which is (2S,3R,4E)-2-aminooctadec-4-ene-1,3-
diol (C18-sphingosine).1 A variety of sphingosines exist
that differ with respect to the lipid chain length and
location of unsaturation, as well as the number of
hydroxy groups. The sphingoid base 5-hydroxy-(3E)-
sphingenine occurs naturally. It was isolated by TLC2 and
HPLC3 from the acid hydrolysate of a human brain
sphingolipid mixture; however, the configuration at C-5
was not established. As part of our interest in analyzing
structure-function relationships of ceramides (N-acyl-
sphingosines) that differ with respect to the location of
the double bond and hydroxy groups in the sphingoid
base,4 we report here the synthesis of ceramides 2 and 3
(see Chart 1). These diastereomers represent regioiso-
mers of (2S,3R)-ceramide (1),5 which occupies the “hub”
of sphingolipid metabolism and serves as a coordinator
of eukaryotic stress responses and other biological activi-
ties.6 In view of the capacity of 1 to regulate various
biological functions, the availability of some of its ana-
logues, such as 2 and 3, would contribute to our under-
standing of the complex structural biology of ceramide.
We report here that 2 and 3 are significantly more
effective than 1 in inhibiting the growth of a breast tumor
cell line, although the mechanism by which they exert
their antiproliferative action is unclear.
* To whom correspondence should be addressed. Phone: (718) 997-
3279. Fax: (718) 997-3349.
† Queens College of The City University of New York.
‡ University of Manitoba.
(1) Smith, W. L.; Merrill, A. H., J r. J . Biol. Chem. 2002, 277, 25841-
25842.
Resu lts a n d Discu ssion
(2) (a) Michalec, C. J . Chromatogr. 1969, 41, 267-269. (b) Michalec,
C.; Ledvinova-Reinisova, J .; Kolman, Z. J . Chromatogr. 1980, 190,
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H. J . Lipid Res. 1984, 25, 209-216. (b) Kadowaki, H.; Bremer, E.; G.;
Evans, J . E.; J ungalwala, F. B.; McCluer, R. H. J . Lipid Res. 1983,
24, 1389-1397.
(4) (a) He, L.; Byun, H.-S.; Smit, J .; Wilschut, J .; Bittman, R. J . Am.
Chem. Soc. 1999, 121, 3897-3903. (b) He, L.; Byun, H.-S.; Bittman,
R. J . Org. Chem. 2000, 65, 7618-7626. (c) He, L.; Byun, H.-S.; Bittman,
R. J . Org. Chem. 2000, 65, 7627-7633. (d) Chun, J .; He, L.; Byun,
H.-S.; Bittman, R. J . Org. Chem. 2000, 65, 7634-7640. (e) Chun, J .;
Li, G.; Byun, H.-S.; Bittman, R. J . Org. Chem. 2002, 67, 2600-2605.
(f) Chun, J .; Byun, H.-S.; Bittman, R. J . Org. Chem. 2002, 67, 348-
354.
Retr osyn th etic An a lysis. Scheme 1 illustrates our
strategy for the preparation of ceramides 2 and 3. The
stereochemistry of the C-5-hydroxy group is generated
by diastereoselective reduction of the key enone inter-
mediates 4 and 5. The stereochemistry of the 3E-double
bond results from the Horner-Wadsworth-Emmons
(HWE) reaction of L-serine-derived aldehydes 6 and 7
(5) Note that the priority sequence at C-2 is reversed in compounds
2 and 3 compared with that in compound 1.
(6) Hannun, Y. A.; Obeid, L. M. J . Biol. Chem. 2002, 277, 25847-
25850.
10.1021/jo026242u CCC: $25.00 © 2003 American Chemical Society
Published on Web 12/06/2002
J . Org. Chem. 2003, 68, 355-359
355

Chun et al.
TABLE 1. Dia ster eoselective Red u ction of En on es 4 a n d 5
reduction of enone 4
reduction of enone 5
reducing agent
reduction conditions
ratio of 15 to 17
overall yield (%)
ratio of 16 to 18
overall yield (%)
NaBH4, CeCl3
DIBAL-H
LiAlH4, Chirald
BH3‚Me2S, oxazaborolidine
L-Selectride
MeOH, 0 °C, 3 h
THF, 0 °C, 1 h
Et2O, 78 °C, 6 h
THF, rt, 1 h
1.0:1.0
1.0:1.0
2.0:1.0
1.4:1.0
8.0:1.0
88
90
86
85
91
1.0:1.8
1.0:1.1
5.2:1.0
2.3:1.0
18.0:1.0
90
89
87
84
90
THF, 0 °C-to rt, 1 h
SCHEME 1. Retr osyn th etic P la n
SCHEME 3. Dep r otection a n d N-Acyla tion a
SCHEME 2. Dia ster eoselective Red u ction of
En on es 4 a n d 5a
a
Reagents and conditions: (a) p-TsOH‚H2O, MeOH, rt; (b) (i)
Li, NH3, -78 °C, (ii) p-O2NC6H4CO2C7H15-n, THF, rt; (c) (i) 1 M
HCl, dioxane, 100 °C, (ii) p-O2NC6H4CO2C7H15-n, THF, rt.
higher selectivity with a variety of reducing agents than
that of N-Cbz-protected enone 4. The tert-butyl group
appears to be more effective than the benzyl group with
respect to blocking one face of the carbonyl group with
all of the reducing agents shown in Table 1 except
DIBAL-H. We found that reduction of 4 and 5 at 0 °C
with NaBH4 in the presence of CeCl3 in methanol gave
alcohols 15 and 16 in low stereoselectivity. Reduction
with LiAlH4 in the presence of a Chirald (a chiral ligand)9
provided diastereomers 15 and 16 as the major products
in ∼2:1 and 5.2:1 ratios of isomers, respectively. Similar
results were obtained with oxazaborolidine-catalyzed
reduction.10 Fortunately, high stereoselectivity was
achieved by using the bulky L-Selectride in THF (1 h,
0 °C to rt), and high ratios of 15 to 17 (8:1) and 16 to
18 (18:1) were obtained. The two diastereoisomers were
readily separated by column chromatography. Thus,
L-Selectride is the reducing agent of choice for the
synthesis of ceramide 2 (via 16), whereas NaBH4/CeCl3
is preferred for the preparation of ceramide 3 (via 18).
a
Reagents and conditions: (a) Cs2CO3, 2-PrOH, rt; (b) see
Table 1.
with ketophosphonate 14. The configuration at C-2 is
derived from L-serine as the chiral precursor.
Syn th esis of Ald eh yd es 6 a n d 7. Aldehyde 6 was
prepared by oxazolidine formation of N-Cbz-L-serine 8
(2,2-dimethoxypropane, catalytic p-TsOH, benzene),7 fol-
lowed by reduction of acetonide 10 with DIBAL-H in
toluene at -78 °C. Similarly, (S)-Garner aldehyde (7) was
prepared from N-Boc-L-serine 9 via acetonide 11.8
In sta lla tion of th e Lip id Ch a in a n d Dia ster eo-
selective Red u ction of En on es 4 a n d 5 (Sch em e 2).
Treatment of dimethyl methanephosphonate (12) with
n-BuLi in THF at -78 °C and reaction with methyl tetra-
decanoate (13) provided â-ketophosphonate 14 in 95%
yield. As shown in Scheme 2, HWE reaction of 14 with
L-(N-Cbz)- and L-(N-Boc)serinals 6 and 7 gave enones
4 and 5, respectively, in high yield. Several reducing
agents were screened for the attempted diastereoselective
reduction of the ketone. Table 1 shows that reduction of
N-Boc-protected enone 5 generally showed modestly
Dep r otection a n d N-Acyla tion (Sch em e 3). Acid
hydrolysis of 15 (p-TsOH, MeOH) provided 19 in 95%
yield; similarly, 20 was obtained from 17 in 92% yield.
After the corresponding regioisomeric sphingosine ana-
logues were obtained by removal of the Cbz group with
lithium in liquid NH3, N-acylation with p-nitrophenyl
octanoate gave the diastereoisomeric ceramide analogues
2 and 3 in 82% and 80% yield, respectively. Ceramides 2
and 3 were also obtained in good yield from 16 and 18,
respectively, by removal of the N-Boc and isopropylidene
(9) (a) Marshall, J . A.; Gung, W. Y. Tetrahedron Lett. 1989, 30,
2183-2186. (b) Cheon, S. H.; Christ, W. J .; Hawkins, L. D.; J in, H.;
Kishi, Y.; Taniguchi, M. Tetrahedron Lett. 1986, 27, 4759-4762.
(10) (a) Wallbaum, S.; Martens, J . Tetrahedron: Asymmetry 1992,
3, 1475-1504. (b) Corey, E. J .; Helal, C. J . Angew. Chem., Int. Ed.
1998, 37, 1986-2012.
(7) (a) Marshall, J . A.; Beaudoin, S. J . Org. Chem. 1996, 61, 581-
586. (b) Beaulieu, P. L.; Schiller, P. W. Tetrahedron Lett. 1988, 29,
2019-2022.
(8) Garner, P.; Park, J . M. Org. Synth. 1991, 70, 18-27.
356 J . Org. Chem., Vol. 68, No. 2, 2003

Asymmetric Synthesis of 5-Hydroxy-(3E)-sphingenines
SCHEME 4. Con figu r a tion a l Assign m en t by 1H
NMR An a lysis of th e Dia ster eom er ic (S)- a n d
(R)-MTP A Ester sa
F IGURE 1. Effects of ceramides 1-3 on the proliferation of
MCF-7 cells. Cells were grown in medium containing 5%
serum and 1-3 (0-20 µM) for 48 h. The cell numbers were
determined as described in the Experimental Section. Key: (b)
1; (9) 2; (2) 3.
effects of 1-3 on MCF-7 cell growth. Surprisingly, we
found that ceramides 2 and 3 possessed significantly
higher antiproliferative activity than 1, which is known
to induce apoptosis in many cells.13 The IC50 value (the
drug concentration required to inhibit growth by 50%)
for 2 and 3 was ∼15 µM, indicating that the configuration
at C-5 did not affect the activity, whereas the IC50 value
of 1 was .20 µM. Thus, MCF-7 cells are significantly
more resistant to C8-ceramides with the prevalent
3-hydroxy-(4E)-sphingenine backbone than with a 5-
hydroxy-(3E)-sphingoid backbone. No information is
available concerning the metabolism and intracellular
localization of lipids containing the unusual 5-hydroxy-
(3E)-sphingenine core. Further studies are planned to
clarify the mechanisms by which ceramides with an
altered alkenyl sphingoid chain such as 2 and 3 exert
their antiproliferative action.
a
Reagents and conditions: (a) (S)-(+)- or (R)-(-)-MTPA chlo-
ride, CH2Cl2, DMAP, rt.
protecting groups (1 M HCl, dioxane, 100 °C) and
N-acylation with p-nitrophenyl octanoate.
Con figu r a tion a l Assign m en t (Sch em e 4). The as-
signment of the configuration at C-5 of 15 and 17 was
made by 1H NMR analysis11 of the corresponding (S)- and
(R)-Mosher esters, which were prepared by the reaction
of (S)-(+)-R-methoxy-R-(trifluoromethyl)phenylacetic acid
(MTPA) chloride or (R)-MTPA chloride with 15 and 17
in the presence of DMAP. The difference between the
chemical shifts of the vinyl proton Ha in the (R)- and (S)-
MTPA ester derivatives was used to determine the
absolute configuration at C-5.11,12 The upfield signal of
Ha in the (S)-MTPA ester 22 (δ 5.51 ppm) compared to
that in the (R)-MTPA ester 21 (δ 5.56 ppm) indicates that
15 has the R configuration (∆δHa ) δS - δR ) -0.05 ppm).
Similarly, the S configuration was assigned to 17 by the
downfield shift of Ha in 24 (δ 5.53 ppm) compared with
25 (δ 5.43 ppm) (∆δHa ) δS - δR ) +0.10 ppm).
Biologica l Eva lu a tion of Com p ou n d s 2 a n d 3
(F igu r e 1). Synthetic ceramides with a short N-acyl
chain (such as octyl) have been widely used for in vitro
studies because they tend to be more cell permeable than
the long-chain endogenous ceramides. To assess whether
C8-ceramides having a 5-hydroxy-(3E)-sphingenine back-
bone show antiproliferative activity against epithelial
tumor cells, we treated exponentially growing MCF-7
cells with varying concentrations of compounds 2 and 3
(0-20 µM) for 48 h. Figure 1 shows a comparison of the
Con clu sion
In summary, the first synthesis of 5R and 5S diastereo-
isomeric ceramides 2 and 3, which have the naturally
occurring but unusual 5-hydroxy-(3E)-sphingenine long-
chain base and an N-octanoyl residue, was achieved in
several steps from serinal derivatives 6 and 7. A higher
degree of diastereoselectivity was achieved in the L-
Selectride reduction of N-Boc-enone 5 than in that of
N-Cbz-enone 4. The in vitro antiproliferative activity of
3-alkenylceramides 2 and 3 in the breast tumor cell line
MCF-7 was much higher than that of D-erythro-N-C8-
ceramide 1.
Exp er im en ta l Section 14
(11) (a) Dale, A. J .; Mosher, S. H. J . Am. Chem. Soc. 1973, 95, 512-
519. (b) Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa, H. J . Am.
Chem. Soc. 1991, 113, 4092-4096. (c) Rieser, M. J .; Hui, Y.; Rupprecht,
J . K.; Kozlowski, J . F.; Wood, K. V.; McLaughlin, J . L.; Hanson, P. R.;
Zhuang, Z.; Hoye, T. R. J . Am. Chem. Soc. 1992, 114, 10203-10213.
(d) Seepersaud, M.; Blumenstein, M.; Mootoo, D. R. Tetrahedron 1997,
53, 5711-5724.
(12) Ha is affected by the anisotropic magnetic field around the
phenyl ring (see ref 11). The chemical shifts reported here for Ha are
for the central line of the double doublet.
Gen er a l In for m a t ion . Chirald (Darvon alcohol), L-
Selectride, L-(N-Cbz)serine methyl ester (8), L-(N-Boc)serine
methyl ester (9), and (R)-(-)- and (S)-(+)-R-methoxy-R-(tri-
(13) For a recent review of apoptosis induced by 1, see: Andrieu-
Abadie, N.; Gouaze, V.; Salvayre, R.; Levade, T. Free Radical Biol. Med.
2001, 31, 717-728.
(14) General experimental methods have been described; see, for
example, ref 4.
J . Org. Chem, Vol. 68, No. 2, 2003 357

Chun et al.
fluoromethyl)phenylacetic acid (MTPA) chloride were used
directly as obtained commercially. NMR spectra (400 MHz for
1H, 100 MHz for 13C) were recorded in CDCl3 unless otherwise
noted. IR spectra were recorded in chloroform. The preparation
of starting materials N-Cbz-L-serine oxazolidine 10, N-Cbz-L-
serinaldehyde 6, N-Boc-L-serine oxazolidine 11, and (S)-Garner
aldehyde (7) is described in the Supporting Information.
Dim eth yl 2-Oxop en ta d eca n ep h osp h on a te (14). To a
solution of 1.5 g (12.0 mmol) of dimethyl methanephosphonate
(12) in 30 mL of dry THF was added 4.8 mL (12.0 mmol) of
n-BuLi (a 2.5 M solution in hexanes) at -78 °C under N2. After
the mixture was stirred for 30 min at -78 °C, a solution of
2.4 g (10.0 mmol) of methyl tetradecanoate (13) in 10 mL of
THF was added dropwise with stirring. The mixture was kept
at -78 °C for 1 h and then allowed to warm to 0 °C for 1 h.
The reaction was quenched with saturated aqueous NH4Cl
solution, extracted with CHCl3 (3 × 30 mL), dried (MgSO4),
and concentrated under reduced pressure. The residue was
recrystallized (EtOAc/hexane) to give 2.8 g (92%) of ketophos-
phonate 14 as a white solid: mp 40.2-41.5 °C; 1H NMR δ 0.88
(t, 3H, J ) 6.6 Hz), 1.25 (m, 20H), 1.57 (t, 2H, J ) 7.0 Hz),
2.61 (t, 2H, J ) 7.3 Hz), 3.10 (d, 2H, J ) 22.7 Hz), 3.76 (d, 2H,
J ) 10.8 Hz); 13C NMR δ 14.1, 22.7, 23.4, 29.0, 29.4, 29.6, 29.7,
31.9, 40.6 (J ) 128.3 Hz), 44.2, 53.0, 202.1; HR-MS [DEI, M+]
m/z calcd for C17H35PO4 334.2273, found 334.2271.
N-Ben zyloxyca r bon yl-4(R)-[3′-oxo-(1′E)-h exa d ecen yl]-
2,2-d im eth yl-1,3-oxa zolid in e [(-)-4]. To a suspension of
ketophosphonate 14 (3.70 g, 11.1 mmol) and Cs2CO3 (3.58 g,
11.0 mmol) in 40 mL of 2-propanol was added a solution of
aldehyde 6 (2.63 g, 10.0 mmol) in 10 mL of 2-propanol at 0
°C. After being stirred at rt overnight, the mixture was diluted
with 200 mL of EtOAc and washed with water and brine. The
organic layer was dried (Na2SO4) and concentrated. The
residue was purified by chromatography (hexane/EtOAc, 4:1)
to give 4.30 g (91%) of enone 4 as a colorless oil: [R]25D -35.6°
(c 5.0, CHCl3); IR 1702, 1631, 1467, 1408, 1349, 1256, 1094
cm-1; 1H NMR δ 0.88 (t, 3H, J ) 6.6 Hz), 1.26 (s, 20H), 1.49-
1.60 (m, 2H), 1.57 (s, 3H) 1.67 (s, 3H), 2.54 and 2.40 (two sets
of t, 2H, J ) 7.2 Hz), 3.83 (dd, 1H, J ) 9.2, 2.2 Hz), 4.12 (dd,
1H, J ) 9.2, 6.4 Hz), 4.45-4.55 and 4.55-4.65 (two sets of m,
1H), 5.00-5.16 (m, 2H), 6.06 (d, 0.68H, J ) 15.7 Hz), 6.24 (d,
0.32H, J ) 15.7 Hz), 6.68 and 6.64 (two sets of dd, 1H, J )
15.7, 7.0 Hz), 7.26-7.36 (m, 5H); 13C NMR δ 14.1, 22.6 (23.9),15
24.4 (24.7), 26.3 (27.3), 28.9, 29.1, 29.2, 29.3, 29.36, 29.4, 29.56,
29.58 (29.60), 31.9 (33.8), 58.0 (58.6), 66.8, (67.4) 67.7, 76.69
(77.00), 77.3, (94.3) 94.9, 128.0, 128.4, 130.5, 136.1, (142.3)
142.7, 152.1 (152.5), 200.0; HR-MS [DCI/NH3, MH+] m/z calcd
for C29H46NO4 472.3426, found 472.3421.
2.50 mmol) in 10 mL of MeOH at 0 °C. The mixture was stirred
for 3 h at 0 °C, then diluted with 100 mL of EtOAc, and filtered
through a pad of silica gel, which was rinsed with 100 mL of
EtOAc. The filtrate was concentrated under reduced pressure.
The residue was purified by chromatography (hexane/EtOAc,
3:1) to give diastereoisomers 15 (0.53 g, 45%) and 17 (0.51 g,
43%) as colorless oils. Data for 15: [R]25D -9.8° (c 1.3, CHCl3);
1
IR 1698, 1467, 1410, 1350, 1253, 1095 cm-1; H NMR δ 0.88
(t, 3H, J ) 6.6 Hz); 1.25 (s, 22H), 1.40-1.60 (m, 2H), 1.55 (s,
3H), 1.65 (s, 3H), 1.84 (br s, 2H), 3.75 (dd, 1H, J ) 2.2, 8.9
Hz), 3.99 (br s, 1H), 4.05 (dd, 1H, J ) 6.1, 8.9 Hz), 4.30-4.40
and 4.40-4.50 (two sets of m, 1H), 4.97-5.19 (m, 2H), 5.30-
5.70 (m, 2H), 7.29-7.39 (m, 5H); 13C NMR δ 14.1, 22.7 (23.6),
24.9 (25.4), 26.4, 27.27, (29.33) 29.5, 29.56, 29.59, 29.63, 29.65,
29.66 (30.9), 31.9, 37.0 (37.1), 58.5, (66.5) 66.9, 68.3 (68.6),
(94.0) 94.4, 128.0, 128.1, (128.3) 128.5, 129.1, 135.3 (135.4),
136.4, 136.6, 152.4; HR-MS [DCI/NH3, MH+] m/z calcd for
C
29H48NO4 474.3583, found 474.3561. Data for 17: [R]25
D
-18.9° (c 2.0, CHCl3); IR 1698, 1466, 1410, 1350, 1254, 968
cm-1; 1H NMR δ 0.88 (t, 3H, J ) 6.6 Hz), 1.25 (s, 22H) 1.43-
1.50 (m, 2H), 1.55 (s, 3H), 1.65 (s, 3H), 1.73 (br s, 1H), 3.77
(dd, 1H, J ) 2.1, 8.9 Hz), 4.01 (br s, 1H), 4.06 (dd, 1H, J )
6.0, 8.9 Hz), 4.34-4.43 and 4.45-4.51 (two sets of m, 1H),
4.95-5.35 (m, 2H), 5.50-5.90 (m, 2H), 7.28-7.40 (m, 5H); 13
C
NMR δ 14.1, 22.7 (23.6), 25.3 (26.4), 29.5, 29.58, 29.60, 29.63,
29.65, 29.7, 30.9, 31.9, 37.1 (37.2), 58.5 (59.1), 66.5 (66.9), (68.3)
68.6, 72.0, 94.4 (94.5), 127.95 (128.02), 128.4 (128.5), 129.0,
135.5 (135.7), (136.4) 136.5, 153.4; HR-MS [DCI/NH3, MH+]
m/z calcd for C29H48NO4 474.3583, found 474.3583.
N-ter t-Bu toxyca r bon yl-4(R)-[3′-h yd r oxy-(1′E)-h exa d e-
cen yl]-2,2-d im eth yl-1,3-oxa zolid in e (16, 18). Compounds
16 and 18 was prepared in 90% overall yield by the same
procedure as described for 15 and 17 (NaBH4 reduction).
Purification by chromatography (hexane/EtOAc, 2:1) afforded
diastereoisomers 16 (32%) and 18 (58%) as colorless oils. Data
for 16: [R]25D -22.5° (c 1.0, CHCl3); IR 1690, 1601, 1392, 1171,
1
1099 cm-1; H NMR δ (C6D6, 70 °C) 0.88 (t, 3H, J ) 6.1 Hz),
1.20-1.80 (m, 39H), 3.54 (dd, 1H, J ) 8.7, 2.4 Hz), 3.75 (dd,
1H, J ) 8.7, 6.2 Hz), 3.98 (d, 1H, J ) 5.2 Hz), 4.19 (br s, 1H),
5.62 (m, 2H); 13C NMR δ (C6D6) 14.3, 23.1, 23.8, 25.2, 26.0,
27.0, 27.5, 28.5, 29.8, 30.1, 32.3, 37.8, 38.0, 59.0 (59.3), 68.4,
72.0 (72.2), 79.2 (79.8), (93.6) 94.2, (129.0) 129.3, 135.7 (136.7),
152.0; HR-MS [FAB, MNa+] m/z calcd for C26H49NO4Na
462.3559, found 462.3579. Data for 18: [R]25 -40.0° (c 1.0,
D
CHCl3); IR 1689, 1602, 1392, 1253, 1171 cm-1; 1H NMR (C6D6,
70 °C) δ 0.88 (t, 3H, J ) 6.6 Hz), 1.20-1.80 (m, 39H), 3.54
(dd, 1H, J ) 6.3, 2.3 Hz), 3.75 (dd, 1H, J ) 8.7, 6.1 Hz), 4.00
(dd, 1H, J ) 11.3, 5.8 Hz), 4.19 (br s, 1H), 5.62 (m, 2H); 13C
NMR (C6D6) δ 14.3, 23.1, 23.8, 25.0, 26.0, 27.1, 27.7, 28.5, 29.8,
30.1, 30.2, 32.3, 37.7, 38.0, 59.0, (68.2) 68.5, 72.0 (72.2), 79.1,
79.1 (79.7), (93.6) 94.2, (128.6) 129.6, 135.8 (136.2), 152.0; HR-
MS [FAB, MNa+] m/z calcd for C26H49NO4Na 462.3559, found
462.3566.
N-ter t-Bu toxycar bon yl-4(R)-[3′-oxo-(1′E)-h exadecen yl]-
2,2-d im eth yl-1,3-oxa zolid in e [(-)-5]. This compound was
prepared from aldehyde 7 in 90% yield by the same procedure
as described for 4: mp 38.0-39.0 °C; [R]25 -43.7° (c 1.0,
D
CHCl3); IR 1693, 1633, 1456, 1391, 1255, 1172, 1098 cm-1; 1H
NMR (C6D6, 70 °C)16 δ 0.88 (t, 3H, J ) 6.7 Hz), 1.0-1.3 (m,
20H), 1.37 (s, 9H), 1.48 (s, 3H), 1.67 (m, 5H), 2.29 (t, 2H, J )
7.2 Hz), 3.43 (dd, 1H, J ) 9.0, 2.7 Hz), 3.67 (dd, 1H, J ) 9.0,
6.4 Hz), 4.15 (br s, 1H), 6.13 (d, 1H, J ) 15.8 Hz), 6.60 (dd,
1H, J ) 15.8, 7.0 Hz); 13C NMR (C6D6) δ 14.3, 23.1, 23.7, 24.4,
25.8, 26.8, 27.6, 28.4, 29.6, 29.8, 29.9, 30.1, 32.3, 40.5, 58.5,
(67.2) 67.5, 79.6 (79.9), (93.8) 94.6, 130.7, (142.7) 143.4, 151.7,
198.6; HR-MS [DCI/NH3, MH+] m/z calcd for C26H48NO4
438.3583, found 438.3588.
L-Selectr id e Red u ction of En on e 5. To a solution of
enone 5 (101 mg, 0.23 mmol) in 10 mL of dry THF was added
0.46 mL (0.46 mmol) of L-Selectride (lithium tri-sec-butylboro-
hydride; a 1.0 M solution in THF) dropwise at 0 °C. The
reaction mixture was stirred for 0.5 h at 0 °C and then allowed
to warm to rt for another 0.5 h. The mixture was then diluted
with 100 mL of EtOAc and filtered through a pad of silica gel,
which was rinsed with 100 mL of EtOAc. The filtrate was
concentrated under reduced pressure. The residue was purified
by chromatography (hexane/EtOAc, 3:1) to give diastereoiso-
mers 16 (86 mg, 85%) and 18 (4.8 mg, 4.7%) as colorless oils
(the ratio of 16 to 18 was 18:1).
N-Ben zyloxyca r b on yl-4(R)-[3′-h yd r oxy-(1′E)-h exa d e-
cen yl]-2,2-d im eth yl-1,3-oxa zolid in e (15, 17). To a solution
of CeCl3 (0.81 g, 3.29 mmol) and NaBH4 (0.12 g, 3.71 mmol)
in 30 mL of MeOH was added a solution of enone 4 (1.18 g,
(2R,5R)-2-[(Ben zyloxycar bon yl)am in o]-(3E)-octadecen e-
1,5-d iol [(+)-19]. A solution of oxazolidine 15 (0.45 g, 0.95
mmol) and p-TsOH‚H2O (0.18 g, 10 µmol) in 20 mL of MeOH
was stirred overnight at rt. The reaction mixture was concen-
trated under reduced pressure. The residue was purified by
column chromatography (CHCl3/MeOH, 25:1) to give 0.39 g
(15) The 13C NMR chemical shifts in parentheses indicate the small
peaks arising from the minor rotamers in the dynamic equilibrium of
the oxazolidine system, which is slow at ambient temperature.
(16) Proton NMR spectra of N-Boc-containing compounds were
recorded at elevated temperature to facilitate the interconversion of
the rotamers and thus simplify the spectra.
(95%) of 19 as a white solid: mp 92.0-93.0 °C; [R]25 +8.6° (c
D
358 J . Org. Chem., Vol. 68, No. 2, 2003

Asymmetric Synthesis of 5-Hydroxy-(3E)-sphingenines
2.5, CHCl3); IR 3435, 1720, 1601, 1503, 1467, 1232 cm-1; H
NMR δ 0.88 (t, 3H, J ) 6.6 Hz), 1.25 (s, 22H), 1.45-1.50 (m,
2H), 2.85 (br s, 2H), 3.60 (dd, 1H, J ) 11.0, 4.0 Hz), 3.66 (dd,
1H, J ) 11.0, 3.8 Hz), 4.06 (q, 1H, J ) 6.1 Hz), 4.27 (br s, 1H),
5.09 (s, 2H), 5.42 (br s, 1H), 5.59 (dd, 1H, J ) 15.7, 4.5 Hz),
5.66 (dd, 1H, J ) 15.7, 5.8 Hz), 7.27-7.36 (m, 5H); 13C NMR
δ 14.1, 22.7, 25.4, 29.3, 29.5, 29.6, 29.7, 31.9, 37.1, 53.9, 64.9,
66.9, 72.3, 128.1, 128.2, 128.5, 135.2, 136.3, 158.0; HR-MS
[FAB, MNa+] m/z calcd for C26H43NO4Na 456.3090, found
456.3089.
solid: mp 69.0-70.0 °C; [R]25 +4.3° (c 1.0, CHCl3); IR 3437,
1
D
1659, 1602, 1503, 1466, 1219 cm-1; 1H NMR δ 0.88 (t, 6H, J )
6.5 Hz), 1.30 (s, 32H), 1.48-1.52 (m, 2H), 1.62-1.65 (m, 2H),
2.31 (t, 2H, J ) 7.5 Hz), 3.41 (br s, 2H), 3.64 (dd, 1H, J )
11.5, 4.3 Hz), 3.68 (dd, 1H, J ) 11.5, 3.7 Hz), 4.08 (q, 1H, J )
6.0 Hz), 4.30-4.53 (m, 1H), 5.62 (dd, 1H, J ) 15.8, 4.1 Hz),
5.67 (dd, 1H, J ) 15.8, 4.9 Hz), 6.35 (d, 1H, J ) 7.5 Hz); 13C
NMR δ 14.0, 14.1, 22.6, 22.7, 24.9, 25.5, 25.7, 25.8, 29.0, 29.1,
29.25, 29.3, 29.59, 29.61, 29.63, 29.65, 29.67, 31.7, 33.9, 36.8,
37.1, 52.6, 65.0, 72.3, 127.8, 135.4, 173.5; HR-MS [DCI/NH3,
MH+] m/z calcd for C26H52NO3 426.3947, found 426.3938.
(2R,5S)-2-[Octan oylam ido]-(3E)-octadecen e-1,5-diol [(-)-
3]. This compound was prepared from 18 and 20 by the
procedure described above (methods A and B): mp 78.5-79.5
(2R,5S)-2-[(Ben zyloxycar bon yl)am in o]-(3E)-octadecen e-
1,5-d iol [(-)-20]. This compound was prepared from 17 in 92%
yield by the procedure described above: mp 93.5-95.0 °C;
[R]25 -2.5° (c 2.5, CHCl3); IR 3437, 1719, 1602, 1503, 1467,
D
1232 cm-1; 1H NMR (CDCl3/MeOD) δ 0.88 (t, 3H, J ) 6.5 Hz),
1.10-1.70 (m, 24H), 3.59 (d, 2H, J ) 4.0 Hz), 4.02 (m, 1H),
4.20 (m, 1H), 5.09 (br s, 1H), 5.62 (m, 2H); 13C NMR (CDCl3/
MeOD) δ 14.2, 22.9, 25.7, 29.6, 29.8, 29.9, 32.1, 37.1, 54.5, 64.5,
67.0, 72.1, 127.8, 128.1, 128.3, 128.7, 135.7, 136.7, 157.0; HR-
MS [FAB, MNa+] m/z calcd for C26H43NO4Na 456.3090, found
456.3090.
°C; [R]25 -11.0° (c 1.0, CHCl3); IR 3435, 1657, 1503, 1466,
D
1218 cm-1; 1H NMR δ 0.85 (t, 6H, J ) 6.5 Hz), 1.10-1.70 (m,
34H), 2.19 (t, 2H, J ) 7.4 Hz), 3.63 (d, 2H, J ) 4.5 Hz), 4.06
(dd, 1H, J ) 12.2, 5.9 Hz), 4.49 (m, 1H), 5.62 (m, 2H), 6.04 (d,
1H, J ) 7.7 Hz); 13C NMR δ 14.0, 22.6, 22.7, 25.5, 25.7, 29.0,
29.2, 29.3, 29.6, 29.7, 31.7, 31.9, 36.8, 37.1, 40.4, 52.6, 65.1,
72.1, 127.3, 135.7, 173.7; HR-MS [DCI/NH3, MH+] m/z calcd
for C26H52NO3 426.3947, found 426.3955.
(2R,5R)-2-[Oct a n oyla m id o]-(3E)-oct a d ecen e-1,5-d iol
[(+)-2]. Meth od A. To the blue solution prepared by addition
of 0.10 g (14.4 mmol) of Li metal to 20 mL of liquid NH3 was
added a solution of 19 (0.26 g, 0.60 mmol) in 10 mL of dry
THF at -78 °C. After the mixture was stirred for 30 min, the
reaction was quenched by addition of NH4Cl (0.78 g, 14.6
mmol). After removal of NH3 by a stream of N2, the mixture
was diluted with 100 mL of Et2O and washed with brine. The
organic layer was dried over Na2SO4 and concentrated. The
residue was dissolved in 20 mL of THF, and p-nitrophenyl
octanoate (0.16 g, 0.60 mmol) was added. After the mixture
was stirred overnight, it was concentrated, and the product
was purified by column chromatography (CHCl3/MeOH, 9:1)
to give 0.21 g (82%) of 2 as a white solid.
Cell Cu ltu r es. MCF-7 (breast cancer) cells, originally
obtained from the American Type Culture Collection, were
grown to the exponential phase in medium supplemented with
5% fetal bovine serum and antibiotics, as described previ-
ously.17 The cells were treated with compounds 1-3 (0-20 µM)
for 48 h, and the increase in cell numbers after 48 h was
determined17 and expressed as a percentage of the controls,
which received no drug. The results are the means of experi-
ments made with quadruplicate wells. The standard deviations
from the means were <10%. Stock solutions of the ceramides
were made in EtOH. The final concentration of EtOH was
<0.1%.
Meth od B. A solution of 44 mg (0.1 mmol) of 16 in 5 mL of
1 M HCl and 5 mL of dioxane was heated at 100 °C with
stirring for 1 h under N2. The reaction mixture was cooled to
rt and neutralized with 1 M NaOH (5 mL). The product was
extracted with EtOAc (3 × 20 mL), and the combined organic
layers were washed with brine and dried (Na2SO4). Removal
of the solvent provided a crude sphingosine analogue as a
white solid, which was dissolved in 6 mL of dry THF. After 54
mg (0.20 mmol) of p-nitrophenyl octanoate was added at rt,
the reaction mixture was stirred for 24 h and concentrated.
Purification by flash chromatography (CHCl3/MeOH, 9:1)
afforded 34 mg (80%, two steps) of ceramide 2 as a white
Ack n ow led gm en t. This work was supported by
National Institutes of Health Grant HL 16660.
Su p p or tin g In for m a tion Ava ila ble: Preparation of start-
ing materials (6, 7, 10, 11) and 1H and 13C NMR spectra for
compounds 2-5 and 14-20. This material is available free of
charge via the Internet at http://pubs.acs.org.
J O026242U
(17) Samadder, P.; Byun, H.-S.; Bittman, R.; Arthur, G. Anticancer
Res. 1998, 18, 465-470.
J . Org. Chem, Vol. 68, No. 2, 2003 359