Synthesis of a novel ceramide analogue via Tebbe methylenation and evaluation of its antiproliferative activity

Article abstract of DOI:10.1021/ol0503440

(Chemical Equation Presented) A new analogue of (2S,3R)-ceramide (2) with a methylene group at C4 has been synthesized from D-tartaric acid (3) by using Tebbe methylenation as the key step. Compound 2 exhibited markedly higher antiproliferative activity o

Full text of DOI:10.1021/ol0503440

ORGANIC
LETTERS
2005
Vol. 7, No. 8
1645-1648
Synthesis of a Novel Ceramide
Analogue via Tebbe Methylenation and
Evaluation of Its Antiproliferative
Activity
Xuequan Lu,† 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 February 18, 2005
ABSTRACT
A new analogue of (2S,3R)-ceramide (2) with a methylene group at C4 has been synthesized from D-tartaric acid (3) by using Tebbe methylenation
as the key step. Compound 2 exhibited markedly higher antiproliferative activity on mouse embryonic fibroblast (MEF) cells than natural
(2S,3R,4E)-ceramide (1).
Ceramide (N-acylsphingosine, 1) is a long-chain aliphatic
2-amido-1,3-diol with a C(4),C(5)-trans double bond (Figure
1).1 As a key intermediate in the biosynthesis of many
sphingolipid mediators, ceramide has been implicated in
many physiological events, including the regulation of cell
growth and differentiation, inflammation, and in cellular
responses to stress stimuli (such as exposure to heat,
radiation, oxidative conditions, and chemotherapeutic agents).2
Ceramide is a messenger for induction of apoptosis, the cell’s
intrinsic death program.3
functions. In a recent monolayer study of 14 synthetic
analogues of 1, we demonstrated that the (E)-C(4)-C(5)
double bond of 1 regulates its dipole potential, elastic
properties, and packing behavior4 and appears to be crucial
for ceramide’s capacity to modulate various fundamental
biological functions. Analogues that lack this double bond
have reduced apoptotic activity.5 The ability of 1 to induce
apoptosis has been postulated to also require the allylic
alcohol group at C3.6,7
A deficiency in induction of apoptosis may result in
aberrant cell proliferation, and may culminate in tumor
formation.8 An experimental approach in anticancer chemo-
The well-known biological importance of ceramide has
inspired the chemical synthesis of analogues to examine the
structural features responsible for some of its physiological
* To whom correspondence should be addressed. Phone: (718) 997-
3279. Fax: (718) 997-3349.
† City University of New York.
‡ University of Manitoba.
(1) Karlsson, K.-A. Chem. Phys. Lipids 1970, 5, 6-43.
(2) (a) Hannun, Y. A. Science 1996, 274, 1855-1859. (b) Ariga, T.;
Jarvis, W. D.; Yu, R. K. J. Lipid Res. 1998, 39, 1-16. (c) Perry, D. K.;
Hannun, Y. A. Biochim. Biophys. Acta 1998, 1436, 233-243. (d) Hannun,
Y. A.; Luberto, C. Trends Cell Biol. 2000, 10, 73-80.
(3) (a) Pettus, B. J.; Chalfant, C. E.; Hannun, Y. A. Biochim. Biophys.
Acta 2002, 1585, 114-125. (b) Reynolds, C. P.; Maurer, B. J.; Kolesnick,
R. N. Cancer Lett. 2004, 206, 169-180.
Figure 1. Structures of D-erythro-N-octanoylceramide (1) and Its
C4-methylene analogue (2).
10.1021/ol0503440 CCC: $30.25
© 2005 American Chemical Society
Published on Web 03/18/2005

Table 1. Methylenation of Ketone 6
overall yield
(%)
entry
conditions
CH2PPh3, THF, -78 °C to reflux
ratio of 7:8
1
2
Wittig reaction
Tebbe reagent
1.0:0
1.0:1.0
29
84
2 equiv of Tebbe reagent added to ketone 6 at -78 °C,
then warmed to room temperature, 3 h
3
4
Tebbe reagent
Tebbe reagent
2 equiv of Tebbe reagent added to ketone 6 at room temperature, 3 h
ketone 6 added to 2 equiv of Tebbe reagent at room temperature, 3 h
3.0:1.0
9.0:1.0
81
81
therapy is to employ unnatural analogues of 1 that escape
recognition by endogenous ceramide-metabolizing enzymes,
and thus are not converted to sphingolipids that stimulate
cell proliferation,9 or inhibit enzymes involved in ceramide
turnover.10 The resulting high intracellular levels of ceramide
or its analogues are expected to amplify apoptosis.11 Our
interest in cell-permeable (N-octanoyl) analogues of ceramide
that can be added exogenously to cells to stimulate
apoptosis5b,12 prompted us to prepare the novel C4-exo-
methylene ceramide analogue 2, in which both the unsat-
uration at C4 and allylic nature of the C3-hydroxy group
are preserved. We also examined the properties of 2 as an
antiproliferative agent in cells having normal or dysfunctional
apoptosis.13
acid as previously reported,14 reacted with tetradecylmag-
nesium bromide to give a diastereomeric mixture of alcohol
5. After the mixture was oxidized to ketone 6 with the Dess-
Martin reagent,15 various methods were tested to carry out
the methylenation reaction (Table 1). Wittig reaction gave a
low yield of (2R,3R)-alkene 7 (without any accompanying
(2R,3S)-diastereomer), and changing the base (KOBu-t or
NaH) or refluxing the reaction mixture did not improve the
yield (entry 1). When 2 equiv of Tebbe reagent16 was added
slowly to a solution of ketone 6 in THF at -78 °C, and the
reaction mixture was allowed to warm with stirring for 3 h,
the yield was high but epimerization occurred at C3 to give
a mixture of alkenes 7 and 8 in a 1:1 ratio (entry 2). Since
epimerization may have resulted from the slow addition of
the Tebbe reagent to the carbonyl group of compound 6 at
low temperature, we added 2 equiv of Tebbe reagent to
ketone 7 at room temperature. Entry 3 shows that the ratio
of 7 to 8 was increased to 3:1. High diastereoselectivity was
attained when ketone 6 was added to 2 equiv of Tebbe
reagent slowly at room temperature for 30 min, with stirring
at room temperature for an additional 3 h; a 9:1 ratio of
alkenes 7 and 8 was obtained (entry 4). The mixture of 7/8
was treated with 5% H2SO4 to give diols 9 and 10 in 81%
yield for the two steps.17 Then, diol 9 was converted to azido
alcohol 11 in a one-pot reaction.18 This was accomplished
by adding the diol to a mixture of diisopropyl azodicarboxyl-
ate (DIAD) and Ph3P at 0 °C. After 3 h, TMSN3, was added
to accomplish the azide substitution reaction. Hydrolysis of
Scheme 1. Synthesis of C4-Methylene-ceramide Analogue 2
(7) (a) An allylic hydroxy group or ketone is present in many anticancer
agents, as reported in: Radin, N. S. Bioorg. Med. Chem. 2003, 11, 2123-
2142. (b) The allylic hydroxyl group of 1 is postulated to be oxidized in
cells to an R,â-unsaturated ketone, which undergoes Michael addition with
glutathione.
(8) Debatin, K. M.; Krammer, P. H. Oncogene 2004, 23, 2950-2966.
(9) (a) Maceyka, M.; Payne, S. G.; Milstien, S.; Spiegel, S. Biochim.
Biophys. Acta 2002, 1585, 193-201. (b) Bektas, M.; Jolly, P. S.; Mu¨ller,
C.; Eberle, J.; Spiegel, S.; Geilen, C. C. Oncogene 2005, 24, 178-187.
(10) Selzner, M.; Bielawska, A.; Morse, M. A.; Rudiger, H. A.; Sindram,
D.; Hannun, Y. A.; Clavien, P. A. Cancer Res. 2001, 61, 1233-1240.
(11) Bieberich, E.; Hu, B.; Silva, J.; MacKinnon, S.; Yu, R. K.; Fillmore,
H.; Broaddus, W. C.; Ottenbrite, R. M. Cancer Lett. 2002, 181, 55-64.
(12) (a) Bittman, R. Chem. Phys. Lipids 2004, 129, 111-131. (b)
Struckhoff, A. P.; Bittman, R.; Burow, M. E.; Clejan, S.; Elliott, S.;
Hammond, T.; Tang, Y.; Beckman, B. S. J. Pharmacol. Exp. Ther. 2004,
309, 523-532.
Scheme 1 outlines the synthesis of C4-methylene-ceramide
analogue 2. Aldehyde 4, which was prepared from D-tartaric
(4) Brockman, H. L.; Momsen, M. M.; Brown, R. E.; He, L.; Chun, J.;
Byun, H.-S.; Bittman, R. Biophys. J. 2004, 87, 1722-1731.
(5) (a) Obeid, L. M.; Linardic, C. M.; Karolak, L. A.; Hannun, Y. A.
Science 1993, 259, 1769-1771. (b) Karasavvas, N.; Erukulla, R. K.;
Bittman, R.; Lockshin, R.; Zakeri, Z. Eur. J. Biochem. 1996, 236, 729-
737. (c) Ahn, E. H.; Schroeder, J. J. Exp. Biol. Med. (Maywood) 2002,
227, 345-353. (d) Chang, Y.-T.; Choi, J.; Ding, S.; Prieschl, E. E.;
Baumruker, T.; Lee, J.-M.; Chung, S.-K.; Schultz, P. G. J. Am. Chem. Soc.
2002, 124, 1856-1857.
(13) (a) Cain, K. Drug Metab. ReV. 2003, 35, 337-363. (b) Sanchis,
D.; Mayorga, M.; Ballester, M.; Comella, J. X. Cell Death Differ. 2003,
10, 977-986. (c) Hajra, K. M.; Liu, J. R. Apoptosis 2004, 9, 691-704.
(14) Lu, X.; Byun, H.-S.; Bittman, R. J. Org. Chem. 2004, 69, 5433-
5438. NMR data of 4 are identical to the previously reported data.
(15) For a review of the Dess-Martin oxidation, see: Tohma, H.; Kita,
Y. AdV. Synth. Catal. 2004, 346, 111-124.
(16) For a review of Tebbe methylenation, see: Hartley, R. C.;
McKiernan, G. J. J. Chem. Soc., Perkin Trans. 1 2002, 2763-2793.
(6) Radin, N. S. Eksp. Onkol. 2004, 26, 3-10.
1646
Org. Lett., Vol. 7, No. 8, 2005

Figure 2. Antiproliferative activities of 1 and 2.23
the concomitant silyl ether of the primary hydroxy group
and purification by column chromatography provided azido
alcohol 11 in 56% yield. Reduction of the azido group with
PPh3, followed by amide formation in a one-pot reaction,
gave amide 12 in 92% yield. The benzyl group of amide 12
was removed by Birch reduction (Na, liquid NH3, 30 min)
to give product 2 in 88% yield.
Activation of the cellular apoptosis machinery with ex-
ogenous agents may provide a viable therapeutic strategy
for the elimination of tumor cells.19 Since the antiproliferative
effects of exogenous short-chain, cell-permeable ceramides
have been widely reported,20 we investigated the effect of
2 on the proliferation of wild-type mouse embryonic fibro-
blasts (MEFs) and MEFs lacking Apaf-1, a component of
the apoptosome (a large complex that activates the caspase
cascade in the intrinsic mitochondrial apoptosis path-
way).13c,21 As shown in Figure 2, analogue 2 inhibited the
growth of wild-type MEFs with an IC50 of 5.9 µM and
Apaf-1 -/- cells with an IC50 of 11 µM. (The loss of Apaf-1
in the latter cells leads to defects in the execution of cell
death by the intrinsic apoptosis pathway.)13c,21 In contrast,
the IC50 value of 1 was .20 µM for both the wild-type and
Apaf-1 null cells. The higher antiproliferative activity of
ceramide analogue 2 against these cell lines indicates that
variations in the structural features of 1 can lead to
significantly enhanced apoptogenic activity.22 While the
sensitivity of the Apaf-1 -/- cells to 2 was less than that
(22) Results shown in Figure 2 were obtained with a 9:1 mixture of
(2S,3R):(2S,3S)-2 vs (2S,3R)-1. To address the possible contribution of the
C3 diastereomer to the potency of 2, we make note of several recent studies
that suggest a minimal role of the configuration at C3 in the activation of
apoptotic cell death by 1: (1) One of the best characterized direct targets
of ceramide-mediated apoptosis, ceramide-activated protein phosphatase
(CAPP), was activated by (2S,3R)-1 but not by the (2S,3S)-diastereomer of
1.24 (2) Ceramidase, which regulates the endogenous level of 1 and its
subsequent apoptotic responses in cells, catalyzed the hydrolysis of the amide
linkage in the natural (2S,3R)-isomer of 1 but not in the other three
stereoisomers; moreover, only a modest stereoselectivity was found for
inhibition of ceramidase by the three unnatural isomers.25 (3) Ceramide-
activated protein kinase (CAPK, which has been identified as the kinase
suppressor of Ras, KSR, an activator of stress pathways mediated by MAP
kinase cascades) was activated by the four stereoisomers of 1 to an equal
extent.5b In addition, CERT, a cytosolic protein that facilitates intracellular
trafficking of (2S,3R)-1, did not recognize the three unnatural stereoisomers
of ceramide in a cell-free assay system.26 These findings indicate that there
is no marked selectivity with respect to the configuration at C3 of 1 for
interaction with several target proteins and suggest that the presence of the
minor amount of the C3 epimer does not account for the improved
antiproliferative potency of 2.
(23) Proliferating cells in 48 well plates were incubated with compounds
1 and 2 at 37 °C for 48 h in a CO2 incubator. The medium was removed,
and the plates were frozen at -80 °C for 3 days. The cell numbers at day
0 and after 48 h of incubation with the ceramides were determined by the
CyQuant assay (Molecular Probes). Each point is the average of six
independent experiments.
(24) Usta, J.; El Bawab, S.; Roddy, P.; Szulc, Z. M.; Hannun, Y. A.;
Bielawska, A. Biochemistry 2001, 40, 9657-9668.
(25) (a) Chalfant, C. E.; Kishikawa, K.; Bielawska, A.; Hannun, Y. A.
Methods Enzymol. 2000, 312, 420-428. (b) Chalfant, C. E.; Szulc, Z.;
Roddy, P.; Bielawska, A.; Hannun, Y. A. J. Lipid Res. 2004, 45, 496-
506.
(17) Representative Procedure for the Preparation of 9. A solution
of 474 mg (1.0 mmol) of ketone 6 in 25 mL of THF was added dropwise
over 30 min to a solution of Tebbe reagent (a 0.5 M solution in toluene, 2
mmol) in 25 mL of toluene at room temperature. After the solution had
been stirred for 3 h at room temperature, 50 mL of a 1 M aqueous NaOH
solution was added; the mixture was extracted with Et2O (3 × 20 mL), and
the extracts were dried (Na2SO4). The solvent was evaporated, and the
resulting residue was dissolved in 100 mL of MeOH and treated with 5
mL of 5% aqueous H2SO4. After the mixture had stirred overnight at room
temperature, 5.0 g (36 mmol) of K2CO3 was added, and the mixture was
filtered; the filtrate was evaporated, and the residue was purified by column
chromatography on silica gel (hexane/EtOAc 3:1) to give 328 mg (81%
for the two steps) of a 9:1 mixture of 9 and 10 as a colorless oil.
(18) (a) He, L.; Wanunu, M.; Byun, H.-S.; Bittman, R. J. Org. Chem.
1999, 64, 6049-6055. (b) A mechanism for the conversion of a 1,2-diol to
a 2-azido-1-ol with inversion has been proposed; see Scheme 1 of ref 18a
and: Mathieu-Pelta, I.; Evans, S. A., Jr. J. Org. Chem. 1992, 57, 3409-
3413.
(19) (a) Lowe, S. W.; Lin, A. W. Carcinogenesis 2000, 21, 485-495.
(b) Johnstone, R. W.; Ruefli, A. A.; Lowe, S. W. Cell 2002, 108, 153-
164.
(20) Ogretmen, B.; Hannun, Y. A. Nature ReV. Cancer 2004, 4, 604-
616.
(21) (a) Volm, M.; Rittgen, W. Anticancer Res. 2002, 20, 3449-3458.
(b) Gallego, M.-A.; Joseph, B.; Hemstrom, T. H.; Tamiji, S.; Mortier, L.;
Kroemer, G.; Formstecher, P.; Zhivotovsky, B.; Marchetti, P. Oncogene
2004, 23, 6282-6291.
(26) Kumagai, K.; Yasuda, S.; Okemoto, K.; Nishijima, M.; Kobayashi,
S.; Hanada, K. J. Biol. Chem. 2005, 280, 6488-6495.
Org. Lett., Vol. 7, No. 8, 2005
1647

of the wild type, the ability of 2 to inhibit the growth of
these cells suggests that its mechanism of action is not
critically dependent on the formation of the apoptosome to
induce apoptosis.
Acknowledgment. This work was supported in part by
National Institutes of Health Grant HL16660 (R.B.) and the
Canadian Institute of Health Research (G.A.). We thank Dr.
Tak Mak for providing the MEF cell lines.
In summary, (2S,3R)-ceramide analogue 2, which bears
an exomethylene group at C4 and an allylic hydroxyl group
at C3, was synthesized from D-threitol acetal aldehyde 4.
The key step was Tebbe methylenation of ketone 6 with
careful control of the conditions to minimize epimerization
at C3. The utility of 2 for inhibition of growth was
demonstrated by using mouse embryonic fibroblast cells.
Supporting Information Available: Experimental pro-
cedures for the preparation of all new compounds and their
1H and 13C NMR spectra. This material is available free of
charge via the Internet at http://pubs.acs.org.
OL0503440
1648
Org. Lett., Vol. 7, No. 8, 2005