(3Z)-2-Acetylamino-3-octadecen-1-ol as a potent apoptotic agent against HL-60 cells

Article abstract of DOI:10.1016/j.bmc.2003.10.040

(2R,3Z)-, (2R,3E)-, (2S,3Z) and (2S,3E)-2-Acetylamino-3-octadecen-1-ol, and (2R)- and (2S)-2-acetylamino-octadecan-1-ol were prepared using the Wittig olefination of Garner's aldehyde (N-Boc-N,O-isopropylidene-L- or D-serinal) from L- or D-serine. The apoptotic activities of these saturated and unsaturated 2-acetylaminoalcohols were examined in human leukemia HL-60 cells using MTT assay. Among the newly synthesized compounds, the cis-isomers were the most potent. Despite their simple structures, (2R,3Z)- and (2S,3Z)-2-acetylamino-3-octadecen-1-ol showed high and comparable apoptotic activities compared with N-acetyl-D-erythro-sphingosine (D-e-C2-Cer, a well-known inducer of apoptosis). Their apoptotic activities were in the order D-e-C2-Cer≈L-e-C2-Cer≈(2R,3Z)-≈(2S,3Z)-?(2R,3E)-≈(2S,3E)-≈(2R) -≈(2S)-derivative. Qualitative analysis of DNA fragmentation caused by these compounds was conducted using agarose gel electrophoresis, and typical DNA fragmentation was found in the cases of (2R,3Z)- and (2S,3Z)-isomers such as C2-Cer, but not trans and saturated isomers. The morphological features of the cells, the proteolytic processing of pro-caspase-3, and the cleavage of PARP as a result of exogenous treatment with (2R,3Z)- and (2S,3Z)-isomers indicated that cell death induced by these compounds was apoptosis. These observations suggest that these newly synthesized compounds, (3Z)-2-Acetylamino-3-octadecen- 1-ol, have similar characteristics and apoptosis-inducing activities against HL-60 cells with C2-Cer.

Full text of DOI:10.1016/j.bmc.2003.10.040

Bioorganic & Medicinal Chemistry 12 (2004) 45–51
(3Z)-2-Acetylamino-3-octadecen-1-ol as a potent apoptotic agent
against HL-60 cells
Hayato Niiro, Hideki Azuma,* Shinsuke Tanago, Kiyohiro Matsumura, Keiji Shikata,
Taro Tachibana and Kenji Ogino*
Department of Applied & Bioapplied Chemistry, Graduate School of Engineering, Osaka City University, Sugimoto 3-3-138,
Sumiyoshi-ku, Osaka 558-8585, Japan
Received 17 September 2003; revised 20 October 2003; accepted 21 October 2003
Abstract—(2R,3Z)-, (2R,3E)-, (2S,3Z) and (2S,3E)-2-Acetylamino-3-octadecen-1-ol, and (2R)- and (2S)-2-acetylamino-octadecan-
1-ol were prepared using the Wittig olefination of Garner’s aldehyde (N-Boc-N,O-isopropylidene-l- or d-serinal) from l- or d-
serine. The apoptotic activities of these saturated and unsaturated 2-acetylaminoalcohols were examined in human leukemia HL-60
cells using MTT assay. Among the newly synthesized compounds, the cis-isomers were the most potent. Despite their simple
structures, (2R,3Z)- and (2S,3Z)-2-acetylamino-3-octadecen-1-ol showed high and comparable apoptotic activities compared with
N-acetyl-d-erythro-sphingosine (D-e-C2-Cer, a well-known inducer of apoptosis). Their apoptotic activities were in the order d-e-
C2-Cerꢀl-e-C2-Cerꢀ(2R,3Z)-ꢀ(2S,3Z)-> >(2R,3E)-ꢀ(2S,3E)-ꢀ(2R)-ꢀ(2S)-derivative. Qualitative analysis of DNA fragment-
ation caused by these compounds was conducted using agarose gel electrophoresis, and typical DNA fragmentation was found in
the cases of (2R,3Z)- and (2S,3Z)-isomers such as C2-Cer, but not trans and saturated isomers. The morphological features of the
cells, the proteolytic processing of pro-caspase-3, and the cleavage of PARP as a result of exogenous treatment with (2R,3Z)- and
(2S,3Z)-isomers indicated that cell death induced by these compounds was apoptosis. These observations suggest that these newly
synthesized compounds, (3Z)-2-Acetylamino-3-octadecen-1-ol, have similar characteristics and apoptosis-inducing activities against
HL-60 cells with C2-Cer.
# 2003 Elsevier Ltd. All rights reserved.
1. Introduction
ceramides, and they are used as apoptosis-inducing
agents for a wide variety of cancer cells.6 It has recently
been reported that C2-Cer treatment activates a family
of asparate-specific cysteine proteases, called caspases,
which are intimately associated with apoptosis and which
cleave a number of substrates including poly(ADP-ribose)
polymerase (PARP).7
Ceramide (N-acyl-d-erythro-sphingosine) is an impor-
tant lipid messenger involved in mediating a variety of
cell functions including apoptosis, cell cycle arrest and
cell senescence.1 Apoptosis induced by a variety of
inducers such as tumor necrosis factor-a (TNF-a), Fas
ligation, chemotherapeutic agents, and environmental
stress is associated with the hydrolysis of sphingomyelin
accompanied by the accumulation of ceramide.2ꢁ5
Although the downstream of ceramide signaling is still
not known, ceramide-induced apoptosis has been well
characterized, and a growing body of work is in pro-
gress to identify the targets of ceramide (ceramide
binding proteins) that can mediate ceramide action. C2-
Ceramide (N-acetyl-d-erythro-sphingosine, (2S,3R,4E)-
2-acetylamino-4-octadecen-1,3-diol, D-e-C2-Cer) and
Many syntheses of ceramide and its isomers as well as of
various analogues have been reported in the past few
decades.8 For examples, Chang et al. reported the
synthesis and biological characterization of the cer-
amide library (528 ceramide analogues) against human
leukemia U937 cells,8a and Kishida et al. studied the
structural requirements of 4,5-cis-, 4,5-trans- or the 4,5-
acetylene-type analogues of C6-ceramide (4 analogues) for
apoptosis-inducing activity against HL-60 cells.8b In these
studies, the fundamental structures inducing activities had
2-acylamino-4,5-unsaturated-1,3-alkandiol.8a,b Both the
cis isomer and acetylene-type derivative of C6-ceramide
analogues were more active than the trans isomer in HL-60
cells.8b Furthermore, synthetic C6 - phytoceramide
C8-Ceramide
(N-octanoyl)
are
cell-permeable
Keywords: Ceramide; Apoptosis; HL-60 cells; Antileukimic activity.
* Corresponding authors. Tel.: +81-6-6605-2799; fax: +81-6-6605-
2167; e-mail: ogino@bioa.eng.osaka-cu.ac.jp
0968-0896/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2003.10.040

46
H. Niiro et al. / Bioorg. Med. Chem. 12 (2004) 45–51
(N-hexanoyl-d-ribo-phytosphingosine) induced higher
levels of apoptosis than C6-Cer in SK-N-BE(2)C cate-
cholaminergic neuroblastoma cells.9
2.2. Biological properties
To examine the antileukemic activities of the analogues
of C2-ceramide, we analyzed cell death by using HL-60
cells. The percentages of cell death were determined
using MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-
2H-tetrazolium bromide] assay.14 Time- and dose-
dependent cell death induced by the incubation of
HL-60 cells with these synthesized (2R)-compounds are
shown in Figure 2. RZ-2- and D-e-C2-Cer- (positive
control for the cell death) induced cell death depended
on incubation time and concentration. Despite its sim-
ple structure, RZ-2 showed comparable activity com-
pared with C2-Cer. Its trans-isomer RE-3 and saturated
analogue R-1 showed no cell death. These results indi-
cate that cis configuration, not trans, of double bond on
the 3,4-position of their structures is necessary for anti-
leukemic activity.
We reported recently the apoptotic activities of
C2-homo-ceramide and C2-bishomo-ceramide,10 sym-
bioramide derivatives11 and N-lactyl-sphingosine12 to
elucidate structure–reactivity relationships in ceramide-
mediated apoptosis.
From a preliminary screening in our laboratory, we
found that a new categorical compound, (2R,3Z)-2-
acetylamino-3-octadecen-1-ol, RZ-2, showed high
apoptotic activity against human leukemia HL-60 cells.
Surprisingly, this compound does not have a similar
structure to (2S,3R,4E)-2-acetylamino-4-octadecen-1,3-
diol (D-e-C2-Cer, a well-known inducer of apoptosis).
Thus, in this paper, we report the syntheses and anti-
leukemic activities of the stereo- and regio-isomers of
RZ-2 as well as their dihydro-analogues.
These structures of 2-acetylamino-3-alken-1-ols, RZ-2,
RE-3, SZ-2 and SE-3 are simpler than that of D-e-C2-
Cer. RZ-2 is not structurally similar to D-e-C2-Cer, but
the structure of RE-3 seems to lack the hydroxy-
methylene moiety of the 3 position that is present in that
of D-e-C2-Cer. In addition, these compounds have only
one chiral center on the 2-acetylaminomethylene carbon
and lack the secondary hydroxyl group. The antileu-
kemic activities of these synthesized compounds were
measured using HL-60 cells, and RZ-2 and SZ-2 indi-
cate high and comparable apoptotic activities compared
with D-e-C2-Cer. The characteristic behaviors of RZ-2
and SZ-2 against HL-60 cells indicate that cell death is
caused by the apoptotic pathway.
2. Results and discussion
2.1. Chemistry
Scheme 1. Reagents and conditions; (a) TFA-H2O (20:1), rt, then
Ac2O, pyridine, rt; (b) K2CO3, EtOH–H2O (4:1), rt; (c) PtO2, H2,
EtOH, rt.
In this study, (2R,3Z)- RZ-2, (2R,3E)- RE-3, (2S,3Z)-
SZ-2, and (2S,3E)-2-acetylamino-3-octadecen-1-ol SE-3
and (2R)- R-1 and (2S)-2-acetylaminooctadecan-1-ol S-
1 were prepared using our previous method.13 A syn-
thetic approach to R-derivatives is outlined in Scheme 1.
From the Wittig olefination of Garner’s aldehyde (N-
Boc-N,O-isopropylidene-l-serinal) from l-serine with
C15H31PPh3Br/LiHMDS, we obtained an 88:12 mixture
of (Z)- and (E)-isomers (RZ-4 and RE-5, respectively)
1
based on H NMR analysis. RZ-4 and RE-5 were care-
fully separated using column chromatography. 2-
Acetylamino compounds RZ-2 and RE-3 were obtained
by the acidic hydrolyses of the corresponding acetonide
derivatives, by acetylation with acetic anhydride in pyr-
idine, followed by the alkaline hydrolysis of the primary
acetoxy groups of RZ-6 and RE-7, respectively. Satu-
rated compound R-1 was prepared as described above,
after the catalytic hydrogenation of RZ-4.
Their corresponding (2S)-stereo-isomers, S-1, SZ-2 and
SE-3 were also synthesized using similar methods from
d-serine.
Figure 1.

H. Niiro et al. / Bioorg. Med. Chem. 12 (2004) 45–51
47
To confirm that cell death induced by these compounds
was apoptosis, we analyzed DNA fragmentation. Figure 3
shows the electrophoretic analysis of extracted DNA
from the cells cultured with 20 mM of R-compounds for
7 h. Treatments with D-e-C2-Cer and RZ-2 showed the
typical DNA ladder. Consistent with the results shown
in Figure 2, RZ-2 induced DNA fragmentation as large
as D-e-C2-Cer did. Therefore, RZ-2 showed comparable
apoptotic activity compared with D-e-C2-Cer.
is important for apoptotic activity. We synthesized S-1,
SZ-2, SE-3 and L-e-C2-Cer, and we analyzed the
apoptotic activities of these compounds. Cell death was
determined by treatment with 20 mM compounds
against HL-60 cells after 8 h (Fig. 4), and nuclear frag-
mentation of HL-60 cells was determined after 6 h (Fig. 5).
As D-e-C2-Cer, L-e-C2-Cer, RZ-2 and SZ-2 had almost
equal apoptotic activities, cell death showed a similar
trend to the R- or S-conformation of the 2 position. It is
reported that the cell toxicity of L-e-C2-Cer is similar to
D-e-C2-Cer.8a,g These results show that the stereo-
chemistry of the 2 position of the C2-ceramide analo-
gues, RZ-2 and SZ-2, is not necessarily essential for
apoptosis-inducing activity against HL-60 cells as well
As these compounds such as C2-Cer have one chiral
center on the 2-acetylaminomethylene position, we
hypothesized that the stereo-chemistry of the 2 position
Figure 3. Agarose gel electrophoresis of DNA following treatment
of HL-60 cells with ceramide analogues. HL-60 cells (1ꢂ106 cells/mL)
were exposed to R-analogue of C2-ceramide (20 mM) or ethanol
vehicle. After 7 h, genomic DNA was subjected to agarose gel
electrophoresis.
Figure 2. Dose- and time-dependent HL-60 cell death induced by R-
analogues of C2-ceramide. (A) Cells were incubated with 5–20 mM
concentrations for 7 h. (B) Cells were incubated with 20 mM of R-
analogues of C2-ceramide and values are the average of at least three
separate experiments.
Figure 4. Percent of cell death after a 8 h treatment with 20 mM R-
and S-analogue of C2-ceramide in HL-60 cells. Values are the average
of at least three separate experiments.

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H. Niiro et al. / Bioorg. Med. Chem. 12 (2004) 45–51
as C2-Ceramide. The trans isomers, RE-3 and SE-3, and
the dihydro-isomers, R-1 and S-1, show almost no
activities.
ment.16 To elucidate the involvement of caspases in
these compound-induced apoptotic processes, the acti-
vation of caspase-3 and the cleavage of PARP was
analyzed in HL-60 cells. The proteolytic processing of
pro-caspase-3 and the cleavage of PARP in response to
the exogenous analogues of C2-ceramide was examined
by Western blotting using a polyclonal anti-caspase-3
antibody or an anti-PARP antibody. The cells by treat-
ments with D-e-C2-Cer and RZ-2 generated the active
form of caspase-3 and the cleaved form of PARP time
dependently (Fig. 6). These results show that RZ-2
induced apoptosis as well as C2-ceramide.
These finding indicate that the ceramide-mediated
apoptotic pathway includes caspase-3 activation. Cas-
pase-3 is the most efficient PARP-cleaving caspase. This
caspase is synthesized as a 32 kDa precursor that, after
cleavage, results in the 17 kDa subunit (the active
form).15 PARP is cleaved to generate an 85 kDa frag-
The structure–reactivity relationships for the biological
action of ceramide are being elucidated. In the case of
C6-ceramide, it has been clarified that the apoptosis
activities of the cis isomer and triple bond-compound
are stronger than the activity of the trans isomer, and it
is important for activities to have unsaturated bonds.8b
Furthermore, it has been reported that the direction of
the 2-acetylamino group in C2-Cer is not always
important for apoptotic activities.6,10 This paper shows,
we showed that newly synthesized compounds,
(2R,3Z)- RZ-2 and (2S,3Z)-2-acetylamino-3-octadecen-
1-ol SZ-2, have similar characteristics and apoptotic
activities to C2-Cer. As the synthesis of these com-
pounds is simpler than that of C2-Cer, RZ-2 and SZ-2
may be used to create new anti-cancer agents and for
the research of ceramide-induced apoptosis, although
the reason for the high apoptotic activities of RZ-2 and
SZ-2 and no activity of RE-3 and SE-3 are unclear at
present.
Furthermore, 2-acetylamino-3-octadecen-1-ol is not an
intermediate of the biosynthetic process, and neither is
it a not the degraded product in the metabolic process
of sphingolipids. Therefore, these compounds may be
possible inhibitors of biosynthetic and/or metabolic
enzymes of sphingolipids.
The findings of this study are summarized as follows:
(2R,3Z)- (RZ-2) and (2S,3Z)-2-Acetylamino-3-octade-
cen-1-ol (SZ-2), not ceramide, strongly induces cell death
with similar activities to D-e-C2-Cer and L-e-C2-Cer at
low concentrations. These cis isomers, RZ-2 and SZ-2
were more active than the trans isomers, RE-3 and SE-3.
RZ-2 and SZ-2, which have only one chiral center at the
2 position showed almost the same apoptotic activities;
therefore, the activity of this new class of compounds
Figure 5. Morphological features of HL-60 cells after treatment with
(A) D-e-C2-Cer; (B) L-e-C2-Cer; (C) RZ-2; (D) SZ-2; (E) ethanol
vehicle after 6 h.
Figure 6. Time-dependent Western blot analysis of caspase-3 and PARP in HL-60 cells with the R-analogue of C2-ceramide. Cell extracts after
treatment with each ceramide analogues (20 mM) were resolved by SDS-PAGE and probed with anti-caspase-3 or anti-PARP antibody.

H. Niiro et al. / Bioorg. Med. Chem. 12 (2004) 45–51
49
depends on geometrical stereochemistry rather than
optical stereochemistry.
3.3. (2R,3Z)-2-acetamino-3-octadecen-1-ol (RZ-2)
The diacetate RZ-6 (650 mg, 1.77 mmol) was stirred
with anhydrous K2CO3 (294 mg, 1.2 equiv) in EtOH (12
mL) and water (3 mL) for 24 h at room temperature.
The reaction mixture was diluted with water and
extracted with CHCl3. The organic phase was dried with
Na2SO4, and concentrated in vacuo. The residue was
purified by column chromatography with CHCl3–
MeOH (10:1) and recrystallized from n-hexane–AcOEt
to give RZ-2 (490 mg, 85%) as a powdery solid; mp
74 ꢃC; [a]D25=+7.68ꢃ (c 1.61, CHCl3); IR (NaCl) 3283,
2918, 2851, 1645, 1589, 1464, 1375, 1310, 1271, 1101,
The morphological changes in cells, DNA degradation,
and the formation of the activated caspase-3 by treat-
ment with RZ-2 and SZ-2 clearly indicates that cell death
is induced by apoptosis as in the case of D-e-C2-Cer.
3. Experimental
All materials were obtained commercially (guaranteed
reagent grade) and used without further purification.
Column chromatography was performed on silica gel.
1
1061, 1028, 1061, 752, 605 cmꢁ1; H NMR (400 MHz,
CDCl3); d 0.88 (t, 3H, J=6.8 Hz), 1.26 (brs, 22H), 1.34–
1.42 (m, 2H), 2.01 (s, 3H), 2.12 (dt, 2H, J=7.3, 6.8 Hz),
3.25 (t, 1H, J=5.9 Hz), 3.56–3.70 (m, 2H), 4.72–4.82
(m, 1H), 5.30 (dd,1H, J=9.3, 10.7 Hz), 5.61 (dt, 1H,
J=10.7, 7.8 Hz), 5.86 (d, 1H, J=5.9 Hz); HRMS (FAB,
direct) calcd for C20H40O2N: [M+H]+ 326.3059.
Found: 326.3058 (100%). Anal. calcd: C, 73.79; H,
12.08; N, 4.3. Found: C, 73.85; H, 12.13; N, 4.25; The
enantiomer SZ-2 was also prepared using compound
SZ-6; [a]2D5=ꢁ7.51ꢃ (c 1.4, CHCl3); HRMS (FAB,
direct) calcd for C20H40O2N: [M+H]+ 326.3059.
Found: 326.3056 (100%). Anal. calcd: C, 73.79; H,
12.08; N, 4.3. Found: C, 73.76; H, 12.06; N, 4.29.
3.1. (2R,3Z)-1-acetoxy-2-acetamino-3-octadecene (RZ-6)
To the oxazolidine compound RZ-413 (1g, 2.36 mmol)
was added a solution of trifluoroacetic acid (16 mL) and
water (0.8 mL). After 1h, the solvent was evaporated in
vacuo and a saturated aqueous NaHCO3 was added.
The mixture was extracted with CHCl3, dried with
Na2SO4 and concentrated. To the residue dissolved in
pyridine (10 mL) was added acetic anhydride (964 mg,
9.44 mmol). After a day of stirring at room tempera-
ture, the solvent was removed in vacuo. A saturated
aqueous NaHCO3 was added to the residue and the mix-
ture was extracted with CHCl3. The organic layer was
dried with anhydrous Na2SO4 and concentrated. Pur-
ification by column chromatography with CHCl3 and
recrystallized from n-hexane–AcOEt to give RZ-6 (0.77 g,
89%) as a solid; mp 64 ꢃC; [a]D25=+8.95ꢃ (c 1.42, CHCl3);
IR (NaCl) 3273, 2918, 2849, 1746, 1647, 1558, 1537, 1466,
3.4. (2R,3E)-2-acetamino-3-octadecen-1-ol (RE-3)
The reaction was carried out as described above, using
RE-7 (500 mg, 1.36 mmol) to give RE-3 (400 mg, 90%)
as a powdery solid; mp 85–86 ꢃC; [a]D25=ꢁ15.67ꢃ (c
1.862, CHCl3); IR (NaCl) 3270, 2918, 2851, 1647, 1614,
1
1373, 1304, 1259, 1043, 743, 721, 604 cmꢁ1; H NMR
(400MHz, CDCl3); d 0.88 (t, 3H, J=6.8 Hz), 1.25 (brs,
24H), 1.98 (s, 3H), 2.07 (s, 3H), 2.14 (q, 2H, J=6.8 Hz),
4.05 (dd, 1H, J=4.9, 11.2 Hz), 4.11 (dd, 1H, J=6.4, 11.2
Hz), 4.95–5.04 (m, 1H), 5.25 (dd,1H, J=9.3, 10.2 Hz),
5.60 (dt, 1H, J=10.2, 7.8 Hz), 5.61 (d, 1H, J=7.3 Hz);
HRMS (FAB, direct) calcd for C22H42O3N: [M+H]+
368.3165. Found: 368.3167 (100%); The enantiomerSZ-6
was also prepared using compound SZ-4; [a]2D5=ꢁ9.02ꢃ (c
1.27, CHCl3); HRMS (FAB, direct) calcd for C22H42O3N:
[M+H]+ 368.3165. Found: 368.3168 (100%).
1564, 1470, 1373, 1086, 1067, 1043, 978, 719, 625 cmꢁ1
;
1H NMR (400 MHz, CDCl3); d 0.88 (t, 3H, J=6.8 Hz),
1.26 (brs, 22H), 1.34–1.42 (m, 2H), 1.97–2.07 (m, 2H),
2.03 (s, 3H), 3.03 (t, 1H, J=5.9 Hz), 3.57–3.65 (m,1H),
3.65–3.72 (m, 1H), 4.45–4.52 (m, 1H), 5.39 (dd,1H,
J=5.9, 15.6 Hz), 5.69 (dt, 1H, J=15.6, 6.4 Hz), 5.99 (d,
1H, J=7.3 Hz); HRMS (FAB, direct) calcd for
C20H40O2N: [M+H]+ 326.3059. Found: 326.3056
(100%). Anal. calcd: C, 73.79; H, 12.08; N, 4.3. Found:
C, 73.68; H, 12.10; N, 4.30; The enantiomer SE-3 was
also prepared using compound SE-7; [a]2D5=+15.0ꢃ (c
1.12, CHCl3); HRMS (FAB, direct) calcd for
C20H40O2N: [M+H]+ 326.3059. Found: 326.3053
(100%). Anal. calcd: C, 73.79; H, 12.08; N, 4.3. Found:
C, 73.80; H, 12.09; N, 4.27.
3.2. (2R,3E)-1-acetoxy-2-acetamino-3-octadecene (RE-7)
The reaction was carried out as described above, using
RE-5 (1g, 2.36 mmol) to give RE-7 (0.73 g, 84%) as a
solid; mp 100 ꢃC; [a]D25=ꢁ7.45ꢃ (c 1.01, CHCl3); IR
(NaCl) 3285, 2918, 2851, 1723, 1653, 1558, 1474, 1464,
3.5. (2R)-1-acetoxy-2-acetamino-3-octadecane (R-9) via
(2R)-2-[(tert-Butoxycarbonyl)amino]-1,2-O,N-isopropyl-
ideneoctadecan-1-ol (RZ-4)
1391, 1373, 1370, 1165, 1042, 976, 762, 719, 600 cmꢁ1
;
1H NMR (400 MHz, CDCl3); d 0.88 (t, 3H, J=6.8 Hz),
1.26 (brs, 22H), 1.30–1.40 (m, 2H), 1.98–2.06 (m, 2H),
1.99 (s, 3H), 2.06 (s, 3H), 4.06 (dd, 1H, J=4.4, 11.2 Hz),
4.17 (dd,1H, J=6.3, 11.2 Hz), 4.66–4.76 (m, 1H), 5.35
(dd,1H, J=6.3, 15.6 Hz), 5.67 (dt, 1H, J=15.6, 6.8 Hz),
5.68 (d, 1H, J=8.3 Hz); HRMS (FAB, direct) calcd for
C22H42O3N: [M+H]+ 368.3165. Found: 368.3168
(56%); The enantiomer SE-7 was also prepared using
compound SE-5; [a]D25=+ 7.86ꢃ (c 1.1, CHCl3); HRMS
(FAB, direct) calcd for C22H42O3N: [M+H]+ 368.3165.
Found: 368.3169 (76%).
To the solution of RZ-4 (1g, 2.36 mmol) in EtOH (20
mL) was added a catalytic amount of PtO2 (25 mg, 0.11
mmol) and the mixture was stirred overnight at room
temperature under H2. The reaction mixture was filtered
over Celite and concentrated in vacuo. The obtained
crude R-8 was used without further purification. The
next reaction was carried out as described above to give
R-9 (690 mg, 79%) as a powdery solid; mp 109.5 ꢃC;
[a]2D5=+22ꢃ (c 1.09, CHCl3); IR (KBr) 3287, 2918,

50
H. Niiro et al. / Bioorg. Med. Chem. 12 (2004) 45–51
2851, 1784, 1651, 1558, 1462, 1373, 1254, 1159, 1043,
1
3.9. DNA Fragmentation
719 cmꢁ1; H NMR (400 MHz, CDCl3); d 0.88 (t, 3H,
J=6.8 Hz), 1.25 (brs, 28H), 1.36–1.54 (m, 2H), 1.99 (s,
3H), 2.08 (s, 3H), 4.03 (dd, 1H, J=3.4, 10.7 Hz), 4.15
(dd, 1H, J=5.4, 10.7 Hz), 4.14–4.21 (m, 1H), 5.45 (d,
1H, J=8.3 Hz); HRMS (FAB, direct) calcd for
C22H44O3N: [M+H]+ 370.3321. Found: 370.3318
(22%); The enantiomer S-9 was also prepared using
compound SZ-4; [a]D25=ꢁ20.3ꢃ (c 1.18, CHCl3); HRMS
(FAB, direct) calcd for C22H44O3N: [M+H]+ 370.3321.
Found: 370.3325 (67%).
HL-60 cells (2ꢂ106 cells/2 mL) were plated in 6-well
dishes. Compounds were dissolved in ethanol at stock
concentration of 20 mM and then diluted with serum
free medium. After an 7 h incubation, the cells were
collected by centrifugation at 2500 rpm for 5 min at
4 ꢃC. Cells were lysed in 100 mL of lysis buffer (10 mM
Tris–HCl; pH 7.4, 10 mM EDTA; pH 8.0, and 0.5%
Triton X-100). Soluble cell lyses were collected by cen-
trifugation at 15,000 rpm for 5 min. Cell lysates were
treated for 1h at 37 ꢃC with RNase A (0.2 mg/mL).
Proteinase K (0.2 mg/mL) was added, and sample was
incubated at 50 ꢃC for 30 min. 5 M NaCl (20 mL) and
isopropanol (120 mL) were added and the sample was
incubated at ꢁ20 ꢃC for one night. DNA pellets were
collected by centrifugation at 15,000 rpm for 15 min,
and dissolved in 20 mL of a TE buffer (10 mM Tris–
HCl; pH 7.4, 1mM EDTA; pH 8.0). The DNA was
then electrophoresed at 50 V through 2.0% agarose gel.
The DNA bands were visualized under UV light after
staining with ethidium bromide.
3.6. (2R)-2-acetamino-octadecan-1-ol (R-1)
The reaction was carried out as described above for RZ-
2, using compound R-9 (440 mg, 1.19 mmol) to give R-1
(360 mg, 92%) as a powdery solid; mp 104 ꢃC;
[a]2D5=+13.0ꢃ (c 2.37, CHCl3); IR (KBr) 3280, 2910,
2849, 1649, 1622, 1556, 1472, 1443, 1373, 1323, 1153,
1086, 721 cmꢁ1; 1H NMR (400 MHz, CDCl3); d 0.88 (t,
3H, J=6.8 Hz), 1.26 (brs, 28H), 1.36–1.57 (m, 2H), 2.00
(s, 3H), 2.62–2.68 (m, 1H), 3.52–3.59 (m, 1H), 3.64–3.71
(m, 1H), 3.87–3.96 (m, 1H) 5.62 (d, 1H, J=5.9 Hz).
Anal. calcd: C, 73.34; H, 12.62; N, 4.28. Found: C,
73.26; H, 12.57; N, 4.25; HRMS (FAB, direct) calcd for
C20H42O2N: [M+H]+ 328.3216. Found: 328.3216
(100%). Anal. calcd: C, 73.34; H, 12.62; N, 4.28.
Found: C, 73.34; H, 12.57; N, 4.25; The enantiomer S-1
was also prepared using compound S-9; [a]2D5=ꢁ13.15ꢃ
(c 1.68, CHCl3); HRMS (FAB, direct) calcd for
C20H42O2N: [M+H]+ 328.3216. Found: 328.3219
(100%). Anal. calcd: C, 73.34; H, 12.62; N, 4.28.
Found: C, 73.13; H, 12.69; N, 4.25.
3.10. Staining of apoptotic nuclei
Morphological changes in the nuclear chromatin of cells
undergoing apoptosis were detected by staining with the
40,6-diamidino-2-phenylindole (DAPI). HL-60 cells
were treated with compounds as described above. After
an 6 h incubation, cells were fixed with 3.7% formalde-
hyde in phosphate buffered saline (PBS) for 30 min at
room temperature. Thereafter, the cells were stained
with the DAPI (1 mg/mL) in PBS for 30 min at room
temperature. Condensed nuclei were detected by Olym-
pus IX-71fluorescence microscopy.
3.7. Cell culture
Human pronyelocytic leukemia HL-60 cells were grown
in RPMI 1640 medium (Sigma) containing 5% heat-
incubated fetal bovine serum supplemented with 100
units/mL penicillin and 100 mg/mL streptomycin. On
the day of the experiment, cells were washed once in
serum-free RPMI 1640 and resuspended in the serum-free
medium (0.4–1ꢂ106 cells/mL). The cells were treated with
various compounds at 37 ꢃC, and an MTT assay was run
or DNA fragmentation was observed. All the compounds
were dissolved in ethanol. Control experiments were per-
formed with ethanol (0.1%) as the vehicle.
3.11. Immunoblot analysis
HL-60 cells were treated with compounds as described
‘DNA Fragmentation’. After an incubation, cells were
collected by centrifugation at 4000 rpm for 5 min at
4 ꢃC, washed twice with PBS and lysed in 100 mL of SDS
sample buffer (62.5 mM Tris–HCl; pH 6.8, 6 M urea, 10%
glycerol, 2% SDS, 0.00125% bromophenol blue, 5% b-
mercaptoethanol). Cell lysates were boiled for 5 min and
separated on 12% SDS-polyacrylamide gels, transferred to
Immobilon-P transfer membrane (Millipore), and probed
with rabbit polyclonal anti-caspase-3 antibody (Santa
Cruz Biotechnology), followed by goat anti-rabbit anti-
body coupled to alkaline phosphatase (Sigma).
3.8. MTT assay
The cell death was assessed by the reduction of 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT). MTT is a water-soluble tetrazolium salt that is
reduced by metabolically viable cells to a colored, water
insoluble formazan salt. 4ꢂ104 cells/100 mL were plated
in 96-well dishes. The cells were treated with various
compounds dissolved in ethanol. The final concentration
of ethanol was 0.1%. 10 mL of 5 mg/mL MTT was
added to each well before 2 h from the end of the cul-
ture, and reactions were stopped by adding 100 mL of
0.04 N HCl in isopropanol. The absorbance at a wave-
length of 570 nm was measured. All results were deter-
mined in triplicate.
For PARP immnoblots, cell lysates were separated on
8% SDS-polyacrylamide gels, transferred to Immobi-
lon-P transfer membrane, and probed with monoclonal
mouse anti-human PARP (Trevigen), followed by goat
anti-mouse antibody coupled to alkaline phosphate
(Sigma).
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