Synthesis of novel phytosphingosine derivatives and their preliminary biological evaluation for enhancing radiation therapy

Article abstract of DOI:10.1016/j.bmcl.2007.09.037

Eight d-ribo-phytosphingosine derivatives were synthesized from d-ribo-phytosphingosine and diverse acyl chlorides with N,N-diisopropylethylamine in tetrahydrofuran for 1 h at room temperature. Effect of these compounds on IR-induced cell death was evaluated on blood cancer cells (Jurkat). Among these, 3d showed the highest enhancement of radiosensitizing effect.

Full text of DOI:10.1016/j.bmcl.2007.09.037

Bioorganic & Medicinal Chemistry Letters 17 (2007) 6643–6646
Synthesis of novel phytosphingosine derivatives and their
preliminary biological evaluation for enhancing radiation therapy
Byung Seok Moon,a,d Moon-Taek Park,b Jeong Hoon Park,c Sang Wook Kim,c
Kyo Chul Lee,a Gwang Il An,a Seung Dae Yang,c Dae Yoon Chi,d
Gi Jeong Cheona,* and Su-Jae Leeb,e,
*
aLaboratory of Radiopharmaceuticals, Korea Institute of Radiological and Medical Sciences, 215-4 Gongneungdong, Nowongu,
Seoul 139-706, Republic of Korea
bLaboratory of Radiation Experimental Therapeutics, Korea Institute of Radiological and Medical Sciences, 215-4 Gongneungdong,
Nowongu, Seoul 139-706, Republic of Korea
cRadiation Application Division, Korea Atomic Energy Research Institute, Jeonggeup-si, Jeollabuk-do 580-185, Republic of Korea
dDepartment of Chemistry, Inha University, 253 Yonghyundong Namgu, Inchon 402-751, Republic of Korea
eDepartment of Chemistry, Hanyang University, 17 Haengdangdong, Seongdonggu, Seoul 133-791, Republic of Korea
Received 23 May 2007; revised 31 August 2007; accepted 8 September 2007
Available online 14 September 2007
Abstract—Eight D-ribo-phytosphingosine derivatives were synthesized from D-ribo-phytosphingosine and diverse acyl chlorides with
N,N-diisopropylethylamine in tetrahydrofuran for 1 h at room temperature. Effect of these compounds on IR-induced cell death was
evaluated on blood cancer cells (Jurkat). Among these, 3d showed the highest enhancement of radiosensitizing effect.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Anticancer therapy is largely classified into surgery,
radiation, and chemotherapy. Alkylating agents, antibi-
otics, antimetabolites, plant derivatives, and steroids are
used in anticancer chemotherapy, for example, Cis-
platin, Doxorubicin, Pentostatin, Taxol, and Dexameth-
asone. It is known that these anticancer drugs, however,
have a limited activity against the common solid tumors
due to side effects that can damage normal cells. In addi-
tion, chemoresistance or recurrence of solid tumors
brings about serious problems in cancer therapy.1
Therefore, radiation therapy is widely utilized for cancer
treatment.2,3 Radiation therapy, however, leads to some
problems such as radioresistance of cancer cells and
damage to normal cells, which result in decreasing radi-
ation therapy efficiency. In this regard, considerable
efforts have been made to develop radiosensitizers for
increasing the radiation therapy efficiency and
attempted to increase radiosensitivity in several solid
tumors such as breast cancer, uterine cervical cancer,
lung cancer, gastric cancer, and large intestine cancer
using Taxol, 5-FU, and Cisplatin that are presently
known as radiosensitizing agents.4 These radiosensitiz-
ing agents, however, have a serious side effect and/or
can be applied only to specific cancer cells.
Phytosphingosine (1, (2S,3S,4R)-2-aminooctadecan-
1,3,4-triol) consists of a long-chain base with a 2-ami-
no-1,3,4-triol and was found to be widely distributed
in fungal, plants, and mammalian tissues such as brain,
kidney, skin, liver, uterus, etc.5,6 In addition to its struc-
tural feature of the long-chain base of sphingolipids in
membranes, phytosphingosine (1) is associated with
the heat stress response of yeast cells and induction of
apoptosis in some cancer cells, and some of its deriva-
tives exhibit important physiological activities such as
high tumor inhibitory potency.7
Phytosphingosine is a plant-derived, cell membrane lipid
metabolite. The precise physiological metabolism and
the function of phytosphingosine or phytoceramide as
an anticancer agent have not been known until recently.
We previously reported the anticancer and radiosensitiz-
ing effects of phytosphingosine and a phytoceramide
Keywords:
Phytosphingosine;
Radiosensitizer;
Sphingolipids;
Phytoceramide.
*
Corresponding authors. Tel.: +82 2 2220 2557; fax: +82 2 2299 0762
(S.L.); e-mail: sj0420@hanyang.ac.kr
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.09.037

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B. S. Moon et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6643–6646
effect with irradiation. Jurkat human T-cell lymphoma
OH
(Type II) was obtained from the American Type Culture
Collection (Manassas, VA). Cells were grown in RPMI
1640 medium supplemented with 10% fetal bovine serum
(FBS; Invitrogen, Carlsbad, CA), penicillin, and strepto-
mycin at 37 ꢁC in a humidified incubator with 5% CO2.
After cells were plated onto 60 mm dishes at a density
of 2 · 106 cells/dish and exposed to 10 lM of phytosphin-
gosine derivatives (1 and 3a–h) for 30 min, cells were
exposed to c-rays from a 137Cs c-ray source (Atomic
Energy of Canada, Canada, located in Korea Institute
of Radiological and Medical Sciences, Seoul, Korea) at
a dose rate of 3.81 Gy/min. Cells were fixed with 4%
para-formaldehyde for 30 min at room temperature and
then washed once with phosphate-buffered saline (PBS).
Hoechst 33258 (50 ng/mL) was added to the fixed cells,
incubated for 30 min at room temperature, and then
washed with PBS. Cells were mounted and examined by
fluorescence microscopy. Apoptotic cells were identified
by the condensation and fragmentation calculated from
the ratio of apoptotic cells to total cells counted. At min-
imum, 500 cells were counted for each treatment. The
radiation sensitizing enhancement ratio is defined as [va-
lue of combination with drug and ionizing radiation-in-
duced cell death (%) ꢀ value of drug-induced cell death
(%)/value of ionizing radiation-induced cell death (%)].
n-C14H29
OH
OH NHR1
D
-ribo-phytosphingosine, 1, R1 = H
D-ribo-phytoceramide,
2a, C4PS, R1 = CO(CH2)2CH3
2b, C6PS, R1 = CO(CH2)4CH3
2c, C8PS, R1 = CO(CH2)6CH3
2d, C12PS, R1 = CO(CH2)10CH3
Figure 1. Structure of phytosphingosine and phytoceramide.
(Fig. 1) containing butanoyl (C4PS, 2a), hexanoyl
(C6PS, 2b), octanoyl (C8PS, 2c), and dodecanoyl
(C12PS, 2d) group on various cancer cells.8 According
to these results, phytosphingosine and its derivatives in-
duced the apoptosis of uterine cervical cancer, breast
cancer, blood cancer, and lung cancer cells without side
effects, and exhibited a concentration- and post-treat-
ment time-dependent increase. Moreover, as phyto-
sphingosine and their derivatives were administered to
various cancer cells in combination with ionizing radia-
tion, the apoptotic rate of cancer cells was significantly
enhanced compared to radiation or 1 and 2a–d alone.
Therefore, it seems that the administration of 1 and
2a–d causes to improve the radiation therapy efficiency.
In the current study, we describe the synthesis of novel
eight phytosphingosine derivatives and their preliminary
biological evaluation for enhancing radiation therapy.
The biological properties of these new derivatives were
evaluated in blood cancer cells (Jurkat).
We examined whether the treatment of phytosphingo-
sine derivatives in combination with ionizing radiation
had a sensitizing effect on cell death in Jurkat human
T-cell lymphoma or not. As shown in Figure 2, phyto-
sphingosine (1) did not show synergistic effect on cell
death when simultaneously treated with ionizing radia-
tion. Instead, phytosphingosine treatment alone showed
a significant cytotoxic effect. In addition, 3h did not
show ionizing radiation sensitizing effect. the treatment
of 3h also showed a significant cytotoxic effect. In the
treatments of 3a, 3c, and 3d in combination with ioniz-
ing radiation (IR), however, these phytosphingosine
derivatives had synergistic effects on cell death. In com-
parison with control, the treatment of 3a (11%), 3c
(12%), and 3d (19%) have a synergistic effect on cell
death in combination with ionizing radiation more than
cell death ratio of IR plus cell death ratio of phytosphin-
gosine derivatives. At this point, natural ceramide con-
Eight novel phytosphingosine derivatives 3a–h were pre-
pared in one step from D-ribo-phytosphingosine as
shown in Scheme 1. The phytosphingosine derivatives
were obtained by acylation of an amino group of phyto-
sphingosine with acid scavanger, N,N-diisopropylethyl-
amine (DIEA), at 0 ꢁC to room temperature for 1 h in
35–78%9 (structure analysis of 3a, 3c and 3d: see Ref.
10). Acyl chlorides are commercially available (for 3a–
d) or obtained from diverse acid using thionyl chloride
(for 3e–h).9,10
The prepared phytosphingosine derivatives were evalu-
ated in blood cancer cells for radiation enhancement
OH
OH
a
n-C14H29
n-C14H29
OH
R1
OH
OH HN
OH NH2
O
3a, R1 = -CH2OBn
1
3b, R1 = -CH2COOCH2CH3
3c, R1 = -(CH2)2COOCH2CH3
3d, R1 = -(CH2)3COOCH3
3e, R1 = -(CH2)4COOCH2CH3
3f, R1 = -(CH2)6COOCH3
3g, R1 = -(CH2)7COOCH3
3h, R1 = -(CH2)8COOCH3
Scheme 1. Reagents and conditions: (a) diverse acyl chlorides, DIEA, 0 ꢁC to rt, 1 h for 3a–d and diverse acyl chlorides from acid using SOCl2,
DIEA, 0 ꢁC to rt, 1 h for 3e–h.

B. S. Moon et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6643–6646
6645
Figure 2. Effect of 1 and 3a–h on IR induced cell death (IR: 10 Gy, treated concentration of 1 and 3a–h: 10 lM, time: 48 h).
sists of a long chain sphingoid base activated at the
nitrogen atom with an acyl chain. As this highly
lipophilic chain is usually responsible for the low cell
permeability of ceramide, shorter acyl chains are better
(typically, two to eight carbon atoms).6 The lipophilic-
ity, therefore, is one of the most important factor for
radiosensitizing effect in the modification base on phyto-
sphingosine. To inclease cell permeability, it is impor-
tant to decrease the lipophilicity of phytoceramide
derivatives by introduction of ester group in terminal
of diverse acyl chains. As the modification results, 3a,
3c, and 3d with 6–8 atom chain will be lower lipophilic-
ities than 2a–d. Also compounds 3a, 3c, and 3d showed
higher enhancement of radiosensitizing effect than 2a–
d8a in Jurkat cell. All amide modification derivatives,
moreover, could be decreased toxicity of themselves
compared to 1 (Fig. 2) and comparison between 3c–d
and 2b–c with 6–8 atom needs further study because
2b–c showed diverse biological effect varying tumor
cells.
In conclusion, eight novel phytosphingosine derivatives
were synthesized from D-ribo-phytosphingosine with
diverse acyl chlorides and evaluated in blood cancer
cells. Our results demonstrate that the treatment of 3a,
3c, and 3d in combination with ionizing radiation
enhances the cell death of human cancer cells. More-
over, our results suggest that the combination treatment
of novel phytosphingosine derivatives and ionizing radi-
ation is potentially an effective way of treating cancers
refractive to conventional radiation therapy.
Acknowledgments
This study was supported by Korea Science and Engi-
neering Foundation (KOSEF) and Ministry of Science
& Technology (MOST), Republic of Korea, through
its National Nuclear Technology Program.
References and notes
The radiation sensitizing enhancement ratios of 1 and
3a–h are summarized in Figure 3. Enhancement ratios
(Y axis) were calculated by the equation as follows:
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of 1 is <1. The radiation sensitizing enhancement ratios
for 10 lM treatment of 3a, 3c, and 3d were approxi-
mately 1.24, 1.28, or 1.45 times, respectively. But other
derivatives did not show remarkable results in the
enhancement of cell death with ionizing radiation.
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Figure 3. Enhancement ratios of phytosphingosine derivatives in
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B. S. Moon et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6643–6646
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4.25–4.20 (m, 1H), 4.00 (s, 2H), 3.79–3.74 (m, 2H), 3.58–
3.52 (m, 2H), 1.71–1.69 (m, 1H), 1.56–1.53 (m, 1H),
1.43–1.23 (m, 24H), 0.91 (t, J = 6.6 Hz, 3H); 13C NMR d
170.6, 137.2, 128.1, 127.69, 127.74, 74.8, 73.1, 72.0, 68.8,
60.4, 51.8, 32.5, 31.6, 29.4 (m), 29.0, 25.4, 22.3, 13.0; MS
(FAB) m/z: 466 (MH+). HRMS (FAB) calcd for
C27H48NO5 (MH+) 466.3532, found 466.3535. 3c: 1H
NMR (300 MHz, CD3OD) d 7.85 (d, NH), 4.17–4.10 (m,
3H), 3.77–3.71 (m, 2H), 3.60–3.52 (m, 2H), 3.56–3.32 (m,
1H), 2.62–2.53 (m, 4H), 1.68–1.64 (m, 1H), 1.61–1.55 (m,
1H), 1.32–1.23 (m, 27H), 0.91 (t, J = 6.6 Hz, 3H); 13C
NMR d 173.1, 172.7, 74.8, 71.9, 60.7, 60.3, 52.3, 32.0,
31.6, 30.2, 29.4 (m), 29.1, 29.0, 25.5, 22.3, 13.1, 13.0; MS
(FAB) m/z: 446 (MH+). HRMS (FAB) calcd for
C24H48NO6 (MH+) 446.3482, found 446.3482. 3d: 1H
NMR (300 MHz, CD3OD) d 7.82 (d, NH), 4.13–4.11 (m,
1H), 3.80–3.69 (m, 2H), 3.67 (s, 3H), 3.59–3.51 (m, 2H),
2.39 (t, J = 7.5 Hz, 2H), 2.29 (t, J = 7.5 Hz, 2H), 1.92 (qt,
J = 7.5 Hz, 2H), 1.69–1.64 (m, 1H), 1.49–1.43 (m, 1H),
1.34–1.26 (m, 24H), 0.91 (t, J = 6.6 Hz, 3H); 13C NMR d
173.7, 173.6, 74.8, 72.0, 60.7, 34.8, 34.7, 32.6, 32.1, 31.7,
29.4 (m), 29.1, 25.6, 22.3, 20.8, 13.1; MS (FAB) m/z: 446
(MH+). HRMS (FAB) calcd for C27H54NO6 (MH+)
446.3482, found 446.3475.
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9. General procedures. Acyl chlorides for 3a–d were pur-
chased from Aldrich Co. To prepare acid chlorides for
3e–h, diverse acids were converted to corresponding acid
chlorides by dissolving it in 5.0–10.0 equiv of thionyl
chloride (SOCl2). The mixture was stirred at 40–50 ꢁC for
3 h, followed by removal of extra thionyl chloride in
vacuo. After the acid chloride was dissolved in THF, the
mixture was added dropwise in THF solution containing
phytosphingosine and DIEA at 0 ꢁC. The reaction mixture