Asymmetric synthesis and biological evaluation of the enantiomeric isomers of the immunosuppressive FTY720-phosphate

Article abstract of DOI:10.1016/j.bmc.2004.10.008

A practical asymmetric synthesis of both enantiomers of the immunosuppressive FTY720-phosphate (2) was accomplished, and the enantiomers were pharmacologically evaluated. Several lipases showed considerable activity and enantioselectivity for O-acylation

Full text of DOI:10.1016/j.bmc.2004.10.008

Bioorganic & Medicinal Chemistry 13 (2005) 425–432
Asymmetric synthesis and biological evaluation of the
enantiomeric isomers of the immunosuppressive FTY720-phosphate
Masatoshi Kiuchi,* Kunitomo Adachi, Ayumi Tomatsu, Masao Chino, Shuzo Takeda,
Yoshihito Tanaka, Yasuhiro Maeda, Noriko Sato, Naoko Mitsutomi,
Kunio Sugahara and Kenji Chiba
Research Laboratory III (Immunology), Mitsubishi Pharma Corporation, Kamoshida-cho 1000, Aoba-ku, Yokohama 227-0033, Japan
Received 6 September 2004; revised 5 October 2004; accepted 6 October 2004
Available online 28 October 2004
Abstract—A practical asymmetric synthesis of both enantiomers of the immunosuppressive FTY720-phosphate (2) was accom-
plished, and the enantiomers were pharmacologically evaluated. Several lipases showed considerable activity and enantioselectivity
for O-acylation of N-acetyl FTY720 (3) or N-benzyloxycarbonyl FTY720 (7) in combination with vinyl acetate or benzyl vinyl carb-
onate as the acyl donors. The synthesis using the lipase-catalyzed acylation as the key step produced the enantiomerically pure
(>99.5% ee) enantiomers of 2 in multigram quantities. (S)-Isomer of 2 had more potent binding affinities to S1P1,3,4,5 and inhibitory
activity on lymphocyte migration toward S1P than (R)-2, suggesting that (S)-isomer of 2 is responsible for the immunosuppressive
activity after administration of 1. Severe bradycardia was observed in anesthetized rats when (S)-2 was administered intravenously,
while (R)-2 had no clear effect on heart rate up to 0.3mg/kg.
Ó 2004 Elsevier Ltd. All rights reserved.
1. Introduction
clear is important for drug development and under-
standing a new aspect of immunological system.
FTY720 (1, Fig. 1) is an immunosuppressive com-
pound,1,2 which is efficacious in various models of auto-
immune diseases and transplantation.3 Recently, it was
reported that 1 was effective to prevent graft rejection
in transplanted kidneys in humans.4 Although the detail
of the immunosuppressive mechanism is unclear, it was
suggested that the decrease of the number of lympho-
cytes in peripheral blood and lymph relates the immuno-
suppressive activity.5,6 Making the mechanism of action
Sphingosine-1-phosphate (S1P) is ubiquitous in the
body, and influences multiple physiological systems7
such as endothelial integrity8 and coronary artery blood
flow9 by binding to its G-protein coupled receptors
(S1P1ꢀ5). One of hydroxyl group of 1 is phosphorylated
in vivo to form FTY720-phosphate (2, Fig. 1) acting as
an agonist for four S1P receptors (S1P1,3,4,5) out of five
(S1P1ꢀ5).10,11 It was reported that 2 downregulates S1P1
after its agonistic effect to the receptor, and the tempo-
rary pharmacological S1P1-null state in lymphocytes is
one of the mechanism of lymphopenia caused by admin-
istration of 1.12 While 1 was well tolerated in Phase I
clinical trials, a transient bradycardia was reported as
an adverse event.13 The transient heart rate changes
caused by a close analog of 2 disappeared in S1P3-dele-
tant mice,14 strongly suggesting that the bradycardia
relates to the agonistic activity of 2 to S1P3.
O
NH2
NH2
C8H17
HCl
P
C8H17
HO
HO
O
HO
OH
1 (FTY720)
OH
2 (FTY720-phosphate)
Figure 1. Structures of an immunosuppressant FTY720 (1) and the
active form, FTY720-phosphate (2).
Compound 1 was designed to delete the asymmetric
centers of its lead compound, myriocin,15 having three
asymmetric carbons; however, the phosphorylation of
1 into the active form 2 makes an asymmetric carbon
again. The optically pure forms of 2 are essential to
Keywords: Asymmetric synthesis; Lipase; Immunosuppressant; Sphin-
gosine-1-phosphate.
*
Corresponding author. Tel.: +81 45 963 3483; fax: +81 45 963
3529; e-mail: kiuchi.masatoshi@ms.m-pharma.co.jp
0968-0896/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.10.008

426
M. Kiuchi et al. / Bioorg. Med. Chem. 13 (2005) 425–432
investigate the mechanisms of action and bradycardia of
2. A preparation of enantiomers of 2 by a chiral HPLC
separation of the fully protected 2 and a formal asym-
metric synthesis via a serine-derived oxazolidine were re-
ported recently.16 Although the method was scrupulous,
it should be inadequate for a multigram scale synthesis
of the both enantiomers of 2 because the long scheme
was required for synthesis of each enantiomer. In this
paper, we describe a shorter and highly practical asym-
metric synthesis of both enantiomers of 2 using an asym-
metric acylation by lipase as the key step.
powerful method to synthesize the enantiomeric isomers
of 2; however, the possibility of racemization on 2-ami-
nopropane-1,3-diol monophosphates such as 2 was un-
clear at the beginning of this research, especially under
acidic or basic conditions required to cleave N- and O-
acetyl groups. During the study to synthesize racemic
2, we had also noticed that neither extraction nor chro-
matography was effective to purify 2 due to its poor sol-
ubility. Therefore, we tried to change N-acetyl groups of
4 into carbobenzyloxy groups (Cbz) in order to make it
easier to cleave the acyl groups and purify the
resultant 2.
2. Chemistry
We then tried acetylation of 7, N-benzyloxycarbonated
1, to replace the N-acetyl group of 3 with benzyloxycarb-
onyl, which would be cleaved easily. The most suitable
lipases for the enantioselective O-acetylation of 7 were
Lipase PL and AL, which gave 95% ee of the product
in 96% yield (Table 1). Although some other lipases (Li-
pase AS, PS, AK, M10, F-AP15, AYS, and G50) and
PPL (porcine pancreas lipase) were ineffective like Li-
pase SL, our successive screening of lipase revealed that
immobilized lipase (Toyobo Co.) and Novozyme 435
had excellent catalytic activities. However, they would
be too active to get higher optical yield. These results
prompted us to investigate O-benzylcarbonation be-
cause both of N- and O-benzyloxycarbonyl groups
could be removed by hydrogenolysis. The result of
screening is shown in Table 1. While the conversion
was low in the case of dibenzyl dicarbonate and dibenzyl
carbonate, benzyl vinyl carbonate,19 and acetone
O-(benzyloxy)carbonyloxime20 showed good reactivity.
Between them, benzyl vinyl carbonate was better for
this reaction because O-(benzyloxy)carbonyloxime in
combination with Lipase PL or TL gave O-isopropylid-
eneaminooxycarbonyl derivative of 7 as a by-product,
which was difficult to be removed by a silica gel
column chromatography from the product. The
immobilized lipase (Toyobo Co.) gave the best result,
which was quantitative conversion accompanied with
97% ee.
Although asymmetric acylation of 2,2-disubstituted pro-
pane-1,3-diol such as 1 has scarcely reported, asymmet-
ric acylation of 2-mono-substituted propane-1,3-diols by
acyl donor such as vinyl acetate using enzymes was
investigated well.17,18 Once the enantioselectivity of the
acylation was achieved, it does not make much differ-
ence in terms of efficient synthesis of both enantiomers
which alcohol is acylated by enzymes. It incited us to
try enantioselective O-acylation of N-protected 1 by
lipase. We first tried O-acetylation of compound 3, N-
acetylated 1, to make clear the possibility of asymmetric
acylation for 2,2-disubstituted propane-1,3-diol. Inter-
estingly, Lipase PL, TL, and AL acetylated 3 into diace-
tate (R)-4 in excellent chemical yields and considerable
optical yields (Scheme 1) in spite of the bulkiness of
the substituents, acetylamino and 2-(4-octylphenyl)ethyl,
at the C-2 position. The configuration of 4 was deter-
mined by deriving it into the known compound 6
(Scheme 2). Although the enantiomeric excess of 6
derived from enantiomerically pure 4 was 43.6% due
to the migration of acetyl group during cyclization of
4, the major component of 6 had the R configuration
showing that the configuration of 4 was R. These results
suggested that lipase catalyzing acylation would be a
NHAc
lipase
C8H17
The synthesis of (S)-enantiomer of 2 was carried out as
shown in Scheme 3. Compound 7, which was derived by
quantitative N-benzyloxycarbonation of 1, was O-benz-
ylcarbonated by benzyl vinyl carbonate using the
immobilized lipase to give 9. The reaction gave similar
chemical and optical yields in multigram quantity (5.10g
of 7) to them of the screening scale. The chiral alcohol
9 was phosphorylated into the fully protected intermedi-
ate 10 by a phosphoramidite with 1H-tetrazole followed
by 3-chloroperbenzoic acid oxidation. Hydrogenolysis
of 10 followed by washing in methanol gave 99.5% ee
HO
vinyl acetate
THF
OH
3
NHAc
C8H17
HO
Lipase PL: 99% (94%ee)
Lipase TL: 99% (93%ee)
Lipase AL: 82% (91%ee)
AcO
4
Scheme 1. Acetylation of 3, N-acetylated 1, using lipases and vinyl
acetate.
O
NHAc
HO
AcO
NH2
HO
C8H17
C8H17
C8H17
c, d, e, f
48%
a, b
N
HO
59%
4 (>99.5%ee)
5
6
Scheme 2. Determination of absolute configuration by deriving 4 to a reported compound 6. Reagents and conditions: (a) MsCl, Et3N, CH2Cl2;
(b) 0.1M NaOH, acetone; (c) (i) (COCl)2, DMSO, CH2Cl2, (ii) Et3N; (d) Ph3PEtBr, n-BuLi, Et2O; (e) H2, Pd/C, EtOH; (f) concd HCl, EtOH.

M. Kiuchi et al. / Bioorg. Med. Chem. 13 (2005) 425–432
Table 1. Results of lipase screening using various acyl donors
NHCbz
427
NHCbz
C8H17
C8H17
lipase
HO
HO
acyl donor
OR
OH
8: R = Ac
THF
9: R = Cbz
7
BnO
O
BnO
O
N
Acyl donor (equiv)
Vinyl acetate
(excess)
(BnOCO)2O (5equiv)
(BnO)2CO (10equiv)
O
O
(10equiv)
(5equiv)
Lipase PL
Lipase TL
96 (95 ee)
97 (92 ee)
96 (95 ee)
24
70 (91 ee)
84 (87 ee)
3
81 (89 ee)
25 (83 ee)
16
23
31
Tracea
Trace
––b
––
Lipase AL
Lipase QL
14
NRc
NR
NR
NR
NR
––
Lipase QLM
Lipase SL
40
NR
95d (89 ee)
95 (89 ee)
NR
NR
––
––
NR
99 (91 ee)
Trace
Immobilized lipase
Novozyme 435
99 (97 ee)
73 (49 ee)
33
NR
––
––
Lipases were screened at room temperature overnight. For the primary screening, 0.1mmol of 7 was used. The results are shown as percent of
conversion determined by LC/MS. For the detailed screening to determine the yields and enantiomeric excesses (ee), 1.0mmol of 7 was used. The
results are shown as isolated yields (%) followed by ee (%) determined by HPLC (Chiralcel OJ-RH, CH3CN/H2O) in parentheses.
a The product was less than 3%.
b The reaction was not tried.
c The product was not detected.
d The reaction was done at 0°C.
NHCbz
NHCbz
HO
HO
a
HO
O
O
C8H17
b
98%
c
C8H17
1
Bn
99%
99%
O
7
9
(97.1%ee)
BnO
HO
O
OBn
O
O
OH
O
P
P
O
NHCbz
NH2
d
76%
O
C8H17
C8H17
HO
Bn
O
(S)-2 (99.5%ee)
10
Scheme 3. Synthesis of (S)-2. Reagents and conditions: (a) ClCOOBn, NaHCO3, CHCl3/H2O; (b) BnOCOOCH@CH2, immobilized lipase, THF;
(c) (i) (BnO)2PN(iso-Pr)2, 1H-tetrazole, CH2Cl2/CH3CN, (ii) m-CPBA; (d) (i) H2, Pd/C, MeOH, (ii) wash in MeOH.
of (S)-enantiomer of 2. Any method for quantitation of
the enantiomeric excess of 2 had not been reported16 be-
cause general chiral selectors of HPLC column such as
cellulose or crown ether derivatives were ineffective to
separate the enantiomers of 2 due to the low solubility
and its unique structure including amine, phosphate,
alcohol, and highly lipophilic groups. However, our suc-
cessive screening of HPLC column revealed that both
enantiomers of 2 were completely separable on a
SumichiralÒ OA-7000 HPLC column (CH3CN/pH3
phosphate buffer), which has b-cyclodextrin as the chiral
selector. We confirmed that the treatment of (S)-2 in 1M
HCl or 2M NaOH at room temperature for 12h did not
change the enantiomeric excess.
1-propene as an acetal,21 which would be removed by
hydrogenolysis, then O-acetyl group was cleaved to give
an alcohol 11. Phosphorylation followed by removal of
the protecting groups yielded optically pure (R)-2. In the
last acidifying step, adjusting the pH of the reaction
mixture to around 1 was important to get a free form
of (R)-2 unlike the method where pH was adjusted to
7.16 The reported method gave a mono-sodium salt of
(R)-2 in our case.22
3. Biology
To confirm which enantiomer is responsible for the
activity of compound 2, and investigate their S1P recep-
tor selectivity, both enantiomers were tested against
human S1P receptors expressed in CHO (S1P1,2,4) or
RH7777 (S1P3,5) cells in competitive ligand binding
assay using [32P]S1P as the ligand (Table 2). As reported
results for rac-2,10,11,16 both enantiomers had no ability
The (R)-enantiomer of 2 was synthesized as shown in
Scheme 4. Acetylation of compound 3 by Lipase PL
gave 92% ee of the product, which was purified by
recrystallization to give optically pure compound 4.
The remaining alcohol was protected by 2-benzyloxy-

428
M. Kiuchi et al. / Bioorg. Med. Chem. 13 (2005) 425–432
NHAc
NHAc
HO
HO
HO
AcO
a
b, c
92%
C8H17
C8H17
d, e
83%
1
97%
4 (>99.5%ee)
3
BnO
BnO
NHAc
f
85%
NHAc
g, h
O
HO
O
O
C8H17
O
C8H17
BnO P
69%
OBn
11
12
HO
O
NH2
O
C8H17
HO P
OH
(R)-2 (>99.5%ee)
Scheme 4. Synthesis of (R)-2. Reagents and conditions: (a) Ac2O, NaHCO3, CHCl3/H2O; (b) vinyl acetate, Lipase PL, THF; (c) recrystallization; (d)
CH2@C(CH3)OBn, PdCl2(COD), benzene; (e) K2CO3, MeOH/acetone/H2O; (f) (i) (BnO)2PN(iso-Pr)2, 1H-tetrazole, CH2Cl2/CH3CN, (ii) m-CPBA;
(g) H2, Pd/C, MeOH; (h) (i) 2M NaOH, MeOH, (ii) 1M HCl.
Table 2. Binding affinities (Ki SEM,nM) to S1P receptors
Compound
S1P1
S1P2
S1P3
S1P4
S1P5
S1P
1.2 0.04
4.1 0.63
277 29.0
2.1 0.12
3.4 0.18
>1000
>1000
5.4 0.23
13 1.6
2.7 0.28
37 2.1
0.92 0.05
4.2 0.71
121 9.5
2.2 0.25
rac-2
(R)-2
(S)-2
586 66.5
5.9 0.26
105 16.1
23 2.2
>1000
Ki Values were determined by competition of [32P]-S1P binding to stably transfected CHO (S1P1,2,4) or RH7777 (S1P3,5) cells expressing the indicated
S1P receptors. Data represent the mean SE of three experiments.
100
80
60
40
20
0
100
80
60
40
20
0
These data are show that (S)-isomer not only binds to
S1P receptors strongly but also is responsible for
decreasing lymphocytes in blood and lymph. Accord-
ingly, it was confirmed that (S)-isomer is the active form
of 2. This result was supported by our previous stud-
ies,2,23 in which pro-(S) hydroxymethyl between two
hydroxymethyl groups of 1 was essential for the immu-
nosuppressive activity. As to the bradycardia, we evalu-
ated both enantiomers of 2 in anesthetized Sprague-
Dawley rats.24 After intravenous bolus administration
of (S)-2, the marked decrease of heart rate was observed
at doses of 0.003 or 0.03mg/kg in a dose dependent
manner (Table 3). In contrast to the severe bradycardia
induced by (S)-isomer, (R)-2 had no clear effect on heart
rate up to a dose of 0.3mg/kg. This result revealed not
only that (S)-isomer is closely related to bradycardia in-
duced by administration of 1, also that S1P/S1P recep-
tors are significant to regulate the heart rate. In a
preliminary attempt to identify the ratio of (R)- and
(S)-2 in rat serum after oral administration of 1
(1mg/kg, 8h), (S)-2 was the only isomer detected by
0.1
1
10
100
Concentration (nM)
(S)-2 : IC50 = 0.137 nM (0.0414—0.451)
(R)-2 : IC50 = 10.98 nM (3.28—36.7)
Figure 2. Inhibitory activity of (S)- and (R)-2 on lymphocyte migra-
tion toward 10nM of S1P. IC50 values are followed by 95%
confidential limits.
to bind S1P2. While (R)-2 bound to S1P1,3,4,5 only at
high concentrations, (S)-2 had the ability to bind them
in nanomolar or subnanomolar ranges. Figure 2 shows
the inhibitory activity of both enantiomers of 2 on T-
lymphocyte chemotaxis toward S1P. It was reported
that peripheral lymphocytes taken from FTY720-trea-
ted mice failed to migrate in response to S1P, and that
this activity could explain the blocking effect of 1 on
lymphocyte egress.12 (S)-Isomer of 2 inhibited T cell
chemotactic response to 10nM of S1P completely at
1nM or higher concentrations (IC50 = 0.137nM). On
the other hand, (R)-2 required approximately 100 times
higher concentration to inhibit it (IC50 = 11.0nM).
Table 3. Bradycardia induced by enantiomers of 2 in anesthetized rats
Compound
Dose (mg/kg)
Heart rate (%)
(S)-2
(S)-2
0.003
0.03
0.3
46.6 13.0
16.4 3.9
91.8 1.7
97.1 2.1
(R)-2
Vehicle
Heart rate was monitored on an electrocardiogram. Data were taken
one minute after intravenous administration of the test compound, and
expressed as the percentage against before administration. Results are
the mean SE of three animals (Sprague-Dawley rats).

M. Kiuchi et al. / Bioorg. Med. Chem. 13 (2005) 425–432
429
LC/MS.25 In the future, the more detail of the bioactive
mechanisms of 1 would be made clear by using the enan-
tiomerically pure form of 2.
5.3. N-[1,1-Bis(hydroxymethyl)-3-(4-octylphenyl)propyl]-
acetamide (3)
A mixture of 1 (10.0g, 29.1mmol), chloroform (800mL),
and saturated sodium bicarbonate (500mL) was stirred
vigorously. After adding of acetic anhydride (3.29mL,
34.9mmol) to the mixture eight times at every 30min,
the organic layer was separated, washed with brine,
and dried over MgSO4. The solution was evaporated,
and the residue was crystallized in diethyl ether to give
9.82g (97%) of 3 as a white crystalline powder. 1H
NMR (CDCl3): d 7.10 (4H, s), 5.75 (1H, s), 3.88
(1H, d, J = 8.6Hz), 3.86 (1H, d, J = 8.6Hz), 3.65–
3.60 (4H, m), 2.64–2.61 (2H, m), 2.56 (2H, t,
J = 7.8Hz), 1.98–1.94 (2H, m), 1.93 (3H, s), 1.58
(2H, quint, J = 7.4Hz), 1.33–1.24 (10H, m), 0.88
(3H, t, J = 6.8Hz). MS (ESI): m/z 350 (M+H), 372
(M+Na).
4. Conclusion
A practical asymmetric synthesis of both enantiomers of
the immunosuppressive FTY720-phosphate (2) was
accomplished using the lipase-catalyzed acylation as
the key step. (S)-Isomer of 2 had more potent binding
affinities to S1P1,3,4,5 and inhibitory activity on lympho-
cyte migration toward S1P than (R)-2, suggesting that
(S)-isomer of 2 is responsible for the immunosuppressive
activity after administration of 1. Severe bradycardia
was observed in anesthetized rats when (S)-2 was admin-
istered intravenously, while (R)-2 had no clear effect on
heart rate up to 0.3mg/kg. The more detail of the bioac-
tive mechanisms of 1 would be made clear by using the
enantiomerically pure form of 2.
5.4. (R)-2-Acetamido-2-hydroxymethyl-4-(4-octylphenyl)-
butyl acetate (4)
5. Experimental
5.1. Chemistry
A mixture of 3 (9.82g, 28.1mmol), THF (130mL), vinyl
acetate (130mL), and Lipase PL (4.91g) was stirred at
room temperature for 140min. The mixture was passed
through a Celite pad and concentrated to give 94% ee of
4. Crystallization in diethyl ether and hexane afforded
10.2g (93%) of enantiomerically pure (>99.5% ee) 4 as
Lipase PL, TL, AL, QL, QLM, and SL were obtained
from Meito Sangyo Co., Ltd. Immobilized lipase (Toy-
obo) and PPL were purchased from Nacalai tesque.
Novozyme 435 and other lipases (lipase AS, PS, AK,
M10, F-AP15, AYS, and G50) were purchased from
Sigma-Aldrich Japan K. K. and Wako Pure Chemical
Industries, Ltd, respectively. Silica gel column chroma-
tography was performed on Fuji Silysia PSQ100B.
NMR spectra were recorded on a Bruker AVANCE
400 (400MHz for proton and 162MHz for phosphorus)
spectrometer. Chemical shifts are expressed in ppm rel-
ative to TMS as internal standard for proton and
H3PO4 for phosphorus, respectively. Mass spectra were
measured in a combination with a Waters Alliance 2795
HPLC system (0.05% TFA in CH3CN/0.05% TFA in
water), a Chromolith SpeedROD RP-18e column, and
a Micromass ZQ (ESI) spectrometer. Melting points
were obtained on a Buchi 535 melting point apparatus
and are uncorrected. Optical rotations were determined
on a JASCO digital polarimeter DIP-360 in a 10cm path
length cell. Elemental analyses were performed on a
Yanako CHN coder MT-5.
1
a white crystalline powder. H NMR (CDCl3): d 7.10
(4H, s), 5.70 (1H, s), 4.47 (1H, t, J = 7.0Hz), 4.38 (1H,
d, J = 11.4Hz), 4.18 (1H, d, J = 11.4Hz), 3.77 (1H, dd,
J = 12.2, 7.4Hz), 3.72 (1H, dd, J = 12.0, 6.8Hz), 2.70–
2.62 (1H, m), 2.59–2.52 (1H, m), 2.56 (2H, t,
J = 7.8Hz), 2.18–2.10 (1H, m), 2.11 (3H, s), 1.97–1.90
(1H, m), 1.93 (3H, s), 1.58 (2H, quint, J = 7.2Hz),
1.33–1.24 (10H, m), 0.88 (3H, t, J = 6.8Hz). MS (ESI):
24
D
m/z 392 (M+H), 414 (M+Na). ½aꢁ ꢀ0.9 (c 1.01,
CHCl3).
5.5. (S)-[2-Methyl-4-[2-(4-octylphenyl)ethyl]oxazolin-4-yl]
methanol (5)
To a mixture of 4 (3.0g, 7.62mmol), Et3N (1.16mL,
8.38mmol) in CH2Cl2 (25mL) was added methane-
sulfonyl chloride (0.619mL, 8.00mmol) at 0°C. The
mixture was stirred for 15min, extracted with CH2Cl2,
and washed with brine. The solution was dried over
MgSO4 and evaporated. The residue was purified by sil-
ica gel column chromatography (hexane/EtOAc = 1/1)
to afford acetate of 5 (1.82g, 64%) as a colorless oil.
To a solution of this intermediate in acetone (50mL)
was added 0.25M NaOH (19.2mL, 4.80mmol) at 0°C.
The mixture was stirred at 0°C for 1h, and concentrated
to remove acetone. The aqueous residue was extracted
with EtOAc. The organic layer was washed with brine,
dried over MgSO4, and evaporated. The residue was
purified by silica gel column chromatography (hexane/
EtOAc = 1/4) to afford 5 (1.47g, 92%) as a colorless
oil. 1H NMR (CDCl3): d 7.08 (4H, s), 4.22 (1H, d,
J = 8.4Hz), 4.06 (1H, d, J = 8.4Hz), 3.68 (1H, br d,
J = 11.0Hz), 3.44 (1H, dd, J = 11.0, 7.8Hz), 2.61–2.49
(4H, m), 2.10–2.04 (1H, m), 2.02 (3H, s), 1.88 (1H,
ddd, J = 13.6, 11.4, 5.8Hz), 1.75 (1H, ddd, J = 13.6,
5.2. Lipase screening
Unless otherwise stated, all primary screening reactions
were done at room temperature in 0.1mmol scale. A part
of reaction solution was diluted with DMSO and ana-
lyzed by LC/MS to determine the rate of conversion. Lip-
ases showing considerable activity were further tested in
1.0mmol scale to determine the isolated yields and enan-
tiomeric excess (ee). At the end of reaction, the reaction
mixture was passed through a Celite pad, concentrated,
and purified by a silica gel column chromatography.
The ee values were determined by HPLC using a Chiralcel
OJ-RH (CH3CN/H2O). All lipase catalyzed products had
R configuration (a shorter retention time).

430
M. Kiuchi et al. / Bioorg. Med. Chem. 13 (2005) 425–432
11.0, 5.8Hz), 1.58 (2H, quint, J = 7.5Hz), 1.34–1.23
(10H, m), 0.87 (3H, t, J = 6.8Hz). MS (ESI): m/z 332
(M+H). ½aꢁ +15.9 (c 0.57, CHCl3).
5.8. (R)-Acetic acid 2-(benzyloxycarbonyl)amino-2-
hydroxymethyl-4-(4-octylphenyl)butyl ester (8)
24
D
A mixture of 7 (442mg, 1.0mmol), lipase PL (220mg),
vinyl acetate (10mL), and THF (10mL) was stirred at
room temperature for 5h. The mixture was passed
through a Celite pad, and concentrated. The residue
was purified by silica gel column chromatography (hex-
ane/EtOAc = 4/1) to afford 8 (462mg, 96%, 95.3% ee by
HPLC (Chiralcel OJ-RH, CH3CN/H2O, shorter reten-
tion time)) as a colorless oil. 1H NMR (CDCl3): d
7.39–7.31 (5H, m), 7.07 (2H, d, J = 8.2Hz), 7.05 (2H,
d, J = 8.2Hz), 5.10 (1H, s), 5.09 (2H, s), 4.36 (1H, d,
J = 11.4Hz), 4.19 (1H, d, J = 11.4Hz), 3.76 (1H, dd,
J = 12.0, 7.6Hz), 3.70 (1H, dd, J = 12.0, 6.8Hz), 3.46
(1H, br s), 2.62–2.49 (2H, m), 2.55 (2H, t, J = 7.8Hz),
2.15–2.08 (1H, m), 2.08 (3H, s), 1.94–1.87 (1H, m),
1.58 (2H, quint, J = 7.4Hz), 1.34–1.24 (10H, m), 0.88
5.6. (R)-2-Amino-2-[2-(4-octylphenyl)ethyl]pentanol (6)
To a solution of oxalyl chloride (0.748mL, 8.58mmol)
and DMSO (0.915mL, 12.9mmol) in CH2Cl2 (20mL)
was added a solution of 5 (1.43g, 4.29mmol) in CH2Cl2
(15mL) at ꢀ70°C. After stirring the mixture for 2h at
the same temperature, Et3N (4.46mL, 32.2mmol) was
added. The mixture was stirred at ꢀ55°C for 30min, di-
luted with saturated NH4Cl, and extracted with CH2Cl2.
The organic layer was washed with brine, dried over
MgSO4, and concentrated to afford an aldehyde
(1.43g, quant.) as a yellow oil. To a suspension of
(ethyl)triphenylphosphonium bromide (3.19g, 8.58
mmol) in Et2O (20mL) was added 1.6M n-BuLi in hex-
anes (4.69mL, 7.50mmol), and the mixture was stirred
for 1h. A solution of the aldehyde obtained above in
Et2O (15mL) was added to the mixture, and the mixture
was stirred for 14h. After adding saturated NaHCO3,
the mixture was extracted with EtOAc. The organic layer
was washed with brine, dried over MgSO4, and evapo-
rated. The residue was purified by silica gel column
chromatography (hexane/EtOAc = 4/1) to afford (S)-[2-
methyl-4-[2-(4-octylphenyl)ethyl]-4-(1-propenyl)oxazo-
line (791mg, 54%) as a yellow oil. A mixture of the
product and 10% Pd/C (100mg) in EtOH (25mL) was
stirred under hydrogen atmosphere for 7h, passed
through a Celite pad, and concentrated. The residue
was dissolved in EtOH (15mL) and concentrated HCl
(5mL), and the mixture was heated at 70°C for 30min,
and concentrated. After adding saturated NaHCO3,
the mixture was extracted with EtOAc. The organic layer
was washed with brine, dried over MgSO4, and evapo-
(3H, t, J = 6.8Hz). MS (ESI): m/z 484 (M+H), 506
24
D
(M+Na). ½aꢁ ꢀ3.27 (c 0.94, CHCl3).
5.9. (R)-2-(Benzyloxycarbonyl)amino-2-(benzyloxycarbon-
yloxy)methyl-4-(4-octylphenyl)butanol (9)
A mixture of 7 (5.10g, 11.5mmol), benzyl vinyl carbon-
ate (5.12g, 28.8mmol), immobilized lipase (Toyobo,
5.10g), and THF (90mL) was stirred at room tempera-
ture for 16h. The mixture was passed through a Celite
pad, and concentrated. The residue was purified by silica
gel column chromatography (hexane/EtOAc = 95/5–8/2)
to afford 9 (6.52g, 99%, 97.1% ee by HPLC (Chiralcel
OJ-RH, CH3CN/H2O, shorter retention time)) as a col-
orless oil. 1H NMR (CDCl3): d 7.38–7.30 (10H, m), 7.06
(2H, d, J = 8.2Hz), 7.04 (2H, d, J = 8.2Hz), 5.16 (2H, s),
5.11 (1H, s), 5.07 (2H, s), 4.41 (1H, d, J = 11.0Hz), 4.30
(1H, d, J = 11.0Hz), 3.79 (1H, dd, J = 12.0, 7.2Hz), 3.73
(1H, dd, J = 12.0, 7.2Hz), 3.40 (1H, br s), 2.62–2.49
(2H, m), 2.54 (2H, t, J = 7.6Hz), 2.10 (1H, ddd,
J = 14.0, 11.4, 5.2Hz), 1.89 (1H, ddd, J = 14.0, 11.8,
5.6Hz), 1.57 (2H, quint, J = 7.2Hz), 1.34–1.23 (10H,
1
rated to afford 6 (506mg, 63%) as a white powder. H
NMR (CDCl3): d 7.09 (4H, s), 3.36 (2H, s), 2.56 (4H,
t, J = 8.1Hz), 1.64–1.56 (6H, m), 1.47–1.43 (1H, m),
1.36–1.26 (14H, m), 0.95 (3H, t, J = 6.9Hz), 0.88 (3H,
24
t, J = 6.8Hz). MS (ESI): m/z 320 (M+H). ½aꢁ +0.81 (c
m), 0.88 (3H, t, J = 6.8Hz). MS (ESI): m/z 576
D
24
D
0.50, CHCl3). The retention time of N-3,5-di-
nitrobenzoyl derivative of 6 was identical with that of
(R)-6 (shorter retention time on Chiralcel OD (hexane/
EtOH), 43.6% ee).23
(M+H), 598 (M+Na). ½aꢁ ꢀ2.34 (c 0.96, CHCl3).
5.10. (S)-2-(Benzyloxycarbonyl)amino-2-(benzyloxycarb-
onyloxy)methyl-1-(dibenzyl)phosphoryloxy-4-(4-octyl-
phenyl)butane (10)
5.7. 2-(Benzyloxycarbonyl)amino-2-[2-(4-octylphenyl)-
ethyl]propane-1,3-diol (7)
To a solution of 9 (6.50g, 11.3mmol) and 1H-tetrazole
(953mg, 13.6mmol) in CH2Cl2 (70mL) and CH3CN
(70mL) was added dibenzyl diisopropylphosphorami-
dite (4.17mL, 12.4mmol) at 0°C. After stirring at 0°C
for 1h, m-CPBA (65% purity, 3.61g, 13.6mmol) was
added. The mixture was stirred at 0°C for 20min, di-
luted with saturated NaHCO3, and extracted with
CH2Cl2. The organic layer was washed with brine, dried
over MgSO4, and evaporated. The residue was purified
by silica gel column chromatography (hexane/
EtOAc = 9/1–3/2) to afford 10 (9.39g, 99%) as a color-
A mixture of 1 (4.03g, 11.7mmol) and benzyl chlorofor-
mate (5.98mL, 41.9mmol) in CHCl3 (500mL) and satu-
rated NaHCO3 (250mL) was stirred at room
temperature for 18h. The organic layer was separated,
washed with brine, and dried over MgSO4. The solution
was concentrated, and the residue was crystallized in
1
Et2O to afford 7 (5.10g, 99%) as a white powder. H
NMR (CDCl3): d 7.37–7.31 (5H, m), 7.07 (4H, s), 5.27
(1H, s), 5.09 (2H, s), 3.91 (2H, dd, J = 11.6, 6.0Hz),
3.68 (2H, dd, J = 11.6, 6.8Hz), 3.13 (2H, br s),
2.60–2.56 (2H, m), 2.55 (2H, t, J = 8.0Hz), 1.92–1.88
(2H, m), 1.61–1.56 (2H, m), 1.33–1.26 (10H, m), 0.88
(3H, t, J = 6.8Hz). MS (ESI): m/z 442 (M+H), 464
(M+Na).
1
less oil. H NMR (CDCl3): d 7.35–7.28 (20H, m), 7.04
(2H, d, J = 7.8Hz), 6.97 (2H, d, J = 7.8Hz), 5.12 (3H,
br s), 5.04 (2H, s), 5.01 (4H, d, J = 8.8Hz), 4.34 (1H,
d, J = 11.0Hz), 4.29 (1H, d, J = 11.0Hz), 4.14 (2H, d,
J = 6.0Hz), 2.54 (2H, t, J = 7.6Hz), 2.50–2.41 (2H, m),

M. Kiuchi et al. / Bioorg. Med. Chem. 13 (2005) 425–432
431
2.11–1.94 (2H, m), 1.59–1.54 (2H, m), 1.33–1.25
(10H, m), 0.88 (3H, t, J = 6.8Hz). MS (ESI): m/z 858
(M+Na).
5.98 (1H, s), 4.73 (1H, t, J = 6.4Hz), 4.53 (1H, d, J =
11.6Hz), 4.51 (1H, d, J = 11.6Hz), 3.73 (1H, d,
J = 9.4Hz), 3.72 (2H, d, J = 6.4Hz), 3.45 (1H, d, J =
9.4Hz), 2.69–2.62 (1H, m), 2.55 (2H, t, J = 7.6Hz),
2.55–2.47 (1H, m), 2.14 (1H, ddd, J = 14.0, 11.6,
5.2Hz), 1.94–1.85 (1H, m), 1.89 (3H, s), 1.57 (2H, quint,
J = 7.4Hz), 1.47 (3H, s), 1.46 (3H, s), 1.33–1.24 (10H,
m), 0.88 (3H, t, J = 6.6Hz). MS (ESI): m/z 350
(MꢀBnOC(CH3)2+H).
5.11. (S)-2-Amino-2-(phosphoryloxy)methyl-4-(4-octyl-
phenyl)butanol ((S)-2)
A mixture of 10 (9.35g, 11.2mmol) and 10% Pd/C (5.0g)
in MeOH (200mL) was stirred under hydrogen atmos-
phere for 7h. The mixture was passed through a Celite
pad, and concentrated. The residue was crystallized in
EtOH/H2O. The white powder was suspended in MeOH
(200mL), and stirred at room temperature for 20h. The
suspending solid was collected to afford (S)-2 (3.31g,
76%, 99.5% ee by HPLC (Sumichiral OA-7000,
CH3CN/pH3 phosphate buffer, slower eluting
enantiomer)) as a white powder; mp 198–199°C. 1H
NMR (CD3OD): d 7.14 (2H, d, J = 7.8Hz), 7.08 (2H,
d, J = 7.8Hz), 4.01 (1H, dd, J = 11.6, 5.6Hz), 3.99
(1H, dd, J = 11.6, 5.6Hz), 3.71 (1H, d, J = 11.6Hz),
3.69 (1H, d, J = 11.6Hz), 2.71–2.60 (2H, m), 2.56 (2H,
t, J = 7.6Hz), 2.01–1.92 (2H, m), 1.58 (2H, quint, J =
7.2Hz), 1.34–1.25 (10H, m), 0.89 (3H, t, J = 6.4Hz).
5.13. (R)-N-[1-(Dibenzylphosphoryloxy)methyl-1-(1-benz-
yloxy-1-methylethoxy)methyl-3-(4-octylphenyl)propyl]-
acetamide (12)
To a solution of 11 (10.5g, 21.1mmol) and 1H-tetrazole
(1.77g, 25.3mmol) in CH2Cl2 (120mL) and CH3CN
(120mL) was added dibenzyl diisopropylphosphorami-
dite (7.80mL, 23.2mmol) at 0°C. After stirring at 0°C
for 1h, m-CPBA (65% purity, 6.72g, 25.3mmol) was
added. The mixture was stirred at 0°C for 20min, di-
luted with saturated NaHCO3, and extracted with
CH2Cl2. The organic layer was washed with brine, dried
over MgSO4, and evaporated. The residue was purified
by silica gel chromatography (hexane/EtOAc = 9/1–1/1)
31P NMR (CD3OD): d ꢀ0.04 (t, J = 5.5Hz). MS
24
D
1
(ESI): m/z 388 (M+H). ½aꢁ +1.26 (c <0.1, MeOH.
to afford 12 (13.5g, 84%) as a colorless oil. H NMR
(CDCl3):
The number of optical rotation was lower reliability due
to the poor solubility.). Anal. Calcd for C19H34NO5PÆ
0.5H2O: C, 57.56; H, 8.90; N, 3.53. Found: C, 57.69;
H, 9.18; N, 3.53.
d
7.34–7.21 (15H, m), 7.05 (2H, d,
J = 7.8Hz), 7.03 (2H, d, J = 7.8Hz), 5.83 (1H, s), 4.98
(4H, d, J = 8.4Hz), 4.46 (2H, s), 4.25 (1H, dd,
J = 10.0, 6.4Hz), 4.21 (1H, dd, J = 10.0, 6.4Hz),
3.70 (1H, d, J = 9.2Hz), 3.61 (1H, d, J = 9.2Hz),
2.55 (2H, t, J = 7.8Hz), 2.52–2.45 (2H, m), 2.22–
2.07 (2H, m), 1.82 (3H, s), 1.57 (2H, quint,
J = 7.4Hz), 1.40 (3H, s), 1.39 (3H, s), 1.33–1.24 (10H,
m), 0.87 (3H, t, J = 6.8Hz). MS (ESI): m/z 610
(MꢀBnOC(CH3)2+H).
5.12. (S)-N-[1-(1-Benzyloxy-1-methylethoxy)methyl-1-
hydroxymethyl-3-(4-octylphenyl)propyl]acetamide (11)
A mixture of 4 (10.1g, 25.8mmol), dichloro(1,5-cyclo-
octadiene)palladium(II) (737mg, 2.58mmol), and benzyl
isopropenyl ether (7.65g, 51.6mmol) in benzene
(170mL) was stirred at room temperature for 18h.
The mixture was diluted with EtOAc, washed with satu-
rated NaHCO3 and then brine, and dried over MgSO4.
The solution was concentrated, and the residue was
purified by silica gel column chromatography (hexane/
EtOAc = 9/1–1/1) to afford an intermediate (13.7g,
98%), (R)-2-acetamido-2-(1-benzyloxy-1-methylethoxy-
methyl)-4-(4-octylphenyl)butyl acetate, as a colorless
5.14. (R)-2-Amino-2-(phosphoryloxy)methyl-4-(4-octyl-
phenyl)butanol ((R)-2)
A mixture of 12 (13.2g, 17.4mmol) and 10% Pd/C (6.5g)
in MeOH (200mL) was stirred under hydrogen atmos-
phere for 9h. The mixture was passed through a Celite
pad, and concentrated. The residue was dissolved in
MeOH (20mL) and 1M NaOH (60mL), and refluxed
for 10h. The reaction mixture was cooled to room tem-
perature, and acidified with 1M HCl to adjust pH1. The
yielded solid was collected, and washed with water to af-
ford (R)-2 (4.67g, 69%, >99.5% ee by HPLC (Sumichiral
OA-7000, CH3CN/pH3 phosphate buffer, faster eluting
enantiomer)) as a white powder; mp 199–200°C. 1H and
1
oil. H NMR (CDCl3): d 7.34–7.31 (4H, m), 7.29–7.24
(1H, m), 7.08 (4H, s), 5.61 (1H, s), 4.49 (2H, s), 4.38
(1H, d, J = 11.2Hz), 4.36 (1H, d, J = 11.2Hz),
3.66 (1H, d, J = 9.4Hz), 2.62–2.50 (4H, m), 2.36–2.28
(1H, m), 2.11–2.03 (1H, m), 1.93 (3H, s), 1.90 (3H, s),
1.61–1.56 (2H, m), 1.45 (6H, s), 1.33–1.24 (10H, m),
0.88 (3H, t, J = 7.0Hz). MS (ESI): m/z 392
(MꢀBnOC(CH3)2+H). To a solution of the above inter-
mediate (13.7g, 25.4mmol) in acetone (100mL), water
(60mL), and MeOH (30mL) was added K2CO3
(3.51g, 25.4mmol) at 0°C. The mixture was stirred at
room temperature for 11h, and concentrated to remove
organic solvents. The residue was diluted with water,
and extracted with EtOAc. The extract was washed with
brine, and dried over MgSO4. The solution was concen-
trated, and the residue was purified by silica gel column
chromatography (hexane/EtOAc = 4/1–1/1) to afford 11
31P NMR spectra were identical with those of (S)-2. MS
24
D
(ESI): m/z 388 (M+H). ½aꢁ ꢀ9.99 (c <0.1, MeOH. The
number of optical rotation was lower reliability due to
the poor solubility.). Anal. Calcd for C19H34NO5PÆ0.5-
H2O: C, 57.56; H, 8.90; N, 3.53. Found: C, 57.79; H,
9.22; N, 3.56.
Acknowledgements
Authors thank Sumika Chemical Analysis Service, Ltd
(Osaka, Japan) for their HPLC column screening effort
on enantiomeric separation of compound 2, and Meito
1
(10.6g, 84%) as a colorless oil. H NMR (CDCl3): d
7.36–7.32 (4H, m), 7.29–7.25 (1H, m), 7.07 (4H, s),

432
M. Kiuchi et al. / Bioorg. Med. Chem. 13 (2005) 425–432
peripheral lymphoid organs is dependent on S1P receptor
1. Nature 2004, 427, 355–360.
Sangyo Co., Ltd (Nagoya, Japan) for donating us their
lipases.
13. Budde, K.; Schmouder, R. L.; Nashan, B.; Brunkhorst,
R.; Lucker, P. W.; Mayer, T.; Brookman, L.; Nedelman,
J.; Skerjanec, A.; Bohler, T.; Neumayer, H.-H. Pharmaco-
dynamics of single doses of the novel immunosuppressant
FTY720 in stable renal transplant patients. Am. J.
Transplant. 2003, 3, 846–854.
14. Sanna, M. G.; Liao, J.; Jo, E.; Alfonso, C.; Ahn, M.-Y.;
Peterson, M. S.; Webb, B.; Lefebvre, S.; Chun, J.; Gray,
N.; Rosen, H. Sphingosine-1-phosphate (S1P) receptor
subtypes S1P1 and S1P3, respectively, regulate lymphocyte
recirculation and heart rate. J. Biol. Chem. 2004, 279,
13839–13848.
References and notes
1. Adachi, K.; Kohara, T.; Nakao, N.; Arita, M.; Chiba, K.;
Mishina, T.; Sasaki, S.; Fujita, T. Design, synthesis, and
structure–activity relationships of 2-substituted-2-amino-
1,3-propanediols: discovery of a novel immunosuppres-
sant, FTY720. Bioorg. Med. Chem. Lett. 1995, 5, 853–
856.
2. Kiuchi, M.; Adachi, K.; Kohara, T.; Minoguchi, M.;
Hanano, T.; Aoki, Y.; Mishina, T.; Arita, M.; Nakao, N.;
Ohtsuki, M.; Hoshino, Y.; Teshima, K.; Chiba, K.;
Sasaki, S.; Fujita, T. Synthesis and immunosuppressive
activity of 2-substituted 2-aminopropane-1,3-diols and
2-aminoethanols. J. Med. Chem. 2000, 43, 2946–2961.
3. Chiba, K.; Adachi, K. FTY720, immunosuppressant.
Drugs Future 1997, 22, 18–22.
4. Tedesco-Silva, H.; Mourad, G.; Kahan, B. D.; Boira, J.
G.; Weimar, W.; Mulgaonkar, S.; Nashan, B.; Madsen, S.;
Charpentier, B.; Pellet, P.; Vanrenterghem, Y. FTY720, a
novel immunomodulator: efficacy and safety results from
the first phase 2A study in de novo renal transplantation.
Transplantation 2004, 77, 1826–1833.
5. Chiba, K.; Yanagawa, Y.; Masubuchi, Y.; Kataoka, H.;
Kawaguchi, T.; Ohtsuki, M.; Hoshino, Y. FTY720, a
novel immunosuppressant, induces sequestration of circu-
lating mature lymphocytes by acceleration of lymphocyte
homing in rats. I. FTY720 selectively decreases the
number of circulating mature lymphocytes by acceleration
of lymphocyte homing. J. Immunol. 1998, 160, 5037–
5044.
15. Fujita, T.; Hirose, R.; Yoneta, M.; Sasaki, S.; Inoue, K.;
Kiuchi, M.; Hirase, S.; Chiba, K.; Sakamoto, H.; Arita,
M. Potent immunosuppressants, 2-alkyl-2-aminopropane-
1,3-diols. J. Med. Chem. 1996, 39, 4451–4459.
16. Hale, J. J.; Yan, L.; Neway, W. E.; Hajdu, R.; Bergstrom,
J. D.; Milligan, J. A.; Shei, G. J.; Chrebet, G. L.;
Thornton, R. A.; Card, D.; Rosenbach, M.; Rosen, H.;
Mandala, S. Synthesis, stereochemical determination and
biochemical characterization of the enantiomeric phos-
phate esters of the novel immunosuppressive agent
FTY720. Bioorg. Med. Chem. 2004, 12, 4803–4807.
17. Wang, Y.-F.; Lalonde, J. J.; Momongan, M.; Bergbreiter,
D. E.; Wong, C.-H. Lipase-catalyzed irreversible trans-
esterifications using enol esters as acylating reagents:
preparative enantio- and regioselective syntheses of alcoh-
ols, glycerol derivatives, sugars, and organometallics. J.
Am. Chem. Soc. 1988, 110, 7200–7205.
18. Terradas, F.; Teston-Henry, M.; Fitzpatrick, P. A.;
Klibanov, A. M. Marked dependence of enzyme prochiral
selectivity on the solvent. J. Am. Chem. Soc. 1993, 115,
390–396.
19. Pozo, M.; Pulido, R.; Gotor, V. Vinyl carbonates as novel
alkoxycarbonylation reagents in enzymatic synthesis of
carbonates. Tetrahedron 1992, 48, 6477–6484.
6. Yanagawa, Y.; Sugahara, K.; Kataoka, H.; Kawaguchi,
T.; Masubuchi, Y.; Chiba, K. FTY720, a novel immuno-
suppressant, induces sequestration of circulating mature
lymphocytes by acceleration of lymphocyte homing in
rats. II. FTY720 prolongs skin allograft survival by
decreasing T cell infiltration into grafts but not cytokine
production in vivo. J. Immunol. 1998, 160, 5493–5499.
7. Spiegel, S.; Milstien, S. Sphingosine-1-phosphate: an
enigmatic signalling lipid. Nat. Rev. Mol. Cell Biol. 2003,
4, 397–407.
20. Moris, F.; Gotor, V. Lipase-mediated alkoxycarbonyl-
ation of nucleosides with oxime carbonates. Tetrahedron
1992, 48, 9869–9876.
21. Mukaiyama, T.; Ohshima, M.; Murakami, M. 2-Benzyl-
oxy-1-propene: a novel protective reagent of hydroxyl
groups. Chem. Lett. 1984, 265–266.
8. English, D.; Brindley, D. N.; Spiegel, S.; Garcia, J. G. N.
Lipid mediators of angiogenesis and the signalling path-
ways they initiate. Biochim. Biophys. Acta 2002, 1582, 228–
239.
22. Data of mono-sodium salt of 2: 1H NMR (CD3OD): d
7.13 (2H, d, J = 8.0Hz), 7.07 (2H, d, J = 8.0Hz), 3.93 (1H,
dd, J = 11.4, 7.4Hz), 3.85 (1H, dd, J = 11.4, 7.4Hz), 3.65
(1H, d, J = 11.2Hz), 3.62 (1H, d, J = 11.2Hz), 2.68–2.60
(2H, m), 2.55 (2H, t, J = 7.6Hz), 1.89–1.82 (2H, m), 1.61–
1.54 (2H, m), 1.34–1.24 (10H, m), 0.89 (3H, t, J = 6.8Hz).
Anal. Calcd for C19H33NO5PNaÆH2O: C, 53.39; H, 7.78;
N, 3.28. Found: C, 53.08; H, 7.89; N, 3.02.
23. Kiuchi, M.; Adachi, K.; Kohara, T.; Teshima, K.;
Masubuchi, Y.; Mishina, T.; Fujita, T. Synthesis and
biological evaluation of 2,2-disubstituted 2-aminoethan-
ols: analogues of FTY720. Bioorg. Med. Chem. Lett. 1998,
8, 101–106.
24. Hale, J. J.; Doherty, G.; Toth, L.; Mills, S. G.; Hajdu, R.;
Keohane, C. A.; Rosenbach, M.; Milligan, J.; Shei, G. J.;
Chrebet, G.; Bergstrom, J.; Card, D.; Forrest, M.; Sun, S.
Y.; West, S.; Xie, H.; Nomura, N.; Rosen, H.; Mandala, S.
Selecting against S1P3 enhances the acute cardiovascular
tolerability of 3-(N-benzyl)aminopropylphosphonic acid
S1P receptor agonists. Bioorg. Med. Chem. Lett. 2004, 14,
3501–3505.
9. Ohmori, T.; Yatomi, Y.; Osada, M.; Kazama, F.; Taka-
futa, T.; Ikeda, H.; Ozaki, Y. Sphingosine 1-phosphate
induces contraction of coronary artery smooth muscle
cells via S1P2. Cardiovasc. Res. 2003, 58, 170–177.
10. Mandala, S.; Hajdu, R.; Bergstrom, J.; Quackenbush, E.;
Xie, J.; Milligam, J.; Thornton, R.; Shei, G.-J.; Card, D.;
Keohane, C.; Rosenbach, M.; Hale, J.; Lynch, C. L.;
Rupprecht, K.; Parsons, W.; Rosen, H. Alteration of
lymphocyte trafficking by sphingosine-1-phosphate recep-
tor agonists. Science 2002, 296, 346–349.
11. Brinkmann, V.; Davis, M. D.; Heise, C. E.; Albert, R.;
Cottens, S.; Hof, R.; Bruns, C.; Prieschl, E.; Baumruker,
T.; Hiestand, P.; Foster, C. A.; Zollinger, M.; Lynch, K.
R. The immune modulator FTY720 Targets sphingosine
1-phosphate receptors. J. Biol. Chem. 2002, 277, 21453–
21457.
12. Matloubian, M.; Lo, C. G.; Cinamon, G.; Lesneski, M. J.;
Xu, Y.; Brinkmann, V.; Allende, M. L.; Proia, R. L.;
Cyster, J. G. Lymphocyte egress from thymus and
25. Our pharmacokinetic study using the enantiomers of 2 will
be reported in due course.