Novel immunomodulator FTY720 is phosphorylated in rats and humans to form a single stereoisomer. Identification, chemical proof, and biological characterization of the biologically active species and its enantiomer

Article abstract of DOI:10.1021/jm050242f

In vivo phosphorylation of FTY720 (1) in rats and humans resulted exclusively in the biologically active (S)-configured enantiomer, which was proven by an ex vivo o-phthaldialdehyde derivatization protocol especially elaborated for phosphates of 1. Starting from the prochiral amino alcohol 1, racemic and enantiomerically pure phosphates of 1 were synthesized. Pure enantiomers were obtained after purification of a partially protected key intermediate on an enantioselective support. The absolute stereochemistry was determined by X-ray diffraction.

Full text of DOI:10.1021/jm050242f

J. Med. Chem. 2005, 48, 5373-5377
5373
Novel Immunomodulator FTY720 Is Phosphorylated in Rats and Humans To
Form a Single Stereoisomer. Identification, Chemical Proof, and Biological
Characterization of the Biologically Active Species and Its Enantiomer
Rainer Albert,* Klaus Hinterding, Volker Brinkmann, Danilo Guerini, Constanze Mu¨ller-Hartwieg,
Helmut Knecht, Corinne Simeon, Markus Streiff, Trixie Wagner, Karl Welzenbach, Fre´de´ric Ze´cri,
Markus Zollinger, Nigel Cooke, and Eric Francotte
Novartis Institutes for BioMedical Research, Lichtstrasse 35, WSJ-507.3.02, CH-4056 Basel, Switzerland
Received March 16, 2005
In vivo phosphorylation of FTY720 (1) in rats and humans resulted exclusively in the biologically
active (S)-configured enantiomer, which was proven by an ex vivo o-phthaldialdehyde
derivatization protocol especially elaborated for phosphates of 1. Starting from the prochiral
amino alcohol 1, racemic and enantiomerically pure phosphates of 1 were synthesized. Pure
enantiomers were obtained after purification of a partially protected key intermediate on an
enantioselective support. The absolute stereochemistry was determined by X-ray diffraction.
Scheme 1. General Phosphorylation Pathway of 1
Introduction
FTY720 (1) is a novel immunomodulator that is highly
effective in animal models of transplantation and au-
toimmunity.1 The drug has recently been shown to be
active in preventing kidney allograft rejection in hu-
mans (phase III clinical trials) and may represent a
novel modality for immunosuppressive therapy.2 Unlike
other agents used in transplantation to prevent acute
rejection, 1 does not inhibit proliferation or activation
of lymphocytes. Instead, it leads to their sequestration
from the periphery into secondary lymphoid organs and
inhibits recirculation of effector T cells to the trans-
planted graft, thereby preventing its rejection.3
Scheme 2 Synthesis of (S)-3, (R)-3, and (rac)-5a
The amino alcohol 1 is phosphorylated in vitro by
sphingosine kinases,4 and only the phosphorylated
molecule (S)-5, signals as an agonist through four of five
S1P receptors (formerly known as EDG receptors),5,6
whereas the second enantiomer (R)-5 shows weak or no
agonistic activity on these receptors. It has been con-
vincingly demonstrated that S1P 1-receptor agonism is
responsible for the observed pharmacological action of
the drug.7 While 1 itself is prochiral (Scheme 1), in vivo
phosphorylation generates a chiral phosphate of 1 with
hitherto unknown stereoisomeric composition.8
We report herein proof that the amino alcohol 1 is
phosphorylated in rats and humans to form a single
stereoisomer, namely, the biologically active (S)-enan-
tiomer (S)-5. To this end, enantiomers (S)-5 and (R)-5
were synthesized with high optical purity and charac-
terized in biological assays. An ex vivo derivatization
of the phosphate (S)-5 was used to determine the
enantiomeric excess of the in vivo generated phospho-
rylated analogue of 1 by enantioselective HPLC. The
absolute configuration was determined by X-ray diffrac-
tion.
a (a) Benzyl chloroformate, NaOH, rt, 48 h; (b) 3-(diethylamino)-
1,5-dihydro-2,4,3-benzodioxaphosphepintriphenyl phosphite, tet-
razole, CH2Cl2/THF, rt, 18 h; then H2O2, rt, 90 min; (c) Pd/C10%
,
H2, MeOH, rt, 90 min; (d) LiOH (10% in water), EtOH, reflux, 18
h.
Chemistry
and 3. After protection of the prochiral 1 as an oxazo-
lidinone9 to give (rac)-2, phosphorylation of the remain-
ing free hydroxyl group was performed with 3-(diethyl-
amino)-1,5-dihydro-2,4,3-benzodioxaphosphepin10 and
subsequent oxidation gave 56% overall yield. From this
The synthesis of (rac)-5 and the synthesis of (S)-5 and
(R)-5 in optically pure form are outlined in Schemes 2
* To whom correspondence should be addressed. Phone: ++41 61
3247113. Fax: ++41 61 3247821. E-mail: rainer.albert@novartis.com.
10.1021/jm050242f CCC: $30.25 © 2005 American Chemical Society
Published on Web 07/09/2005

5374 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 16
Brief Articles
Table 1. Agonistic Activities of Selected Compounds on S1P
Receptors 1 and 5
Scheme 3. Synthesis of Enantiomerically Pure (S)-5
and (R)-5a
EC50 (nM)
compd
S1P-1
S1P-3
S1P-2
S1P-4
S1P-5
1
>10000
1.2
>10000
1.4
>10000
>10000
>10000
>10000
>10000
2.4
>10000
4.9
(rac)-5
(S)-5
(R)-5
0.3
3.1
0.6
0.3
218
28.9
80.1
>10000
Scheme 4. OPA Derivatization Protocol for (rac)-5,
(S)-5, and (R)-5a
a (a) Pd/C10%, H2, MeOH, rt, 90 min; (b) LiOH (10% in water),
EtOH, reflux, 18 h.
a (a) EDTA (10 mM in water), boric acid (3% in water), KOH
(10% in water), o-phthaldialdehyde, rt, 1 h.
binding assays for all five known S1P receptors. This
scintillation proximity assay measures agonist-induced
GTP[γ-35S] binding as functional assay for human S1P
receptors (EDG receptors). For this assay, membrane
protein of CHO cells expressing S1P receptor N-terminal
c-myc was prepared at large scale and stored frozen,
and 10 µg/well of the protein was immobilized onto 1
mg/well SPA beads. GTP[γ-35S] binding was conducted
with 200 pM GTP[γ-35S] in the presence of 50 mM
Hepes, 100 mM NaCl, 10 mM MgCl2, pH 7.4, 10 µM
GDP, 20 µg/mL saponin for 90-120 min. After a
centrifugation step at 1500 rpm, plates were read with
a TOPcount. The reference compound for all measure-
ments was the natural ligand S1P.
Results in Table 1 indicate a striking difference in
the biological activities of (S)-5 and (R)-5 and provided
a first biological evidence for the optical purity of the
synthesized samples. Only (S)-5 acts as an agonist on
S1P receptors at low nanomolar concentrations, and
these results are in agreement with published data of
closely related structures.8,14
Figure 1. X-ray structure of (R)-3.
key intermediate (rac)-3, (rac)-5 was obtained by re-
moval of phosphate protecting group by hydrogenation
in MeOH over 10% palladium on carbon, and subse-
quent hydrolysis of the oxazolidinone with 10% aqueous
lithium hydroxide solution under reflux and final neu-
tralization with solid carbon dioxide gave 80% overall
yield. Pure enantiomers of 3 were accessible after
separation of intermediate (rac)-3 using Chiralpak AS11
as an enantioselective support using n-hexane/ethanol,
40:60 (volume), as the mobile phase. Deprotection of
(S)-3 and (R)-3 was performed under the same condi-
tions as applied for the key intermediate (rac)-3, which
was proven not to cause racemization by phosphate
migration, yielding enantiomers (S)-5 and (R)-5 in pure
form.
Absolute stereochemical assignment was performed
on the basis of the crystal structure of intermediate (R)-
3. Figure 1 shows the X-ray structure of (R)-3 in the
crystal.12 Atomic displacement ellipsoids are drawn at
the 50% probability level, and hydrogen atoms are
drawn as spheres of arbitrary radius. The Flack x
parameter for (R)-3 is refined to 0.06(4).13
Derivatization and Chiral Separation
Since our efforts to separate the enantiomers of 5
directly on enantioselective support were met with
limited success,15 the optical purity of both enantiomers
was determined by analyzing the corresponding deriva-
tives that were prepared according to an OPA deriva-
tization protocol.16 Derivatization (Scheme 4) resulted
in the formation of the lactams (S)-6 and (R)-6 by
heterocycle condensation.
HPLC separation of the enantiomeric OPA derivatives
(S)-6 and (R)-6 could be successfully achieved on an ion
exchange quinine-based column (Prontosil AXQN 1) and
revealed that (S)-5 and (R)-5 were formed with >99%
Biological Activity
The biological activity of all analogues of 1 was
assessed in a S1P (hEDG) dependent SPA based GTPγS

Brief Articles
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 16 5375
Figure 3. Enantioselective HPLC analysis of 14C-labeled
phosphate of 1 from human blood (12 h) following OPA
derivatization.
synthesis of both enantiomers of phosphorylated 1 in
their optically pure form and determined their biological
activity. We defined the absolute configuration of both
enantiomers based on the crystal structure of a pro-
tected intermediate (R)-3 and successfully used a chemi-
cal derivatization of phosphorylated 1 ex vivo followed
by enantioselective HPLC to determine the enantio-
meric excess of the biologically formed species in rats
and humans.
Figure 2. Chiral separation of lactams (S)-6 and (R)-6.
Experimental Section
1. Chemistry. General Methods. All reactions were
carried out at room temperature if not otherwise stated.
Commercial reagents and absolute solvents were used. The
1H, 13C, and 31P NMR spectra were determined with a Bruker
DRX 400 MHz spectrometer and referenced to the solvent.
Chemical shifts are expressed in ppm. HRMS results were
obtained on a 9.4 T APEX-III FT-MS (Bruker Daltonics), and
ESI+ and ESI- mass spectra were obtained on a Micromass
Platform LCZ LC-MS. Preparative flash column chromatog-
raphies were performed using silica gel (Merck) G60 230-240
mesh. Analytical thin layer chromatography was performed
on precoated silica gel 60 F254 GLP glass plates (Merck
1.05702).
2 Synthesis. (R/S)-4-Hydroxymethyl-4-[2-(4-octylphe-
nyl)ethyl]oxazolidin-2-one ((rac)-2). Benzyl chloroformate
(0.45 mL, 3.2 mmol) was added to a suspension of 1 (1.03 g, 3
mmol) in 2 N NaOH (20 mL). The mixture was kept at room
temperature overnight, and to complete the reaction, more
benzyl chloroformate (0.9 mL, 6.4 mmol) was added. After 2
days at room temperature the mixture was acidified with 1 N
HCl, extracted with methylene chloride, and purified on a silica
optical purity (Figure 2), providing additional evidence
that no racemization occurs during the deprotection
protocol (Scheme 2).
Ex Vivo Analysis
We used the OPA derivatization protocol to investi-
gate the stereoselectivity of the biological phosphoryla-
tion of 1 in rats and humans. The enantiomeric com-
position of the 14C-labeled phosphate of 1 formed in vivo
after single oral administration of 14C-labeled 1 to rats
or humans was determined according to the protocols
described in refs 17 and 18, respectively. 14C-labeled 1
was synthesized according to a published route using
14C-labeled diethyl acetamidomalonate.19
Briefly, 14C-labeled phosphate of 1 was isolated from
blood taken from rats and humans at different times
postdose by extraction with methanol, followed by
nonenantioselective HPLC purification. The isolated
metabolite was derivatized with OPA and subjected to
enantioselective HPLC with radioactivity detection. In
rats and humans, only the OPA derivative of the
pharmacologically active (S)-5 was detected in blood.
The OPA derivative of the pharmacologically inactive
(R)-5 was below the detection limit (estimated to be <3%
of total FTY720-P). Figure 3 shows the results of the
enantioselective HPLC analysis of 14C-labeled phos-
phate of 1 isolated from human blood at 12 h postdose.
The results demonstrate that a single stereoisomer of
phosphorylated 1 is formed in vivo and suggest a very
high stereoselectivity of the rat and human kinase(s)
involved in the formation of phosphorylated 1.
gel column using methylene chloride/methanol/acetic acid50%
,
9:1:0.125 (volume), as the mobile phase. ESI-MS (ESI+), m/z:
334 (M + H)+. HRMS (FT), m/z: 334.2377 (M + H)+, 356.2196
(M + Na)+. Anal. (C20H31NO3) C, H, N, O.
(R/S)-4-[2-(4-Octylphenyl)ethyl]-4-(3-oxo-1,5-dihydro-
3λ5-benzo[e][1,3,2]dioxaphosphepin-3-yloxymethyl)ox-
azolidin-2-one ((rac)-3). To a solution of (rac)-2 (2.4 g, 7.2
mmol) in methylene chloride/THF, 1:1 (100 mL), at 0 °C was
added tetrazole (recrystallized, 2.52 g, 36 mmol) and 3-(di-
ethylamino)-1,5-dihydro-2,4,3-benzodioxaphosphepintriphe-
nyl phosphite (5.17 g, 21.6 mmol). After 18 h at room
temperature, H2O2 (8.2 mL [30% in water], 72 mmol) was
added (cooling) to the solution and kept at room temperature
for an additional 90 min. After the reaction was quenched with
saturated Na2S2O3 (100 mL), the mixture was extracted with
ethyl acetate (3×). The organic layer was dried over Na2SO4,
and the compound was purified on silica gel using cyclohexane/
ethyl acetate, 1:1 (volume), as the mobile phase. HRMS (FT),
m/z: 538.2331 (M + Na)+, 547.2934 (M + MeNH3)+, 554.2070
Conclusion
We have demonstrated that FTY720 is phosphory-
lated in vivo in rats and humans to form a single
stereoisomer, namely, the biologically active (S)-enan-
tiomer. For this purpose we developed an efficient
(M + K)+ Anal. (C28H38NO6P) C, H, N, O, P.
.
Phosphoric Acid Mono[(R/S)-2-amino-2-hydroxymethyl-
4-(4-octylphenyl)butyl] Ester ((rac)-5). Step 1. (rac)-3 (1.03
g, 2 mmol) was hydrogenated at normal pressure (Pd/C10%, 50

5376 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 16
Brief Articles
mg) over 90 min. After filtration, the solution was concentrated
and used in step 2 without further purification. ESI-MS (ESI-),
m/z: 412 (M - H)-.
Protocol for Rats. Blood samples from male Wistar rats
were taken after single oral (7.5 mg/kg, 8 and 72 h) adminis-
tration of [14C]FTY720 and stored at -20 °C until analysis.
Each blood sample (pool of three animals) was extracted with
methanol, and [14C]FTY720-P was isolated by nonenantiose-
lective HPLC. The isolated [14C]FTY720-P was derivatized
with OPA. The derivative was spiked with unlabeled (S)-6 and
(R)-6 as retention time markers and subjected to enantiose-
lective HPLC separation on a 150 mm × 4.0 mm Prontosil
AXQN 1 column packed with 5 µm particles (Bischoff Analy-
sentechnik, Leonberg, Germany). The components were eluted
isocratically with a 4:6 (v/v) mixture of 50 mM aqueous sodium
phosphate buffer, pH 5.0, and 2-propanol at a total flow rate
of 0.4 mL/min. Derivatized [14C]FTY720-P was monitored by
radioactivity detection. The unlabeled reference compounds
(S)-6 and (R)-6 were monitored by UV detection at 215 nm.
Protocol for Humans. Blood samples from healthy male
humans were taken after single oral administration of 4.5 mg
of [14C]FTY720 (12 and 72 h) and stored at -80 °C until
analysis. Each blood sample (pool of four subjects) was
extracted with methanol, [14C]FTY720-P was isolated by
nonenantioselective HPLC, derivatized with OPA, spiked with
unlabeled (S)-6 and (R)-6, and analyzed by enantioselective
HPLC as described in ref 17.
Step 2. To a solution of the end product of step 1 in ethanol
(20 mL), LiOH (20 mL, 10% solution in water) was added. After
24 h at reflux, the mixture was neutralized with HCl (1 N in
water) and concentrated. The residue was treated with glacial
acetic acid (5 mL), and precipitation of the end product
occurred after addition of water (50 mL). After the mixture
was filtered, washed (water), and dried, pure (rac)-5 was
obtained as a white amorphous powder in an overall yield of
80%. ESI-MS (ESI-), m/z: 386 (M - H)-.
(S)-4-[2-(4-Octylphenyl)ethyl]-4-(3-oxo-1,5-dihydro-3λ5-
benzo[e][1,3,2]dioxaphosphepin-3-yloxymethyl)oxazoli-
din-2-one and (R)-4-[2-(4-Octylphenyl)ethyl]-4-(3-oxo-1,5-
dihydro-3λ5-benzo[e][1,3,2]dioxaphosphepin-3-yloxy-
methyl)oxazolidin-2-one ((S)-3 and (R)-3). Both enantiomers
were isolated in optically pure form after preparative enan-
tiselective HPLC. Preparative separation of (S)-3 and (R)-3
was performed by chromatography on a Chiralpak AS-V (20
µm) column, 5 cm × 20 cm, with n-hexane/ethanol, 60:40
(volume), as the mobile phase at a flow rate of 50 mL/min.
Analytical HPLC: Chiralpak AS (4.6 mm × 250 mm), n-
hexane/ethanol, 40:60, 1.5 mL/min. UV: 210 nm. For (S)-3:
>99% ee, [R]20D -2.1° (c 1.75, CHCl3); tR ) 13.01 min; HRMS,
m/z 516.2512 (M + H)+, 538.2331 (M + Na)+, 547.2934 (M +
MeNH3)+. For (R)-3: >99% ee; [R]20D +1.9° (c 1.75, CHCl3); tR
) 6.31 min; HRMS, m/z 516.2512 (M + H)+, 538.2331 (M +
Na)+, 547.2934 (M + MeNH3)+.
Acknowledgment. We gratefully acknowledge Jean-
Pierre Baldeck for performing the enantioselective
analysis of the phosphate of 1 in blood, Hans-Peter
Gschwind for organizing the human ADME study in
vivo, Hansrudolf Walter for the preparation of crystals
for the X-ray studies, and Caroline Radoch and Gabri-
elle Lecis for the enantioselective separation of the OPA
analogues of (rac)-5, (S)-5, and (R)-5.
Phosphoric Acid Mono[(S)-2-amino-2-hydroxymethyl-
4-(4-octylphenyl)butyl] Ester ((S)-5). The compound was
synthesized according to the procedure given for (rac)-5
starting with (S)-3 as intermediate. HRMS, m/z: 386.2103 (M
- H)-. Anal. (C19H34NO5P) C, H, N, O, P.
Phosphoric Acid Mono[(R)-2-amino-2-hydroxymethyl-
4-(4-octylphenyl)butyl] Ester, (R)-FTY720-phosphate ((R)-
5). The compound was synthesized according to the procedure
given for (rac)-5 starting with (R)-3 as intermediate. HRMS,
m/z: 386.2103 (M - H)-. Anal. (C19H34NO5P) C, H, N, O, P.
Appendix
Abbreviation. FTY720, 2-amino-2-[2-(4-octylphenyl)-
ethyl]propane-1,3-diol; FTY720-P, phosphoric acid mono-
[2-amino-2-hydroxymethyl-4-(4-octylphenyl)butyl] ester;
S1P, sphingosine-1-phosphate; hEDG, human endothe-
lial cell differentiation gene; OPA, o-phthalaldehyde;
HPLC, high-performance liquid chromatography; GTPγS,
guanosine 5′-(γ-thio)triphosphate; CHO, Chinese ham-
ster ovary; GDP, guanosine diphosphate; SPA, scintil-
lation proximity assays; NMR, nuclear magnetic reso-
nance; HRMS, high-resolution mass spectrometry; ESI-
MS, electrospray ionization mass spectrometry; rt, room
temperature; MeOH, methanol; THF, tetrahydrofuran;
DMSO, dimethyl sulfoxide; TMS, tetramethylsilane;
EDTA, ethylenediaminetetraacetic acid).
Phosphoric Acid Mono[(R/S)-2-hydroxymethyl-4-(4-
octylphenyl)-2-(1-oxo-1,3-dihydroisoindol-2-yl)butyl] Es-
ter (OPA Derivatization) ((rac)-6). (rac)-5 (50 mg, 0.125
mmol) was suspended in a solution of EDTA (0.5 mL, 10 mM
in water) and aqueous boric acid (0.5 mL, 3% in water,
adjusted to pH 10.5 with aqueous 10% KOH). After addition
of OPA (33 mg, 0.25 mmol) dissolved in ethanol (0.5 mL), the
mixture was kept at room temperature for 1 h (ultrasound).
After that the pH was adjusted to 3.5 (aqueous 1 N HCl) and
the mixture was extracted with ethyl acetate (3×). The organic
layer was dried over Na2SO4, and the compound was purified
on silica gel using methylene chloride/methanol (95:5 f 0:100)
as the mobile phase. ESI-MS (ESI-), m/z: 502.5 (M - H)-.
Enantioselective HPLC: Prontosil AXQN 1 (150 mm × 4.0
mm), NaH2PO4-Na2HPO4 (0.05 M, pH 5.0)/2-propanol, 40:60
f 60:40, 0.4 mL/min, rt, DAD, tR ) 65.39 and 74.08 min.
Phosphoric Acid Mono[(S)-2-hydroxymethyl-4-(4-oc-
tylphenyl)-2-(1-oxo-1,3-dihydroisoindol-2-yl)butyl] Ester
((S)-6). The compound was prepared similarly to (rac)-6 with
(S)-5 as starting material. HRMS, m/z: 502.2368 (M - H)-.
Enantioselective HPLC: Prontosil AXQN 1 (150 mm × 4.0
mm) (Bischoff Analysentechnik, Leonberg, Germany), NaH2-
PO4-Na2HPO4 (0.05 M, pH 5.0)/2-propanol, 40:60 f 60:40, 0.4
mL/min, rt, DAD, tR ) 64.88 min, >99% ee, [R]20D +4.6° (c 0.48,
CHCl3).
Supporting Information Available: 1H NMR, HRMS,
elemental analysis, and HPLC results of selected coumpounds
described in the Experimental Section and a crystallographic
file in CIF format. This material is available free of charge
via the Internet at http://pubs.acs.org.
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Brief Articles
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 16 5377
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(17) Rat protocol; see Experimental Section.
(18) Human protocol; see Experimental Section.
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