Asymmetric synthesis of conformationally constrained Fingolimod analogues - Discovery of an orally active sphingosine 1-phosphate receptor type-1 agonist and receptor type-3 antagonist

Article abstract of DOI:10.1021/jm7010172

Compound 1 (FTY720, Fingolimod) represents a new generation of immunosuppressant that modulates lymphocyte trafficking by interacting with the S1P₁ receptor. Compound 1 also provides a template molecule for studying the molecular biology of S1P

Full text of DOI:10.1021/jm7010172

6428
J. Med. Chem. 2007, 50, 6428–6435
Asymmetric Synthesis of Conformationally Constrained Fingolimod Analogues—Discovery of
an Orally Active Sphingosine 1-Phosphate Receptor Type-1 Agonist and Receptor Type-3
Antagonist
Ran Zhu,† Ashley H. Snyder,‡ Yugesh Kharel,§ Lisa Schaffter,| Qin Sun,† Perry C. Kennedy,§ Kevin R. Lynch,‡,§ and
Timothy L. Macdonald*,†
Department of Chemistry, Department of Biochemistry and Molecular Genetics, and Department of Pharmacology, UniVersity of Virginia,
McCormick Road, CharlottesVille, Virginia 22904-4319, and Department of Medicinal Chemistry, Abbott Laboratories,
Worcester, Massachusetts 01605
ReceiVed August 14, 2007
Compound 1 (FTY720, Fingolimod) represents a new generation of immunosuppressant that modulates
lymphocyte trafficking by interacting with the S1P1 receptor. Compound 1 also provides a template molecule
for studying the molecular biology of S1P receptors and related enzymes (kinases and phosphatases). In
this study, two conformationally constrained analogues of 1 (3a and 3c) were asymmetrically synthesized
in high optical purity. In Vitro assessment documented that both analogues are Sphk2 substrates, their
phosphorylated species are potent S1P1 receptor agonists, and 3a-P is a potent S1P3 antagonist. After oral
administration in mice, both compounds evoked lymphopenia, but their duration of action differed markedly.
Chart 1. Structures of 1 and 1-P S-Phosphate, 2 Sphingosine
and 2-P, and 3 and its four stereoisomers
Introduction
Drug-induced modulation of the immune system is a corner-
stone of the treatment of autoimmune diseases and allows organ
allografting. In addition to biological (e.g., monoclonal antibody)
immunosuppressants, the small-molecule drugs used commonly
are cytotoxins (e.g., cyclophosphamide), corticosteroids, purine
synthesis inhibitors, and immunophilin-binding compounds
[cyclosporine A (CsA), rapamycin,1 and tacrolimus (FK506)2].3
All of these chemical agents function by either depleting
lymphocytes or preventing lymphocyte activation. Although
these small-molecule drugs have enabled widespread organ
transplantation, they also have significant adverse effects as
consequences of both the therapeutic target and often alternative,
“off” targets. Thus, additional drugs are needed to modulate
the immune system.
A new modality for the treatment of immunological disorders
arose from the discovery of 1 (FTY720, Fingolimod).4 This
compound, which is a sphingosine (2) analogue, modulates the
immune system by a unique mechanism, i.e., disruption of
normal lymphocyte trafficking. Specifically, 1 inhibits egress
of lymphocytes from secondary lymphoid tissues, such as lymph
nodes;5 the biomarker of 1 is thus lymphopenia (abnormally
low numbers of circulating lymphocytes). Preventing effector
lymphocytes from traveling to sites of inflammation might
underlie the efficacy of 1 in prolonging allograft survival and
in autoimmune disease models. Compound 1 has completed a
phase-III human renal transplantation trial, and phase-III trials
for relapsing-remitting multiple sclerosis (MS) are underway
currently.
tion of 1 by Sphk2a produces the S-phosphate species 1-P
specifically,7 which mimics much of S1P’s (2-P in Chart 1 is
an important extracellular lipid mediator) biological behavior.
Compound 2-P signals cells via five GPCRs: S1P1–5 (formerly,
EDG1, EDG5, EDG3, EDG6, and EDG8).8 Compound 1-P is
an agonist at four of the five receptors: S1P1, S1P3, S1P4, and
S1P5. The physiological activities of the S1P family of receptors
are currently under intense investigation through both genetic
and chemical biological approaches. Current evidence docu-
ments that in ViVo activation of the S1P1 receptor by agonists
(e.g., 2-P and 1-P) induces lymphopenia, but the precise
mechanism is disputed.9–11 Thus, 1 occupies an interesting
chemical space; it is a substrate for phosphorylation by Sphk2,
avoids the phosphatase activities relative to 2-P, and interacts
with a subset of S1P receptors, including those necessary for
lymphopenia.
Compound 1 is a prodrug; it enters cells and invades the
sphingolipid catabolic pathway, proceeding through cycles of
phosphorylation and dephosphorylation.6 In ViVo phosphoryla-
An adverse event associated with 1-P administration is
bradycardia; in rodents and perhaps other mammalian species,
* To whom correspondence should be addressed. Telephone: 434-924-
7718. Fax: 434-982-2302. E-mail: tlm@virginia.edu.
†
Department of Chemistry, University of Virginia.
Department of Biochemistry and Molecular Genetics, University of
aAbbreviations: S1P, sphingosine 1-phosphate; Sphk1, sphingosine
1-phosphate kinase type I; Sphk2, sphingosine 1-phosphate kinase type II;
S1Pn, sphingosine 1-phosphate receptor subtype n (n ) 1–5); GPCR,
G-protein coupled receptor; EDG, endothelial differentiation gene; SAR,
structure–activity relationship; PPA, pyrophosphoric acid.
‡
Virginia.
§
Department of Pharmacology, University of Virginia.
Abbott Laboratories.
|
10.1021/jm7010172 CCC: $37.00
2007 American Chemical Society
Published on Web 11/10/2007

Conformationally Constrained Fingolimod Analogues
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 25 6429
Scheme 1
this response is mapped to the S1P3 receptor.12 Activation of
the S1P3 receptor is also suspected to cause pulmonary
leakage.13 Thus, S1P3 receptor activity may limit the usefulness
of 1 as a therapeutic agent.
We intended to use the hydroxymethyl group to direct
reduction selectively to each face of the cyclopentene, thereby
inducing chirality at the phenyl-bearing carbon. The Crabtree
catalyst efficiently hydrogenated the alkene from the same face
of alcohol, producing the S stereochemistry of the octylphenyl
group (see 10). However, generation of the R stereochemistry
was more challenging. Little stereoselectivity was obtained with
most common heterogeneous catalysts, and many homogeneous
catalysts were unreactive, for example, Pfaltz’s catalyst.18 Only
after installation of the bulky TIPS group on the alcohol could
hydrogenation on the opposite face of the hydroxymethyl group
be effective with acceptable selectivity (see 9-12).
The critical transformation in the syntheses was the rhodium-
catalyzed C-H bond amination developed by Du Bois (Scheme
2).19 After transforming the 1(S)-hydroxymethyl-3-octylphenyl
alcohol 13 (13 represents either 10 or 12) to carbamate 14, the
carbamate amine inserted into the tertiary C-H bond on the
cyclopentyl ring via a rhodium-mediated nitrene mechanism
with retained stereochemistry to give oxazolidinone 15. Intro-
duction of the Boc group on the oxazolidinone nitrogen
facilitated the hydrolysis of carbamate to the N-Boc diastere-
omers 16.20 After removal of the N-Boc group with trif-
louroacetic acid, the final products 3a and 3c could be obtained
as their hydrochloride salts. For in Vitro receptor assays, we
treated 3 with PPA to transform the alcohol into phosphates
3-P. Ultimately, this was found to work equally well on both
diastereomers 10 and 12.
To confirm the validity of this synthesis, we compared the
stereoselectively synthetic isomers with racemic 3 by chiral
chromatography (Figure 1) and nuclear magnetic resonance
(NMR). The asymmetrically synthesized 3a and 3c proved to
be the first and third eluent, respectively.
Biology. We phosphorylated the four isomers of 3 obtained
either by chiral chromatography or by our asymmetric synthetic
routes. Because of the greater degree of purity of the synthetic
material, we present the data obtained from that set of
phosphorylated compounds. In a broken-cell assay of ligand
efficacy and potency, both 3a-P and 3c-P proved to be partial
The study reported herein was undertaken in efforts to define
further the molecular pharmacology of the S1P receptor family
and 2-P’s activating (Sphks) and inactivating (phosphatases)
enzymes. Using 1 as a template, we sought to restrict the
conformations of the three bonds that “link” the (hydroxy-
methyl)amino-substituted carbon “head group” and the oc-
tylphenyl “tail group” (see 1 in Chart 1). Our initial efforts
focused on two-dimensional or planar “linker” groups to restrict
conformations.14 Subsequent studies have restricted the sp3
hybridized “linker” backbone of the parent molecule through
ring formation. These studies led to the identification of 3
(VPC0109115), which evokes a profound, long lasting lym-
phopenia in rodents without a reduction in heart rate (data not
shown). However, our lead compound 3 was a mixture of four
stereoisomers. To elucidate the structure-activity relationships
(SARs) of the stereoisomers, it was necessary to isolate and
identify the active isomer(s) from the 4-component mixture
(3a-3d). This work presents the activities of each of the four
stereoisomers and selective asymmetric syntheses of the isomers
active in ViVo.
Results
Chemistry. Asymmetric synthesis (Scheme 1) began with a
modified Kumada cross-coupling of 2-chloropropenol 4 to (p-
octyl)phenyl magnesium bromide to produce hydroxymethyl
styrene 5,16 which was subsequently transformed into the
corresponding iodide 6 in two steps. The C-1 chiral center was
generated by alkylation of 4-pentenoic oxazolidinone using the
Evans’ chiral auxiliary technology.17 Cyclization using a second-
generation Grubbs’ catalyst furnished the cyclopentene ring with
the retention of stereochemistry at the hydroxymethyl-bearing
carbon, and reductive removal of the chiral auxiliary gave the
first key intermediate, 1-(4′-Octyl)phenyl-4(S)-hydromethyl cy-
clopent-1-ene (9).

6430 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 25
Zhu et al.
Scheme 2
agonists at the S1P1 receptor, with potency comparable to 2-P
(Figure 2a). Interestingly, their respective enantiomers, 3d-P
and 3b-P exhibited minimal, if any, agonist activity at the S1P1
receptor. Conversely, all four isomers lack agonist activity at
the S1P3 receptor. Because 3a-P (derived from the parent
alcohol-isolated chiral chromatography) showed inverse agonism
(i.e., negative efficacy) at the S1P3 receptor (not shown), we
collided each of the phosphorylated synthetic isomers with 2-P
at the S1P3 receptor. As documented in Figure 2b, 3a-P, 3b-P,
and 3d-P but not 3c-P behaved as surmountable antagonists at
the S1P3 receptor. We did not test the individual phosphorylated
synthetic isomers at the S1P2, S1P4, or S1P5 receptors, but the
racemate, 3-P, is inactive at S1P2, a full agonist at S1P4, and a
partial agonist at S1P5 (not shown).
Compounds 3a and 3c but not 3b or 3d were substrates of
recombinant Sphk2 (Figure 3). None of the isomers is either a
substrate for or an inhibitor of Sphk1.
From reference to 1, we expect that 3 is also a prodrug, i.e.,
requiring phosphorylation to achieve activity (indeed, 3 is not
active in a Sphk2 null mouse22). To test the hypothesis that
only the Sphk2 substrates 3a and 3c evoke lymphopenia, each
of the isomers was introduced into mice by oral gavage. After
a single dose (1 mg/kg body weight), both 3a and 3c evoked
lymphopenia and maintained a low level of circulating lym-
phocytes for at least 48 h after dosing (Figure 4). Interestingly,
8 days after dosing, the mice receiving 3c remained lym-
phopenic, while the blood lymphocyte counts in the 3a-treated
mice had returned to normal. As expected from the in Vitro
Figure 1. Chiral chromatographic separation of racemic 3 (top), 3c (middle), and 3a (bottom) synthesized according to Schemes 1 and 2. Separation conditions:
Chiralpak AD 4.6 mm i.d. × 250 mm at 35 °C, isocratic mobile phase of 95% A and 5% B at 0.8 mL/min, detection by UV absorption (254 nm). Mobile
phase A was heptane with a 0.2% diethylamine modifier. Mobile phase B was 50:50 methanol/ethanol with a 0.2% diethylamine modifier.

Conformationally Constrained Fingolimod Analogues
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 25 6431
Figure 4. Blood lymphocyte counts following administration of 3a
and 3c to C57Bl/6j mice (female, n ) 3 per treatment group).
Compounds were dissolved in vehicle (2% hydroxypropyl ꢀ-cyclo-
dextrin) and administered once by oral gavage (1 mg/kg) on day 0. On
the indicated days, mice were lightly anesthetized and blood was drawn
from the orbital sinus. Lymphocyte counts were determined with a
Hemavet model 950 blood analyzer.21
gous to 1, and have similar kinase and receptor profiles, they exhibit
very different duration in ViVo. Thus, these compounds may have
significant utility in further parsing out the roles that these receptors
play in normal and pathologic physiology.
These compounds (3a and 3c) were designed as conforma-
tionally restricted analogues of 1, and analysis of the limited
conformational space of each diastereomer is facilitating the
definition of the binding site for the S1P1 and S1P3 receptors.
The compounds also have three preferred properties of 1-like
prodrugs: first, the potency and efficacy at S1P1 is retained;
second, the new analogues are substrates for either Sphk1 or
Sphk2 (or possibly other lipid kinases); and third, the activity
at the S1P3 receptor is minimized.
Figure 2. In Vitro evaluation of 3a-P-3d-P. (a) Concentration effect
curves of 2-P and 3a-P-3d-P at the recombinant human S1P1 receptor.
The parameter measured is membrane-bound GTP[γ-35S] (see methods
for details).21 (b) Concentration effect curve for 2-P and 2-P in the
presence of 1 µM 3a-P-3d-P. The parameter measured is calcium
mobilization (see methods for details).21 The images shown are
representative of three independent experiments.
Because of their highly hydrophobic structures, it was
challenging to isolate the four isomers in quantities sufficient
for broad biological evaluation. Milligram scale pure isomers
could be isolated through high-performance liquid chromatog-
raphy (HPLC) on chiral columns, and a preliminary assay of
these four isomers suggested that 3a and 3c are the two active
species in ViVo (named in order of the chiral HPLC eluting
sequence). These two compounds proved to be diastereomers
(at the C-3 position) instead of enantiomers (as assessed by
optical rotation and NMR spectroscopy).
To establish definitively the stereochemistry of 3a and 3c,
we chemically synthesized the two isomers through a short
sequence involving an intramolecular nitrene C-H insertion.
It was suspected that the active species would have the R
stereocenter at the quaternary amino-bearing carbon,7,14a,24 and
thus, our synthesis was designed with that stereochemical
outcome anticipated. The stereochemical configuration of the
benzylic carbon remained undefined until this work was done.
Using several established asymmetric synthesis methods, we
constructed the two stereocenters predicted for 3a with high
selectivity and 3c with >85% optical purity (with ∼15% 3a).
Figure 3. 2 and 3a-3d were incubated with homogenates of HEK293T
cells, wherein mouse Sphk2 expression had been forced. n ) 2 (see
methods for details).22
activity profiles at both the kinase and the S1P1 receptor, neither
3b nor 3d evoked lymphopenia (not shown).
The pharmacological profiles of the isomers are different in
important ways, particularly in ViVo. As determined using the
GTP[γ-35S] binding assay, compound 3c was slightly more
efficacious than 3a. The greater efficacy of 3c is perhaps
reflected in the lower lymphocyte counts in 3c- versus 3a-treated
mice at 24 and 48 h. However, 3c is also a somewhat better
substrate at mouse Sphk2; this may further enhance the
lymphopenic effect of 3c. In contrast, there was no indication
from in Vitro analysis that the 3a/3c enantiomers would differ
greatly in their duration of action. It will be interesting to learn
Discussion
Understanding the molecular pharmacology of the S1P
receptors is evolving rapidly. Although an increasing number
of compounds have been developed to study these receptors,14,23
additional molecules of varying behavior will be necessary to
fully elucidate the pharmacology of these new targets. Here,
we report the asymmetric syntheses of two compounds that are
S1P1 agonists, but one is an S1P3 antagonist; a profile heretofore
unreported. Although both diastereomers are prodrugs, are analo-

6432 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 25
Zhu et al.
mmol (1.34 g) of MsCl and 11 mmol (1.11 g) of TEA at 0 °C for
1 h. After removal of the solvent and excess reagents, the mixture
was suspended in 30 mL of acetone and filtered through a Celite
pad. After another two washes, 10 mmol (1.50 g) of NaI was added
to the combined filtrate and stirred overnight. Removal of the
solvent was followed by hexane washes (30 mL × 3). When the
filtrate was concentrated, 3.00 g (85%) of light brown oil was
collected and used for the next step without further purification.
1H NMR (300 MHz, CDCl3) δ: 7.52–7.20 (m, 4H), 5.50 (s, 1H),
5.48 (s, 1H), 4.33 (s, 2H), 2.63 (app t, J ) 7.52 Hz 2H), 1.64 (m,
2H), 1.36 (m, 10H), 0.90 (t, J ) 6.71 Hz, 3H). 13C NMR (75 MHz,
CDCl3) δ: 145.5, 143.5, 135.2, 128.8, 126.1, 115.1, 35.9, 32.1, 31.6,
29.7, 29.6, 29.5, 22.9, 14.4, 7.6.
whether the pharmacokinetic profile of these compounds paral-
lels this pharmacodynamic property.
Perhaps the most salient property of compound 3a-P is the
antagonism at the S1P3 receptor. The lead compound in this
class, 1, is a potent S1P3 agonist. In rodents, agonist activity at
the S1P3 receptor is associated with toxicities.12,13 If this is true
in humans also, the more selective 3-type compounds might be
superior because of reduced toxicity. Finally, 3a-P is 1 log order
more potent than previously reported S1P3 antagonists.25
However, we do not know at present whether antagonism at
the S1P3 receptor would have any therapeutic benefit.
Conclusion
(R)-4-Benzyl-3-((S)-2-(2-(4-octylphenyl)allyl)pent-4-enoyl)-
oxazolidin-2-one (7). A total of 9 mmol (2.33 g) of (R)-4-benzyl-
3-pent-4-enoyloxazolidin-2-one in 60 mL of dry THF was cooled
to -78 °C, and 9 mmol (9 mL) of 1 N NaHMDS (in THF) was
added slowly. After 30 min, 9 mmol (3.18 g) of 6 was added
dropwisely through a synringe. The reaction was maintained at -78
°C for 1.5 h, then warmed up to 0 °C, and quenched with half-
saturated NH4Cl solution. After removal of THF, the residue was
patitioned between NH4Cl solution (25 mL) and CH2Cl2 (25 mL
× 2). The combined organic layer was dried over NaSO4 and
concentrated. Flash column eluting with 10% EtOAc in hexanes
(Rf ) 0.35) gave 3.5 g (7.2 mmol, 80%) of clear oil. 1H NMR
(300 MHz, CDCl3) δ: 7.50–6.97 (m, 9H), 5.91–5.62 (m, 1H), 5.33
(d, J ) 1.04 Hz, 1H), 5.20–4.92 (m, 3H), 4.61 (m, 1H), 4.13 (m,
3H), 3.25–2.90 (m, 2H), 2.73 (m, 1H), 2.57 (m, 3H), 2.41 (m, 1H),
2.33 (m, 1H), 1.58 (m, 1H), 1.27 (s, 10H), 0.89 (t, J ) 6.69 Hz,
3H). 13C NMR (75 MHz, CDCl3) δ: 175.6, 153.2, 145.9, 142.7,
138.2, 135.7, 135.4, 129.6, 129.1, 128.6, 127.5, 126.4, 117.5, 114.0,
66.1, 55.6, 41.7, 38.1, 37.2, 37.0, 35.8, 32.1, 31.6, 29.7, 29.6, 29.5,
22.9, 14.3.
We asymmetrically synthesized all of the four stereoisomers
of a 4-component immunomodulatory S1P prodrug. Two of the
isomers, 3a and 3c, are substrates for Sphk2 and, following
phosphorylation, are potent partial agonists at the S1P1 receptor.
Further, one isomer is the most potent S1P3 antagonist reported
to date. The unique biological properties of 3 make it a useful
chemical tool for the study of S1P signaling.
Experimental Section
General Procedure. All chemicals and solvents were purchased
from Aldrich or Fisher unless specified; anhydrous solvents and
amine bases were used without further distillation. All reactions
were performed under inert gas protection in oven-dried reaction
vessels unless specified. Analytical thin-layer chromatography
(TLC) was carried out on a Merck silica gel 60F254 aluminum-
based plate, and preparative TLC separation was performed on
Analtech 02013 (1000 µm) or a similar plate with a varied thickness
of coating. UV absorption was detected at 254 nm. All NMR raw
data were collected on a Varian Mercury Plus or Unity Inova 300
MHz (75 MHz carbon) and processed by MestReC 4.9.9.6 with
the default setting. Optical rotation was obtained on a Perkin-Elmer
343 plus polarimeter. Low-resolution mass spectrometry (MS)
[electrospray ionization (ESI)] were recorded on ThermoFinnigan
LCQ, and high-resolution MS (ESI-TOF) analysis was performed
by the University of Illinois MS laboratory. Elemental analysis was
performed by the Atlantic Micro Laboratory. Chiral HPLC separa-
tions were obtained from a Chiralpak AD 4.6 mm i.d. × 250 mm
column using isocratic conditions with 95% A and 5% B at a flow
rate of 0.8 mL/min and monitoring at a wavelength of 254 nm or
with an evaporative light scattering detector (ELSD). The column
was maintained at 35 °C using a column heater. Mobile phase A
was heptane with a 0.2% diethylamine modifier. Mobile phase B
was 50:50 methanol/ethanol with a 0.2% diethylamine modifier.
Chemistry. 2-(4-Octylphenyl)prop-2-en-1-ol (5). In a 250 mL
round-bottom flask, 24 mmol (0.576 g) of magnesium turnings and
20 mmol (5.36 g) of p-octylbromobenzene (TCI Chemicals) in 80
mL of dry tetrahydrofuran (THF) were refluxed for 1 h. In another
100 mL flask, 16 mmol (1.472 g) of 2-chloro-2-propen-1-ol (4,
90% tech grade) in 40 mL of dry THF was cooled to 0 °C and 17
mmol (6.8 mL) of 2.5 M n-butyl lithium (in hexane) was added
dropwise. After 10 min, the deprotonated chloride was syringed
into the flask containing the Grignard reagent. After the addition
of 1 mmol (0.542 g) NiDPPPCl2, the reaction mixture was refluxed
for 3 h. After removal of the solvent, the mixture was partitioned
between half-saturated NH4Cl solution (25 mL) and CH2Cl2 (25
mL × 2). The combined organic solution was dried over NaSO4,
concentrated, and columned with CH2Cl2 (Rf ) 0.25). A total of
(R)-4-Benzyl-3-((S)-3-(4-octylphenyl)cyclopentx-3-xenecar-
bonyl)oxazolidin-2-one (8). A total of 3.5 g (11.2 mmol) of 7 was
refluxed in 40 mL of CH2Cl2 with 20 mg of second-generation
Grubbs catalyst for 2 h. Flash column with 10% EtOAc in hexanes
(Rf ) 0.3) gave 2.70 g (9.5 mmol, 85%) of clear oil. 1H NMR
(300 MHz, CDCl3) δ: 7.55–6.93 (m, 9H), 6.08 (m, 1H), 4.72 (m,
1H), 4.41 (m, 1H), 4.33–4.10 (m, 2H), 3.32 (dd, J ) 13.35, 3.12
Hz, 1H), 3.23 (m, 1H), 3.16–3.03 (m, 1H), 2.98–2.89 (m, 2H), 2.82
(dd, J ) 13.34, 9.61 Hz, 1H), 2.62 (t, J ) 7.66 Hz, 2H), 1.62 (m,
2H), 1.32 (m, 10H), 0.91 (t, J ) 6.67 Hz, 3H). 13C NMR (75 MHz,
CDCl3) δ: 175.7, 153.4, 142.4, 140.4, 135.6, 133.5, 129.7, 129.2,
128.6, 127.6, 125.8, 122.8, 66.4, 55.7, 41.8, 38.1, 37.3, 36.6, 35.9,
32.2, 31.7, 29.8, 29.6, 29.5, 22.9, 14.4.
(S)-(3-(4-Octylphenyl)cyclopent-3-enyl)methanol (9). A total
of 3.4 mmol (1.55 g) of 8 and 3.5 mmol (0.11 g) of MeOH were
dissolved in 40 mL of THF, and 7 mmol (3.5 mL) of 2 M LiBH4
in THF was added at 0 °C. After 1 h of the reaction at 0 °C, the
mixture was concentrated and partitioned between water and CH2Cl2
(25 mL/25 mL × 2). The combined organic layer was dried over
NaSO4 and concentrated. Flash column eluting with CH2Cl2 (Rf )
0.2) gave 0.61 g (2.1 mmol, 63%) of white flakes (recrystallized
1
from hexanes). H NMR (300 MHz, CDCl3) δ: 7.36 (d, J ) 8.13
Hz, 2H), 7.14 (d, J ) 8.13 Hz, 2H), 6.17–5.99 (m, 1H), 3.64 (d, J
) 6.63 Hz, 2H), 2.96–2.81 (m, 1H), 2.69 (m, 2H), 2.63–2.57 (m,
2H), 2.52 (m, 1H), 2.32 (m, 1H), 1.62 (m, 3H), 1.43–1.17 (m, 10H),
0.90 (t, J ) 6.71 Hz, 3H). 13C NMR (75 MHz, CDCl3) δ: 142.1,
141.3, 134.1, 128.6, 125.6, 123.8, 67.5, 39.9, 36.5, 35.9, 32.1, 31.7,
29.7, 29.6, 29.5, 22.9, 14.4.
((1S,3S)-3-(4-Octylphenyl)cyclopentyl)methanol (10). A total
of 0.20 g of 9 and 5 mg of Crabtree catalyst (Strem) were dissolved
in 50 mL of dry CH2Cl2 in an oven-dried 100 mL flask under
nitrogen protection. The mixture reacted under a hydrogen balloon
for 1 day. After removal of the solvent, the product was used for
the next step without purification. 1H NMR (300 MHz, CDCl3) δ:
7.15 (q, J ) 8.17 Hz, 4H), 3.58 (d, J ) 7.02 Hz, 2H), 3.17–2.98
(m, 1H), 2.66–2.52 (app t, J ) 7.55 Hz, 2H), 2.48–2.30 (m, 1H),
2.21–1.54 (m, 8H), 1.47–1.19 (m, 10H), 0.91 (t, J ) 6.66 Hz, 3H).
1
2.51 g (10.2 mmol, 71%) of white solid was collected. H NMR
(300 MHz, CDCl3) δ: 7.50–7.08 (m, 4H), 5.46 (s, 1H), 5.31 (m,
1H), 4.54 (d, J ) 5.66 Hz, 1H), 2.62 (app t, J ) 8.00 Hz 2H), 1.61
(m, 1H), 1.31 (m, 10H), 0.90 (t, J ) 6.69 Hz, 3H). 13C NMR (75
MHz, CDCl3) δ: 147.3, 143.2, 135.9, 128.8, 126.1, 112.1, 65.3,
35.9, 32.2, 31.7, 29.8, 29.6, 29.5, 22.9, 14.4.
1-(3-Iodoprop-1-en-2-yl)-4-octylbenzene (6). A total of 9.55
mmol (2.35 g) of 5 in 40 mL of dry CH2Cl2 was reacted with 10

Conformationally Constrained Fingolimod Analogues
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 25 6433
13C NMR (75 MHz, CDCl3) δ: 143.2, 140.7, 128.5, 127.1, 67.7,
44.5, 41.5, 37.1, 35.8, 35.0, 32.2, 31.9, 29.8, 29.7, 29.6, 29.6, 23.0,
14.4.
5 mL of MeOH and 2 drops of water and 0.1 g of K2CO3 was
added and reacted overnight. The reaction mixture was then TLC-
purified in acetone/CHCl3 (5:95, v/v, Rf ) 0.3, weak UV). A total
of 42 mg (0.1 mmol, 74%) of product was collected. 1H NMR
(300 MHz, CDCl3) δ: 7.18–7.07 (m, 4H), 4.93 (s, 1H), 3.72 (q, J
) 11.27 Hz, 1H), 3.08 (m, 1H), 2.57 (app t, J ) 7.75 Hz, 1H),
2.46 (dd, J ) 13.29, 7.55 Hz, 1H), 2.16–2.03 (m, 1H), 2.00–1.80
(m, 3H), 1.73 (dd, J ) 13.16, 11.13 Hz, 1H), 1.59 (m, 2H), 1.45
(s, 9H), 1.38–1.18 (m, 10H), 0.88 (t, J ) 6.74 Hz, 3H). 13C NMR
(75 MHz, CDCl3) δ: 156.6, 141.8, 141.0, 128.6, 127.1, 80.2, 69.4,
65.0, 44.4, 44.0, 43.3, 35.9, 35.8, 32.9, 32.1, 31.8, 29.7, 29.6, 29.5,
22.9, 14.4.
((1S,3R)-3-(4-Octylphenyl)cyclopentyl)methanol (12). A total
of 1.33 mmol (0.380 g) of 9, 1.6 mmol (0.307 g) of TIPSCl, and
2.0 mmol (0.136 g) of imidazole were dissolved in 15 mL of CH2Cl2
and reacted overnight. Upon removal of the solvent, the mixture
was suspended in 20 mL of hexane and filtered through a Celite
pad. The filtrate was concentrated and redissolved in 50 mL of
EtOAc; this solution was hydrogenated under a H2 balloon overnight
with Pd/C (10%, wt). When strong UV absorption disappeared on
TLC, the Pd/C was filtered off followed by removal of EtOAc.
The mixture was deprotected with 4 mmol (1.046 g) of TBAF in
40 mL of THF overnight. After removal of most of the solvent,
the mixture was separated on a 1000 µm preparative TLC (or a
small column) with CH2Cl2 (Rf ) 0.2). A total of 0.285 g (9.9
mmol, 75%) of clear oil was collected. 1H NMR (300 MHz, CDCl3)
δ: 7.24–7.08 (m, 4H), 3.61 (dd, J ) 6.58, 1.51 Hz, 2H), 3.06 (m,
1H), 2.59 (app t, J ) 7.58 Hz, 3H), 2.41–1.81 (m, 6H), 1.75–1.51
(m, 4H), 1.45–1.21 (m, 10H), 0.91 (t, J ) 6.85 Hz, 3H). 13C NMR
(75 MHz, CDCl3) δ: 142.8, 140.7, 128.5, 127.1, 67.7, 45.8, 42.2,
38.5, 35.8, 33.6, 32.2, 31.9, 29.8, 29.7, 29.6, 28.6, 23.0, 14.4.
((1S,3S)-3-(4-Octylphenyl)cyclopentyl)methyl Carbamate (14a).
A total of 0.42 mmol (0.120 g) of 10 was dissolved in 15 mL of
dry CH2Cl2, and 0.50 mmol (0.094 g) of Cl3CC(O)NCO was added
at 0 °C. After about 0.5–1 h (track by TLC in CH2Cl2), the solvent
was removed and the mixture was redissolved in 10 mL of MeOH
and 1 mL of H2O. A total of 0.5 g of K2CO3 was added and reacted
for 5 h. Upon removal of most solvents, the residue was loaded on
preparative TLC and developed in CH2Cl2 (just above the starting
point, weak UV absorption). A total of 0.130 g (3.9 mmol, 93%)
of white solid was collected. 1H NMR (300 MHz, CDCl3) δ:
7.22–7.04 (m, 4H), 5.05 (b, 2 H), 4.08–3.96 (m, 2H), 3.18–3.00
(m, 1H), 2.59 (app t, J ) 7.76 Hz, 2H), 2.54–2.42 (m, 1H),
2.19–1.54 (m, 8H), 1.48–1.16 (m, 10H), 0.91 (t, J ) 7.47 Hz, 3H).
13C NMR (75 MHz, CDCl3) δ: 157.8, 143.0, 140.7, 128.6, 127.1,
69.4, 44.3, 38.2, 37.3, 35.8, 34.9, 32.2, 31.9, 29.8, 29.7, 29.6, 23.0,
14.4.
Tert-butyl (1R,3R)-1-(Hydroxymethyl)-3-(4-octylphenyl)cyclo-
pentylcarbamate (16c). This compound was prepared in a manner
similar to 16a (78%). 1H NMR (300 MHz, CDCl3) δ: 7.13 (m,
4H), 4.89 (s, 1H), 3.73 (dd, J ) 23.07, 11.08 Hz, 2H), 3.30 (m,
0.86H), 3.13–3.01 (m, trace amount), 2.57 (app t, J ) 7.77 Hz,
1H), 2.29 (m, 1H), 2.23–2.03 (m, 2H), 1.78 m, 3H), 1.58 (m, 2H),
1.53–1.38 (m, 9H), 1.28 (m, 10H), 0.88 (t, J ) 6.69 Hz, 3H). 13
C
NMR (75 MHz, CDCl3) δ: 156.5, 141.7, 141.0, 128.6, 127.1, 80.1,
69.9, 65.1, 43.8, 39.7, 36.4, 35.8, 33.3, 32.1, 31.8, 29.7, 29.6, 29.5,
28.7, 22.9, 14.4.
((1R,3S)-1-Amino-3-(4-octylphenyl)cyclopentyl)methanol Hy-
drochloride (3a). A total of 100 mg (0.25mmol) of 16a was stirred
in 5 mL of CH2Cl2 and 1.5 mL of TFA at 0 °C for 1 h; after
quenching with 5 mL of methanol and evaporation of excess
reagents, the product was partitioned between CH2Cl2 and 1 N
NaOH (10 mL/10 mL × 3). The combined organic layer was dried
over Na2SO4 and concentrated to 15 mL; this solution was treated
with 1 drop of concentrated hydrochloric acid, and a fine needle
product precipitated within minutes. A total of 73 mg (0.22 mmol,
88%) of HCl salt was collected. NMR, elemental analysis, and
rotation was performed on the free amine form of the product. 1H
NMR (300 MHz, CDCl3) δ: 7.14 (dd, J ) 17.60, 8.04 Hz, 4H),
3.46 (m, 2H), 3.06 (m, 1H), 2.65–2.49 (m, 2H), 2.28 (m, 4H),
2.14–2.00 (m, 1H), 1.90 (m, 1H), 1.70 (m, 2H), 1.65–1.55 (m, 2H),
1.50 (m, 1H), 1.29 (m, 10H), 0.88 (t, J ) 6.33 Hz, 3H). 13C NMR
(75 MHz, CDCl3) δ: 142.2, 140.9, 128.6, 127.0, 70.2, 62.3, 46.5,
44.8, 38.6, 35.8, 33.3, 32.1, 31.8, 29.7, 29.7, 29.5, 22.9, 14.4. HRMS
(ESI+) (m/z): calcd for C20H34NO (MH+), 304.2640; found,
304.2641. Anal. Calcd for C20H33NO: C, 79.15; H, 10.96; N, 4.62.
Found: C, 78.86; H, 11.01; N, 4.60. [R]2D0 -2.8 (c 1.05, CHCl3).
((1R,3R)-1-Amino-3-(4-octylphenyl)cyclopentyl)methanol Hy-
drochloride (3c). This compound was prepared in a manner similar
((1S,3R)-3-(4-Octylphenyl)cyclopentyl)methyl Carbamate (14c).
This compound was prepared in a manner similar to 14a (88%).
1H NMR (300 MHz, CDCl3) δ: 7.13 (q, J ) 8.20 Hz, 4H), 4.87
(b, 1H), 4.03 (m, 2H), 3.14–2.96 (m, 1H), 2.58 (app t, J ) 7.75
Hz, 2H), 2.40 (m, 1H), 2.24 (m, 1H), 2.15–2.02 (m, 1H), 1.98–1.51
(m, 6H), 1.46–1.15 (m, 10H), 0.89 (t, J ) 6.63 Hz, 3H). 13C NMR
(75 MHz, CDCl3) δ: 157.6, 142.6, 140.8, 128.5, 127.1, 69.5, 45.7,
38.8, 38.6, 35.8, 33.5, 32.2, 31.9, 29.8, 29.7, 29.5, 28.7, 22.9, 14.4.
15a. A total of 0.78 mmol (0.26 g) of 14a, 1.10 mmol (0.354 g)
of BAIB, 1.79 mmol (0.072 g) of MgO, and 0.039 mmol (0.017 g)
of [Rh(CH3COO)2]2 ·2H2O were refluxed in 10 mL of CH2Cl2 for
14 h. The reaction mixture was TLC-separated in acetone/CHCl3
(5:95, v/v, Rf ) 0.3, weak UV). A total of 0.14 g (0.44 mmol,
1
to 3a (67%). H NMR (300 MHz, CDCl3) δ: 7.13 (m, 4H), 3.46
(s, 2H), 3.40–3.21 (m, 0.85H), 3.09–2.97 (m, 0.15H), 2.54 (m, 2H),
2.11 (m, 4H), 2.04–1.83 (m, 2H), 1.78–1.64 (m, 2H), 1.58 (m, 4H),
1.27 (m, 10H), 0.88 (t, J ) 8.27 Hz, 3H). 13C NMR (75 MHz,
CDCl3) δ: 142.2, 141.0, 128.6, 127.0, 70.9, 62.4, 46.9, 43.8, 37.9,
35.8, 33.7, 32.1, 31.8, 29.7, 29.6, 29.5, 22.9, 14.4. HRMS (ESI+)
(m/z): calcd for C20H34NO (MH+), 304.2640; found, 304.2641.
1
57%) of white solid was collected. H NMR (300 MHz, CDCl3)
((1R,3S)-1-Amino-3-(4-octylphenyl)cyclopentyl)methyl
Di-
δ: 7.13 (m, 1H), 6.64 (s, 1H), 4.29 (dd, J ) 17.60, 8.40 Hz, 2H),
3.01 (m, 1H), 2.62–2.47 (app t, J ) 7.75 Hz, 2H), 2.29 (dd, J )
13.23, 6.98 Hz, 1H), 2.20–2.03 (m, 3H), 2.03–1.73 (m, 3H), 1.59
(m, 2H), 1.40–1.16 (m, 10H), 0.88 (t, J ) 6.70 Hz, 3H). 13C NMR
(75 MHz, CDCl3) δ: 159.6, 141.4, 140.8, 128.8, 126., 65.0, 47.2,
43.3, 38.7, 35.8, 32.4, 32.1, 31.8, 29.7, 29.6, 29.5, 22.9, 14.4.
15c. This compound was prepared in a manner similar to 15a
hydrogen Phosphate (3a-P). A total of 10.0 mg (0.03 mmol) of 3a
was vigorously stirred in ∼2 mL of freshly prepared pyrophosphoric
acid (PPA) at 100 °C for 2 h. Upon cooling, ∼9 mL of ice was
added to the reaction mixture and the product precipitated as a white
solid. The product was collected on a Buchner funnel, further
washed by water (8 mL × 2), and then vaccuum-dried. A total of
6.3 mg (0.016 mmol, 56%) of white solid was collected. 1H NMR
(300 MHz, CD3OD) δ: 7.27–7.12 (m, 4H), 4.04–3.86 (app q, J )
5.62 Hz, 1H), 2.94–2.72 (b, 2H), 2.66–2.57 (m, 2H), 2.57–2.48
(m, 1H), 2.23–2.13 (m, 1H), 2.13–1.89 (m, 2H), 1.82–1.69 (m, 1H),
1.68–1.55 (m, 2H), 1.33 (m, 10H), 0.93 (t, J ) 6.60 Hz, 1H). HRMS
(ESI+) (m/z): calcd for C20H35NO4P (MH+), 384.2304; found,
384.2305.
1
(66%). H NMR (300 MHz, CDCl3) δ: 7.19–7.06 (m, 4H), 4.32
(q, J ) 8.50 Hz, 2H), 3.38–3.15 (m, 0.86H), 3.11–2.92 (m, 0.14H),
2.63–2.50 (app t, J ) 7.75 Hz, 2H), 2.38 (dd, J ) 13.51, 7.49 Hz,
1H), 2.31–1.94 (m, 4H), 1.88–1.65 (m, 2H), 1.59 (m, 2H), 1.28
(m, 10H), 0.88 (t, J ) 6.62 Hz, 3H). 13C NMR (75 MHz, CDCl3)
δ: 159.9, 141.3, 141.2, 128.7, 126.9, 65.4, 47.3, 43.5, 39.7, 35.8,
32.6, 32.1, 31.8, 29.7, 29.6, 29.5, 22.9, 14.4.
((1R,3R)-1-Amino-3-(4-octylphenyl)cyclopentyl)methyl
Di-
Tert-butyl (1R,3S)-1-(Hydroxymethyl)-3-(4-octylphenyl)cyclo-
pentylcarbamate (16a). A total of 0.14 mmol (46 mg) of 15a, 0.2
mmol (44 mg) of Boc2O, 0.3 mmol (30 mg) of triethylamine, and
a catalytic amount of DMAP were reacted in 5 mL of CH2Cl2 for
4 h. After removal of the solvent, the mixture was redissolved in
hydrogen Phosphate (3c-P). A total of 10.0 mg (0.03 mmol) of 3c
was treated similarly to PPA as 3a. After hydrolysis, the gummy
product was briefly centrifuged; after removal of phosphoric acid,
the product was further washed and centrifuged in water (8 mL ×
2). A total of 5.5 mg (0.014 mmol, 50%) of white solid was

6434 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 25
Zhu et al.
collected. 1H NMR (300 MHz, CD3OD) δ: 7.18 (m, 4H), 4.21–4.07
(m, 1H), 4.05–3.92 (m, 1H), 2.60 (app t, J ) 7.65 Hz, 2H),
2.39–2.14 (m, 3H), 2.07–1.75 (m, 3H), 1.62 (m, 2H), 1.43–1.26
(m, 10H), 0.93 (t, J ) 6.65 Hz, 3H). HRMS (ESI+) (m/z): calcd
for C20H35NO4P (MH+), 384.2304; found, 384.2311.
(2) Tanaka, H.; Kuroda, A.; Marusawa, H.; Hatanaka, H.; Kino, T.; Goto,
T.; Hashimoto, M.; Taga, T. Structure of FK506, A Novel Immuno-
suppressant Isolated from Streptomyces. J. Am. Chem. Soc. 1987, 109,
5031–5033.
(3) Halloran, P. F. Immunosuppressive Drugs for Kidney Transplantation.
N. Engl. J. Med. 2004, 351, 2715–2729.
Biology. [γ-35S]GTP Binding Assay.21 A plasmid encoding the
human S1P1 receptor was introduced into CHO K1 cells by DNA-
mediated transfection, and a clonal line expressing high levels of
the receptor was isolated. Crude membrane fractions from these
cells containing ca. 5 µg of protein were incubated in 0.1 mL of
binding buffer [50 mM N-2-hydroxyethylpiperazine-N′-2-ethane-
sulfonic acid (HEPES), 100 mM NaCl, and 10 mM MgCl2 at pH
7.5] containing 5 µg of saponin, 0.10 mM GDP, 0.1 nM [γ-35S]GTP
(1200 Ci/mmol), and the indicated compounds for 30 min at 30
°C. Membranes were collected on GF/C filters using a Brandel Cell
Harvester (Gaithersburg, MD), and samples were analyzed for
bound radionuclide using a TopCount ꢀ scintillation counter. Each
of the eight points on a concentration effect curve were determined
in triplicate and are presented as standard errors of the mean.
Calcium Mobilization.21 A plasmid encoding the human S1P3
receptor containing an amino-terminal HA epitope tag was intro-
duced into CHO K1 cells by DNA-mediated transfection, and a
clonal line expressing high levels of the receptor was isolated using
a fluorescence-activated cell sorter. Cells were plated on a clear
bottom, black wall 96-well plate; the cell monolayers were grown
to confluence, washed, and loaded with a calcium-sensitive
fluorescent dye; and calcium transients in response to compound
addition were monitored using a Molecular Devices FLEXStation.
Each of the eight points on a concentration effect curve were
determined in triplicate and are presented as standard errors of the
mean.
(4) (a) Fujita, T.; Inoue, K.; Yamamoto, S.; Ikumoto, T.; Sasaki, S.;
Toyama, R.; Chiba, K.; Hoshino, Y.; Okumoto, T. Fungal Metabolites.
Part 11. A Potent Immunosuppressive Activity Found in Isaria
sinclairii Metabolite. J. Antibiot. 1994, 47, 208–215. (b) 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 Im-
munosuppressant, FTY720. Bioorg. Med. Chem. Lett. 1995, 5, 853–
856.
(5) Mandala, S.; Hajdu, R.; Bergstrom, J.; Quackenbush, E.; Xie, J.;
Milligan, 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 Receptor Agonists. Science 2002, 296, 346–349.
(6) Spiegel, S.; Milstien, S. Sphingosine 1-Phosphate: An Enigmatic
Signalling Lipid. Nat. ReV. Mol. Cell Biol. 2003, 4, 397.
(7) Albert, R.; Hinterding, K.; Brinkmann, V.; Guerini, D.; Muller-
Hartwieg, C.; Knecht, H.; Simeon, C.; Streiff, M.; Wagner, T.;
Welzenbach, K.; Zecri, F.; Zollinger, M.; Cooke, N.; Francotte, E.
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. J. Med. Chem. 2005, 48, 5373–5377.
(8) Hla, T.; Lee, M.-J.; Ancellin, N.; Paik, J. H.; Kluk, M. J. Lysophospho-
lipids—Receptor Revelations. Science 2001, 294, 1875–1878.
(9) 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 Peripheral Lymphoid Organs Is Dependent
on S1P Receptor 1. Nature 2004, 427, 355–360.
(10) Sanna, M. G.; Wang, S. K.; Gonzalez-Cabrera, P. J.; Don, A.;
Marsolais, D.; Matheu, M. P.; Wei, S. H.; Parker, I.; Jo, E.; Cheng,
W. C.; Cahalan, M. D.; Wong, C. H.; Rosen, H. Enhancement of
Capillary Leakage and Restoration of Lymphocyte Egress by a Chiral
S1P1 Antagonist in ViVo. Nat. Chem. Biol. 2006, 2, 434–441.
(11) Pappu, R.; Schwab, S. R.; Cornelissen, I.; Pereira, J. P.; Regard, J. B.;
Xu, Y.; Camerer, E.; Zheng, Y.-W.; Huang, Y.; Cyster, J. G.; Coughlin,
S. R. Promotion of Lymphocyte Egress into Blood and Lymph by Distinct
Sources of Sphingosine 1-Phosphate. Science 2007, 316, 295–298.
(12) Forrest, M.; Sun, S. Y.; Hajdu, R.; Bergstrom, J.; Card, D.; Doherty,
G.; Hale, J.; Keohane, C.; Meyers, C.; Milligan, J.; Mills, S.; Nomura,
N.; Rosen, H.; Rosenbach, M.; Shei, G. J.; Singer, I. I.; Tian, M.;
West, S.; White, V.; Xie, J.; Proia, R. L.; Mandala, S. Immune Cell
Regulation and Cardiovascular Effects of Sphingosine 1-Phosphate
Receptor Agonists in Rodents Are Mediated via Distinct Receptor
Subtypes. J. Pharmacol. Exp. Ther. 2004, 309, 758–768.
Sphk Assay.22 Mouse Sphk2 was expressed by the introduction
of plasmid DNA into HEK293T cells. Sphk2 activity was measured
in a solution that consisted of 20 mM Tris-Cl (pH 7.4), 1 mM
2-mercaptoethanol, 1 mM ethylenediaminetetraacetic acid (EDTA),
5 mM sodium orthovanadate, 40 mM ꢀ-glycerophosphate, 15 mM
NaF, 1 mM phenylmethylsulfonyl fluoride, 10 mM MgCl2, 0.5 mM
4-deoxypyridoxine, 10% glycerol, and 0.01 mg/mL each of
leupeptin, aprotinin, and soybean trypsin inhibitor. To determine
the fractional activity of Sphk1 versus Sphk2, the buffer was
supplemented with either 0.5% Triton X-100 or 1 M KCl,
respectively. The buffer was supplemented with substrate (D-
erythro-sphingosine, 15 µM; 3a-3d, 50 µM), [γ32P]ATP (10 µM,
specific activity ) 8.3 Ci/mmol), and cell extract (0.02–0.03 mg
of total protein). After 30 min at 37 °C, the reaction mixture was
extracted with 2 volumes of chloroform/methanol/HCl (100:200:
1), and the components in the organic phase were separated by
normal-phase TLC using a 1-butanol/acetic acid/water (3:1:1)
solvent system. Radiolabeled enzyme products were detected by
autoradiography and identified by migration, relative to authentic
standards. For quantification, the silica gel containing radiolabeled
lipid was scraped into a scintillation vial and counted.
(13) Gon, Y.; Wood, M. R.; Kiosses, W. B.; Jo, E.; Sanna, M. G.; Chun,
J.; Rosen, H. S1P3 Receptor-Induced Reorganization of Epithelial Tight
Junctions Compromises Lung Barrier Integrity and Is Potentiated by
TNF. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 9270–9275.
(14) (a) Clemens, J. J.; Davis, M. D.; Lynch, K. R.; Macdonald, T. L.
Synthesis of para-Alkyl Aryl Amide Analogues of Sphingosine
1-Phosphate: Discovery of Potent S1P Receptor Agonists. Bioorg. Med.
Chem. Lett. 2003, 13, 3401–3404. (b) Clemens, J. J.; Davis, M. D.;
Lynch, K. R.; Macdonald, T. L. Synthesis of 4(5)-Phenylimidazole-
Based Analogues of Sphingosine 1-Phosphate and FTY720: Discovery
of Potent S1P1 Receptor Agonists. Bioorg. Med. Chem. Lett. 2005,
15, 3568–3572. (c) Foss, F. W., Jr.; Snyder, A. H.; Davis, M. D.;
Rouse, M.; Okusa, M. D.; Lynch, K. R.; Macdonald, T. L. Synthesis
and Biological Evaluation of γ-Aminophosphonates as Potent, Subtype-
Selective Sphingosine 1-Phosphate Receptor Agonists and Antagonists.
Bioorg. Med. Chem. 2007, 15, 663–677.
Lymphopenia Assay. Mice were dosed by oral gavage with
isomers of 3 (1 mg/kg), dissolved in 2% hydroxypropyl ꢀ-cyclo-
dextrin. Peripheral blood was collected from the orbital sinus of
lightly anesthetized mice, and total lymphocytes were counted using
a Hemavet 950 blood analyzer (Drew Scientific, Oxford, CT).
(15) Lynch, K. R.; Macdonald, T. L. Sphingosine 1-Phosphate Agonists
Comprising Cycloalkanes and 5-Membered Heterocycles Substituted by
Amino and Phenyl Groups. 2006-US5304 2006088944, 20060214, 2006.
(16) Organ, M. G.; Murray, A. P. Ni-Catalyzed Cross Coupling of
Alkoxide-Containing Vinyl Halides with Grignard Reagents. A “One-
Pot” Synthesis of 2-[(Trimethylsilyl)methyl]-2-propen-1-yl Acetate.
J. Org. Chem. 1997, 62, 1523–1526.
(17) (a) Evans, D. A.; Britton, T. C.; Dorow, R. L.; Dellaria, J. F., Jr.
Tetrahedron 1988, 44 (17), 5525–5540. (b) Evans, D. A.; Ennis, M. D.;
Mathre, D. J. J. Am. Chem. Soc. 1982, 104 (6), 1737–1739. (c) Evans,
D. A.; Ng, H. P.; Rieger, D. L. J. Am. Chem. Soc. 1993, 115 (24),
11446–11459.
Acknowledgment. This work was supported in part by a
research grant from the NIH (R01 GM067958). We also thank
the UIUC mass spec lab for HRMS (NSF DBI-0100085) and
the Atlantic Micro Lab for elemental analysis.
Supporting Information Available: Proton and carbon NMR
of new compounds. This material is available free of charge via
the Internet at http://pubs.acs.org.
References
(18) Frederik Menges, A. P. Threonine-Derived Phosphinite-Oxazoline
Ligands for the Ir-Catalyzed Enantioselective Hydrogenation. AdV.
Synth. Catal. 2002, 344, 40–44.
(1) Vezina, C.; Kudelski, A.; Sehgal, S. N. Rapamycin (AY-22,989), A
New Antifungal Antibiotic. I. Taxonomy of the Producing Strepto-
mycete and Isolation of the Active Principle. J. Antibiot. 1975, 28,
721–726.
(19) Espino, C. G.; Du Bois, J. A. Rh-Catalyzed C-H Insertion Reaction

Conformationally Constrained Fingolimod Analogues
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 25 6435
for the Oxidative Conversion of Carbamates to Oxazolidinones. Angew.
Chem., Int. Ed. 2001, 40 (3), 598–600.
(20) Hinman, A.; Du Bois, J. J. Am. Chem. Soc. 2003, 125 (38), 11510–
11511.
(21) Davis, M. D.; Clemens, J. J.; Macdonald, T. L.; Lynch, K. R.
Sphingosine 1-Phosphate Analogs as Receptor Antagonists. J. Biol.
Chem. 2005, 280, 9833–9841.
(22) Kharel, Y.; Lee, S.; Snyder, A. H.; Sheasley-O’Neill, S. L.; Morris,
M. A.; Setiady, Y.; Zhu, R.; Zigler, M. A.; Burcin, T. L.; Ley, K.;
Tung, K. S.; Engelhard, V. H.; Macdonald, T. L.; Pearson-White, S.;
Lynch, K. R. Sphingosine Kinase 2 Is Required for Modulation of
Lymphocyte Traffic by FTY720. J. Biol. Chem. 2005, 280, 36865–
36872.
(23) (a) 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. (b)
Hale, J. J.; Doherty, G.; Toth, L.; Li, Z.; Mills, S. G.; Hajdu, R.;
Keohane, C. A.; Rosenbach, M.; Milligan, J.; Shei, G. J.; Chrebet,
G.; Bergstrom, J.; Card, D.; Rosen, H.; Mandala, S. The Discovery
of 3-(N-Alkyl)aminopropylphosphonic Acids as Potent S1P Receptor
Agonists. Bioorg. Med. Chem. Lett. 2004, 14, 3495–3499. (c) Hale,
J. J.; Lynch, C. L.; Neway, W.; Mills, S. G.; Hajdu, R.; Keohane,
C. A.; Rosenbach, M. J.; Milligan, J. A.; Shei, G. J.; Parent, S. A.;
Chrebet, G.; Bergstrom, J.; Card, D.; Ferrer, M.; Hodder, P.; Strulovici,
B.; Rosen, H.; Mandala, S. A Rational Utilization of High-Throughput
Screening Affords Selective, Orally Bioavailable 1-Benzyl-3-car-
boxyazetidine Sphingosine 1-Phosphate-1 Receptor Agonists. J. Med.
Chem. 2004, 47, 6662–6665. (d) Hale, J. J.; Neway, W.; Mills, S. G.;
Hajdu, R.; Keohane, C. A.; Rosenbach, M.; Milligan, J.; Shei, G. J.;
Chrebet, G.; Bergstrom, J.; Card, D.; Koo, G. C.; Koprak, S. L.;
Jackson, J. J.; Rosen, H.; Mandala, S. Potent S1P Receptor Agonists
Replicate the Pharmacologic Actions of the Novel Immune Modulator
FTY720. Bioorg. Med. Chem. Lett. 2004, 14, 3351–3355. (e) Pan, S.;
Mi, Y.; Pally, C.; Beerli, C.; Chen, A.; Guerini, D.; Hinterding, K.;
Nuesslein-Hildesheim, B.; Tuntland, T.; Lefebvre, S.; Liu, Y.; Gao,
W.; Chu, A.; Brinkmann, V.; Bruns, C.; Streiff, M.; Cannet, C.; Cooke,
N.; Gray, N. A Monoselective Sphingosine 1-Phosphate Receptor-1
Agonist Prevents Allograft Rejection in a Stringent Rat Heart
Transplantation Model. Chem. Biol. 2006, 13, 1227–1234.
(24) 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.; HughRosen; Mandala, S. Synthesis, Stereochem-
ical Determination and Biochemical Characterization of the Enantio-
meric Phosphate Esters of the Novel Immunosuppressive Agent
FTY720. Bioorg. Med. Chem. 2004, 12, 4803–4807.
(25) Koide, Y.; Uemoto, K.; Hasegawa, T.; Sada, T.; Murakami, A.;
Takasugi, H.; Sakurai, A.; Mochizuki, N.; Takahashi, A.; Nishida, A.
Pharmacophore-Based Design of Sphingosine 1-Phosphate-3 Receptor
Antagonists That Include a 3,4-Dialkoxybenzophenone Scaffold.
J. Med. Chem. 2007, 50, 442–454.
JM7010172