Short-chain 3-ketoceramides, strong apoptosis inducers against human leukemia HL-60 cells

Article abstract of DOI:10.1016/j.bmc.2007.02.008

Ceramides act as a second messenger of the apoptotic signaling process. The allylic alcohol portion comprising the C-3, C-4, and C-5 carbons is essential for this function. The suggestion has been made that this alcohol moiety is oxidized in mitochondria to a carbonyl moiety, with the generation of reactive oxygen species. However, there is no established precedent for the apoptotic performance of 3-ketoceramides thus presumed. In this work, we have synthesized three different types of short-chain 3-ketoceramides, that is, (2S, 4E)-2-acetylamino-3-oxo-4-octadecen-1-ol (A), (2S, 4E, 6E)-2-acetylamino-3-oxo-4,6-octadecadien-1-ol (B), and (2S, 4E)-2-acetylamino-1-methoxy-3-oxo-4-octadecene (C), and demonstrated that these 3-ketoceramides are capable of inducing effective apoptosis in human leukemia HL-60 cells. In particular, the two monoenoic compounds, A and C, are far more powerful than the corresponding alcoholic analogue, N-acetyl-d-erythro-sphingosine. Observations of DNA fragmentation, caspase-3 activation, and cytochrome c release from mitochondria provide substantiated evidence for mitochondrial apoptosis and the effects of exogenous glutathione on these phenomena are also discussed.

Full text of DOI:10.1016/j.bmc.2007.02.008

Bioorganic & Medicinal Chemistry 15 (2007) 2860–2867
Short-chain 3-ketoceramides, strong apoptosis inducers
against human leukemia HL-60 cells
*
Hideki Azuma, So Ijichi, Mayuko Kataoka, Akira Masuda, Takayuki Izumi,
Tetsuya Yoshimoto and Taro Tachibana
Department of Applied and Bioapplied Chemistry, Graduate School of Engineering, Osaka City University,
Sugimoto 3-3-138, Sumiyoshi-ku, Osaka 558 8585, Japan
Received 10 November 2006; revised 8 February 2007; accepted 9 February 2007
Available online 13 February 2007
Abstract—Ceramides act as a second messenger of the apoptotic signaling process. The allylic alcohol portion comprising the C-3,
C-4, and C-5 carbons is essential for this function. The suggestion has been made that this alcohol moiety is oxidized in mitochon-
dria to a carbonyl moiety, with the generation of reactive oxygen species. However, there is no established precedent for the apop-
totic performance of 3-ketoceramides thus presumed. In this work, we have synthesized three different types of short-chain
3
-ketoceramides, that is, (2S,4E)-2-acetylamino-3-oxo-4-octadecen-1-ol (A), (2S,4E,6E)-2-acetylamino-3-oxo-4,6-octadecadien-1-
ol (B), and (2S,4E)-2-acetylamino-1-methoxy-3-oxo-4-octadecene (C), and demonstrated that these 3-ketoceramides are capable
of inducing effective apoptosis in human leukemia HL-60 cells. In particular, the two monoenoic compounds, A and C, are far more
powerful than the corresponding alcoholic analogue, N-acetyl-D-erythro-sphingosine. Observations of DNA fragmentation, cas-
pase-3 activation, and cytochrome c release from mitochondria provide substantiated evidence for mitochondrial apoptosis and
the effects of exogenous glutathione on these phenomena are also discussed.
ꢀ
2007 Elsevier Ltd. All rights reserved.
1. Introduction
stoylamino)-1-phenyl-1-propanol, N-oreoylethanolamine,
8
etc. could also increase endogenous ceramide levels.
,9
Sphingolipids are ubiquitous membrane components of
all eukaryotic cells and play a physiologically important
role in bioorganisms. The metabolites or precursors
Some exogenous cell-permeable ceramides such as N-ac-
etyl- (C2-Cer) and N-octanoyl-D-erythro-sphingosine
(C8-Cer) induce apoptosis, even when exogenously add-
1
of sphingolipids, ceramides (N-acyl-D-erythro-sphingo-
sines), are an important second messenger involved in
mediating a variety of cell functions, especially, apopto-
1
0
ed. In particular, C2-Cer is widely used as a standard
tool for the study of apoptosis. Related dihydrocera-
mides, which lack the 4,5-trans C–C double bond, are
2
sis. Some biosubstances and environmental stresses,
3
such as the anti-Fas antibody, tumor necrosis factor,
10
biologically inactive. Meanwhile, the mitochondrial
4
5
a and ionizing radiation, promote apoptotic ceramide
generation by enhancing the mitochondrial sphingomy-
elinase-mediated hydrolysis of sphingomyelin (the
oxidation of the C-3 hydroxy group of ceramides to pro-
duce the corresponding 3-ketoceramides together with
the reactive oxygen species (ROS) has been proposed
6
11
so-called ‘sphingomyelin cycle’). Exogenously added
bacterial sphingomyelinase is capable of inducing apop-
as the primary step of ceramide-induced apoptosis.
For the purpose of proving this assumption, Chun
et al. synthesized 4,6-diene-ceramide which, compared
to normal ceramide, is expected to be readily oxidized
7
tosis by increasing endogenous ceramide levels. In addi-
tion, daunorubicin, an anti-cancer reagent, which
activates neutral sphingomyelinase, or glucosylcera-
mide-synthase inhibitors such as D-erythro-2-(N-myri-
1
2
to the dienone analogue in mitochondria, and Struck-
hoff et al. indicated that 4,6-diene-ceramides are more
potent than ceramide in inducing apoptosis via the mito-
1
3
chondrial pathway in MCF-7 cells. It is implied,
accordingly, that the Michael addition of mitochondrial
glutathione (GSH) to the dienone product would cause
a decreased concentration of the ROS scavenger GSH,
and then an increase in ROS concentration, which
Keywords: 3-Ketoceramides; C2-ceramide; Apoptosis; Mitochondria;
Antileukemic; HL-60 cells.
*
Corresponding author. Tel.: +81 6 6605 2168; fax: +81 6 6605
167; e-mail: azumah@bioa.eng.osaka-cu.ac.jp
2
0
968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.02.008

H. Azuma et al. / Bioorg. Med. Chem. 15 (2007) 2860–2867
2861
increase is responsible for apoptotic cell death. Howev-
O
O
a)
er, no detailed study has been reported so far, for not
only the cell-death inducing ability of the 3-ketocera-
mide but also the formation of the 3-ketoceramide-
GSH adduct.
O
C11H23
HO
C11H23
NBoc
NH Cl
3
1
2
O
b)
In this work, we examined whether the allylic ketone part
of 3-ketoceramides is critical for the cell death event in the
human leukemia HL-60 cell. For this purpose, three new
HO
C11H23
NHAc
c)
B
3
-ketoceramides with a short acyl-chain were prepared:
2S,4E)-2-acetylamino-3-oxo-4-octadecen-1-ol (A, Fig. 1),
2S,4E,6E)-2-acetylamino-3-oxo-4,6-octadecadien-1-ol (B),
and (2S,4E)-2-acetylamino-1-methoxy-3-oxo-4-octadecene
C).
O
O
(
(
d)
HO
OMe
MeO
OMe
NHBoc
3
NHBoc
(
4
O
O
P
O
OMe
OMe
e)
MeO
MeO
C11H23
2. Syntheses
NHBoc
NHBoc
5
6
The short-chain 3-ketoceramide
through its precursor, (2S,4E)-3-ketosphingosine
A
was obtained
O
f)
hydrochloride (prepared according to a previous meth-
1
MeO
C11H23
4
od ). The N-acetylation of the HCl salt with acetic
acid using WSC in the presence of N-hydroxybenzo-
NHAc
C
triazole (HOBt) and Et N gave compound A in 57%
3
Scheme 1. Reagents and conditions: (a) 10% HCl, MeOH, reflux; (b)
AcOH, WSC, HOBt, Et N, CH Cl , rt; (c) MeI, Ag O, acetone, reflux;
d) LiCH PO(OMe) N, LiCl,
, THF, ꢀ78 ꢁC; (e) tetradecanal, Et
THF, rt; (f) TFA, rt, then Ac O, pyridine, rt.
yield. The diene analogue B was derived from N,O-
protected dienone 1 (prepared according to a previous
3
2
2
2
(
2
2
3
1
2
method ) by its O-deprotection with 10% HCl fol-
lowed by N-acetylation according to the procedure
mentioned above, giving the desired compound B in
2
3
4% yield (Scheme 1).
The 1-methoxy analogue C was prepared following
1
5
Scheme 1. (2S)-N-Boc-serine methyl ester 3
was
O-methylated with methyl iodide in the presence of sil-
ver oxide to provide the N,O-protected serine ester 4
in 61% yield. The obtained serine ester 4 was treated
with excess lithium dimethyl methylphosphonate to be
converted to the b-ketophosphonate 5 in 69% yield.
The Horner–Wadsworth–Emmons (HWE) olefination
of the phosphonate 5 with tetradecyl aldehyde using
OH
OH
HO
C13H27 HO
C13H27
HN
C H
HN
C H
n 2n+1
n
2n+1
O
O
Et N as a base in the presence of anhydrous LiCl at
3
Ceramides
n = 1, C2-Cer)
n = 7, C8-Cer)
Dihydroceramdes
ꢀ
78 ꢁC in THF afforded N-Boc-1-O-methyl-3-ketosp-
(
(
hingosine 6 in 70% yield. After removal of the Boc
protection with trifluoroacetic acid (TFA), the resulting
amine was immediately acetylated to give compound C
in 72% yield.
O
HO
C11H23
NHAc
3. Biological evaluations and discussion
A
B
C
3
.1. Cell death
O
Evaluation of the antileukemic property of the newly
synthesized C2-3-ketoceramides (compounds A, B, and
C) was made by MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-
diphenyl-2H-tetrazolium bromide] assay. HL-60 cells
were treated for 6 h with several different concentrations
HO
C11H23
NHAc
(1–20 lM) of each analogue. As Figure 2 shows, the C2-
O
3-ketoceramides were all high in activity compared with
C2-Cer, a widely used apoptosis inducer. In particular,
compounds A and C maintained high levels of activity
even at a low concentration such as 1 lM, where about
MeO
C11H23
NHAc
70% of the cells were brought to death in 6 h. The fact
that both A and its 1-methoxy derivative C exhibit near-
ly the same activity suggests that the hydroxy moiety at
Figure 1. Structures of short-chain ceramides and 3-ketoceramides.

2862
H. Azuma et al. / Bioorg. Med. Chem. 15 (2007) 2860–2867
1
20
00
The dienone B caused no fragmentation even after 2 h.
These results are consistent with those of the MTT assay
mentioned above.
1
1 μM
5
5 μM
1
1
0
0 μM
20 μM
20
1
3
.3. Caspase-3 activation
8
6
4
2
0
0
0
0
0
Caspases, a family of aspartate-specific cysteine proteas-
es, play a crucial role in apoptotic cell death by cleaving a
specific site of numerous cellular targets in the execution
phase; in particular, caspase-3 could be activated by the
proteolytic processing of pro-caspase-3 in response
to exogenous apoptosis inducers. In order to elucidate
the involvement of caspase-3 in the active form in the
C2-3-ketoceramides-induced cell death process in the
HL-60 cell, we attempted to analyze the amount of
caspase-3 activated in response to exogenously added
C2-3-ketoceramides by the Western blot analysis
employing a polyclonal anti-caspase-3 antibody. The
upper panels of Figure 4a display time courses of 2–6 h
for the generation of active caspase-3 induced by the
addition of the three C2-3-ketoceramides (5 lM) or
C2-Cer (5 or 20 lM), indicating that all of the 3-keto-
ceramides can activate caspase-3 within 2 h and their
activity exceeds that of C2-Cer; in particular, caspase-3
activation treated with compound A or C was completed
in 2 h. Figure 5 shows time courses of 30–90 min for
caspase-3 activation by 5 lM of the C2-3-ketoceramides.
The activated caspase-3 appeared after 1 h treatment
with compounds A and C. We also investigated whether
poly(ADP-ribose) polymerase (PARP), a proteolytic
target of the activated caspase-3, could be cleaved by
the 3-ketoceramides. As the lower panels of Figure 4a
indicate, any analogue can cleave PARP after a delay
of the caspase-3 activation.
A
B
C
C2-Cer EtOH
Figure 2. Antileukemic effects of short-chain ceramide and 3-keto-
ceramides against HL-60 cells. Cells were treated with the indicated
concentrations of each compound for 6 h.
the 1-position plays no critical role against the antileu-
kemic cell death event. The activity of the dienone B
was intermediate between those of A and C2-Cer, and
fairly close to that of the former rather than the latter.
3
.2. DNA fragmentation
Next, we investigated whether C2-3-ketoceramides
could induce DNA fragmentation, a typical phenome-
non accompanying apoptosis. Figure 3 shows that
DNA fragmentation with 5 lM of A and C was com-
pleted within just 2 h, which leads us to expect that com-
pounds A and C will be promising, practical apoptosis
inducers. However, C2-Cer was so much less active as
to require four times the concentration and more than
It has been generally accepted that ceramides induce
apoptosis in HL-60 cells through a mitochondrial sig-
naling pathway. Although C2-3-ketoceramides acti-
vate caspase-3, there is the possibility that the executor
caspase-3 could be directly activated by the initiator cas-
pase-8 in such a way that the Fas-mediated apoptosis
takes the non-mitochondrial pathway. To determine
whether C2-3-ketoceramide-induced apoptosis adopts
the non-mitochondrial pathway, HL-60 cells were pre-
treated with the caspase-8-specific inhibitor Ac-IETD-
cho or the broad caspase inhibitor Z-VAD-fmk for
4
h to accomplish the same extent of fragmentation.
1
6
1
7
3
0 min before addition of compound A. Caspase-3 acti-
vation and subsequent PARP cleavage were completely
inhibited by Z-VAD-fmk, but not inhibited at all by Ac-
IETD-cho (Fig. 4b). Although caspase-8 can also acti-
vate the mitochondrial pathway of apoptosis by cleaving
Bid, this result demonstrates that the caspase-3 media-
tion by 3-ketoceramide A does not pass through cas-
pase-8 activation.
3
.4. Mitochondrial pathway
In order to determine the contribution of the mitochon-
drial pathway when HL-60 cells were treated with the
C2-3-ketoceramides as well as C2-Cer, Western blot
analysis was performed. The results are summarized in
Figure 6a, which shows that the mitochondrial cyto-
chrome c release from the mitochondria to the cytoplasm
Figure 3. Agarose gel electrophoresis of DNA following treatment of
HL-60 cells with short-chain ceramide and 3-ketoceramides. HL-60
6
cells (1 · 10 cells/mL) were exposed to C2-3-ketoceramides (5 lM) or
C2-Cer (20 lM) for various periods. Each genomic DNA was
subjected to agarose gel electrophoresis; M, marker; E, ethanol vehicle.

H. Azuma et al. / Bioorg. Med. Chem. 15 (2007) 2860–2867
2863
a
C2-Cer
EtOH
4
A
B
C
5 μM
20 μM
2
6
2
4
6
2
4
6
2
4
6
2
4
6
2
4
6 (h)
Pro-caspase-3
Active caspase-3
PARP
Cleaved PARP
b
A
+ Ac-IETD-cho
+ Z-VAD-fmk
2
5 μM
50 μM
25 μM
50 μM
3
0 60 90 30 60 90 30 60 90
30 60 90 30 60 90 (min)
Pro-caspase-3
Active caspase-3
PARP
Cleaved PARP
Figure 4. Activation of caspase-3 and PARP cleavage induced by C2-3-ketoceramides. (a) HL-60 cells were treated with C2-3-ketoceramides (5 lM)
or C2-Cer (5 or 20 lM) for various periods (2–6 h); (b) HL-60 cells were preincubated with Ac-IETD-cho or Z-VAD-fmk for 30 min and then
incubated with compound A (5 lM). Each cell extract was subjected to Western blot analysis using anti-caspase-3 or anti-PARP antibody.
In fact, markedly enhanced apoptosis of HL-60 cells
E
A
B
by extracellularly added C2-3-ketoceramides appears
to support the above scenario. Furthermore, in order
to understand the vital role of GSH in C2-3-ketocera-
mide-mediated apoptosis in more detail, we examined
the influence of pretreatment of the cells with GSH on
the dynamics of two successive processes, that is, the
mitochondria-to-cytoplasm cytochrome c release and
the caspase-3 activation. The results are displayed in
Figures 5 and 6, which indicate that GSH pretreatment
indeed was able to largely suppress both of those consec-
utive processes in the C2-3-ketoceramide-driven apopto-
sis. However, GSH cannot be a depressor for releasing
mitochondrial cytochrome c, because the C2-Cer-driven
apoptosis was not affected by GSH pretreatment at all,
as anticipated also from the above scenario.
GSH
+
–
+
–
+
1.5 0.5 1 1.5 0.5 1 1.5 0.5 1 1.5 0.5 1 1.5 (h)
Pro-caspase-3
Active caspase-3
E
C
C2-Cer
GSH
+
–
+
–
4
+
4
6
0.5 1 1.5 0.5 1 1.5
2
6
2
6 (h)
Pro-caspase-3
Active caspase-3
Figure 5. Inhibition effect of C2-3-ketoceramides-induced caspase-3
activation by extracellularly supplemented GSH. HL-60 cells were
pretreated with GSH (10 mM) for 30 min and then incubated with C2-
3
-ketoceramides (5 lM) or C2-Cer (20 lM). Each cell extract was
One possible explanation for these suppressions is as fol-
lows: unlike C2-Cer having no keto group, 3-ketocera-
mides undergo Michael addition with GSH during
their migration to the mitochondria, and then, the
Michael addition lowers the effective concentration of
the 3-ketoceramides which triggers the cell death
signaling. This explanation might have been supported
by the result of Schultz et al. that 0.1375 mM GSH
underwent Michael addition to simple a,b-enone
compounds at rather high concentrations of the milli-
subjected to Western blot analysis using anti-caspase-3; E, ethanol
vehicle.
takes place in a time-dependent manner, as with the re-
sult of caspase-3 activation. Thus, the immunoblot dem-
onstrates also that the C2-3-ketoceramides can give rise
to rapid mitochondrial apoptosis of HL-60 cells.
1
8
3
.5. Exogenous GSH effect
molar order in pH 7.4 aqueous buffered solution.
However, no remarkable data have been presented for
the Michael addition to 3-ketoceramides themselves.
Figure 7a shows a considerable decreasing change in
230 nm absorbance due to the enone A in the presence
of GSH, strongly suggesting that enone A has the capa-
bility of reacting with GSH even under the conditions of
UV measurements (0–2 mM GSH in pH 7.4 buffered
solution at 37 ꢁC). Thus, we could acquire chemical
Radin has recently proposed that the oxidation of the
C-3 hydroxy group of ceramides by mitochondrial ubi-
quinone in complex III could produce the corresponding
ketoceramides together with ROS. The 3-ketocera-
mide thus formed would trap GSH through Michael
addition. As a result of the GSH deletion, ROS would
be allowed to increase, eventually leading to apoptosis.
1
1

2864
H. Azuma et al. / Bioorg. Med. Chem. 15 (2007) 2860–2867
a
b
E
A
B
C
E
C2-Cer
6 (h)
2
0.5 1 2 (h)
0.5 1 2 (h)
0.5 1 2 (h)
6
2
4
Cytochrome
Cytochrome
β-actin
c
c
(
C)
(M
)
Cytochrome
Cytochrome
β-actin
c
c
(
C)
(M
)
Figure 6. Cytochrome c release from mitochondria to the cytoplasm induced by C2-3-ketoceramides. (a) HL-60 cells were treated with C2-3-
ketoceramides (5 lM) or C2-Cer (20 lM) for various periods, and both the cytosolic (C) and mitochondrial (M) fractions were subjected to Western
blot analysis using anti-cytochrome c antibody; (b) the same experiments were carried out after pretreatment with 10 mM GSH for 30 min; E, ethanol
vehicle.
On the other hand, the observed diminution in the die-
none B absorbance at 280 nm, the results being revealed
a 0.30
Compound A
0.25
0.20
0.15
0.10
0.05
0.00
also from Figure 7b, is not attributed to the Michael
addition, but merely to a self-decomposition of the die-
none substrate in buffer, because time-tracing of absor-
bance change is independent of the GSH concentration.
It is evident, therefore, that the relatively low activity of
compound B versus A can be favorably accounted for
entirely by this instability.
+
+
+
+
GSH 0 mM
GSH 0.2 mM
GSH 1 mM
GSH 2 mM
Thus, we acquired definite, chemical evidence for the
Michael addition of enone A and GSH in aqueous buf-
fered solution. However, it is still unclear whether the
anti-apoptotic effect of additional GSH on enone A-in-
duced apoptosis is due to the reduction in effective
0
1
2
3
4
5
6
7
Times (h)
3
-ketoceramide level by the Michael addition, because
0
0
0
0
0
0
0
0
0
.16
.14
.12
.10
.08
.06
.04
.02
.00
b
the anti-apoptotic effect of exogenous GSH was also
observed in the case of dienone B. Further pursuing
of the detailed mechanism of the 3-ketoceramide-
induced apoptosis is currently in progress in this
laboratory.
Compound B
3
.6. Summary
+
+
+
GSH 0 mM
GSH 2 mM
GSH 10 mM
We demonstrated, for the first time, that 3-ketocera-
mides have a high apoptotic activity against HL-60 cells,
and are now studying apoptosis by C2-3-ketoceramides
against other cell lines. For example, compound A also
shows a great activity over C2-Cer in HeLa cells (data
not shown). Some synthetic methods for N,O-protected
0
10
20
30
Times (min)
Figure 7. Effects of GSH on C2-3-ketoceramides. Compounds A
(0.2 mM) and B (0.05 mM) were incubated with GSH (0–10 mM) in
3
cursors of D-erythro-sphingosine,
-ketosphingosines have been reported as utilizable pre-
14,19
because, as com-
aqueous buffer solutions (pH 7.4) for the indicated times at 37 ꢁC and
each absorbance (a: 230 nm, b: 280 nm) was plotted.
pared with a normal ceramide (or sphingosine) having
two chiral centers, 3-ketoceramide (or 3-ketosphingo-
sine) having only one chiral center is easily prepared.
3-Ketoceramides may be a better motif than normal
ceramides for developing new ceramide-like anti-cancer
agents, accordingly.
evidence for the Michael reaction of the enone A and
GSH in such concentrations that are close to the intra-
cellular GSH concentration.

H. Azuma et al. / Bioorg. Med. Chem. 15 (2007) 2860–2867
2865
4
. Experimental
15.4 Hz); HRMS (FAB, direct) calcd for C H NO ,
1
8
34
2
+
[
MꢀCl] 296.259; found: 296.2586.
All materials obtained commercially (guaranteed re-
agent grade) were used without further purification.
All solvents were freshly distilled under nitrogen before
use; THF and CH Cl were distilled from LiAlH and
4.3. (2S,4E,6E)-2-acetylamido-3-oxo-4,6-octadecadien-
1-ol (B)
2
2
4
CaH , respectively. Column chromatography was per-
2
The reaction was carried out as described for C2-keto-
Cer, using 2 (50 mg, 151 lmol) to give compound B as
a powdery solid (22 mg, 44%). The specific rotation of
B could not be measured accurately, because the com-
pound was decomposed by irradiation with a sodium
formed on silica gel. C2-Cer was prepared according
2
0
to our previous report. Protease inhibitors, Z-VAD-
fmk and Ac-IETD-CHO, were purchased from the Pep-
tide Institute (Osaka, Japan) and Sigma Chemical Co.
(
St. Louis, MO, USA), respectively. Polyclonal antibody
lamp in CHCl ; mp 79 ꢁC; IR (KBr) 3233, 2927, 2851,
3
for caspase-3 and monoclonal antibody for cytochrome
c were purchased from Santa Cruz Biotechnology (San-
ta Cruz, CA, USA). Monoclonal antibodies for PARP
and for b-actin were purchased from Trevigen (Gai-
thersburg, MD, USA) and Abcam (Cambridge, UK),
respectively. Anti-rabbit and anti-mouse IgGs coupled
to alkaline phosphatase were purchased from Sigma
Chemical Co. (St. Louis, MO, USA).
1684, 1630, 1752, 1466, 1379, 1283, 1144, 1069, 995,
ꢀ
1 1
723 cm ; H NMR (400 MHz, CDCl ); d 0.88 (t, 3H,
3
J = 6.8 Hz), 1.26 (br s, 16H), 1.39–1.75 (m, 2H), 2.09
(s, 3H), 2.21 (q, 2H, J = 7.1 Hz), 3.4 (br s, 1H), 3.81–
3.89 (m, 1H), 3.92–4.02 (m, 1H), 4.9 (ddd, 1H, J = 3.2,
4.6, 6.1 Hz), 6.22 (dd, 1H, J = 10.5, 15.1 Hz), 6.24 (d,
1H, J = 15.1 Hz), 6.32 (dt, 1H, J = 15.1, 6.8 Hz), 6.83
(d, 1H, J = 6.1 Hz), 7.44 (dd, 1H, J = 10.5, 15.1 Hz)+;
HRMS (FAB, direct) calcd for C H NO , [M+H]
2
0
36
3
4
.1. (2S,4E)-2-acetylamido-3-oxo-4-octadecen-1-ol (A)
338.2695; found: 338.2691; Anal. Calcd: C, 71.18; H,
0.45; N, 4.15. Found: C, 70.92; H, 10.51; N, 4.15.
1
To a suspension of acetic acid (14 mg, 0.234 mmol),
WSC (45 mg, 0.234 mmol), and HOBt (36 mg,
4.4. (S)-(N-Boc-O-methyl)serine methyl ester (4)
0
.234 mmol) in dry CH Cl (10 mL), a solution of
2 2
1
4
15
(
2S,4E)-2-amino-3-oxo-4-octadecen-1-ol Æ HCl (60 mg,
.18 mmol) and Et N (27 mg, 0.27 mmol) in dry CH Cl
2
(S)-N-Boc-serine methyl ester 3 (2.3 g, 10.5 mmol) was
dissolved in dry acetone (50 mL) containing methyl
0
3
2
(
2 mL) was added dropwise, and the mixture was stirred
iodide (15 mL) and then Ag O (3.77 g, 16.3 mmol) was
2
overnight at room temperature. The resulting mixture
was washed with saturated brine, dried with Na SO ,
and then concentrated. The obtained residue was puri-
fied by column chromatography with hexane/EtOAc
added. The mixture was refluxed and stirred overnight.
The solids were removed by filtration and the solution
was concentrated. The residue was purified by column
chromatography with hexane/EtOAc (4:1) to give 4
2
4
2
0
(
1:3) and recrystallized from hexane to give compound
(1.31 g, 54%) as colorless oil; ½aꢁ +12.2 (c 1.76, CHCl3);
D
IR (NaCl) 3447, 2980, 1749, 1720, 1506, 1456, 1367,
2
0
A (35 mg, 57%) as a colorless powder; mp 64 ꢁC; ½aꢁ
D
9.26 (c 1.05, CHCl ); IR (KBr) 2918, 2851, 2350,
697, 1682, 1568, 1470, 1379, 1302, 1267, 1161, 1067,
ꢀ1
1
+
1167, 1119, 1063, 980, 874, 779 cm
;
H NMR
3
1
9
3
2
3
(400 MHz, CDCl ); d 1.45 (s, 9H), 3.34 (s, 3H), 3.59
3
ꢀ
1 1
70, 719 cm ; H NMR (400 MHz, CDCl ); d 0.88 (t,
(dd, 1H, J = 3.2, 9.3 Hz), 3.77 (s, 3H) 3.8 (dd, 1H,
J = 3.2, 9.3 Hz), 4.38–4.44 (m, 1H), 5.37 (d, 1H,
J = 8 Hz); HRMS (FAB, direct) calcd for C H NO ,
3
H, J = 6.8 Hz), 1.26 (br s, 20H), 1.44–1.52 (m, 2H),
.08 (s, 3H), 2.26 (q, 2H, J = 6.8 Hz), 3.36 (br s, 1H),
.8–3.88 (m, 1H), 3.93–4.01 (m, 1H), 4.91 (ddd, 1H,
1
0
20
5
+
[M+H] 234.1341; found: 234.134.
J = 3.6, 4.8, 6 Hz), 6.27 (d, 1H, J = 15.6 Hz), 6.8 (d,
H, J = 6 Hz), 7.11 (dt, 1H, J = 15.6, 7.2 Hz); HRMS
1
4.5. (3S)-[3-(tert-butoxycarbonyl)amino-4-methoxy-2-
oxo-butyl]phosphonic acid methyl ester (5)
+
FAB, direct) calcd for C H NO : [M+H] 340.2852;
(
found: 340.2852; Anal. Calcd: C, 70.75; H, 10.98; N,
2
0
38
3
4
.13. Found: C, 70.45; H, 11.05; N, 4.13.
To a solution of methylphosphonic acid dimethyl ester
6.14 g, 49.5 mmol) in dry THF (150 mL), n-BuLi
(1.6 M in hexane, 30.9 mL, 49.5 mmol) was added drop-
(
4.2. (2S,4E,6E)-2-amino-3-oxo-4,6-octadecadien-1-ol
hydrochloride (2)
wise at ꢀ78 ꢁC under N . After the mixture was stirred
2
for 30 min at ꢀ78 ꢁC, a solution of 4 (3.85 g, 16.5 mmol)
in dry THF (20 mL) was added dropwise. The mixture
was stirred for 1 h at ꢀ78 ꢁC and then warmed up to
0 ꢁC. The reaction mixture was poured into ice-cooled
1 M HCl and extracted with EtOAc. After drying with
Na SO , the solution was concentrated, and the residue
1
2
To a solution of 1 (210 mg, 0.48 mmol), in MeOH
20 mL), 10% HCl (1.2 mL) was added, and the mixture
(
was stirred for 1 h at reflux. After cooling, the resulting
mixture was washed with hexane and then concentrated.
The crude product was recrystallized from i-PrOH/Et O
2
2
4
to give 2 (80 mg, 50%) as a colorless powder; mp 130 ꢁC;
was purified by column chromatography with CHCl3/
MeOH (20:1) to give 5 (1.18 g, 65%) as colorless oil;
2
5
½
aꢁ +7.35 (c 1, MeOH); IR (KBr) 3420, 2922, 2853,
D
435, 1684, 1636, 1601, 1468, 1213, 1057, 1005,
2
0
2
7
3
2
1
1
½aꢁ +35.3 (c 1.8, CHCl ); IR (NaCl) 3300, 2977, 1711,
D
1520, 1367, 1252, 1169, 1117, 1032, 866, 812 cm ; H
3
ꢀ
1
1
ꢀ1
1
21 cm
;
H NMR (400 MHz, CD OD); d 0.88 (t,
3
H, J = 6.8 Hz), 1.29 (br s, 16H), 1.42–1.52 (m, 2H),
.24 (q, 2H, J = 6.8 Hz), 3.95 (dd, 1H, J = 4.9,
2.2 Hz), 4.02 (dd, 1H, J = 3.7, 12.2 Hz), 4.39–4.43 (m,
H), 6.28–6.46 (m, 3H), 7.44 (dd, 1H, J = 10.2,
NMR (400 MHz, CDCl ); d 1.45 (s, 9H), 3.18 (dd, 1H,
3
J = 14.8, 22 Hz), 3.35 (s, 3H), 3.38 (dd, 1H, J = 14.8,
22 Hz), 3.59 (dd, 1H, J = 4.4, 9.6 Hz), 3.78 (dd, 3H,
J = 1.2, 2.8 Hz), 3.81 (dd, 3H, J = 1.2, 2.8 Hz), 3.85

2866
H. Azuma et al. / Bioorg. Med. Chem. 15 (2007) 2860–2867
(
dd, 1H, J = 4.0, 9.6 Hz), 4.47–4.53 (m, 1H), 5.58 (d, 1H,
A, B, C and C2-Cer were dissolved in EtOH at 1–
20 mM, and added to the cells as ethanolic solutions.
The final concentration of EtOH was 0.1%. Control
experiments were performed with EtOH (0.1%) as the
vehicle.
J = 7.2 Hz); HRMS+ (FAB, direct) calcd for
C H NO P, [M+H] 326.1369; found: 326.1362.
1
2
25
7
4
3
.6. (2S,4E)-2-[(tert-butoxycarbonyl)amino]-1-methoxy-
-oxo-4-octadecene (6)
4
.9. MTT assay
To a solution of dimethyl methanephosphonate 3 (1.6 g,
.92 mmol), tetradecanal (2.09 g, 9.84 mmol) and
anhydrous LiCl (625 mg, 14.8 mmol), Et N (1.49 g,
4
The cell viability was assessed by the reduction of MTT
[3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium
bromide]. MTT is a water-soluble tetrazolium salt that is
reduced by metabolically viable cells to a colored, water
3
14.8 mmol) was added dropwise at room temperature
under N . The mixture was stirred overnight at room
temperature. The reaction mixture was treated with
2
5
insoluble formazan salt. 4 · 10 cells/100 lL were plated
1
was dried with Na SO and concentrated. The residue
M HCl and extracted with EtOAc. The organic layer
in 96-well dishes. All of the ketoceramide analogues and
C2-Cer were diluted into each well at the indicated con-
centrations. Ten microliters of 5 mg/mL MTT was add-
ed to each well 2 h before the end of the culture, and
reaction was stopped by adding 100 lL of 0.04 M HCl
in isopropanol. Two optical densities of the mixture
were measured at 570 and 650 nm. Final values were
obtained by subtracting 650 nm from 570 nm. All
data were the average of at least three separate
determinations.
2
4
was purified by column chromatography with hexane/
EtOAc (10:1) to give 6 (1.42 g, 70%) as a waxy solid;
20
mp 39 ꢁC; ½aꢁ +10.4 (c 1.84, CHCl ); IR (NaCl) 3325,
D
918, 2853, 1722, 1693, 1472, 1366, 1173, 1123, 1043,
3
2
1
0
1
ꢀ
1 1
026, 978, 716 cm ; H NMR (400 MHz, CDCl ); d
3
.88 (t, 3H, J = 6.8 Hz), 1.26 (br s, 20H), 1.45 (br s,
1H), 2.23 (dq, 2H, J = 1.4, 7.1 Hz), 3.32 (s, 3H), 3.63
(
dd, 1H, J = 4.4, 9.8 Hz), 3.75 (dd, 1H, J = 3.6,
9
6
7
.8 Hz), 4.59–4.65 (m, 1H), 5.57 (d, 1H, J = 7.6 Hz),
.28 (d, 1H, J = 15.6 Hz), 7.01 (dt, 1H, J = 15.6,
.1 Hz); HRMS (FAB, direct) calcd for C H NO ,
4.10. DNA fragmentation
2
4
46
4
+
6
[
M+H] 412.343; found: 412.3427.
HL-60 cells (2 · 10 cells/2 mL) were treated with C2-
ketoceramides (5 lM) or C2-Cer (20 lM) for various
periods. Cells were collected by centrifugation at 200g
for 5 min at 4 ꢁC. Cells were lysed in 100 lL of a lysis
buffer [10 mM Tris–HCl (pH 7.4), 10 mM EDTA, and
0.5% Triton X-100]. Soluble cell lysates were collected
by centrifugation at 14,000g for 5 min. Cell lysates were
treated for 1 h at 37 ꢁC with RNase A (0.2 mg/mL). Pro-
teinase K (0.2 mg/mL) was added, and the sample was
incubated at 50 ꢁC for 30 min. 5 M NaCl (20 lL) and
isopropanol (120 lL) were added and the sample was
incubated at ꢀ20 ꢁC for one night. DNA pellets were
collected by centrifugation at 14,000g for 15 min and
dissolved in 20 lL of a TE buffer [10 mM Tris–HCl
(pH 7.4), 1 mM EDTA]. The DNA was then electropho-
resed at 50 V through 2.0% agarose gel. The DNA
bands were visualized under UV light after staining with
ethidium bromide.
4.7. (2S,4E)-2-acetylamido-1-methoxy-3-oxo-4-octade-
cene (C)
The carbamate 6 (200 mg, 0.486 mmol) was dissolved in
TFA (2 mL). After stirring for 2 h at room temperature,
the solvent was evaporated in vacuo. The resulting ami-
ne Æ TFA salt was suspended in dry CH Cl (5 mL). Ace-
2
2
tic anhydride (149 mg, 3 equiv) and Et N (1 mL) were
3
added dropwise to the suspension, and then the mixture
was stirred for 2 h at room temperature. The reaction
mixture was washed with 1 M HCl, and the organic
layer was dried with Na SO and concentrated. The res-
2
4
idue was purified by column chromatography with
CHCl /MeOH (20:1) and recrystallized from hexane to
give compound C (124 mg, 72%) as a powdery solid;
3
20
mp 79 ꢁC; ½aꢁ +7.87 (c 1.06, CHCl ); IR (KBr) 3321,
D
922, 2849, 1699, 1632, 1529, 1468, 1379, 1188, 1121,
3
2
1
0
2
3
1
1
1
ꢀ
1 1
086, 972, 723 cm ; H NMR (400 MHz, CDCl ); d
4.11. Immunoblot analysis for whole cell extraction
3
.88 (t, 3H, J = 6.8 Hz), 1.26 (br s, 20H), 1.42–1.52 (m,
H), 2.06 (s, 3H), 2.25 (q, 2H, J = 6.8 Hz), 3.31 (s,
H), 3.67 (dd, 1H, J = 4, 10 Hz), 3.77 (dd, 1H, J = 3.6,
0 Hz), 4.94 (ddd, 1H, J = 3.6, 4.0, 7.2 Hz), 6.26 (d,
H, J = 15.6 Hz), 6.58 (d, 1H, J = 7.2 Hz), 7.04 (dt,
6
HL-60 cells (2 · 10 cells/2 mL) were treated with C2-3-
ketoceramides (5 lM) or C2-Cer (5 or 20 lM) for
various periods. Cells were collected by centrifugation
at 200g for 5 min at 4 ꢁC, washed twice with phosphate
buffer (pH 7.4), and lysed in 100 mL of lysis buffer
[62.5 mM Tris–HCl (pH 6.8), 6 M urea, 10% glycerol,
2% SDS, 0.00125% bromophenol blue, and 5% b-
mercaptoethanol]. Cell lysates were boiled for 3 min
and separated on 8% (for PARP) or 14% (for cas-
pase-3) SDS–polyacrylamide gels, transferred to a
polyvinylidene difluoride membrane, and probed with
rabbit polyclonal anti-caspase-3 antibody or monoclo-
nal anti-PARP antibody, followed by probing with
goat anti-rabbit antibody (for Caspase-3) or anti-
mouse antibody (for PARP) coupled to alkaline
phosphatase.
H, J = 15.6, 7.2 Hz); HRMS (FAB, direct) calcd for
+
C H NO , [M+H] 354.3008; found: 354.3004; Anal.
2
1
40
3
Calcd: C, 71.34; H, 11.12; N, 3.96. Found: C, 71.26;
H, 11.14; N, 3.95.
4
.8. Cell culture
Human leukemia HL-60 cells were grown in RPMI 1640
medium containing 10% heat-incubated fetal bovine ser-
um. On the day of the experiment, cells were washed
twice in serum-free RPMI 1640 and resuspended in the
6
serum-free medium (0.4–1 · 10 cells/mL). Compounds

H. Azuma et al. / Bioorg. Med. Chem. 15 (2007) 2860–2867
2867
4.12. Preparation of cytosolic and mitochondrial fractions
and immunoblot analysis
800; (b) Okazaki, T.; Kondo, T.; Kitano, T.; Tashima, M.
Cell Signal. 1998, 10, 685; (c) Manna, S. K.; Sah, N. K.;
Aggarwal, B. B. J. Biol. Chem. 2000, 275, 13297; (d)
Birbes, H.; Bawab, S. E.; Obeid, L. M.; Hannun, Y. A.
Advan. Enzyme Regul. 2002, 42, 113; (e) Tomassini, B.;
Testi, R. Biochimie 2002, 84, 123.
. Cifone, M. G.; Maria, R. D.; Roncaioli, P.; Rippo, M. R.;
Azuma, M.; Lanier, L. L.; Santoni, A.; Testi, R. J. Exp.
Med. 1994, 180, 1547.
We prepared cytosolic and mitochondrial fractions
1
2
6
according to Lee’s method. HL-60 cells (6 · 10 cells/
mL) were treated as described above, collected by cen-
6
3
trifugation at 200g for 5 min at 4 ꢁC, and washed twice
with phosphate buffer (pH 7.4). The cell pellet was resus-
pended in ice-cold cell extraction buffer [20 mM HEPES
4. Kim, M.; Linardic, C.; Obeid, L.; Hannun, Y. J. Biol.
Chem. 1991, 266, 484.
(
pH 7.5), 10 mM KCl, 1.5 mM mgCl , 1 mM EDTA,
2
1
mM EGTA, 1 mM dithiothreitol, PMSF 100 lM,
5. Haimovitz-Friedman, A.; Kan, C. C.; Ehleiter, D.;
Persaud, R. S.; McLoughlin, M.; Fuks, Z.; Kolesnick,
R. N. J. Exp. Med. 1994, 180, 525.
including protease inhibitor cocktail], and then homoge-
nized with a glass Dounce B-type pestle (120 strokes).
Unbroken cells, pellet nuclei, and heavy membranes were
removed by centrifuging the homogenates at 1000g for
5
3
vy membrane fraction, and the resulting supernatant was
further centrifuged at 100,000g to obtain the cytosolic
fraction. The mitochondria-rich fraction was washed
once with a cell extraction buffer and then solubilized in
a lysis buffer [150 mM NaCl, 50 mM Tris–HCl (pH 7.4),
6. Hannun, Y. A. J. Biol. Chem. 1994, 269, 3125.
7. Jarvis, W. D.; Kolesnick, R. N.; Fornari, F. A.; Traylor,
R. S.; Gewirtz, D. A. Proc. Natl. Acad. Sci. U.S.A. 1994,
min. The supernatant was centrifuged at 14,000g for
0 min to provide a pellet of mitochondria-enriched hea-
9
1, 73.
8
. Jaffrezou, J. P.; Levade, T.; Bettaieb, A.; Andrieu, N.;
Bezombes, C.; Maestre, N.; Vermeersch, S.; Rousse, A.;
Laurent, G. EMBO J. 1996, 15, 2417.
9. Bielawska, A.; Greenberg, M. S.; Perry, D.; Jayadev, S.;
Shayman, J. A.; McKay, C.; Hannun, Y. A. J. Biol. Chem.
1
996, 271, 12646.
1
0. (a) Karasavvas, N.; Erukulla, R. K.; Bittman, R.;
Lockshin, R. Eur. J. Biochem. 1996, 236, 729; (b)
Jarvis, W. D.; Fornari, F. A., Jr.; Traylor, R. S.;
Martin, H. A.; Kramer, L. B.; Erukulla, R. K.; Bittman,
R.; Grant, S. J. Biol. Chem. 1996, 271, 8275; (c) Chang,
Y. T.; Choi, J.; Ding, S.; Prieschl, E. E.; Baumruker, T.;
Lee, J. A.; Chung, S. K.; Schultz, P. G. J. Am. Chem.
Soc. 2002, 124, 1856.
1
% NP-40, 0.25% sodium deoxycholate, and 1 mM
EGTA]. Cytosolic and mitochondrial fractions remained
frozen in tubes at ꢀ80 ꢁC until use. Western blot analysis
was carried out by using mouse monoclonal anti-cyto-
chrome c antibody, as described above.
4
.13. UV spectrometric analysis
1
1
1
1. (a) Radin, N. S. Med. Hypotheses 2001, 57, 96; (b) Radin,
N. S. Bioorg. Med. Chem. 2003, 11, 2123.
2. Chun, J.; Li, G.; Byun, H. S.; Bittman, R. J. Org. Chem.
Freshly prepared before use were 0.1 M HEPES buffers
pH 7.4) containing 0.2, 2, and 10 mM of reduced GSH.
The aliquots of the GSH solutions were taken up in
(
2
002, 67, 2600.
3. 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.
0
3
.5 mL-Eppendorf tubes, incubated at 37 ꢁC. The
-ketoceramides were diluted into each tube at the indi-
cated concentrations. The 3-ketoceramide-free GSH
solutions were also incubated and used as the blank.
After incubating at 37 ꢁC for the indicated times, each
absorbance at 230 nm (for compound A) or 280 nm
14. Lee, J. M.; Lim, H. S.; Chung, S. K. Tetrahedron:
Asymmetry 2002, 13, 343.
15. Garner, P.; Park, J. M. J. Org. Chem. 1987, 52, 2361.
16. Kim, H. J.; Mun, J. Y.; Chun, Y. J.; Choi, K. H.; Kim, M.
Y. FEBS Lett. 2001, 505, 264.
17. Choi, C.; Benveniste, E. N. Brain Res. Brain Res. Rev.
(
1
for compound B) was measured on a NanoDrop ND-
000 spectrophotometer.
2
004, 44, 65.
1
1
8. Schultz, T. W.; Yarbrough, J. W.; Johnson, E. L. SAR
QSAR Environ. Res. 2005, 16, 313.
9. Koskinen, P. M.; Koskinen, A. M. Methods Enzymol.
2000, 311, 458.
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2
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