Ceramides: Branched alkyl chains in the sphingolipid siblings of diacylglycerol improve biological potency

Article abstract of DOI:10.1016/j.bmc.2009.01.005

The synthesis of a small number of ceramide analogues containing a combination of linear and highly branched alkyl chains on either the d-sphingosine or the N-acyl core of the molecule is reported. Regardless of location, the presence of the branched chain improves potency relative to the positive control, C2 ceramide; however, the most potent compound (4) has the branched side chain as part of the d-sphingosine core. The induction of apoptosis by 4 in terms of Annexin V binding and DiOC₆ labeling was superior to that achieved with C2 ceramide.

Full text of DOI:10.1016/j.bmc.2009.01.005

Bioorganic & Medicinal Chemistry 17 (2009) 1498–1505
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Ceramides: Branched alkyl chains in the sphingolipid siblings
of diacylglycerol improve biological potency
a
b
a
b
Ji-Hye Kang , Himanshu Garg , Dina M. Sigano , Nicholas Francella ,
b,
a,
*
*
Robert Blumenthal , Victor E. Marquez
Laboratory of Medicinal Chemistry, National Cancer Institute, National Institutes of Health, Bldg 376/104, Frederick, MD 21702, United States
a
b
Nanobiology Program, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bldg 469/152, Frederick, MD 21702, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis of a small number of ceramide analogues containing a combination of linear and highly
Received 14 November 2008
Revised 7 January 2009
Accepted 8 January 2009
Available online 11 January 2009
branched alkyl chains on either the
less of location, the presence of the branched chain improves potency relative to the positive control, C2
ceramide; however, the most potent compound (4) has the branched side chain as part of the -sphingo-
labeling was superior
D-sphingosine or the N-acyl core of the molecule is reported. Regard-
D
sine core. The induction of apoptosis by 4 in terms of Annexin V binding and DiOC
6
to that achieved with C2 ceramide.
Keywords:
Ó 2009 Elsevier Ltd. All rights reserved.
Alkyl branched ceramides
D-Sphingosine
Apoptosis
SupT1 cells
Annexin V
DiOC6
1
. Introduction
prove its biological potency. DAG is a second messenger derived
from the phospholipase-C cleavage of phosphatidyl inositol 4,5-
biphosphate. Once released, DAG exerts its biological activity
by binding to specific membrane targeting domains (C1 do-
The design of ceramide derivatives that mimic the activities of
natural ceramide is an important area of research aimed at devel-
oping novel therapeutics for the treatment of cancer, allergy, and
other diseases originating from cell regulation disorders.1
1
0,11
mains) present in PKC and other proteins.
Ceramide is simi-
–5
larly generated through phospholipase-C-type reaction by
a
For many years our laboratory has been engaged in the design
of novel, more effective diacylglycerol (DAG) analogues as activa-
tors of protein kinase C (PKC). In converting the micromolar active,
naturally occurring DAG analogues into more potent and pharma-
cologically effective compounds, we have pursued two indepen-
dent strategies in modifying the structure of DAG. These involve
sphingomyelinases acting on the lipid precursor sphingomyelin
(Fig. 1). The fate and function of the released ceramide, however,
is less well understood and although its function as a second
1
2
messenger has been challenged, evidence for its role in apopto-
1
3
sis is experimentally sound. While the consensus is that cera-
mide indeed operates as a unique second messenger for the
induction of apoptosis, as originally proposed by Hannun and
(
1) the conversion of the DAG backbone into a pentonolactone,
1
4
more specifically a 5,5-bis(hydroxymethyl)-3,4,5-trihydrofuran-2-
one and (2) the use of branched alkyl chains to replace the natural,
co-workers, diverse effects on cell differentiation, senescence
and proliferation are more complex and depend highly on the
6
15,16
linear aliphatic hydrocarbons. These changes, singly or in combi-
origin and cellular compartmentalization of ceramide.
In
nation, have resulted in compounds with increased potencies,
which have become useful probes and potential drug candidates.
addition to its production from the hydrolysis of sphingomyelin
(Fig. 1), ceramide can also be generated by de novo synthesis in
response to the cellular stress resulting from radiation and can-
7
,8
Because ceramide can be considered to be the sphingolipid
9
17–19
sibling of DAG, we reasoned that introducing similar structural
cer chemotherapeutic agents.
changes in ceramide as those implemented with DAG might im-
Since natural ceramide is poorly soluble in physiologically
acceptable solvents and does not enter cells, short chain ceramides,
like C2 ceramide (1), have been used as surrogates. These com-
pounds, albeit effective as apoptosis inducers, may not be ideal
mimics of ceramide because of their different physicochemical
*
Corresponding authors. Tel.: +1 301 846 5532; fax: +1 301 846 5598 (R.
Blumenthal); tel.: +1 301 846 5954; fax: +1 301 846 6033 (V.E. Marquez).
E-mail addresses: blumen@helix.nih.gov (R. Blumenthal), marquezv@mail.nih.
gov (V.E. Marquez).
2
0
properties.
0
968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.01.005

J.-H. Kang et al. / Bioorg. Med. Chem. 17 (2009) 1498–1505
1499
O
O
R
O
O
R
O
O
O
R'
O
R'
OH
P
OH
HO
O
O
O
O
DAG
HO
OP(OH)2
OP(OH)2
+
Inositol-1,4,5-triphosphate
O
R and R' = alkyl chains
O
O
R
NH
R
NH
R'
OH
O
P
R'
O
O
N(CH3)3
OH
Ceramide
OH
O
+
Phosphorylcholine
Figure 1. Generation of DAG and ceramide by the hydrolysis of phosphatidylinositol-4,5-biphosphate and sphingomyelin, respectively.
Nevertheless, these lower molecular weight ceramides have
been used extensively as biochemical tools to study the role of cer-
amide in apoptosis.
was chosen because of the significant improvement in potency it
produced compared to equivalent linear alkyl chains in DAG ana-
2
1
24–27
logues.
Because compound 3 was almost as cytotoxic as C2
ceramide (Table 1), we decided to increase the number of carbons
on the N-acyl side to 11. The resulting compound (4) with a total
distribution of 26 carbons (15DES-N-acyl11) showed an activity
approaching 3 times the potency of C2 ceramide. We then synthe-
sized 5, an isomer of 4, to test the importance of the location of the
branched chain by swapping the components on each side while
maintaining a similar 15DES-N-acyl11 distribution. This arrange-
ment proved to be less effective, and although the compound
was about 1.5 times more potent than C2 ceramide, it displayed
half the potency of 4. Because the activity of ceramides is lost when
the double bond is reduced, as confirmed by the inactivity of dihy-
dro-C2 ceramide (2), the double bond in compound 5 was reduced
to give compound 6 which was to be compared to 2. Remarkably,
despite a 3-fold loss in potency the compound still retained a sig-
nificant level of cytotoxicity in the same range of C2 ceramide,
showing only about a 2-fold decrease in potency. The cytotoxic
activity for the dihydroceramide analogue of compound 4 (not
shown) was also reduced by 3-fold.
2
. Activity guided design of ceramide analogues
In a recent study Schultz and co-workers,3 described the syn-
thesis and apoptotic activity of an extensive ceramide library,
and concluded that one of the key structural elements required
for activity was the sum of the carbon length in the two cores—
the sphingosine and the N-acyl chain. The most potent analogue
of such a library was a compound with a total length of 22 carbons
containing a sphingosine unit of 12 carbons and an N-acyl chain 10
carbons long. Keeping this concept in mind, we decided to explore
some structural changes on ceramides, similar to those imple-
mented with DAG, beginning with the use of combined linear
3
and branched alkyl chains on both the D-sphingosine (DES) and
the N-acyl cores. To gauge the progress of our strategy, we mea-
sured cytotoxicity (IC50) in cultured Sup T1 cells relative to C2 cer-
amide as a positive control (Table 1). Although C6 ceramide with
the longer acyl chain had an IC50 value similar to C2 (1) in this as-
say (48.01 lM and 41.44 lM, respectively), C2 ceramide (1) was
chosen to be the positive control because of its extensive use as
3. Chemistry
a reference. Both analogues have been reported in the literature
2
2
as nearly equivalent.
The branched chain substituents were derived from a common
intermediate 7, which could be converted to its acyl chloride (8)
OH
OH
2
5–27
using previously published methodology.
Alternatively, this
HO
(CH ) CH3
same intermediate (7) could also be homologated to alkyne 9 using
a one-pot procedure developed by Bestmann and co-workers by
treating the aldehyde with dimethyl acetyldiazophosphonate un-
HO
(CH2)12CH3
2 12
HN
O
HN
O
2
8
der basic conditions (Scheme 1).
The ceramide analogues were synthesized as shown in Scheme
. Commercially available N-Boc- -serine methyl ester was pro-
tected as the oxazolidine derivative 10. This compound was con-
C2 Ceramide (1)
Log P = 4.10
C6 Ceramide
Log P = 5.66
IC50 = 48.01 µM
2
L
IC50 = 41.44 µM
29–31
verted into Garner’s aldehyde 11
Williams et al.
according to the method of
3
1
C2 ceramide (1) has an unbalanced distribution of 20 carbons
between the DES and N-acyl core (18DES-N-acyl2); so for our first
target (3) with a matching logP,23 we provided a more balanced
distribution of 21 carbons (15DES-N-acyl6) with a branched chain
as part of the DES core. This type of branched hydrocarbon chain
involving a tandem reduction–oxidation se-
quence. Nucleophilic addition by the deprotonated alkynes affor-
ded 12a–b which, following deprotection of the oxazolidine ring,
gave 13a–b. Partial reduction of the triple bond to the major trans
3
2,33
double bond isomers (14a–b)
was followed by removal of the

1
500
J.-H. Kang et al. / Bioorg. Med. Chem. 17 (2009) 1498–1505
Table 1
Ceramide analogues with mixed linear and branched alkyl chains and cytotoxicity (IC50) in SupT1 cells
Compound #
Ceramides
IC50
(
lM)
Formula
S logP23
OH
HO
HN
(CH ) CH3
2 12
1
41.44 ± 5.04
C
C
20
H
39NO
3
4.1016
C2 ceramide
O
OH
HO
HN
(CH ) CH3
2 12
2
>200
20
H
41NO
3
4.3256
4.0594
Dihydro-C2 ceramide
O
OH
3
HO
HN
52.89 ± 7.92
21 3
C H41NO
O
(
CH ) CH
2 4 3
OH
4
HO
HN
15.45 ± 6.22
C
26
H
51NO
3
6.0099
O
(
CH ) CH
2 9 3
OH
HO
HN
(CH ) CH
2 9 3
O
5
29.08 ± 3.1
C
26
H
51NO
3
6.0099
OH
HO
HN
(CH ) CH
2 9 3
O
6
76.30 ± 13.78
C
26
H
53NO
3
6.2339
COCl
CHO
a
b
8
7
9
2 3
Scheme 1. Reagents and conditions: (a) see Refs. 25–27; (b) dimethyl acetyldiazophosphonate, K CO , MeOH.
Boc group and MgO-promoted acylation34 to give the final targets
3–5). Further reduction of 5 gave 6.
ratio of fluorescence at 560/590 nm. In terms of cytotoxicity, com-
pounds 4 and 5 displayed IC50 values lower than C2 ceramide un-
der similar conditions. Both compounds have the same molecular
weight, logP and distribution of carbons between the two cores
(
4
. Biological results and discussion
(15DES-N-acyl11). Therefore, their nearly 2-fold difference sug-
gests that the role of the branched side chain is more effective
when incorporated into the DES core (Table 1). The lower potency
of compound 3, which has a similar disposition of the branched
As discussed above, preliminary cellular activity was measured
in terms of cytotoxicity in cultured Sup T1 cells (Table 1) using a
3
5
cell titer blue assay. This was accomplished by measuring the

J.-H. Kang et al. / Bioorg. Med. Chem. 17 (2009) 1498–1505
1501
O
O
O
OH
HO
OMe
NHBoc
a
OMe
O
b
H
O
c
d
O
O
O
N
N
N
O
R
O-tBu
0
O-tBu
O-tBu
12a-b
1
11
a: R = C10H21
b: R =
OH
OH
OH
OH
HO
e
HO
R
f
HO
R
g
HO
R
HN
O
R
HN
O
HN
O
HN
O
O-tBu
3a-b
O-tBu
14a-b
R'
3-5
R'
6
1
Scheme 2. Reagents and conditions: (a) 2,2-dimethoxypropane, TsOHꢀH
78 °C; (d) pTSOH, MeOH, rt; (e) Red-Al, THF, rt; (f) (i) TFA, CH Cl
2
O, C
6
H
6
, 110 °C; (b) (i) NaBH
4
, MeOH/THF, (ii) (COCl)
2
, DMSO, Et
3
N, CH
2
2
Cl ; (c) nBuLi, RC„CH, THF,
0
ꢁ
2
2
, rt, (ii) MgO, R COCl, THF/H
2
O, rt; (g) H
2
, 5% Pd/C, MeOH, rt.
chain as in 4, could simply be due to its lower lipophilicity of 2logP
units. The cytotoxicity of the dihydroceramide analogue 6 is inter-
esting and consistent with the findings reported by Schultz and co-
Compound 4 was selected for further studies in comparison
with C2 ceramide beyond the simple determination of the IC50 val-
ues in Sup T1 cells to determine the effect on apoptosis induction
by two independent markers: (1) Annexin V as an early marker of
3
workers who showed that the lack of apoptotic activity of some
dihydroceramide analogues could be restored when a bulky N-acyl
tail was introduced. Although in such a study all of the synthesized
ceramides had linear alkyl chains on the DES core, our results seem
to indicate that the effect of branching the dihydroceramides is
helpful regardless of whether the branched chain is located on
the DES or N-acyl side of the molecule.
The fact that compound 4 was significantly more cytotoxic than
C2 ceramide in SupT1 cells encouraged us to test the cytotoxicity
of this compound in other cell lines. Since breast cancer remains a
significant threat and the focus of intense research, we used breast
cancer cell lines MCF-7, MDA MB231 and SKBR3 to test the potency
of 4. As seen in Table 2, the compound was significantly more cyto-
toxic than C2 ceramide in all of the cell lines with IC50 values ranging
6
apoptosis and (2) DiOC to detect mitochondrial depolarization.
Apoptosis is characterized by a variety of morphological features
such as loss of membrane asymmetry and attachment, condensa-
tion of the cytoplasm and nucleus, and internucleosomal cleavage
3
6
of DNA. One of the earliest indications of apoptosis is the trans-
location of the membrane phospholipid phosphatidylserine (PS)
from the inner to the outer leaflet of the plasma membrane. Once
exposed to the extracellular environment, binding sites on PS be-
come available for Annexin V, a phospholipid binding protein with
3
7
a high affinity for PS. The translocation of PS is an early event that
normally precedes other apoptotic processes such as loss of plasma
membrane integrity, DNA fragmentation, and chromatin conden-
sation. After the induction of apoptosis by C2 ceramide and com-
pound 4, cells were collected, washed, and stained with Annexin
V conjugated to the fluorochrome FITC. Cells that stain brightly
with Annexin V by flow cytometry are considered apoptotic and
are marked by marker M1 in the histograms (Fig. 2 top).
from 10.15 lM for SKBR-3 cells to 22.36 lM for MDA-MB231 cells.
The increase in activity was almost 5-fold for SKBR-3 although for
other cell lines including SupT1 cells it was 2.7-fold. MDA-MB231
cells only showed a 1.6-fold increase in activity underscoring the dif-
ference between cell lines with regards to the ceramide signaling
pathways. Cytotoxicity against non-cancerous cells, such as normal
human dermal fibroblasts (NHDF), was also compared. The IC50 for
Mitochondria are also critical regulators of apoptosis. The depo-
larization of the outer mitochondrial membrane releases cyto-
chrome c into the cytoplasm. Cytoplasmic cytochrome c forms a
complex with the apoptotic protease activating factor (apaf1) and
caspase 9 to initiate the caspase cascade leading to apoptosis.38
Short chain ceramides are known to induce apoptosis via the mito-
C2 ceramide was 66.5
5.39 M (single determinations). The average IC50 for C2 ceramide
against the panel of cancer cell lines examined was 45.85 M with a
selectivityindex (SI) of just 1.5. On the other hand, the average IC50 of
compound 4 against the same panel was 16.06 M, thus providing a
lM whereas for compound 4 it was
4
l
l
3
9
chondrial pathway. It was thus important to look at mitochon-
l
drial depolarization induced by compound 4 and C2 ceramide in
more favorable SI value of nearly 3-fold. Therefore, compound 4 rep-
resents a novel ceramide analog that shows significantly higher
cytotoxicity and selectivity in a variety of cell lines by the induction
of apoptosis (vide infra).
SupT1 cells. DiOC
drial membrane and the intensity of binding is proportional to the
membrane potential. Hence, in the case of DiOC , less staining with
DiOC represents mitochondrial depolarization and an indicator of
6
is a dye that binds specifically to the mitochon-
6
6
cells undergoing apoptosis as shown by marker M1 in the histo-
grams (Fig. 2 bottom).
As seen in Figure 2, both C2 ceramide and compound 4 induced
apoptosis in SupT1 cells determined by Annexin V binding or
Table 2
IC50 of compound 4 in various tumor cell lines
C2 Ceramide
IC50
Compound 4
IC50
Fold increase
in activity
DiOC
6
labeling. Compound 4 caused more apoptosis determined
M (66.16%) than C2 ceramide at 50
l
M
lM
by Annexin V at 25
l
lM
MCF-7
MDA-MB-231
SKBR-3
45.41 ± 7.24
35.37 ± 1.81
49.20 ± 1.7
41.44 ± 5.04
16.30 ± 7.55
22.36 ± 8.19
10.15 ± 3.32
15.45 ± 6.22
2.78
1.58
4.84
2.68
(42.33%) confirming our cytotoxicity assays. Similar results were
seen for mitochondrial depolarization with C2 ceramide and com-
pound 4 causing 56.14% and 75.15% apoptosis, respectively. Con-
trol cells did not show significant apoptosis in either assay.
SupT1

1
502
J.-H. Kang et al. / Bioorg. Med. Chem. 17 (2009) 1498–1505
Figure 2. Apoptosis induced by compound 4 (25 lM) relative to C2 ceramide (50 lM) and control.
Importantly, a dose-dependent effect was seen on apoptosis
and ionization was effected by a beam of xenon atoms generated
in a saddle-field ion gun at 8.0 ± 0.5 kV. Nominal mass spectra were
obtained at a resolution of 1200, and matrix derived ions were
background subtracted during data system processing. Optical
rotations were recorded on a Jasco P-1010 polarimeter. Elemental
analyses were performed by Atlantic Microlab, Inc., Atlanta, GA.
induction by both compounds with compound 4 being significantly
more potent than C2 ceramide (Fig. 3 A and B). The induction of
apoptosis by both compounds showing morphological features of
apoptosis suggests that compound 4 acts via a mechanism similar
to C2 ceramide albeit with a higher potency.
5
. Conclusions
6.2. Cells and reagents
Herein we report for the first time the synthesis of ceramide
SupT1 cells were maintained in RPMI media supplemented with
10% FBS and penicillin streptomycin (5000 U/mL). Breast cancer
cell lines MCF-7 MDA-MB231 and SKBR3 cells were maintained
in Dulbecco’s modified Eagles Medium supplemented with 10%
FBS and penicillin streptomycin (5000 U/mL). NHDF cells were ob-
tained from Lonza (Walkersville, MD) and cultured in fibroblast
cell medium as recommended by the manufacturer. Stock solu-
tions of compounds were made in DMSO at a concentration of
50 mM. Compounds were diluted in media at 37 °C before addition
to cells.
analogues decked with
branched alkyl chains distributed on either side of the
a
combination of linear and highly
-sphingo-
D
sine (DES) and the N-acyl cores. We have determined that cera-
mide analogue 4, with the branched chain on the DES core, is
more potent than the corresponding isomer (5) that bears the
branched alkyl chain on the N-acyl core. Both isomers 4 and 5 were
more cytotoxic than C2 ceramide. A direct comparison between C2
ceramide and the most potent isomer 4 using Annexin V as an early
6
marker of apoptosis and DiOC to detect mitochondrial depolariza-
tion suggests that compound 4 is more potent than C2 ceramide.
6
.3. Cytotoxicity assay and IC50 calculation
6
6
. Experimental
Cytotoxicity was determined on various cell lines by incubating
.1. General procedures
with serial dilutions of the test compounds in media for 24 h. Cell
viability was determined using the CellTiter-BlueÒ Cell Viability
Assay (Promega Corp. Madison, WI) as per the manufacturer’s
instructions. The CellTiter-BlueÒ Cell Viability Assay is based on
the ability of living cells to reduce resazurin to fluorescent resoru-
fin. The formation of resorufin was measured using fluorescence
plate reader (CytoFluor 4000, Applied Biosytems) at 560/590 exci-
tation/emission. Data was normalized to no drug control and ex-
pressed as percent viability. Dose response curves obtained from
serial dilution of the compounds were fit using sigma plot analysis
software and IC50 values for each experiment were calculated. Each
experiment was repeated three times and the average IC50 value
was calculated along with its standard deviation.
All chemical reagents were commercially available. Column
chromatography was performed on Silica Gel 60, 230–400 mesh
E. Merck) or by CombiFlashÒ instrument. H and C NMR spectra
were recorded on a Varian Unity Inova instrument at 400 and
00 MHz, respectively. Spectra are referenced to the solvent in
which they were run (7.24 ppm for CDCl ). Positive-ion, fast-atom
bombardment mass spectra (FAB-MS) were obtained on a VG
070E-HF double-focusing mass spectrometer operated at an
1
13
(
1
3
7
accelerating voltage of 6 kV under the control of a MASPEC-II32
data system for Windows (Mass Spectrometry Services, Ltd). Either
glycerol or 3-nitrobenzyl alcohol was used as the sample matrix,

J.-H. Kang et al. / Bioorg. Med. Chem. 17 (2009) 1498–1505
1503
1
1
1
40
20
00
6.4.2. tert-Butyl (4S)-4-carbonyl-2,2-dimethyl-1,3-oxazolidine-
3
-carboxylate (11)
This compound, known as Garner’s aldehyde
29,30
1
was synthe-
3
sized according to the method of Williams et al. from 10 involv-
ing a tandem reduction oxidation sequence.
8
6
4
2
0
0
0
0
0
6
.4.3. General procedure A—Alkylation
According to a literature procedure, the alkyne (1.2 equiv)
3
2
was added slowly to a ꢁ70 °C stirring solution of THF (5 mL/mmol
alkyne) and n-BuLi (1.6 M in hexanes,1.5 equiv) under argon. A
suspension of 11 (1 equiv) in THF (30 mL/mmol) was then added
to the resultant acetylide and the mixture allowed to reach rt. After
1 h, the reaction mixture was poured into water and extracted with
diethyl ether. The combined organic extracts were dried over
C2 ceramide
Compound 4
4
MgSO , filtered and concentrated in vacuo. This reaction provides
0
20
40
60
80
100
120
the alcohols as a mixture of erythro and threo isomers from which
the desired, more abundant erythro isomer can be purified by Com-
biflashÒ column chromatography (hexanes/EtOAc, 4:1).
Conc µM
32,33
1
1
20
00
6
.4.3.1. tert-Butyl 4-((1R)-1-hydroxytridec-2-ynyl) (4S)-2,2-
dimethyloxazolidine-3-carboxylate (12a). According to general
procedure A, 11 (800 mg, 3.4 mmol) in THF (10 mL) was added to
a solution of n-BuLi (3.2 mL, 5.1 mmol) and 1-dodecyne (681 mg,
8
6
4
2
0
0
0
0
0
4
.1 mmol) in THF (20 mL) to give 12a (310 mg, 23%) as a colorless
2
D
2
1
oil; ½
a
ꢂ
¼ ꢁ37:07 (c 3.85, CH
2
Cl
and CHOH), 2.16 (td, J = 7.1, 1.8 Hz, 2H,
), 1.55 (br s, 4H, CH and OH), 1.48 (s, 6H, CH ), 1.47 (s,
), 1.23 (m containing s at 1.23, 14H, CH ), 0.85 (t,
) d 154.04, 94.87,
86.59, 81.14, 77.85, 65.03, 64.07, 62.78, 31.84, 29.53, 29.46,
2 3
); H NMR (400 MHz, CDCl ) d
4
4 5
.14 (m, 5H, H , H
C„CCH
9H, CH
J = 6.9 Hz, 3H, CH
2
2
3
3
2
1
3
); C NMR (100 MHz, CDCl
3
C2 ceramide
Compound 4
3
2
1
9.26, 29.08, 28.83, 28.52, 28.33, 25.71, 25.38, 22.62, 18.70,
4.05.; FABMS m/z (relative intensity) 396 (MH , 19), 57 (100);
+
0
20
40
60
80
100
120
Anal. Calcd for C23
Found: C, 69.18; H, 10.26; N, 3.60.
4 2
H41NO ꢀ0.2H O: C, 69.20; H, 10.45; N, 3.51.
Conc µM
Figure 3. Apoptotic dose–responses for compound 4 (d) and C2 ceramide (s) in
terms of Annexin V binding (A) and DiOC labeling (B).
6
.4.3.2. tert-Butyl 4-[(1R)-1-hydroxy-7-methyl-5-(2-methylpro-
pyl)oct-2-ynyl](4S)-2,2-dimethyl-1,3-oxazolidine-3-carboxylate
12b). According to general procedure A, 11 (900 mg, 3.9 mmol)
6
(
in THF (10 mL) was added to a solution of n-BuLi (3.7 mL,
5.9 mmol) and 9 (778 mg, 4.7 mmol) in THF (20 mL) to give 12b
6
.4. Apoptosis detection
Sup T1 cells were incubated with different concentrations of the
2
2
(
320 mg, 20%) as a colorless oil; ½
aꢂ
¼ ꢁ34:61 (c 0.81, CH
2
Cl
2
);
D
1
H NMR (400 MHz, CDCl ) d 4.60 (m, 1H, CHOH), 4.25 (br s, 1H,
3
test compounds for 24 h. After 24 h incubation the cells were col-
lected, washed with PBS and apoptosis was determined as phos-
phatidyl serine exposure by Annexin V FITC staining using
5 4
CHOH), 4.05 (m, 2H, H ), 3.93 (br s, 1H, H ), 2.13 (dd, J = 5.4,
1.9 Hz, 2H, C„CCH ), 1.58 (m, 3H, CH), 1.46 (s, 6H, C(CH ) ), 1.45
2
3 2
(s, 9H, C(CH ) ), 1.19 (m, 2H, CH ), 1.04 (m, 2H, CH ), 0.83 (d,
3
3
2
2
Ò
13
ApoAlert Annexin V Apoptosis Kit (Clontech) or as mitochondrial
J = 6.5 Hz, 6H, CH(CH ) ), 0.82 (d, J = 6.6 Hz, 6H, CH(CH ) );
C
3
2
3 2
depolarization by staining with 10
l
M DiOC
6
followed by flow
NMR (100 MHz, CDCl ) d 153.78, 94.83, 84.99, 81.04, 79.02,
3
cytometry on a BD FACScan flow cytometer (BD Bioscience). At
least 10,000 events were collected and analyzed using CellQuestÒ
software (BD Bioscience).
64.83, 63.71, 62.59, 43.49, 32.00, 28.34, 25.89, 25.02, 23.25,
23.10, 22.44; FABMS m/z (relative intensity) 396 (MH , 13), 57
+
(100); Anal. Calcd for C23H41NO : C, 69.83; H, 10.45; N, 3.54.
4
Found: C, 69.60; H, 10.53; N, 3.64.
6
.4.1. 6-Methyl-4-(2-methylpropyl)hept-1-yne (9)
According to
published procedure,28 dimethyl acetyl
diazophosphonate (5.9 g, 31 mmol) was added to a solution of
a
6.4.4. General procedure B. Deprotection of the oxazolidine
3
2,33
According to the literature procedure,
a catalytic amount of
2
5–27
7
(2.0 g, 12 mmol) and K
50 mL) and stirring continued until the reaction was complete
as indicated by TLC. The reaction mixture was diluted with ether,
washed with an aqueous solution of NaHCO , dried over MgSO
2
CO
3
(5.8 g, 42 mmol) in MeOH
p-toluenesulfonic acid was added to solution of 12a–b in anhy-
drous MeOH and stirred at rt until the reaction was complete by
TLC analysis. The crude mixture was concentrated in vacuo and
purified by CombiflashÒ column chromatography (hexane/EtOAc,
2:1) to give 13a–b.
(
3
4
Ò
and concentrated in vacuo. Purification by Combiflash column
chromatography (hexanes) gave 9 (1.9 g, 92%) as a colorless oil
which was used directly without further purification or character-
6.4.4.1. N-[(1S,2R)-2-Hydroxy-1-(hydroxymethyl)tetradec-3-
ynyl](tert-butoxy)carboxamide (13a). According to general pro-
cedure B, p-toluenesulfonic acid (30 mg) was combined with 12a
1
3
ization. H NMR (400 MHz, CDCl ) d 2.14 (dd, J = 5.3, 2.7 Hz, 2H,
CH
m, 2H, CH
J = 6.5 Hz, 6H); C NMR (100 MHz, CDCl
1.86, 25.06, 23.12, 22.93, 22.49.
2
C„C), 1.89 (t, J = 2.7 Hz, 1H, C„CH), 1.63 (m, 3H, CH), 1.25
), 1.09 (m, 2H, CH ), 0.86 (d, J = 6.6 Hz, 6H), 0.85 (d,
) d 83.06, 69.07, 43.37,
(
2
2
(300 mg, 0.76 mmol) in anhydrous MeOH (15 mL) and stirred for
1
3
22
D
3
27 h to give 13a (155 mg, 58%) as a colorless oil; ½
aꢂ
¼ ꢁ13:50
1
3
2 2 3
(c 0.33, CH Cl ); H NMR (400 MHz, CDCl ) d 5.36 (d, J = 8.3 Hz,

1
504
J.-H. Kang et al. / Bioorg. Med. Chem. 17 (2009) 1498–1505
1
(
H, NH), 4.55 (s, 1H, CHOH), 4.01 (d, J = 7.6 Hz, 1H, CHHOH), 3.65
m, 3H, CHN, CHHOH), 3.57 (s, 1H, OH), 3.11 (s, 1H, OH), 2.16 (t,
J = 7.1 Hz, 2H, C„CCH ), 1.46 (m, 11H, CH and C(CH ), 1.29 (m,
4H, CH ), 0.83 (t, J = 6.5 Hz, 3H, CH ); C NMR (100 MHz, CDCl
d 156.22, 87.92, 79.98, 77.80, 64.36, 62.64, 55.81, 31.83, 29.53,
9.46, 29.25, 29.07, 28.87, 28.49, 28.30, 22.61, 18.65, 14.04; FABMS
2H); 13C NMR (100 MHz, CDCl
) d 158.94, 131.93, 130.46, 79.81,
3
74.72, 62.68, 55.47, 43.68, 36.58, 32.58, 28.37, 25.08, 23.09, 22.65;
+
2
2
3
)
3
FABMS m/z (relative intensity) 358 (MH , 21), 284 (100); Anal. Calcd
13
1
2
3
3
)
for C20
10.56; N, 3.67.
H
39NO
4
2
ꢀ0.3H O: C, 66.19; H, 11.00; N, 3.86. Found: C, 66.19; H,
2
+
m
C
/z (relative intensity) 356 (MH , 39), 57 (100); Anal. Calcd for
ꢀ0.5H O: C, 65.90; H, 10.51; N, 3.84. Found: C, 66.00;
H, 10.42; N, 3.92.
6.4.6. General procedure D: Deprotection/N-acylation
20
H37NO
4
2
2 2
Trifluoroacetic acid was added to a solution of 14a–b in CH Cl
and stirred for 30 min to 1 h at rt. The crude reaction was then con-
centrated in vacuo and purified by CombiflashÒ chromatography
6
6
.4.4.2. N-[(1S,2R)-2-Hydroxy-1-(hydroxymethyl)-8-methyl-
-(2-methoxypropyl)non-3-ynyl](tert-butoxy)carboxamide
(CH
2 2
Cl /MeOH, 6:1 or 4:1) to give the free amine as a colorless
oil which was used immediately without further characterization.
According to a literature procedure, magnesium oxide was added
to a solution of the sphingosine in THF/H O (4:1) and stirred vigor-
ously for 30 min to 1 h at rt. The corresponding acid chloride was
then added and stirring continued until the reaction was complete
by TLC analysis. The reaction mixture was then filtered through a
3
4
(
13b). According to general procedure B, p-toluenesulfonic
acid (30 mg) was combined with 12b (310 mg, 0.78 mmol) in
anhydrous MeOH (10 mL) and stirred for 3 h to give 13b
2
2
D
2
(
131 mg, 47%) as a colorless oil; ½
aꢂ
¼ ꢁ12:96 (c 0.28, CH
2
Cl
) d 5.33 (br d, J = 8.2 Hz, 1H, NH), 4.58
br s, 1H, CHOH), 4.04 (br d, J = 7.7 Hz, 1H, CHHOH), 3.72 (m,
2
);
1
H NMR (400 MHz, CDCl
3
Ò
(
bed of Celite and the filtrate was concentrated in vacuo. Purifica-
2
2
H, CHHOH, CHNH), 3.32 (br s, 1H, OH), 2.90 (br s, 1H, OH),
.16 (dd, J = 5.3, 2.0 Hz, 2H, C„CCH ), 1.59 (m, 3H, CH), 1.42
), 1.18 (m, 2H, CH ), 1.06 (m, 2H, CH ), 0.84 (m,
); C NMR (100 MHz, CDCl ) d 156.25, 86.45,
tion by CombiflashÒ chromatography (100% EtOAc or hexane/
2
EtOAc, 1:1) gave 3–5.
(
s, 9H, C(CH
3
)
3
2
2
1
3
1
8
2
3
6
2H, CH(CH
3
)
2
3
6.4.6.1. N-[(3E)(1S,2R)-2-Hydroxy-1-(hydroxymethyl)-8-methyl-
6-(2-methylpropyl)non-3-enyl]hexanamide (3). According to
general procedure D, trifluoroacetic acid (1 mL) was combined
0.01, 78.84, 64.59, 62.85, 55.87, 43.55, 31.97, 28.31, 25.05,
3.16, 23.13, 22.46, 22.44.; FABMS m/z (relative intensity)
+
56 (MH , 16), 57 (100); Anal. Calcd for C20
H
37NO
4
ꢀ0.3H
2
O: C,
with 14b (40 mg, 0.11 mmol) in CH
oil which was dissolved in THF/H O (4 mL) and treated with mag-
nesium oxide (11 mg, 6.3 mmol) and hexanoyl chloride (41 mg,
0.2 mmol) to give (24.2 mg, 62%) as colorless oil;
); H NMR (400 MHz, CDCl ) d 6.28
2 2
Cl (1 mL) to give a colorless
6.56; H, 10.50; N, 3.88. Found: C, 66.37; H, 10.34; N, 3.87.
2
6
.4.5. General procedure C: Reduction of alkyne to alkene
3
a
According to the literature procedure,32 Red-Al (65%w in tolu-
½
aꢂ
22
D
¼ ꢁ7:50 (c 0.875, CH
1
2
Cl
2
3
ene) was added to solution of alkyne (150 mg, 0.42 mmol) in anhy-
drous THF (20 mL). Stirring at rt continued until TLC analysis
showed the reaction was complete. The mixture was quenched
with water and extracted with diethyl ether. The combined organic
extracts were dried (MgSO
Purification by Combiflash column chromatography (hexane/
EtOAc, 1:1) gave the trans-double bond alkene.
(d, J = 7.3 Hz, 1H, NH), 5.72 (m, 1H, CH@CH), 5.50 (ddt, J = 15.4,
6.2, 1.1 Hz, 1H, CH@CH), 4.31 (irr br t, 1H, CHOH), 3.90 (m, 2H,
CHHOH, CHNH), 3.68 (dd, J = 10.9, 3.0 Hz, 1H, CHHOH), 2.94 (v br
s, 2H, OH), 2.21 (t, J ꢃ 7.7 Hz, 2H, (CO)CH
2
), 2.00 (t, J = 6.3 Hz, 2H,
4
), filtered and concentrated in vacuo.
CH
CH
2
), 1.61 (m, 4H, CH
), 1.03 (m, 4H, CH
2
and CH), 1.52 (m, 1H, CH), 1.30 (m, 4H,
Ò
2
2
), 0.87 (t, J = 7.0 Hz, 3H, CH
3
), 0.84 (d,
1
3
J = 1.9 Hz, 6H, CH
CDCl
3
), 0.83 (d, J = 1.9, 6H, CH
3
); C NMR (100 MHz,
3
) d 173.96, 132.04, 130.33, 112.53, 74.53, 62.47, 54.52,
6
3
.4.5.1. N-[(3E)(1S,2R)-2-Hydroxy-1-(hydroxymethyl)tetradec-
-enyl](tert-butoxy)carboxamide (14a). According to general
43.67, 36.78, 36.59, 32.58, 31.40, 25.41, 25.08, 23.08, 22.66,
22.38, 13.92; FABMS m/z (relative intensity) 356 (MH , 56), 338
+
procedure C, Red-Al (0.26 mL, 0.84 mmol) was combined with
3a (150 mg, 0.42 mmol) in anhydrous THF (20 mL) and stirred
for 18 h to give 14a (100 mg, 67%) as a white amorphous solid;
(100); Anal. Calcd for C21
Found: C, 70.67; H, 11.67; N, 3.94.
3
H41NO : C, 70.94; H, 11.62; N, 3.94.
1
22
1
½
a
ꢂ
¼ ꢁ4:13 (c 1.485, CH
2
Cl
2
); H NMR (400 MHz, CDCl
3
) d 5.75
6.4.6.2. N-[(3E)(1S,2R)-2-Hydroxy-1-(hydroxymethyl)-8-methyl-
6-(2-methylpropyl)non-3-enyl]undecanamide (4). According to
general procedure D, trifluoroacetic acid (2 mL) was combined
D
(
irr dt, 1H, C@CH), 5.50 (ddd, J = 15.4, 6.4, 1.2 Hz, 1H, C@CH), 5.28
(
br d, J = 5.9 Hz, 1H, NH), 4.28 (s, 1H, CHOH), 3.90 (dd, J = 11.3,
3
1
1
.4 Hz, 1H, CHHOH), 3.68 (br d, J = 12.1 Hz, 1H, CHHOH), 3.57 (s,
H, CHNH), 2.66 (s, 2H, OH), 2.03 (q, J = 7.0 Hz, 2H, C@CHCH ),
.43 (s, 9H, CCH ), 1.34 (m, 2H, CH ), 1.23 (s, 14H, CH ), 0.85 (t,
); C NMR (100 MHz, CDCl ) d 165.38, 134.09,
28.92, 79.75, 74.82, 65.83, 55.39, 32.27, 31.88, 29.61, 29.59,
9.46, 29.31, 29.18, 29.09, 28.35, 22.66, 15.23, 14.09; FABMS m/z
with 14b (70 mg, 0.19 mmol) in CH
oil which was dissolved in THF/H O (5 mL) and treated with mag-
nesium oxide (18 mg, 0.43 mmol) and undecanoyl chloride (41 mg,
2 2
Cl (2 mL) to give a colorless
2
2
3
2
2
1
3
22
J = 6.5 Hz, 3H, CH
1
2
3
3
0.2 mmol) to give 4 (39 mg, 48%) as a colorless oil. ½
a
ꢂ
¼ ꢁ7:63 (c
D
1
2 2 3
0.06, CH Cl ); H NMR (400 MHz, CDCl ) d 6.29 (d, J = 7.1 Hz, 1H,
NH), 5.71 (m, 1H, CH@CH), 5.49 (ddd, J = 15.4, 6.1, 1.1 Hz, 1H,
CH@CH), 4.30 (br s, 1H, CHOH), 3.89 (m, 2H, CHHOH, CHNH),
3.67 (br d, J = 9.2 Hz, 1H, CHHOH), 2.91 (v br s, 2H, OH), 2.20 (t,
+
(
relative intensity) 358 (MH , 35), 284 (100); Anal. Calcd for
ꢀ0.1H O: C, 66.85; H, 11.00; N, 3.90. Found: C, 66.95;
H, 10.89; N, 3.90.
C
20
H39NO
4
2
J = 7.1 Hz, 2H, @CHCH
2
), 2.00 (t, J = 6.3 Hz, 2H, C(O)CH
H, CH), 1.50 (m, 2H, CH ), 1.25 (m, 14H, CH ), 1.02 (m, 4H, CH
); C NMR (100 MHz, CDCl ) d 173.99, 131.99,
2
), 1.61 (m,
3
2
2
2
),
1
3
6
6
.4.5.2. N-[(3E)-(1S,2R)-2-Hydroxy-1-(hydroxymethyl)-8-methyl-
-(2-methylpropyl)non-3-enyl](tert-butoxy)carboxamide
0.84 (m, 15H, CH
3
3
130.33, 74.46, 62.44, 54.55, 43.67, 36.83, 36.60, 32.57, 31.87,
29.56, 29.49, 29.35, 29.28, 25.75, 25.08, 23.08, 22.66, 14.09; FABMS
(
14b). According to general procedure C, Red-Al (0.68 mmol,
.2 mL) was combined with 13b (120 mg, 0.34 mmol) in anhydrous
THF (10 mL) and stirred for 16 h to give 14b (80 mg, 65%) as a color-
+
0
m/z (relative intensity) 426 (MH , 26), 408 (100); Anal. Calcd for
C
26
H
51NO
3
ꢀ0.2H
2
O: C, 72.74; H, 12.07; N, 3.26. Found: C, 72.60;
2
D
2
1
less oil; ½
a
ꢂ
¼ ꢁ2:92 (c 2.425, CH
2 2 3
Cl ); H NMR (400 MHz, CDCl ) d
H, 12.06; N, 3.24.
5
.71 (m, 1H, CH@CH), 5.50 (dd, J = 15.4, 6.0 Hz, 1H, CH@CH), 5.30 (br
d, J = 4.7 Hz, 1H, NH), 4.32 (br s, 1H, CHOH), 3.91 (br d, J = 9.4 Hz, 1H,
CHHOH), 3.68 (m, 1H, CHHOH), 3.58 (br s, 1H, CHNH), 2.71 (br s, 2H,
6.4.6.3. N-[(3E)(1S,2R)-2-Hydroxy-1-(hydroxymethyl)tetradec-
3-enyl]-5-methyl-3-(2-methylpropyl)hexanamide (5). Accord-
ing to general procedure D, trifluoroacetic acid (3 mL) was com-
OH), 2.00 (t, J = 6.1 Hz, 2H, CH
2
), 1.61 (sept, J = 6.3 Hz, 2H), 1.49 (m,
), 0.83 (d, J = 6.4 Hz,
1
H, CH), 1.43 (s, 9H, C(CH ), 1.03 (m, 4H, CH
3
)
3
2
2 2
bined with 14a (95 mg, 0.27 mmol) in CH Cl (3 mL) to give a

J.-H. Kang et al. / Bioorg. Med. Chem. 17 (2009) 1498–1505
1505
colorless oil which was dissolved in THF/H
2
O (5 mL) and treated
3. 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.
with magnesium oxide (27 mg, 0.68 mmol) and 3-isobutyl-5-
4.
Bieberich, E.; Hu, B.; Silva, J.; MacKinnon, S.; Yu, R. K.; Fillmore, H.; Broaddus,
methyl-hexanoyl chloride (8)2
(
5–27
(66 mg, 0.32 mmol) to give 5
¼ ꢁ6:05 (c 0.305, CH
W. C.; Ottenbrite, R. M. Cancer Lett. 2002, 181, 55.
2
2
1
44 mg, 37%) as a colorless oil; ½
aꢂ
2
Cl
2
); H
5. Delgado, A.; Casas, J.; Llebaria, A.; Abad, J. L.; Fabrias, G. ChemMedChem 2007, 2,
80.
D
5
3
NMR (400 MHz, CDCl ) d 6.25 (d, J = 6.9 Hz, 1H, NH), 5.75 (m, 1H,
6
7
.
.
Marquez, V. E.; Blumberg, P. M. Acc. Chem. Res. 2003, 36, 434.
CH@CH), 5.49 (dd, J = 15.4, 6.3 Hz, 1H, CH@CH), 4.27 (s, 1H, CHOH),
.88 (m, 2H, CH OH), 3.65 (d, J = 8.1 Hz, 1H, CHNH), 3.15 (br s, 2H,
OH), 2.09 (d, J = 6.6 Hz, 2H, C(O)CH ), 2.03 (m, 2H, CH@CHCH ), 1.94
m, J = 6.7 Hz, 1H, CH), 1.61 (sept, J = 6.6 Hz, 2H, CH(CH ), 1.31 (m,
6H, CH ), 1.10 (m, 4H, CH ), 0.85 (m, 15H, CH
100 MHz, CDCl ) d 173.65, 134.12, 128.78, 74.41, 62.44, 54.69,
4.19, 42.35, 32.29, 31.88, 31.00, 29.61, 29.46, 29.31, 29.22,
9.12, 25.16, 22.87, 22.78, 22.76, 22.65, 14.08; FABMS m/z (relative
Caloca, M. J.; Garcia-Bermejo, M. L.; Blumberg, P. M.; Lewin, N. E.; Kremmer, E.;
Mischak, H.; Wang, S. M.; Nacro, K.; Bienfait, B.; Marquez, V. E.; Kazanietz, M. G.
Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 11854.
3
2
2
2
8.
Garcia-Bermejo, M. L.; Leskow, F. C.; Fujii, T.; Wang, Q.; Blumberg, P. M.;
Ohba, M.; Kuroki, T.; Han, K. C.; Lee, J.; Marquez, V. E.; Kazanietz, M. G. J.
Biol. Chem. 2002, 277, 645 [Published erratum appears in J. Biol. Chem. 2004,
(
3 2
)
1
3
1
2
2
3
); C NMR
2
79, 23846].
(
3
9.
Hannun, Y. A.; Obeid, L. M. Trends Biochem. Sci 1995, 20, 73.
4
2
10. Newton, A. C. Chem. Rev. 2001, 101, 2353.
11. Brose, N.; Rosenmund, C. J. Cell Sci. 2002, 115, 4399.
+
1
1
2. Hofmann, K.; Dixit, V. M. Trends Biochem. Sci. 1998, 23, 374.
3. Levade, T.; Malagarie-Cazenave, S.; Gouaze, V.; Segui, B.; Tardy, C.; Betito, S.;
Andrieu-Abadie, N.; Cuvillier, O. Neurochem. Res. 2002, 27, 601.
4. Obeid, L. M.; Linardic, C. M.; Karolak, L. A.; Hannun, Y. A. Science 1993, 259,
1769.
3
intensity) 426 (MH , 39), 408 (100); Anal. Calcd for C26H51NO : C,
7
3.36; H, 12.08; N, 3.29. Found: C, 73.35; H, 12.16; N, 3.32.
1
6
.4.7. N-[(1S,2R)-2-Hydroxy-1-(hydroxymethyl)tetradecyl]-5-
1
1
5. Okazaki, T.; Kondo, T.; Kitano, T.; Tashima, M. Cell. Signal. 1998, 10, 685.
6. Pettus, B. J.; Chalfant, C. E.; Hannun, Y. A. Biochim. Biophys. Acta Mol. Cell Biol.
Lipids 2002, 1585, 114.
methyl-3-(2-methyl-propyl)hexanamide (6)
The double bond in compound 5 was reduced in MeOH with 5%
Pd/C under and H
2
atmosphere at rt after 30 min. The crude reac-
17. Zheng, W.; Kollmeyer, J.; Symolon, H.; Momin, A.; Munter, E.; Wang, E.; Kelly,
S.; Allegood, J. C.; Liu, Y.; Peng, Q.; Ramaraju, H.; Sullards, M. C.; Cabot, M.;
Merrill, A. H. BBA. Biomembranes 2006, 1758, 1864.
tion was then concentrated in vacuo to give 6 as a colorless oil;
1
H NMR (400 MHz, CDCl
J = 11.3, 3.5 Hz, 1H, CHOH), 3.82 (m, 1H, CHHOH), 3.75 (m, 2H,
CHHOH, CHNH), 2.53 (s, 1H, OH), 2.10 (d, J = 6.7 Hz, 2H, (CO)CH ),
), 1.24 (s,
), 0.87 (d, J = 1.9 Hz, 6H, CH(CH ),
) d
3
) d 6.26 (d, J = 7.7 Hz, 1H, NH), 3.99 (dd,
1
8. Wedeking, A.; van Echten-Deckert, G. Curr. Org. Chem. 2007, 11, 579.
19. Morales, A.; Lee, H.; Goni, F. M.; Kolesnick, R.; Fernandez-Checa, J. C. Apoptosis
007, 12, 923.
2
2
2
2
0. Sot, J.; Goni, F. M.; Alonso, A. BBA. Biomembranes 2005, 1711, 12.
1. Verheij, M.; Bose, R.; Lin, X. H.; Yao, B.; Jarvis, W. D.; Grant, S.; Birrer, M. J.;
Szabo, E.; Zon, L. I.; Kyriakis, J. M.; HaimovitzFriedman, A.; Fuks, Z.; Kolesnick,
R. N. Nature 1996, 380, 75.
1
2
0
1
2
2
.95 (p, J = 6.7 Hz, 1H, CH), 1.58 (m, 7H, CH, OH and CH
1H, CH ), 1.11 (m, 4H, CH
3 2 3
.85 (m, 9H, CH CH and CH(CH ) ); C NMR (100 MHz, CDCl
73.14, 74.26, 62.58, 53.84, 44.25, 42.42, 34.57, 31.91, 31.06,
9.66, 29.63, 29.57, 29.55, 29.35, 25.96, 25.19, 22.91, 22.80,
2
2
2
3 2
)
13
2
3
2
2
2. Phillips, D. C.; Allen, K.; Griffiths, H. R. Arch. Biochem. Biophys. 2002, 407, 15.
3. Wildman, S. A.; Crippen, G. M. J. Chem. Inf. Comput. Sci. 1999, 39, 868.
24. Nacro, K.; Bienfait, B.; Lewin, N. E.; Blumberg, P. M.; Marquez, V. E. Bioorg. Med.
Chem. Lett. 2000, 10, 653.
25. Nacro, K.; Bienfait, B.; Lee, J.; Han, K. C.; Kang, J. H.; Benzaria, S.; Lewin, N. E.;
Bhattacharyya, D. K.; Blumberg, P. M.; Marquez, V. E. J. Med. Chem. 2000, 43,
921.
+
2.69, 14.12; FABMS m/z (relative intensity) 428 (MH , 100); HRMS
+
(
(
FAB) Calcd for C26
M+K ) 466.3662, found 466.3687.
H53NO
3
: (MH ) 428.4104; found 428.4107;
+
26. Lee, J.; Han, K. C.; Kang, J. H.; Pearce, L. L.; Lewin, N. E.; Yan, S.; Benzaria, S.;
Nicklaus, M. C.; Blumberg, P. M.; Marquez, V. E. J. Med. Chem. 2001, 44, 4309
Acknowledgment
[
Erratum published in J. Med. Chem. 2003, 46 (13): 2794].
27. Choi, Y.; Kang, J. H.; Lewin, N. E.; Blumberg, P. M.; Lee, J.; Marquez, V. E. J. Med.
Chem. 2003, 46, 2790.
This research was supported by the Intramural Research Pro-
gram of the NIH, National Cancer Institute, Center for Cancer
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