Synthesis, stereochemical determination and biochemical characterization of the enantiomeric phosphate esters of the novel immunosuppressive agent FTY720

Article abstract of DOI:10.1016/j.bmc.2004.07.020

The novel immunosuppressive agent FTY720 (1) is phosphorylated in vivo in a variety of species yielding an active metabolite that is an agonist of four of the five known G-protein-coupled sphingosine-1-phosphate (S1P) receptors. A synthesis amenable to pr

Full text of DOI:10.1016/j.bmc.2004.07.020

Bioorganic & Medicinal Chemistry 12 (2004) 4803–4807
Synthesis, stereochemical determination and
biochemical characterization of the enantiomeric phosphate
esters of the novel immunosuppressive agent FTY720
Jeffrey J. Hale,a,* Lin Yan,a William E. Neway,a Richard Hajdu,b James D. Bergstrom,b
James A. Milligan,b Gan-Ju Shei,b Gary L. Chrebet,b Rosemary A. Thornton,b
Deborah Card,b Mark Rosenbach,b Hugh Rosenb,y and Suzanne Mandalab
aDepartment of Medicinal Chemistry, Merck Research Laboratories, Rahway, NJ 07065, USA
bDepartment of Immunology and Rheumatology Research, Merck Research Laboratories, Rahway, NJ 07065, USA
Received 23 June 2004; accepted 7 July 2004
Available online 6 August2004
Abstract—The novel immunosuppressive agentFTY720 ( 1) is phosphorylated in vivo in a variety of species yielding an active
metabolite that is an agonist of four of the five known G-protein-coupled sphingosine-1-phosphate (S1P) receptors. A synthesis
amenable to producing gram quantities of the stereoisomeric phosphate esters, a determination of their absolute stereochemistry
via an enantioselective synthesis and their characterization as S1P receptor agonists and antagonists is reported.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
long been known to be potent immunosuppressants,
but exhibit myelotoxicity and, in the case of MMF, gas-
trointestinal toxicity.2 There exists a clear need for
immunosuppressive therapies with improved safety
profiles arising from the enhanced inherent potency
and selectivity of new agents and/or synergisms between
them with existing drugs.
Currentimmunosuppressive agents are in partresponsi-
ble for the recent significant advances in prolonging
allograft survival after organ transplantation.1 The
potential to extend many of these therapies to the treat-
mentof chronic auotimmune disorders and inflamma-
tory diseases, such as rheumatoid arthritis and
multiple sclerosis, has been realized, but suboptimal
therapeutic indices and a lack of immunosuppressive
selectivity currently limits applicability. Calcineurin
inhibitors such as cyclosporin A (CsA, Sandimmuneꢁ,
Neoralꢁ) and FK506 (Prografꢁ) are widely used in
transplantation therapy, but both exhibit a mecha-
nism-based nephrotoxicity at or near therapeutic doses.
The efficacy of rapamycin (Rapamuneꢁ) arises from its
disruption of the cytokine signaling that promotes the
growth and proliferation of lymphocytes, but it also
has demonstrated toxicities (thrombocytopenia, hyper-
lipidemia). Antimetabolites such as mycophenolate mo-
fetil (MMF, Cellceptꢁ) and leflunomide (Aravaꢁ) have
2-Amino-2-(4-octylphenyl)propane-1,3-diol (FTY720,
1) is a novel, orally active immunosuppressive agent first
identified by researchers at Yoshitomi Pharma.3 The un-
ique mode of action of 1 results in immunosuppression
by promoting a lowering of circulating T lymphocytes,
which prevents their infiltration into transplanted or
antigen-bearing nonlymphoid tissues.4 Preclinical stud-
ies have demonstrated the efficacy of 1 in prolonging al-
lograftsurvival in rodens,t dogs and cynomolgus
monkeys; synergy has been demonstrated when CsA,
FK506 or rapamycin was co-administered.5 The ability
of 1 to suppress adjuvant- and collagenase-induced
arthritis,6 experimental autoimmune encephalitis,7 acute
viral myocarditis8 autoimmune diabetes,9 and systemic
lupus erythematosus10 in rodents has been demon-
strated. The mechanism of action of 1 appears to confer
advantages regarding host defense in that it does not im-
pair myelomonocytic cell function; responses to bacte-
rial and fungal challenge in preclinical models appear
*
Corresponding author. Tel.: +1-732-594-2916; fax: +1-732-594-
5966; e-mail: jeffrey_hale@merck.com
y Present address: The Scripps Research Institute, ICND-118, 10550 N.
Torrey Pines Road, La Jolla, CA 92037, USA.
0968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.07.020

4804
J. J. Hale et al. / Bioorg. Med. Chem. 12 (2004) 4803–4807
to be spared.11 In Phase I clinical trials with stable renal
transplant patients, 1 was reported to induce the lower-
ing of circulating lymphocytes that was observed pre-
O
NH2
OCH2Ph
OH
a
c
b
d
HN
OH
Ar
Ar
clinically while being well-tolerated.
A transient,
HO
HO
asymptomatic bradycardia was the most commonly
encountered adverse event.12 Twelve month preliminary
results from phase II clinical trials in de novo kidney
transplant patients indicate that 1, in combination with
low dose CsA and steroids, appears to be at least equally
efficacious as the currently recognized standard of care
(full dose CsA, steroids, MMF).13
1
3
O
O
OCH2Ph
OCH2Ph
OH
HN
HN
O
Ar
Ar
O
Ph
OCH2Ph
4
5
NH2
OH
O
OCH2Ph
HN
OH
e
(R)-6
+
(S)-6
O
PO(CH2Ph)2
Ar
OCH2Ph
1, FTY720
Ar = 4-(octyl)phenyl
6
NH2
O
PO3H2
NH2
O
HO
OH
PO3H2
f
(S)-6
Sphingosine-1-phosphate (S1P)
2
OH
H2N
It was only recently that a molecular target was con-
nected to the immunosuppressive actions of 1.14,15 Com-
pound 1 was shown to be rapidly metabolized in the
blood of a variety of species to the corresponding phos-
phate ester, which is an agonist of four of the five known
G-protein-coupled sphingosine-1-phosphate receptors
(S1P1-5).16 Agonism of S1P receptors by the phosphate
ester of 1 appears to inhibit lymphocyte migration into
lymphatic sinuses; this alteration in lymphocyte traffick-
ing has been postulated to lead to the observed periph-
eral lymphopenia and immunosuppressive efficacy. In
this paper, we wish to describe our efforts to synthesize
and determine the absolute stereochemistries of the
enantiomeric phosphate esters of 1 and to characterize
further the biochemical properties of these compounds.
O
PO3H2
g
(R)-6
7
Scheme 1. (a) PhCH2OCOCl, KHCO3, EtOAc/H2O; (b) PhCHO, cat.
p-TSA, toluene, 100ꢂC (57%, 2 steps); (c) BH3ÆNHMe2, BF3ÆOEt2,
CH2Cl2, À78ꢂC ! À5ꢂC (92%); (d) iPr2N–P(OCH2Ph)2, 1H-tetrazole,
CH2Cl2, 0ꢂC, then MCPBA À78ꢂC ! rt(99%); (e) enaniotmer
resolution (chiral HPLC); (f) TMS-I, CHCl3 (63%); (g) Na, NH3,
THF, À33ꢂC (81%).
enantiomers of both 5 and 6 were found to be readily
separable on a Chiralcelꢁ OD HPLC column. Itwas ini-
tially thought that the global deprotection of 6 could be
readily accomplished using catalytic hydrogenation, but
this was found not to be the case. Reactions using vari-
ous catalysts and solvents were found to require forcing
conditions to proceed and the mixtures obtained were
often difficult to purify. Reduction of 6 with an excess
of sodium metal in ammonia gave much better results
affording product that was determined to be 95% pure
by HPLC. Global deprotection of (S)-6 with trimethyl-
silyliodide proved to be optimal; analytically pure 2
was obtained after HPLC purification.
2. Chemistry
Two factors influenced the route chosen to synthesize
the enantiomeric phosphate esters of 1 (Scheme 1). First,
it was envisioned that these compounds could be ob-
tained via resolution of a late-stage synthetic intermedi-
ate by chiral HPLC and that converting 1 t o an
unsymmetric derivative with multiple aromatic func-
tional groups would aid in accomplishing this. Second,
it was desired that a final global deprotection with a
low potential for phosphate ester scrambling be em-
ployed to give the target compounds. In practice, the
tertiary amino group of 1 was selectively acylated on
treatment with benzyl chloroformate under Schotten–
Baumann conditions to give 3. Crude 3 was converted
to the corresponding benzylidene acetal 4; this was fol-
lowed by selective reduction with borane dimethylamine
complex in the presence of boron trifluoride etherate17
to give the unsymmetric intermediate 5. The free hydr-
oxy group of 5 was dibenzylphosphorylated using the
protocol described by Frasier-Reid18 to give 6. The
An enantioselective route to 5 was executed in order to
establish the absolute configurations of 2 and 7. Oxida-
tive cleavage of the double bond of the known L-serine-
derived oxazolidine 819 afforded aldehyde 9 in excellent
yield. Treatment of 9 with 4-(octyl)phenylmagnesium
bromide resulted in the formation of lactone 10, which
was obtained as a 1:1 mixture of diastereomers. The
stereochemical outcome of the Grignard addition was
without consequence as catalytic reduction of the mix-
ture of lactones afforded a single compound, carboxylic
acid 11. Reduction of 11 to the corresponding alcohol
followed by O-benzylation gave 12. Acid hydrolysis of
the oxazoline ring of 12 and subsequent N-acylation of

J. J. Hale et al. / Bioorg. Med. Chem. 12 (2004) 4803–4807
4805
excesses of 2 and 7 in all likelihood are similar those
of (S)-6 and (R)-6, respectively, they have not yet been
accurately quantitated.
a
b
O
O
N
CHO
N CHO
CO2CH3
CO2CH3
3. In vitro biochemistry
O
8
9
Ligand competition studies with [33P]-S1P were carried
outwiht S1P and compounds 1, 2 and 7 for each of
the five human S1P receptors stably expressed in Chi-
nese Hamster Ovary (CHO) cell membranes.14 Human
S1P receptor agonism by the test compounds was deter-
c
d,e
O
O
N
CHO
O
N
CHO
CO2
35
mined by measurementof ligand-induced [ S]-50-O-3-
O
Ar
Ar
thiotriphosphate (GTPcS) binding for S1P1, S1P3 and
S1P5 in CHO cells membranes14 and ERK activation
for S1P4. (S)-Phosphate ester 2 was found to be the
enantiomer with the greater affinity for all five S1P
receptors. Additionally, GTPcS functional data indi-
11
10
O
f,g
N
CHO
OCH2Ph
OH
cated that 2 was a full agonistof S1P and an agonist
1
PhCH2O2CNH
Ar
of S1P3, S1P4 and S1P5 with 70–90% maximal efficacy
compared to the endogenous ligand, S1P. Calculated
EC50 values agreed well with the IC50 values obtained
from the ligand competition experiments. (R)-Phosphate
ester 7 was found to be a weaker full agonist of S1P1 and
S1P4, buta weak antagonistof S1P 3 and S1P5 based on
the observations that 7 competed with S1P binding to
S1P3 and S1P5, but failed to activate GTPcS binding
to these receptors. The antagonist activity was con-
firmed with further functional experiments; 7 was found
to antagonize GTPcS binding to S1P3 and S1P5 stimu-
lated by 2 (data not shown) and to block Ca++ flux in-
duced by either S1P or 2 in S1P3-bearing CHO cells
with an IC50 = 80nM (Table 1).
OCH2Ph
Ar
(S)-5
12
Ar = 4-(octyl)phenyl
Scheme 2. (a) cat. OsO4, NaIO4, 3:1 v/v dioxane/H2O, rt(85%); (b) 4-
(octyl)phenylmagnesium bromide, THF, À78ꢂC to rt (67%, ds = 1:1);
(c) H2, 10% Pd/C, MeOH, rt(90%); (d) CH CH2OCOCl, TEA, THF,
3
0ꢂC, then NaBH4, THF/H2O, 0ꢂC to rt (69%); (e) PhCH2Br, NaH,
Bu4N+IÀ, DMF, 65ꢂC (93%); (f) 6.0N HCl, MeOH, reflux; (g)
PhCH2OCOCl, KHCO3, EtOAc/H2O, rt(73%, 2 setps).
the resulting amino alcohol afforded (S)-5. Elaboration
of this material (as described for the racemate in the pre-
ceding paragraph) ultimately gave 7, which established
that it is the (R)-enantiomer and that 2 is the (S)-enantio-
mer (Scheme 2).
4. Conclusions
The novel immunosuppressive agentFTY720 ( 1) is
phosphorylated in vivo in a variety of species yielding
an active metabolite that is an agonist of four of the five
known G-protein-coupled S1P receptors. Despite the
advanced clinical developmentof 1, little has appeared
in the literature regarding the synthesis and characteri-
zation of 2 and/or 7. The factthat 2 and 7 have different
S1P receptor profiles indicates that the efficient access to
both of these compounds will be of value for both the
study and understanding of the pharmacology arising
Attempts to develop analytical chiral HPLC methods
suitable to measure the enantiomeric excesses of 2 and
7 have been unsuccessful. Phosphate esters 2 and 7 were
prepared using both of the deprotection methods de-
scribed (Na/NH3, TMS-I) and the receptor profiles
(see below) were found to be the same regardless of
the manner of preparation. Based on this, it appears un-
likely that significant racemization occurs during the de-
protection reactions. However, while the enantiomeric
Table 1. Ligand competitiona and functional activityb
Compd
S1P1
S1P2
S1P3
S1P4
S1P5
S1P
IC50 (nM)
EC50 (nM)
IC50 (nM)
EC50 (nM)
IC50 (nM)
EC50 (nM)
IC50 (nM)
EC50 (nM)
0.67
ndc
840
145d
0.28
0.28
25
0.35
ndc
0.26
ndc
34
ndc
0.55
ndc
1
2
7
>10,000
ndc
>10,000
>10,000
6.3
>10,000
>10,000
15
>10,000
>10,000
0.77
1100
ndc
3.1
120
12
380
2.5
49
300e
>10,000
ndc
32
>10,000
93
a Inhibition of [33P]-S1P binding to stably expressed human S1P1–5 in CHO cell membranes.
b EC50 for ligand-induced GTPcS binding (S1P1, S1P3, S1P5) or ERK activation (S1P4).
c Notdeetrmined.
d 44% maximal efficacy compared to 2.
e 39% maximal efficacy compared to 2. IC50 and EC50 data are the averages from multiple (n P 3) assays; standard deviations were generally 50%
of the average values.

4806
J. J. Hale et al. / Bioorg. Med. Chem. 12 (2004) 4803–4807
from the systemic administration of 1 and the design
and synthesis of future S1P agonists and antagonists.
treated with BF3ÆEt2O (4.70mL, 37.0mmol). The
resulting mixture was allowed to warm to À5ꢂC and
was stirred for 2h. The reaction mixture was poured into
1.0N NaOH (25mL) and the resulting mixture was ex-
tracted with CH2Cl2 (100mL). The extract was sepa-
rated and dried over MgSO4. The aqueous layer was
extracted with ether (200mL). The ether extract was
dried and the two organic extracts were combined and
concentrated. Chromatography on a Biotage 75S car-
tridge using 6:1 v/v heptane/acetone as the eluant af-
5. Experimental
Flash chromatography was carried out using a Biotage
Flash Chromatography apparatus (Dyax Corp.) on sil-
˚
ica gel (32–63mM, 60A pore size) in pre-packed car-
tridges of the size noted. NMR spectra were obtained
in CDCl3 solution unless otherwise noted. Coupling
constants (J) are in hertz (Hz). Analytical HPLC condi-
tions: HPLC A: Analytical Sales and Service Armor C8,
1
forded 3.63g (92%) of 5: H NMR (500MHz) d 0.88
(t, J = 6.5, 3H), 1.26–1.31 (10H), 1.54–1.60 (m, 2H),
1.85–1.89 (m, 1H), 2.05–2.08 (m, 1H), 2.44–2.61 (m,
2H), 2.55 (t, J = 7.5, 2H), 3.58 (AB q, J = 130, 2H),
3.72–3.78 (m, 2H), 4.50 (app s, 2H), 5.08 (s, 2H), 5.45
(s, 1H), 7.04–7.08 (4H), 7.30–7.38 (10H); ESI-MS 532
(M+H+); HPLC A: 5.98min; HPLC B: 5.32min.
˚
5A, 4.6mm · 50mm column, gradient10:90 ! 90:10
v/v CH3CN:H2O + 0.05% TFA over 4min, then hold
at90:10 v/v CH 3CN:H2O + 0.05% TFA for 4min;
˚
2.5mL/min, 210nm. HPLC B: YMC ODS A, 5A,
4.6 · 50mm column, gradient10:90
! 95:5 v/v
CH3CN:H2O + 0.05% TFA over 4.5min, then hold at
95:5 v/v CH3CN:H2O + 0.05% TFA for 1.5min;
2.5mL/min, 210nm.
The enantiomers of 5 were found to be separable by chi-
ral HPLC. Conditions: Chiral Technologies Chiralcele
OD 4.6 · 250mm column, 65:35 v/v hexanes/iPrOH,
1.0mL/min, 210nm. For the faster eluting enantiomer,
t = 5.5min. For the slower eluting (S)-enantiomer,
5.1. 2-Benzyloxycarbonylamino-2-hydroxymethyl-4-(4-
(octyl)phenyl)butanol (3)
t= 9.0min. Elaboraiotn of
L-serine-derived oxazoline
A mixture of 120 (3.07g, 10.0mmol) and KHCO3 (3.00g,
30.0mmol) in EtOAc (200mL) and H2O (150mL) was
treated with benzyl chloroformate (1.50mL, 10.0mmol),
then stirred at room temperature for 2h. The organic
layer of the reaction mixture was separated, dried over
MgSO4 and concentrated to afford 5.29g of crude 3.
This material was used without further purification:
1H NMR (500MHz) d 0.88 (t, J = 6.5, 3H), 1.22–1.34
(12H), 1.55–1.60 (m, 2H), 1.87–1.91 (m, 2H), 2.53–2.59
(4H), 3.23 (br s, 2H), 3.67 (dd, J = 6.5, 11.5, 1H), 3.90
(dd, J = 6.5, 11.5, 1H), 5.08 (s, 2H), 5.30 (s, 1H), 7.05–
7.09 (4H), 7.32–7.36 (5H); HPLC A 5.34min.
8 as described in the text afforded the enantiomer of
5, which corresponded to the slower eluting peak thus
establishing that it was the (S)-enantiomer.
5.4. ( )-1-Dibenzyloxyphosphoryloxy-2-benzyloxycarbon-
ylamino-2-benzyloxymethyl-4-(4-(octyl)phenyl)butane
(6)
A solution of 5 (2.90g, 5.4mmol) and dibenzyl diisopro-
pylphosphoramidite (2.00mL, 6.0mmol) in CH2Cl2
(40mL) at0 ꢂC was treated with 1H-tetrazole (117mg,
8.2mmol). The resulting mixture was stirred at room
temperature for 1.25h, then cooled to À78ꢂC. MCPBA
(2.0g, ꢀ8.2mmol) was added, the cooling bath was re-
moved and the reaction was stirred at ambient tempera-
ture for 45min. The reaction mixture was quenched with
satd NaHCO3 (50mL), then extracted with CH2Cl2
(200mL). The extract was separated and dried over
MgSO4. The aqueous layer was extracted with ether
(200mL). The ether extract was dried and the two or-
ganic extracts were combined and concentrated. Chro-
matography on a Biotage 75S cartridge using 8:1 v/v
heptane/acetone (4.5L), then 4:1 v/v heptane/acetone
(2L) as the eluant afforded 4.25g (99%) of 6: 1H
NMR (500MHz) d 0.88 (t, J = 6.5, 3H), 1.22–1.36
(12H), 1.55–1.61 (m, 2H), 1.94–2.04 (m, 1H), 2.08–2.18
(m, 1H), 2.47 (app t, J = 8.5, 1H), 2.55 (app t, J = 7.5,
1H), 3.54 (AB q, J = 26.0, 2H), 4.18–4.25 (m, 2H),
4.44 (s, 2H), 4.98 (s, 2H), 5.00 (s, 2H), 5.03 (s, 2H),
5.11 (br s, 1H), 6.96–7.06 (8H), 7.24–7.36 (36H); HPLC
A: 6.31min.
5.2. 2-Phenyl-4-(benzyloxycarbonylamino)-4-(4-(octyl)-
phenyl)-1,3-dioxane (4)
A mixture of 3 (5.29g, ꢀ10.0mmol), benzaldehyde
(1.10mL, 11.0mmol) and p-TSA Æ H2O (95mg,
0.05mmol) in toluene (50mL) was stirred at 100ꢂC for
1h. The reaction mixture was treated with additional
benzaldehyde (2mL) and stirring was continued at
100ꢂC for 2h. The mixture cooled to room temperature,
then partitioned between ether (400mL) and 1.0N
NaOH (150mL). The organic layer was separated, dried
over MgSO4 and concentrated. Chromatography on a
Biotage Flash 75S cartridge using 20:1 v/v heptane/
EtOAc (3L), then 10:1 v/v heptane/EtOAc (3L) as the
eluantafforded 3.02g of 4 (57%, 2 steps): 1H NMR
(500MHz) d 0.88 (t, J = 6.5, 3H), 1.22–1.36 (12H),
1.55–1.60 (m, 2H), 1.94–2.06 (m, 2H), 2.54–2.58 (4H),
3.69 (d, J = 11.5, 2H), 4.31 (d, J = 11.5, 2H), 5.13 (s,
2H), 5.41 (br s, 1H), 5.45 (s, 1H), 7.06–7.10 (4H),
7.29–7.40 (8H), 7.46–7.48 (2H); HPLC A 6.08min.
The enantiomers of 6 were resolved using preparative
chiral HPLC. Conditions: Chiral Technologies Chiral-
cele OD 20mm · 250mm column, 60:40 v/v hexanes/
iPrOH, 9.0mL/min, 210nm, ꢀ25mg of 6 per injection.
For the faster eluting enantiomer, t = 16.7min. For the
slower eluting enantiomer, t = 24.0min. Subjecting (S)-
5 to the reaction conditions described above afforded
5.3. ( )-2-Benzyloxycarbonylamino-2-benzyloxymethyl-
4-(4-(octyl)phenyl) butanol (5)
A solution of 4 (3.90g, 7.4mmol) and BH3ÆNHMe2
(2.18g, 37.0mmol) in CH2Cl2 (150mL) at À78ꢂC was

J. J. Hale et al. / Bioorg. Med. Chem. 12 (2004) 4803–4807
4807
the enantiomer of 6 that corresponded to the faster
eluting peak thus establishing that it was the (R)-
enantiomer.
by SDS-PAGE. Compound-induced phosphorylation
of ERK 1/2 was measured by quantitative immunoblot-
ting with a phospho-ERK antibody. HRP-linked anti-
rabbit IgG was used as the secondary antibody with
the ECL Plus chemiluminescent detection kit. Images
were captured using the Storm 860 phosphorimager
and phosphorylated ERK bands were quantitated using
the Imagequant program.
5.5. (S)-2-Amino-2-phosphoryloxymethyl-4-(4-(octyl)phen-
yl)butanol (2)
A solution of (S)-6 (4.20g, 5.3mmol) in CHCl3 (50mL)
at0 ꢂC was treated with iodotrimethylsilane (4.60mL,
32mmol). The cooling was removed and the reaction
mixture was stirred at room temperature for 16h. The
reaction mixture was treated with MeOH (50mL) and
stirred for 1h, then concentrated. The crude product
was purified by preparative reverse-phase HPLC. Con-
ditions: Kromasilꢁ KR100-10-C8 30mm · 100mm col-
umn, gradient10/90 ! 90:10 v/v CH3CN/H2O + 0.05%
TFA over 12min, then hold at 90:10 v/v CH3CN/
H2O + 0.05% TFA for 4min, 20mL/min, 220nM,
ꢀ20mg of crude 2 per injection. Fractions containing
2 were pooled and concentrated to remove the CH3CN.
The solid that precipitated was filtered, washed with
References and notes
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1
water and dried to obtain 1.30g (63%) of pure 2: H
NMR (500MHz, CD3OD) d 0.88 (t, J = 7.0, 3H),
1.22–1.36 (12H), 1.54–1.64 (m, 2H), 1.86–2.04 (m, 2H),
2.55 (app t, J = 7.5, 2H), 2.63–2.68 (m, 2H), 3.66 (app
q, J = 5.5, 1H), 3.95–4.05 (m, 2H), 7.07 (d, J = 8.0,
2H), 7.13 (d, J = 8.0, 2H); 13C NMR (125MHz,
CD3OD) d 14.4, 23.7, 29.6, 30.3, 30.4, 30.6, 32.8, 33.0,
34.9, 36.5, 61.4 (d, J = 6.8), 62.5, 65.8 (d, J = 3.9),
129.2, 129.6, 139.5, 141.9; ESI-MS 388 (M+H+); HPLC
A: 3.96min; HPLC B: 3.83min; Anal. Calcd for
C19H34NO5P: C, 58.90; H, 8.84; N, 3.62. Found: C,
58.90; H, 9.20; N, 3.57.
8. Miyamoto, T.; Matsumori, A.; Hwang, M.-W.; Nishio,
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