Diastereoselective synthesis and bioactivity of long-chain anti-2-amino-3-alkanols

Article abstract of DOI:10.1016/j.ejmech.2011.09.010

An improved four-step approach for the stereoselective synthesis of long-chain anti-2-amino-3-alkanols is described. Using this method, the syntheses of antiproliferative (antitumoral) compounds, spisulosine (ES-285, 2), clavaminols A and B (3 and 4), the deacetylated products of clavaminols H and N (7 and 8), as well as (2S,3R)-2-aminododecan-3-ol (9) and xestoaminol C (10), have been achieved in excellent diastereoselectivities. In vitro study showed that these compounds induced cell death and dose-dependently inhibited cell proliferation in human glioblastoma cell line SHG-44, indicating the anti-tumor property of this series of compounds.

Full text of DOI:10.1016/j.ejmech.2011.09.010

European Journal of Medicinal Chemistry 46 (2011) 5480e5486
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Diastereoselective synthesis and bioactivity of long-chain anti-2-amino-3-
alkanols
,
Bi-Shuang Chena, Long-He Yangb, Jian-Liang Yea, Tao Huanga, Yuan-Ping Ruana, Jin Fub
**
,
,
Pei-Qiang Huanga
*
a Department of Chemistry and The Key Laboratory for Chemical Biology of Fujian Province, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen,
Fujian 361005, PR China
b Department of Pharmaceutical Sciences, Medical College, Xiamen University, Xiamen, Fujian 361005, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
An improved four-step approach for the stereoselective synthesis of long-chain anti-2-amino-3-alkanols
is described. Using this method, the syntheses of antiproliferative (antitumoral) compounds, spisulosine
(ES-285, 2), clavaminols A and B (3 and 4), the deacetylated products of clavaminols H and N (7 and 8), as
well as (2S,3R)-2-aminododecan-3-ol (9) and xestoaminol C (10), have been achieved in excellent dia-
stereoselectivities. In vitro study showed that these compounds induced cell death and dose-dependently
inhibited cell proliferation in human glioblastoma cell line SHG-44, indicating the anti-tumor property of
this series of compounds.
Received 17 May 2011
Received in revised form
6 September 2011
Accepted 7 September 2011
Available online 16 September 2011
Keywords:
Ó 2011 Elsevier Masson SAS. All rights reserved.
Marine compound
Enantioselective synthesis
One-pot reaction
1-Deoxy-sphingoid bases
Cell proliferation
1. Introduction
Specially, spisulosine has shown the inhibition of cell proliferation
in numerous tumor cell lines due to the disruption of actin stress
Sphingoid bases (also called sphingosines) are a class of naturally
occurring long-chain 2-amino-3-alkanols. Since the first isolation of
sphingosine (1) (Fig. 1), hundreds of sphingoid bases with consid-
erable structural diversity have been isolated from plants and
animals [1]. Due to the wide spectrum of biological activities, this
family has attracted a lot of attention by both biologists and
synthetic organic chemists [1e12]. Recently, marine organisms,
ascidians (tunicates) and marine sponges have also been found to be
the enriched sources of 1-deoxy-sphingoid bases [1]. In spite of their
structural simplicity, these long-chain amino alcohols exhibit
remarkable bioactivities, and are promising for the development of
new anti-tumor agents [13]. 1-Deoxy-sphingoid bases have been
shown to be more cytotoxic than sphingosine against HT29 cells
[14]. In this context, spisulosine (2) (also referred as “ES-285”),
isolated from the marine clam Spisula polynyma by Caudros et al.
[15], is considered to be a novel anti-tumor compound and currently
in Phase I clinical trial against solid tumor in Europe [16,17].
fibers through Rho signaling pathway [15]. It has also been found to
activate caspases 3 and 12 and to modify the phosphorylation of p53
[18]. In addition, clavaminols A w N, a family of fourteen members
isolated from the Mediterranean ascidian Clavelina phlegraea, also
displayed biological cytotoxicity. Opposite to the 2S,3R-configura-
tion of sphingosine, clavaminols A w N possess 2R,3S-configuration
in the anti-2-amine-3-alkanol motif [19,20]. Clavaminols AeC and F
were tested for their cytotoxic and pro-apoptotic properties and
clavaminol A (3) was shown to be the most potent cytotoxic
compound of this series in inducing cell death through activation of
the apoptotic machinery. Interestingly, while clavaminols GeN have
shown no significant cytotoxicity, the unnatural deacetylated
product (8) of clavaminol N (6) showed a cytotoxic effect compa-
rable to thatof clavaminol A (IC50 z 5
mg/mL) on twodifferent tumor
cell lines, human lung adenocarcinomic epithelial cells A549 and
human gastric adenocarcinoma epithelial cell AGS. Similarly, the
deacetylated product (7) of clavaminol H (5) showed significant
cytotoxic activity in AGS cells, while it has no effect on lung cancer
cell line A549 [19,20]. Moreover, (2S,3R)-2-aminododecan-3-ol (9),
the enantiomer of clavaminol A (3), is an antifungal agent isolated
from the ascidian Clavelina oblonga in Brazil [21]. This compound
displayed antifungal activity against Candida albicans ATCC 10231
* Corresponding author. Tel.: þ86 592 2182240; fax: þ86 592 2180992.
** Corresponding author. Tel.: þ86 592 2187210; fax: þ86 592 2187868.
E-mail addresses: fujin@xmu.edu.cn (J. Fu), pqhuang@xmu.edu.cn (P.-Q. Huang).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.09.010

B.-S. Chen et al. / European Journal of Medicinal Chemistry 46 (2011) 5480e5486
5481
NP1P2
R1
NH2
NH2
R1M
NP1P2
n-C15H31
HO
n-C13H27
R
R
CHO
OH
OH
OH
2
(2S,3R)-spisulosine ( )
1
sphingosine ( )
(ES-285)
Scheme 1. Anti-diastereoselective addition of organometallic reagents to N-protected
-amino aldehydes.
NH2
NH2
a
n-C9H19
OH
OH
NHX
alcohols [40,44e46], the lability of the a-amino aldehydes towards
4
clavaminol B ( )
3
clavaminol A ( )
partial racemization and side reaction [43,47,48] made it worth-
while to modify to ensue a more reliable CeC bond formation with
high enantioselectivity.
NHX
HO
n-C9H19
On the basis of these considerations, a four-step approach for the
OH
clavaminol N (6. X = Ac)
synthesis of 1-deoxy-sphingoid bases starting from unprotected
L or
OH
D-alanine is developed and shown in Scheme 2. The method features
5
clavaminol H ( . X = Ac)
a tandem Swern oxidation-organometallic reagent addition proce-
dure [48e50], which will allow, on one hand, avoiding the racemi-
zation of the N,N-dibenzyl amino aldehyde, and on the other hand,
simplifying a purification step.
For the synthesis of the natural products spisulosine (2), (2S,3R)-
2-aminododecan-3-ol (9), and xestoaminol C (10) with (2S,3R)
(2R,3S)-2-aminododecan- (2R,3S)-2-aminoisodecan-
7
8
1,3-diol ( . X = H)
NH2
3-ol ( . X = H)
NH2
n-C9H19
n-C11H23
OH
(2S,3R)-2-aminododecan-
3-ol (9)
OH
configuration, L-alanine was the required starting material (Scheme
(2S,3R)-xestoaminol C (10)
2). Compounds (S)-11 and (S)-12 were prepared as previously
described [25,44,46,51]. The key one-pot reaction was run by Swern
oxidation [(COCl)2, DMSO, CH2Cl2; NEt3] at ꢀ78 ꢁC, followed by
addition of a solution of n-pentadecyl magnesium bromide in ether,
which produced the amino alcohol 13a in 68% yield. Removal of the
protecting groups under catalytic hydrogenolytic conditions (10%
Pd/C, H2, MeOH, 28 h) afforded (2S,3R)-spisulosine (2) in 84% yield.
The physical and spectral data of our synthetic compound agreed
Fig. 1. Structure of long-chain anti-2-amino-3-alkanols.
and Candida glabrata with a MIC of 0.7
mg/mL and 30 mg/mL,
respectively. Furthermore, xestoaminol C (10), isolated together
with its congeners xestoaminols A and B from a Fijisponge xesto-
spongia, sp., was extremely active in assay against reverse tran-
scriptase with 95% inhibition at 1 mg/mL [22]. Apart from those
mentioned above, many other bioactive long-chain 2-amino-3-
alkanols have also been reported [23,24].
with those reported by Gálvez et al. [27] {[
a
]
20 þ24.2 (c 1.0, CHCl3);
D
lit. [27] [
a
]
D
25 þ24.0 (c 1.0, CHCl3)}.
Using n-nonyl magnesium bromide in the tandem Swern
oxidation-Grignard reagent addition of (S)-12 led to the formation
of 13b in 67% yield. Catalytic hydrogenolysis (10% Pd/C, H2, MeOH,
In recent years, several synthetic approaches to these amino
alcohols have been reported [8,25e34], including seven for spisulo-
sine (2) [25e31], two for the deacetylated products (7) of clavaminol
H [28,32], and three for xestoaminol C (10) [8,33,34]. As a continua-
tion of our longstanding interest in the synthesis of N-containing
bioactive molecules [35e37], and in conjunction with our recent
interest in the development of anti-tumor agents [38,39], we were
engaged in the synthesis of above-mentioned long-chain amino
alcohols. In present study, we used Reetz approach [40,41] with
modification to synthesize sphingoid bases (Fig. 1), including spisu-
losine (2), clavaminols A and B (3 and 4), as well as the deacetylated
products of clavaminols H and N (7 and 8), antifungal compound (9),
and xestoaminol C (10). Moreover, we tested the effect of these
synthetic compounds on cell toxicity and proliferation to elucidate
their biological and pharmacological roles as anti-cancer agents.
2. Chemistry
All the molecules shown in Fig. 1 are vicinal amino alcohols with
an anti stereochemistry and with either (2S,3R) or (2R,3S) config-
uration. Biosynthetically they are originated from
L or D-alanine or
serine [19e21]. Thus -amino acids would afford a convenient
a
starting point to access these compounds and the homologues and
analogs thereof via the addition of an organometallic reagent to the
a
-amino aldehydes (Scheme 1) [42]. Among the methods available
for the conversion of -amino acids into the corresponding vicinal
a
anti-amino alcohols [43], the Reetz methodology of anti-diaster-
eoselective addition with N,N-dibenzyl amino aldehydes appeared
to be the ideal one [40,44e46]. Although this method has been
applied for the enantioselective synthesis of vicinal anti-amino
Scheme 2. Synthesis of 1-deoxy-sphingoid bases from L or D-alanine.

5482
B.-S. Chen et al. / European Journal of Medicinal Chemistry 46 (2011) 5480e5486
28 h) of the latter afforded (2S,3R)-2-aminododecan-3-ol (9) in 84%
yield. The optical rotation data of the synthetic compound matched
(Scheme 2). For the chemoselective cleavage of the N,N-dibenzyl
groups, the conventional catalytic hydrogenolytic condition could
not be used due to the presence of the double bond. This was ach-
ieved smoothly under 10% Pd/C-catalyzed transfer hydrogenolytic
conditions [54] (HCO2H, NEt3, MeOH), which provided the desired
those reported {[
a
]
20 þ4.2 (c 1.5, MeOH); lit. [21] [
a
]
29 þ4.5 (c 0.22,
D
D
MeOH)}, while differences existed in its 1H and 13C NMR data
comparing with those of the natural product reported [21]. The
structure of the synthetic compound was confirmed via the
synthesis of its dibenzoyl derivative, whose 1H NMR data matched
those reported [21].
20
25
product 4 {[
a
]
ꢀ8.3 (c 0.2, MeOH); lit. [19] [
a
]
ꢀ5.0 (c 0.001,
D
D
MeOH)} in 89% yield. The physical and spectral data, which were not
reported for its synthetic compound or derivative so far, were similar
to those of the obtained compounds described above.
Similarly, tandem Swern oxidation-undecyl magnesium bromide
addition produced 13c in 73% yield. Catalytic hydrogenolysis (10% Pd/
C, H2, MeOH, 28 h) of 13c afforded (2S,3R)-xestoaminol C (10) in 90%
yield. It is worthy to note that the spectral data of our synthetic
compound agreed with those reported by Ooi et al. [33] The physical
and spectral data of the diacetyl derivative matched those reported
As regarding the synthesis of compound 8, the deacetylated
product of clavaminol N (6), the required Grignard reagent 25 was
prepared from 1,5-hexanediol in five steps (Scheme 4). Mono-
silylation of the diol 20 with TBSCl (imidazole, DMAP, CH2Cl2) fol-
lowed by tosylation (p-TsOH, NEt3, DMAP, CH2Cl2, r.t.) gave the
desired tosylate 22 in 76% overall yield. CuI-Mediated coupling of i-
butyl magnesium bromide with tosylate 22 smoothly produced the
coupling product 23 in 90% yield. Treatment of the silyl ether 23 with
triphenylphosphine and bromine [52] produced directly the
bromide 24, which was converted into the Grignard reagent 25. One-
pot Swern oxidation-Grignard addition of 25 then gave anti-amino
alcohol 13f in 72% yield (Scheme 2). Catalytic hydrogenolysis of 13f
produced compound 8 in 86% yield, of which the 13C spectral and
[34] {[
a
]
20 ꢀ22.8 (c 0.8, MeOH); lit. [34] [
a
]
25 ꢀ22.1 (c 0.17, MeOH)}.
D
D
In view of the synthesis of clavaminol A (3) and clavaminol B (4)
with 2R,3S-configuration, amino alcohol (R)-12 was prepared from
D-alanine by the procedure shown in Scheme 2 [44]. One-pot Swern
oxidation-nonyl magnesium bromide addition gave the anti-amino
alcohol ent-13d in 67% yield. 10% Pd/C-Catalyzed hydrogenolysis
then afforded the clavaminol A (3) in 85% yield. The optical rotation
20
of the synthetic compound matched that reported {[
a
]
ꢀ4.2 (c
D
25
0.5, MeOH); lit. [19] [
a
]
ꢀ4.25 (c 0.0094, MeOH)}. The spectral
physical data {[
a
]
20 ꢀ4.5 (c 0.22, MeOH)} were not reported so far.
D
D
data of its diacetyl derivative matched those reported [19].
We next turned our attention to the synthesis of the deacety-
lated product (7) of clavaminol H (5). The synthesis was performed
as shown in Scheme 5. Compounds 26, 27, 28 were prepared as
previously described [45,55e58]. One-pot Swern oxidation-nonyl
magnesium bromide addition gave the anti-amino alcohol 29 in
65% yield. 10% Pd/C-Catalyzed hydrogenation then afforded the
deacetylated clavaminol H (7) in 88% yield. The optical rotation and
For the synthesis of clavaminol B (4), the Grignard reagent 19 was
prepared (Scheme 3). Thus, deprotonation of n-hept-1-yne with n-
butyl lithium followed by coupling of the resultant lithium heptylide
with iodide 14 afforded the coupling product 15 in 81% yield. Lindlar
catalyst-catalyzed partial hydrogenation of the alkyne 15 gave cis-
olefin 16 in 79% yield. Unfortunately, compound 16 could not be
converted directly into the corresponding bromide 18 with triphe-
nylphosphine and bromine [52], as the addition of bromine to
double bond would also happen. Finally, it was necessary that
cleavage of the silyl group (TBAF, THF) yielded the alcohol 17, which
was treated with PPh3/CBr4 [53] to give bromide 18. Tandem Swern
oxidation and addition with the Grignard reagent (19) prepared from
bromide 18 produced the anti-adduct 13e in 70% overall yield
spectral data of the synthetic compound matched those reported
20
by Ferreira/Chemla [28] {[
a]
ꢀ6.3 (c 0.3, MeOH); lit. [28]
D
[a
]
25 ꢀ6.0 (c 0.10, MeOH)}.
D
3. Pharmacology
Cancer therapies predominantly focus on the anti-proliferation
strategy as a number of studies have demonstrated and supported
that tumor growth occurred upon imbalance between cell
n-BuLi,HMPA
OTBS
I
n-C H
14
81%
TBSO
Lindlar cat.
79%
15
TBAF
TBSO
THF
92%
16
Ph P, CBr
HO
88%
17
X
18 (X = Br)
Mg
THF
19 (X =MgBr )
Scheme 3. Preparation of the Grignard reagent 19 for the synthesis of clavaminol B.
Scheme 4. Preparation of the Grignard reagent 25 for the synthesis of compound (8).

B.-S. Chen et al. / European Journal of Medicinal Chemistry 46 (2011) 5480e5486
5483
120
NBn2
CO2Bn
NH2
CO2H
OH
D-serine
TBSCl, imid,
BnCl, K2CO3
EtOH-H2O
DMAP, CH2Cl2, rt
50%
97%
OH
26
NBn2
80
40
0
1. (COCl)2, DMSO
NBn2
NaBH4, CaCl2
CH Cl , 78 °C;
–
2
2
OH
NEt3;
CO2Bn
OTBS
27
THF, EtOH, rt
90%
2. n-C9H19MgBr
OTBS
28
65%
NH2
NBn2
10% Pd/C, H2
HO
n-C9H19
TBSO
n-C9H19
HCl, MeOH
88%
-0.5
0.0
0.5
1.0
1.5
2.0
OH
OH
29
Compouds (Log[µM])
deacetylclavaminol H
(7)
Fig. 3. Dose-dependent inhibition of cell proliferation by compound 7 (open triangle),
9 (open circle) and 10 (closed diamond) with IC50 of 4.41 mM, 7.96 mM, and 4.5 mM,
respectively.
Scheme 5. Synthesis of the deacetylclavaminol H (7).
proliferation and apoptosis. It has been suggested that marine
organisms be the potential sources for antineoplastic agents [59] and
several compounds isolated from marine crops have been portrayed
as anti-cancer candidates [60e63]. Although recent reports showed
that sphingosine and its derivatives can cause cell growth arrest or
death in various tumor cell lines [16,64], their anti-tumor effects on
glioma have not yet been reported. Gliomas, accounting for 30e35%
of the primary malignant brain tumors in adults, are a line of the most
aggressive and invasive tumors that lead to death in most cases.
Treatments for human gliomas nowadays are still ineffective and
mainly palliative [65,66]. Due to their poor prognosis and high
mortality, it is urgent and necessary to develop an effective thera-
peutics for human gliomas. To test whether sphingosine derivatives
effectively affect the proliferation of human gliomas, we used human
glioblastoma cell line SHG-44 and Cell Count Kit-8 (CCK-8) assay to
examine cell viability and toxicity in response to these compounds.
10 inhibited cell growth with 60%, 80% and 100%, respectively)
(Fig. 2). The extended studies showed that compound 7, 9, 10
demonstrated anti-proliferation property in SHG-44 cell with IC50
of 4.41 mM, 7.96 mM and 4.5 mM, respectively (Fig. 3). In contrast,
compounds 4 and 8 were lack of anti-proliferation activity, indi-
cating that additional instauration and branched carbon chain were
detrimental for the cytotoxic activities [67,68].
The detection sensitivity of CCK-8 was greater than other tetra-
zolium salt methods such as MTT, XTTor MTS. Cell proliferation assay
using CCK-8 correlated well with thymidine incorporation assay, the
most common assay to detect chromosomal DNA synthesis during
mitotic cell division. Thus, the CCK-8 assay may directly measure cell
proliferation and determine cell division that has occurred in
response to test agents. However, CCK-8 assay was unable to distin-
guish the two major cell biology pathways, i.e., cell arrest and
apoptosis, which induced cell death. In the future study, we would
like to carry out the specific assay to illustrate the mechanism of anti-
tumor effect exerted by these compounds. To detect cell cytotoxicity,
CCK-8 assay may minimize and facilitate the process to determine
living cell number. Therefore, the cytotoxicity assay using CCK-8 was
suitable for the preliminary screening of chemicals.
4. Results and discussion
In SHG-44 cells, all compounds were capable to induce cell
toxicity at 50 mM and compounds 7, 9, 10 were found of the
strongest effect among these compounds (10
mM of compound 7, 9,
5. Conclusion
120
In summary, by the development of a one-pot Swern oxidation-
organometallic reagent addition procedure, the Reetz’s method for
the asymmetric synthesis of b-amino alcohols has been improved.
2
3
4
7
8
9
10
80
40
0
Using this concise and versatile method, the asymmetric syntheses of
seven naturally occurring or derived cytotoxic long-chain anti-2-
amino-3-alkanols, including the first stereoselective synthesis of
clavaminol B (4) and (2R,3S)-deacetylclavaminol N (8), have been
achieved in ꢂ99% diastereoselectivities and ꢂ99% enantioselectiv-
ities. Bioactivity assay further confirmed in human glioblastoma cells
their anti-proliferation effect, which was previously reported in other
cancer cell lines. Future mechanism studies will be performed on
those compounds that are potential candidates for anti-cancer drugs.
6. Experimental
0
25
50
75
100
Compounds (µM)
6.1. Chemistry
Fig. 2. Effect of synthetic compounds on cell death. Dose-dependent (0.1
mM, 1 mM,
6.1.1. General methods
10 M, 50 M and 100 M) compounds induced cell death in SHG-44 cells. Compound
m
m
m
Melting points (M.p.) were determined on a Yanaco MP-500
micro melting point apparatus and were uncorrected. Infrared
2, closed square; 3, open square; 4, closed triangle; 7, open triangle; 8, closed circle; 9,
open circle; 10, closed diamond.

5484
B.-S. Chen et al. / European Journal of Medicinal Chemistry 46 (2011) 5480e5486
spectra were measured with a Nicolet Avatar 360 FT-IR spectrom-
eter using film KBr pellet techniques. 1H and 13C NMR spectra were
recorded in CDCl3 or CD3OD on a Bruker 400 spectrometer with
tetramethylsilane as an internal standard. Chemical shifts are
25.9, 29.4, 29.6, 29.6, 29.6, 29.6, 29.7, 31.9, 34.3, 54.8, 57.3, 73.7,
126.9, 128.2, 128.8, 140.2; MS (ESI, m/z) 410.2 (M þ Hþ, 100). Anal.
calcd. for C28H43NO: C, 82.09; H, 10.58; N, 3.42. Found: C, 82.45; H,
10.33; N, 3.64.
expressed in
recorded by
d
(ppm) units downfield from TMS. Mass spectra were
Bruker Dalton ESquire 3000 plus liquid
a
6.1.5. (2R,3S)-2-(Dibenzylamino)dodecan-3-ol (13d)
chromatography-mass spectrum (direct injection). Optical rota-
tions were measured with a PerkineElmer 341 automatic polar-
imeter. Diastereoselectivities and enantioselectivities were
determined by chiral HPLC analysis using a Shimadzu LC-10AT VP
series and a Shimadzu SPD-M10Avp photo diode array detector
(190e370 nm) with a Chiralcel OJ-H column using n-hexane/i-
PrOH (98:2, v/v) as a mobile phase. Flash column chromatography
was carried out with silica gel (300e400 mesh). THF was distilled
over sodium benzophenone ketyl under N2.
Using the procedure described for the synthesis of the compound
13a, reaction of (R)-12 [44] (252 mg, 0.96 mmol) with n-C9H19MgBr
(3.84 mmol in 8 mL of Et2O) gave 13d (260 mg, 0.66 mmol, 67%) as
20
a colorless oil. [
a
]
ꢀ28.0 (c 0.2, CHCl3); IR (film) nmax: 3365, 2917,
D
2853, 1607, 1454, 1375, 1119 cmꢀ1; 1H NMR (400 MHz, CDCl3)
d: 0.88
(t, J ¼ 6.9 Hz, 3H), 1.09 (d, J ¼ 6.8 Hz, 3H), 1.14e1.42 (m, 15H),
1.62e1.74 (m, 1H),1.79 (s, 1H), 2.67e2.74 (m, 1H), 3.47 (d, J ¼ 13.8 Hz,
2H), 3.56e3.62 (m, 1H), 3.76 (d, J ¼ 13.8 Hz, 2H), 7.19e7.35 (m, 10H);
13C NMR (100 MHz, CDCl3)
d: 8.7,14.1, 22.7, 25.9, 29.3, 29.6, 29.6, 29.7,
31.9, 34.3, 54.8, 57.3, 73.6, 126.9, 128.2, 128.7, 140.2; MS (ESI, m/z)
382.2 (M þ Hþ, 100). Anal. calcd for C26H39NO: C, 81.84; H, 10.30; N,
3.67. Found: C, 81.81; H, 10.18; N, 3.66.
6.1.2. (2S,3R)-2-(Dibenzylamino)octadecan-3-ol (13a)
To a cooled (ꢀ78 ꢁC) solution of (COCl)2 (0.2 mL, 1.96 mmol) in
CH2Cl2 (2 mL) was added DMSO (0.3 mL, 3.92 mmol). After stirring
for 10 min, (S)-12 [44] (200 mg, 0.78 mmol) in CH2Cl2 (2 mL) was
carefully added. 30 min later, Et3N (0.6 mL, 3.92 mmol) was care-
fully added and the mixture was warmed to room temperature and
stirred for 1 h. Then the mixture was cooled to ꢀ78 ꢁC again and
a solution of n-C15H31MgBr (3.12 mmol in 6 mL of Et2O) was added.
The resultant mixture was stirred for 40 min. After warming
to ꢀ40 ꢁC and stirring for 20 min, the mixture was warmed to room
temperature, stirred for 1 h, before quenching with a saturated
NH4Cl (8 mL). The mixture was extracted with EtOAc (10 mL ꢃ 3).
The organic phases were washed with brine, dried over Na2SO4,
filtered and concentrate in vacuo. The residue was purified by flash
6.1.6. (Z,2R,3S)-2-(Dibenzylamino)dodec-8-en-3-ol (13e)
Using the procedure described for the synthesis of the compound
13a, reaction of (R)-12 (519 mg, 2.02 mmol) with the Grignard
reagent 19 (9.09 mmol in 18 mL of Et2O) gave 13e (561 mg,
1.48 mmol, 73%) as a colorless oil. [
a
]
20 ꢀ22.6 (c 0.2, CHCl3); IR (film)
D
nmax: 3398, 2971, 2930, 2842, 1582, 1457, 1262, 1084, 1030 cmꢀ1; 1H
NMR (400 MHz, CDCl3)
d
: 0.92 (t, J ¼ 7.3 Hz, 3H), 1.13 (d, J ¼ 6.8 Hz,
3H), 1.18e1.31 (m, 5H), 1.31e1.47 (m, 6H), 1.63 (s, 1H), 1.61e1.82 (m,
1H), 2.70e2.78 (m, 1H), 3.49 (d, J ¼ 13.8 Hz, 2H), 3.57e3.66 (m, 1H),
3.79 (d, J ¼ 13.8 Hz, 2H), 5.35e5.42 (m, 2H), 7.21e7.53 (m, 10H); 13C
NMR (100 MHz, CDCl3) d: 8.7, 13.8, 22.9, 25.6, 29.3, 29.7, 29.8, 34.2,
chromatography on silica gel (eluent: EtOAc/PE 1:30) to give 13a
54.8, 57.3, 73.6, 126.9, 128.2, 128.8, 129.8, 129.9, 140.2; MS (ESI, m/z)
380.3 (M þ Hþ, 100). HRMS m/z calcd for C26H37NO (M þ Hþ):
380.2947; found: 380.2941.
20
(248 mg, 0.53 mmol, 68%) as a colorless oil. [
CHCl3); IR (film) nmax
1027 cmꢀ1; 1H NMR (400 MHz, CDCl3)
a
]
þ17.4 (c 0.3,
D
:
3381, 2927, 2841, 1494, 1454, 1375,
d
: 0.88 (t, J ¼ 6.8 Hz, 3H), 1.10
(d, J ¼ 6.8 Hz, 3H),1.13e1.34 (m, 27H),1.64e1.74 (m,1H),1.78 (s,1H),
2.67e2.76 (m, 1H), 3.52 (d, J ¼ 13.8 Hz, 2H), 3.56e3.63 (m, 1H), 3.76
(d, J ¼ 13.8 Hz, 2H), 7.17e7.37 (m, 10H); 13C NMR (100 MHz, CDCl3)
6.1.7. (2R,3S)-2-(Dibenzylamino)-11-methyldodecan-3-ol (13f)
Using the procedure described for the synthesis of the compound
13a, reaction of (R)-12 (508 mg, 1.98 mmol) with the Grignard
reagent 25 (7.92 mmol in 16 mL of Et2O) gave 13f (577 mg,
d
: 8.7, 14.1, 22.7, 25.9, 29.4, 29.6, 29.7, 31.9, 34.3, 54.8, 57.3, 73.7,
126.9, 128.2, 128.8, 140.2; MS (ESI, m/z) 466.3 (M þ Hþ, 100). HRMS
1.43 mmol, 72%) as a colorless oil. [
a
]
25 ꢀ32.0 (c 0.1, CHCl3); IR (film)
D
m/z calcd for C32H51NO (M þ Hþ): 466.4043; found: 466.4041.
nmax: 3396, 3024, 2951, 2927, 2850, 2808, 1942, 1722, 1607, 1494,
1451, 1360 cmꢀ1
;
1H NMR (400 MHz, CDCl3)
d: 0.92 (d, J ¼ 6.6 Hz,
6.1.3. (2S,3R)-2-(Dibenzylamino)dodecan-3-ol (13b)
6H), 1.15 (d, J ¼ 6.8 Hz, 3H), 1.19e1.23 (m, 2H), 1.26e1.37 (m, 11H),
1.58e1.20 (m, 1H), 1.68e1.80 (m, 1H), 1.82 (s, 1H), 2.71e2.81 (m, 1H),
3.52 (d, J ¼ 13.8 Hz, 2H), 3.62e3.68 (m, 1H), 3.82 (d, J ¼ 13.8 Hz, 2H),
Using the procedure described for the synthesis of the compound
13a, reaction of (S)-12 (500 mg, 1.96 mmol) with n-nonyl magne-
sium bromide (8.00 mmol in 16 mL of Et2O) gave 13b (519 mg,
7.21e7.53 (m, 10H); 13C NMR (100 MHz, CDCl3)
d: 8.7, 22.7, 26.0, 27.5,
1.31 mmol, 67%) as a colorless oil. [
a
]
20 þ28.0 (c 0.2, CHCl3); IR (film)
28.0, 29.7, 29.7, 29.9, 34.3, 39.1, 54.8, 57.3, 73.7, 126.9, 128.3, 128.8,
140.2; MS (ESI, m/z) 396.3 (M þ Hþ, 100). HRMS m/z calcd for
C27H41NO (M þ Hþ): 396.3266; found: 396.3264.
D
nmax: 3365, 2917, 2853, 1607, 1454, 1375, 1119 cmꢀ1
;
1H NMR
(400 MHz, CDCl3)
d
: 0.88 (t, J ¼ 6.9 Hz, 3H), 1.09 (d, J ¼ 6.8 Hz, 3H),
1.14e1.42 (m, 15H), 1.62e1.74 (m, 1H), 1.79 (s, 1H), 2.67e2.74 (m, 1H),
3.47 (d, J ¼ 13.8 Hz, 2H), 3.56e3.62 (m, 1H), 3.76 (d, J ¼ 13.8 Hz, 2H),
6.1.8. (2S,3R)-Sphingosine (ES-285, 2)
7.19e7.35 (m, 10H); 13C NMR (100 MHz, CDCl3)
d: 8.7, 14.1, 22.7, 25.9,
A suspension of compound 13a (112 mg, 0.24 mmol) and 10% Pd/
C (30 mg) in MeOH (3 mL) was stirred for 28 h under hydrogen
atmosphere (1 atm) at room temperature. When the reaction was
monitored to be complete by TLC analysis, the mixture was filtered
and the solid phase was washed with methanol 5 times. The filtrate
was concentrated in vacuo and the residue was purified by flash
chromatography on silica gel (eluent: MeOH/CH2Cl2 1:10) to give
compound 2 [27] (56 mg, 0.20 mmol, 84%) as a white solid. M.p.
65ꢀ66 ꢁC (EtOAc/PE) {lit. [27] M.p. 64.5e66 ꢁC (EtOAc/PE)};
29.3, 29.6, 29.6, 29.7, 31.9, 34.4, 54.8, 57.3, 73.7, 126.9, 128.2, 128.8,
140.2; MS (ESI, m/z) 382.2 (M þ Hþ,100). Anal. calcd for C26H39NO: C,
81.84; H, 10.30; N, 3.67. Found: C, 81.81; H, 10.18; N, 3.66.
6.1.4. (2S,3R)-2-(Dibenzylamino)tetradecan-3-ol (13c)
Using the procedure described for the synthesis of the
compound 13a, reaction of (S)-12 (519 mg, 2.02 mmol) with n-
C11H23MgBr (8.08 mmol in 16 mL of Et2O) gave 13c (604 mg,
1.48 mmol, 73%) as a colorless oil. [
a
]
D
20 þ20.4 (c 0.5, CHCl3). IR (film)
[a]
20 þ24.2 (c 1.0, CHCl3) {lit. [27] [
a]
D
25 þ24.0 (c 1.0, CHCl3)}; IR (film)
D
ymax: 3361, 2922, 2847, 1602, 1453, 1366, 1084 cmꢀ1
;
1H NMR
nmax: 3323, 2917, 2844, 1591, 1479, 1366, 1061 cmꢀ1
;
1H NMR
(400 MHz, CDCl3)
d
: 0.92 (t, J ¼ 6.9 Hz, 3H), 1.13 (d, J ¼ 6.8 Hz, 3H),
(400 MHz, CD3OD)
d
: 0.91 (t, J ¼ 6.9 Hz, 3H), 1.05 (d, J ¼ 6.6 Hz, 3H),
1.18e1.32 (m, 19H), 1.68e1.78 (m, 1H), 1.79 (s, 1H), 2.71e2.77 (m,
1H), 3.51 (d, J ¼ 13.8 Hz, 2H), 3.59e3.67 (m, 1H), 3.80 (d, J ¼ 13.8 Hz,
1.28e1.68 (m, 28H), 2.75e2.87 (m, 1H), 3.44 (m, 1H); 13C NMR
(100 MHz, CD3OD) d: 14.5, 17.0, 23.8, 27.4, 30.6, 30.9, 30.9, 30.9, 30.9,
2H), 7.19e7.41 (m, 10H); 13C NMR (100 MHz, CDCl3)
d: 8.7, 14.1, 22.7,
30.9, 30.9, 33.2, 34.1, 52.3, 76.3; MS (ESI, m/z) 286.2 (M þ Hþ, 100).

B.-S. Chen et al. / European Journal of Medicinal Chemistry 46 (2011) 5480e5486
5485
6.1.9. (2S,3R)-2-Aminododecan-3-ol (9)
(m, 11H), 1.46e1.63 (m, 2H), 2.74e2.87 (m, 1H), 3.37e3.51 (m, 1H);
Using the procedure described for the synthesis of the
compound 2, reaction of 13b (65 mg, 0.17 mmol) with 10% Pd/C
(20 mg) gave 9 (29 mg, 0.14 mmol, 84%) as a white solid. M.p.
13C NMR (100 MHz, CD3OD)
d: 14.8, 21.0 (2C), 25.2, 26.5, 27.2, 28.8,
29.0, 32.0, 38.3, 50.2, 74.2; MS (ESI, m/z) 216.2 (M þ Hþ, 100). HRMS
m/z calcd for C12H26NO (M þ Hþ): 216.2327; found: 216.2320.
63ꢀ64 ꢁC (EtOAc/PE); [
a
]
20 þ4.2 (c 1.5, MeOH) {lit. [21] [
a
]
29 þ4.5 (c
D
D
0.22, MeOH)}; IR (film) nmax: 3335, 2914, 2847, 1582, 1481, 1390,
6.1.14. (2R,3S)-1-(tert-Butyldimethylsilyloxy)-2-(dibenzylamino)
dodecan-3-ol (29)
Using the procedure described for the synthesis of the compound
13a, reaction of 28 [58] (302 mg, 0.78 mmol) with n-C9H19MgBr
1079 cmꢀ1
1.05 (d, J ¼ 6.6 Hz, 3H), 1.28e1.60 (m, 16H), 2.82 (ddd, J ¼ 2.1, 4.2,
6.2 Hz, 1H), 3.44 (m, 1H); 13C NMR (100 MHz, CD3OD)
: 12.5, 15.0,
;
1H NMR (400 MHz, CD3OD)
d
: 0.90 (t, J ¼ 6.9 Hz, 3H),
d
21.7, 25.3, 28.5, 28.7, 28.8, 28.8, 31.1, 32.0, 50.2, 74.3; MS (ESI, m/z)
202.1 (M þ Hþ, 100). HRMS m/z calcd for C12H27NO (M þ Hþ):
202.2171; found: 202.2170.
(3.12 mmol in 7 mL of Et2O) gave 29 (258 mg, 0.51 mmol, 65%) as
20
a colorless oil. [
a
]
þ12.8 (c 1.0, CHCl3); IR (film) nmax: 3457, 2930,
D
2850,1597,1448,1250,1088 cmꢀ1; 1H NMR (400 MHz, CDCl3)
d: 0.09
(s, 3H), 0.11 (s, 3H), 0.86e0.98 (m, 12H), 1.18e1.36 (m, 15H),
1.68e1.79 (m, 1H), 2.63e2.69 (m, 1H), 2.97 (s, 1H), 3.62 (d,
J ¼ 13.8 Hz, 2H), 3.82e3.91 (m, 3H), 3.94e4.05 (m, 2H), 7.19e7.35 (m,
6.1.10. (2S,3R)-Xestoaminol C (10)
Using the procedure described for the synthesis of the
compound 2, reaction of 13c (85 mg, 0.21 mmol) with 10% Pd/C
(26 mg) gave 10 (43 mg, 0.19 mmol, 90%) as a white solid. M.p.
10H); 13C NMR (100 MHz, CDCl3)
d: ꢀ5.6, ꢀ5.6, 14.1, 18.1, 22.7, 25.5,
25.9 (2C), 29.4, 29.6, 29.6, 29.7, 31.9, 34.5, 55.3, 61.3, 72.3, 126.9,
128.2, 128.8, 140.1; MS (ESI, m/z) 512.3 (M þ Hþ, 100). HRMS m/z
calcd for C32H53NO2Si (M þ Hþ): 512.3918; found: 512.3915.
63ꢀ64 ꢁC (EtOAc/PE); [
a
]
20 þ 12.0 (c 0.5, MeOH) {lit. [22] [
a
]
20 þ7.0
D
D
(c 0.14, MeOH)}; IR (film) ymax: 3432, 3336, 2913, 2843, 1582, 1462,
1379, 1042 cmꢀ1; 1H NMR (400 MHz, CD3OD)
3H), 0.98 (d, J ¼ 6.6 Hz, 3H), 1.28e1.58 (m, 20H), 2.75 (ddd, J ¼ 2.1,
4.2, 6.2 Hz, 1H), 3.35 (m, 1H); 13C NMR (100 MHz, CD3OD)
: 14.2,
d
: 0.85 (t, J ¼ 6.9 Hz,
6.1.15. (2R,3S)-Aminoldodecan-1,3-diol (7$HCl)
d
Using the procedure described for the synthesis of the compound
2, reaction of 29 (100 mg, 0.19 mmol) and a few drops of 6 N HCl with
10% Pd/C (30 mg) gave 7$HCl (59 mg, 0.14 mmol, 70%) as a white
17.1, 23.5, 27.1, 30.0, 30.3, 30.5, 30.6, 30.6, 30.6, 32.9, 33.8, 51.9, 76.4;
MS (ESI, m/z) 230.2 (M þ Hþ, 100); HRMS m/z calcd for C14H31NO
(M þ Hþ): 230.2484; found: 230.2480.
solid. M.p. 78ꢀ80 ꢁC (EtOAc/PE); [
a
]
20 ꢀ6.3 (c 0.3, MeOH) {lit. [28]
D
20
[a
]
ꢀ6.3 (c 0.3, MeOH)}; IR (film) nmax: 3394, 3312, 2922, 2847,
D
6.1.11. (2R,3S)-Clavaminol A (3)
1587, 1472, 1381, 1060 cmꢀ1; 1H NMR (400 MHz, CD3OD)
d: 0.90 (t,
Using the procedure described for the synthesis of the
compound 2, reaction of 13d (98 mg, 0.24 mmol) with 10% Pd/C
(29 mg) gave 3 (43 mg, 0.21 mmol, 84%) as a white solid. M.p.
J ¼ 6.9 Hz, 3H), 1.26e1.59 (m, 16H), 1.61e2.81 (m, 1H), 3.48 (dd,
J ¼ 10.8, 7.7 Hz,1H), 3.52e3.68 (m,1H), 3.73 (dd, J ¼ 10.8, 4.0 Hz,1H);
13C NMR (100 MHz, CD3OD)
d: 15.2, 24.5, 27.8, 31.2, 31.3, 31.5, 33.8,
63ꢀ64 ꢁC (EtOAc/PE); [
a
]
20 ꢀ4.2 (c 0.5, MeOH) {lit. [19] [
a
]
D
25 ꢀ4.25
35.0, 59.3, 59.7, 71.1; MS (ESI, m/z) 218.1 (M þ Hþ, 100).
D
(c 0.0094, MeOH)}; IR (film) nmax: 3335, 2914, 2847, 1582, 1481,
1390, 1079 cmꢀ1; 1H NMR (400 MHz, CD3OD)
3H), 1.04 (d, J ¼ 6.7 Hz, 3H), 1.28e1.60 (m, 16H), 2.82 (ddd, J ¼ 2.1,
4.2, 6.2 Hz, 1H), 3.44 (m, 1H); 13C NMR (100 MHz, CD3OD)
: 12.46,
d
: 0.90 (t, J ¼ 6.9 Hz,
6.2. Pharmacology
d
6.2.1. Cell culture
15.09, 21.75, 25.28, 28.48, 28.75, 28.80, 28.84, 31.08, 31.98, 50.15,
74.36; MS (ESI, m/z) 202.1 (M þ Hþ, 100). HRMS m/z calcd for
C12H27NO (M þ Hþ): 202.2171; found: 202.2170.
The human glioblastoma cell SHG-44 was purchased from ATCC
(Shanghai, PRC) and cultured in DMEM medium (Thermo Scientific,
Shanghai, PRC) supplemented with 10% fetal bovine serum, peni-
cillin (50 U/mL) and streptomycin (50 mg/mL). Cells were main-
6.1.12. (2R,3S)-Clavaminol B (4)
tained in 37 ꢁC incubator with a 5% CO2 humidified atmosphere. For
viability assay, cells were plated onto 96-well plate with a density
of 5000/well and cultured in DMEM medium containing 1% fetal
bovine serum.
A solution of compound 13e (76 mg, 0.20 mmol) in anhydrous
MeOH (3.5 mL) and HCOOH (0.4 mL) was added to a suspension of
10% Pd/C (20 mg). The reaction mixture was stirred for 3 h at room
temperature and under a nitrogen atmosphere. When the reaction
was monitored to be complete by TLC analysis, the mixture was
filtered and the solid phase was washed with methanol 5 times. The
filtrate was concentrated in vacuo and the residue was purified by
flash chromatography on silica gel (eluent: MeOH/CH2Cl2 1:10) to
give 4 (36 mg, 0.18 mmol, 89%) as a white solid. M.p. 64ꢀ66 ꢁC
6.2.1. Cell viability assay
Cell viability was assessed by using Cell Counting Kit-8 (CCK-8,
Dojindo Laboratories, Japan) following the manufacture instruc-
tion. Briefly, 5000 cells/well in 96-well plate were cultured over-
night, then incubated with compound with different
20
25
(EtOAc/PE); [
MeOH)}; IR (film) nmax: 3387, 2948, 2944, 2920, 1603, 1466, 1396,
1265, 1054, 798 cmꢀ1 1H NMR (400 MHz, CD3OD)
: 0.83 (t,
a
]
ꢀ8.3 (c 0.2, MeOH) {lit. [19] [
a
]
ꢀ5.0 (c 0.001,
concentrations (0.1e100
mM) and vehicle (0.1% Dimethyl sulfoxide,
D
D
DMSO). After 24 h incubation, 10
ml of CCK-8 solution was added
;
d
and incubated at 37 ꢁC for 2 h. Plate was measured for absorbance
at 450 nm using a microplate reader (Kehua Bio-engineering,
Shanghai, PRC). Cell viability data was obtained from triplicate
experiments and expressed as the percentage of compound-treated
relative to vehicle-treated.
J ¼ 6.8 Hz, 3H),1.01 (d, J ¼ 6.6 Hz, 3H),1.21e1.45 (m,12H), 2.85 (ddd,
J ¼ 2.7, 3.8, 6.6 Hz, 1H), 3.45 (m, 1H), 5.38 (m, 2H); 13C NMR
(100 MHz, CD3OD) d: 11.4, 13.0, 20.7, 27.4, 27.7, 27.7, 30.1, 30.9, 49.2,
72.3, 132.9, 132.9; MS (ESI, m/z) 200.2 (M þ Hþ, 100). HRMS m/z
calcd for C12H25NO (M þ Hþ): 200.2014; found: 200.2012.
Conflict of interest
6.1.13. (2R,3S)-2-Amino-11-methyldodecan-3-ol (8)
None.
Using the procedure described for the synthesis of the compound
2, reaction of 13f (91 mg, 0.23 mmol) with 10% Pd/C (27 mg) gave 8
Acknowledgments
(43 mg, 0.20 mmol, 86%) as a white solid. M.p. 65ꢀ67 ꢁC (EtOAc/PE);
25
[
a]
ꢀ4.5 (c 0.22, MeOH); IR (film) nmax: 3389, 2923, 2853, 2521,
The authors are grateful to the National Basic Research Program
(973 Program) of China (Grant No. 2010CB833200), the NSF of
China (20832005), NFFTBS (No. J1030415), Fujian Province Health-
D
1747, 1463, 1383 cmꢀ1
; d: 0.88 (d,
1H NMR (400 MHz, CD3OD)
J ¼ 6.6 Hz, 6H), 1.05 (d, J ¼ 6.6 Hz, 3H), 1.11e1.22 (m, 2H), 1.22e1.45

5486
B.-S. Chen et al. / European Journal of Medicinal Chemistry 46 (2011) 5480e5486
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2597e2599.
Education Research Projects (WKJ2008-2-45) and Xiamen Science
and Technology Key Program Grant (No. 3502Z20100006) for the
financial support.
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Appendix. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.ejmech.2011.09.010.
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