Synthesis and evaluation of fluorinated fingolimod (FTY720) analogues for sphingosine-1-phosphate receptor molecular imaging by positron emission tomography

Article abstract of DOI:10.1021/jm502021d

Sphingosine-1-phosphate (S1P) is a lysophospholipid that evokes a variety of biological responses via stimulation of a set of cognate G-protein coupled receptors (GPCRs): S1P₁-S1P₅. S1P and its receptors (S1PRs) play important roles in the immune, cardiovascular, and central nervous systems and have also been implicated in carcinogenesis. Recently, the S1P analogue Fingolimod (FTY720) has been approved for the treatment of patients with relapsing multiple sclerosis. This work presents the synthesis of various fluorinated structural analogues of FTY720, their in vitro and in vivo biological testing, and their development and application as [¹⁸F]radiotracers for the study of S1PR biodistribution and imaging in mice using small-animal positron emission tomography (PET).

Full text of DOI:10.1021/jm502021d

Article
pubs.acs.org/jmc
Synthesis and Evaluation of Fluorinated Fingolimod (FTY720)
Analogues for Sphingosine-1-Phosphate Receptor Molecular
Imaging by Positron Emission Tomography
Rizwan S. Shaikh,†,‡,◆ Stefanie S. Schilson,†,◆ Stefan Wagner,§ Sven Hermann,∥ Petra Keul,#
Bodo Levkau,# Michael Schafers,§,∥,⊥ and Gunter Haufe*,†,∥,⊥
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†Organisch-Chemisches Institut and International NRW Graduate School of Chemistry, Westfalische Wilhelms-Universitat Munster,
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Corrensstraße 40, D-48149 Munster, Germany
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‡NRW Graduate School of Chemistry, Westfalische Wilhelms-Universitat Munster, Wilhelm-Klemm-Straße 10, D-48149 Munster,
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Germany
§Klinik fur Nuklearmedizin, Universitatsklinikum Munster, Albert-Schweitzer-Campus 1, Gebaude A1, D-48149 Munster, Germany
∥European Institute for Molecular Imaging, Cells-in-Motion Cluster of Excellence, Westfalische Wilhelms-Universitat Munster,
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⊥
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Waldeyerstraße 15, D-48149 Munster, Germany
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#Institute of Pathophysiology, Westdeutsches Herz- und Gefaßzentrum, University Hospital Essen, University of Duisburg-Essen,
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Hufelandstraße 55, D-45122 Essen, Germany
S
* Supporting Information
ABSTRACT: Sphingosine-1-phosphate (S1P) is a lysophospholipid that evokes a variety of biological responses via stimulation
of a set of cognate G-protein coupled receptors (GPCRs): S1P1−S1P5. S1P and its receptors (S1PRs) play important roles in the
immune, cardiovascular, and central nervous systems and have also been implicated in carcinogenesis. Recently, the S1P analogue
Fingolimod (FTY720) has been approved for the treatment of patients with relapsing multiple sclerosis. This work presents the
synthesis of various fluorinated structural analogues of FTY720, their in vitro and in vivo biological testing, and their development
and application as [18F]radiotracers for the study of S1PR biodistribution and imaging in mice using small-animal positron
emission tomography (PET).
linase-catalyzed degradation and converts into ceramide.
Deacylation of ceramide results in sphingosine, which is
phosphorylated by sphingosine kinases (SphK1 and SphK2)
to S1P.2 Both of these enzymes possess broad tissue
distribution, with SphK1 being widely expressed in lungs and
spleen, and SphK2, in heart, brain, and liver.3 Extracellular S1P
acts in autocrine and paracrine manners by engaging a set of
five high-affinity S1P receptors (S1P1−S1P5) that belong to the
family of G-protein coupled receptors (GPCRs).4 The S1P1,
S1P2, and S1P3 receptors are broadly expressed throughout the
body, whereas S1P4 is mostly expressed in immune cells, and
S1P5, in natural killer cells (NKs), lymphocytes, and
oligodendrocytes.5
1. INTRODUCTION
Sphingosine-1-phosphate (S1P) 1 (Figure 1) is a zwitterionic
lysophospholipid synthesized as a part of the sphingomyelin
cycle in yeasts, plants, insects, and animals. S1P plays a vital role
in cell growth, programmed cell death, angiogenesis, immunity,
and the cardiovascular (CVS) and central nervous systems
(CNS).1 In living cells, sphingomyelin undergoes sphingomye-
S1P receptors are therapeutic targets for the treatment of
certain autoimmune diseases such as multiple sclerosis (MS),
the most common inflammatory disorder of the central nervous
system.6,7 Fingolimod (2) is the representative of its class of
compounds approved for the treatment of relapsing multiple
Figure 1. Sphingosine-1-phosphate (S1P, 1) and phosphate of
Fingolimod (FTY720, 2).
Received: December 31, 2014
Published: March 31, 2015
© 2015 American Chemical Society
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Figure 2. Structures of BZM055 (3), W146 (4), and the radiofluorinated analogue of W146 [18F]5.
Scheme 1. Synthesis of 2, Its ω-Fluorinated Analogue 17, and Its Deoxyfluoro Analogue 18
a
a
Reagents: (i) bromoacetyl bromide, AlCl3, DCM, rt, overnight; (ii) diethylacetamidomalonate, Cs2CO3, MeCN, reflux, 4 h; (iii) PBSF, Et3N·3HF,
EtiPr2N, DCM, rt, 2 days; (iv) Et3SiH, TiCl4, DCM, rt, overnight for 10, or Et3SiH, cat. PdCl2, EtOH, rt, overnight for 12; (v) LiCl, NaBH4, THF/
EtOH, rt, 3 days for 13, or LiBH4 solution in THF, rt, overnight for 14; (vi) 1 M NaOH, MeOH, reflux, 5 h; (vii) DAST, DCM, −78 °C → rt,
overnight.
sclerosis by the U.S. Food and Drug Administration (FDA) and
the European Medicines Agency (EMA) in 2010 and 2011,
respectively. Compound 2 was discovered as a structural
analogue of myriocin and has led to great interest in S1P
signaling and physiology.8,9 After phosphorylation of 2 by
SphK2, the formed Fingolimod-phosphate engages four out of
the five S1P receptors (all except S1P2)13 and, most
importantly, acts as a functional antagonist to S1P1,10 thereby
sequestering lymphocytes in secondary lymphoid organs and
leading to a profound reduction of T- and B-cells in peripheral
blood.11,12 The efficacy of 2 in animal models and humans
results from its prevention of lymphocyte infiltration into sites
of autoimmunity, transplanted organs, and the central nervous
system.8,14 Studying organ distribution of 2 and pharmacoki-
netics using an imaging tracer based on 2 in vivo would thus be
an extremely valuable tool.
there.16,17 The presence of iodine resulted in a significantly
increased lipophilicity and decreased rate of phosphorylation as
compared to that of 2. In blood and brain, compound 3 showed
similar properties as those of 2. These favorable properties
encouraged moving forward with 3 as a SPECT tracer for S1PR
imaging in brain. Unfortunately, the much lower penetration
into brain made 3 a less suitable tracer for in vivo imaging
studies.18 In 2014, we reported [18F]5 as a radiolabeled
structural analogue of (R)-{3-amino-4-[(3-hexylphenyl)-
amino]-4-oxobutyl}phosphonic acid (W146, 4) for PET
imaging studies of S1PRs (Figure 2).19 W146 was originally
synthesized by Sanna20 as a chiral S1P1 receptor antagonist that
led to vascular leakage and pulmonary edema after application
in vivo.21 The nonradioactive isotopomer of 5 had equal in vitro
efficacy as that of parent compound 4. Although it was stable
for hours in serum ex vivo, PET images showed fast
defluorination and accumulation of free [18F]fluoride in
bone.19 The in vivo defluorination of [18F]5 prevented its
application as a tracer for PET imaging of S1PRs.
During the past 2 decades, noninvasive molecular imaging
techniques have been developed for application in biomedical
studies and particularly for applications in the early detection of
various diseases such as cancer, cardiovascular diseases, and
CNS related disorders.15 These techniques include positron
emission tomography (PET), single photon emission com-
puted tomography (SPECT), computed tomography (CT),
magnetic resonance imaging (MRI), and ultrasound (US). The
high sensitivity of SPECT and PET have attracted researchers
to develop molecular imaging agents.15
Here, we report the synthesis of different structural analogues
of 2 as [18F]-labeled potential radiotracers for molecular
imaging of S1PRs.
2. RESULTS AND DISCUSSION
Compound 2 possesses a simple structural scaffold consisting
of a 2-amino-1,3-diol polar headgroup, which is phosphorylated
in vivo by SphK, with the 1,4-disubstituted phenyl ring acting as
a rigid linker group, and an eight-carbon chain length aliphatic
tail for interaction with the hydrophobic binding pocket of
There have been no reports on S1PR imaging until the 123I-
radiolabeled SPECT tracer BZM055 (3), an agonist of S1PRs,
was described in 2011. This structural analogue of 2 was used
to image the myelin sheath, as 2 has been shown to accumulate
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a
Scheme 2. Synthesis of Structural analogues of FTY720 Phosphate
a
Reagents: (i) LiCl, NaBH4, THF/EtOH rt, overnight; (ii) 1 M NaOH, MeOH, reflux, 6 h.
a
Scheme 3. Synthesis of ω-Fluorinated Analogues of FTY720
a
Reagents: (i) (a) (Boc)2O, DMF, 25 °C, 2 h, (b) DMP, PTSA, DMF, 25 °C, 12 h; (ii) (COCl)2, DMSO, TEA, DCM, −78 °C → rt, 1 h; (iii) 26,
K2CO3, toluene, 120 °C, 12 h; (iv) hex-5-yn-1-ol or oct-7-yn-1-ol, Pd/C, TPP, CuI, TEA, MeCN, microwave, 80 °C, 90 min; (v) H2, Pd/C, EtOAc,
rt, 2 h; (vi) PBSF, Et3N·3HF, EtiPr2N, THF, rt, 2 days for 30 and DAST, DCM, −78 °C → rt, 8 h for 31; (vii) TFA, DCM, rt, 12 h; (viii) triethyl
orthoacetate, EtiPr2N, DMF, 120 °C, 2 h; (ix) (a) TBDPA, 0.45 M tetrazole, 30% H2O2, rt, 4 h, (b) HCl/ethanol, 1:10, 50 °C, 6 h.
S1PRs. After the synthetic discovery of 2 by Fujita22 and
development of its immunomodulatory activity,23 a number of
other compounds have been synthesized.24−30
borohydride and lithium chloride in THF/EtOH or with
LiBH4 in THF in the case of 14. Hydrolysis of the amide
moiety of compounds 15 and 16 was accomplished with
aqueous sodium hydroxide to give 2 and its fluorinated
structural analogue 17. Fluorination of diol 2 with DAST in
DCM gave the known 1833 in low yield.34
Scheme 2 presents the synthesis of the 4-hydroxy analogue
and the ω-fluorinated 4-hydroxy analogue of 2. Compounds 19
and 20 were synthesized by reduction of 10 and 12 using
sodium borohydride and lithium chloride in THF/EtOH.
Further amide hydrolysis of 19 and 20 with aqueous sodium
hydroxide gave 21 and 22.
Synthesis of ω-fluorinated exact and shorter-chain analogues
of FTY720 starting from commercial triethanolamine (23) is
shown in Scheme 3. One-pot protection of 23 using Boc-
anhydride and 2,2-dimethoxy propane in the presence of a
catalytic amount of p-toluenesulfonic acid gave 24, which was
further oxidized to aldehyde 25 using Swern oxidation.35 Wittig
reaction of 25 with intermediate 26 and potassium carbonate as
base in toluene gave a 3:1 mixture of the Z/E-isomeric iodides
27. Sonogashira coupling of 27 with hex-5-yne-1-ol and oct-7-
yne-1-ol under microwave irradiation led to 28 and 29,
respectively.36 Hydrogenation over palladium on carbon in
2.1. Chemistry. Syntheses of 2 and some structural
analogues including radiofluorinated analogues are depicted in
Schemes 1−4. The routes for the synthesis of FTY720 (2) and
the fluorinated derivative 18, shown in Scheme 1, are analogous
to those described by Durand et al.24 Intermediates 8 and 9
were obtained by Friedel−Crafts acylation with bromoacetyl
bromide of 6 and 7 followed by nucleophilic substitution of the
bromide with diethylacetamidomalonate in the presence of
cesium carbonate to afford 10 and 11. Alcohol 11, in turn, was
treated with perfluoro-1-butanesulfonyl fluoride (PBSF) in the
presence of triethylamine tris-hydrogenfluoride and N,N-
diisopropylethylamine (DIPEA) in DCM to yield the desired
fluoride 12.31 Compound 10 was converted to 13 using
triethylsilane (Et3SiH) and titanium tetrachloride in DCM.24
This protocol was not successful for 12. In the presence of
TiCl4, the terminal fluoride was replaced by chloride.
Therefore, 12 was converted to 14 using Et3SiH and
palladium(II)-chloride in DCM;32 however, this proceeded
with low yield. Compounds 15 and 16 were synthesized by
reduction of the corresponding diester 13 with sodium
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a
Scheme 4. Synthetic Scheme for Radiofluorination
a
Reagents: (i) TsCl, TEA, DCM, 0 °C → rt, overnight; (ii) (a) [18F]F−, K222, K2CO3, CH3CN, 84 °C, 10−12 min; (b) 10:1, TFA/H2O, 40 °C, 5
min.
a
Table 1. Lymphopenia Induced in Vivo by Different Compounds as a Readout for S1P1 Activity
WBC
(106/mL)
CD4+
(106/mL)
CD8+
(106/mL)
0.48 0.05
B cells
(106/mL)
activity
(%)
compound
control
8.94 0.59
3.69 0.63
3.04 0.42
4.91 0.53
3.93 0.39
0.73 0.07
4.72 0.53
0.61 0.12
1.03 0.24
2.23 0.30
1.05 0.19
b
b
b
b
b
b
b
b
b
b
2 (FTY720)
0.02 0.002
0.04 0.01
97
95
59
89
b
b
17
34
36
0.04 0.01
0.03 0.004
b
b
0.30 0.06
0.25 0.07
b
b
0.08 0.01
0.14 0.03
a
Data are presented as mean SEM from at least three independent experiments per compound. Control, untreated mice. Activity was calculated as
b
percent reduction of peripheral blood CD4+ cell number by each compound. p < 0.05 compared to control.
ethyl acetate at room temperature provided intermediates 30
and 31. The OH functions were converted to fluoride either by
reaction with PBSF and Et3N·3HF31 or with DAST in DCM34
to achieve 32 and 33. Then, acid-catalyzed deprotection of Boc
and acetonide led to the ω-fluorinated structural analogues of
FTY720, 34 and 17. Aminodiol 17 was reacted with triethyl
orthoacetate and DIPEA in DMF to afford intermediate 35.37
Finally, 35 was reacted with di-tert-butyl N,N-diethyl-
phosphoramidite (TBDPA) in the presence of tetrazole and
hydrogen peroxide in DCM, and the formed phosphate was
then hydrolyzed with concentrated hydrochloric acid in ethanol
to give the final compound 36.18,37
region at different wavelengths, with the result that
concentrations below 20 μg/mL cannot be detected with the
UV detector. These missing UV signals were observed for the
final solutions of the preparations of [18F]34 and [18F]17,
respectively. Therefore, the AS of the [18F]34 and [18F]17
preparations had to be estimated. Under the assumption that
the concentration of carrier and radiolabeled compound in the
final solution of the radiosyntheses is 20 μg/mL (detection
limit) or lower, the AS of the preparations of [18F]34 and
[18F]17 is calculated to be ≥0.9 Gbq/μmol. Actually, for similar
[18F]-radiolabeling procedures with comparable starting
activities, we achieved AS in the range of 10−127 GBq/μmol,
which is also a realistic range for the syntheses of [18F]34 and
[18F]17.20,38
2.3. Biological Studies. 2.3.1. Biological Activity in Vivo
and in Vitro. The synthesized compounds were tested for
biological activity toward S1P1 in vivo by measuring their ability
to induce peripheral blood lymphopenia as a characteristic of
successful downregulation of the S1P1 receptor (Table 1).
Compounds 17 and 36 showed excellent in vivo activity,
comparable to that of FTY720 (2), as evidenced by the ∼20-
fold reduction of T-cell counts (Table 1). In contrast,
compound 34 was only moderately effective (∼2-fold reduction
in lymphocytes). Compounds 18, 21, and 22 were not tested
because of insolubility in the desired formulation. The similar
effectiveness of 17 and its phosphorylated form 36 is not
surprising given the high efficiency of endogenous phosphor-
ylation known for FTY720 that, apparently, also applies to its
ω-fluorinated analogue 17. Interestingly, compound 34, which
features two fewer carbons in its aliphatic chain than that of 17,
has substantially impaired activity in vivo.
2.2. Radiochemistry. For the syntheses of [18F]-radio-
labeled structural analogues of FTY720, precursors 37 and 38
were synthesized from 30 and 31 by tosylation with tosyl
chloride and triethylamine as base in DCM (Scheme 4).
Radiosyntheses of [18F]34 and [18F]17 were performed over
two steps. In the first step, [18F]32 and [18F]33 were prepared
by heating precursors 37 and 38 in acetonitrile with anhydrous
potassium [18F]fluoride in the presence of Kryptofix 222 (K222
)
at 84 °C for 10 min. Without isolation and purification, the
obtained intermediates were directly hydrolyzed with TFA/
H2O (10:1) at 40 °C for 5 min to yield [18F]34 and [18F]17,
respectively. These products were obtained in radiochemical
yields (rcy) of 29.2
3.3% ([18F]34) and 30.2
4.2%
([18F]17) (decay-corrected based on cyclotron-derived [18F]-
fluoride ions) in 115 12 min ([18F]34) and 115 16 min
([18F]17) from the end of radionuclide production. The target
compounds were isolated in ≥99% radiochemical purity after
HPLC.
A standard procedure to determine specific activities (AS) of
radiolabeled compounds is to generate an UV calibration curve
of the nonradioactive isotopomere with analytical radio-HPLC,
e.g., with concentrations in a range of 1−50 μg/mL. Then, from
the UV signal of the radiolabeled compound and carrier (the
nonradioactive counterpart) in the HPLC quality control (QC)
of the radiochemical preparations, the concentration can be
calculated. Finally, from the concentration, the AS can be
determined. In this case, the nonradioactive counterparts 34
and 17 showed high detection limits (20 μg/mL) in the UV
Subsequently, both the biological activity and the S1P
receptor subtype specificity of compound 36 were tested in
vitro using Chinese hamster ovary (CHO) cells stably
overexpressing the human S1P1 and S1P3 receptors, respec-
tively. The functional readout was phosphorylation of the p44/
42 mitogen activated kinases (MAPK) after stimulation with
compound 36 in direct comparison with that after stimulation
by S1P (1). In both CHO-S1P1 and CHO-S1P3, compound 36
showed a robust time-dependent phosphorylation of MAPK,
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Figure 3. Western blots showing phosphorylation of p44/42 MAP kinases after stimulation of CHO cells overexpressing S1P1 or S1P3 with 1 μM 2-
amino-4-[4-(8-fluorooctyl)phenyl]-2-(hydroxymethyl)butyldihydrogen phosphate (36) and 1 μM S1P.
comparable to that of S1P, indicating its ability to interact with
and activate the S1P1 and S1P3 receptors (Figure 3).
2.3.2. In Vitro Stability. The serum stability of radioligand
[18F]17 was evaluated (Figure 4) by incubation in human
serum at 37 °C for 90 min. Significant radiometabolites or
decomposition products could not be observed.
Figure 4. In vitro stability of [18F]17 (tR = 11.15 min) after incubation
in human blood serum at 37 °C for 10 min (top), 60 min (middle),
and 90 min (bottom) measured using analytical HPLC system B,
method B.
2.3.3. In Vivo Biodistribution/PET Imaging. Motivated by
the in vivo immunosuppressive activity of compounds 17 and
34, 18F-labeled structural analogues (Scheme 4) [18F]34 and
[18F]17 were selected for biodistribution experiments in wild-
Figure 5. In vivo PET/CT studies in healthy C57BL/6 mice of (a)
[18F]34 and (b) [18F]17 from 0 to 90 min postinjection. PET (color
scale) revealed a fast uptake and elimination of the tracer via the liver
(L) and the kidneys (K). In addition, tracer uptake was observed in the
myocardium (M) and the spleen (not displayed on selected slides).
type mice. Whole-body imaging was performed in C57BL/6
wild-type mice (n = 4, male, 13 weeks old) over a 90 min
period after intravenous injection of [18F]34 and [18F]17 (400
KBq/g bodyweight in 100 μL of saline). The in vivo imaging
studies showed similar tracer kinetics and biodistribution for
both [18F]34 (Figure 5a) and [18F]17 (Figure 5b). On the
basis of quantitative PET time−activity curves, a rapid clearance
from blood within 3 min postinjection (p.i.) followed by a
nearly constant blood activity concentration of about ∼4% ID/
mL until the end of the study at 90 min p.i. was confirmed
([18F]34, Figure 6a,b; [18F]17, Figure 6c,d). The decrease in
blood concentration coincides with an intense uptake of both
tracers into the liver, with a maximum of 25% ID/mL at about
10 min p.i. and a less pronounced increase of tracer signal in the
kidneys up to 20% ID/mL, reflecting the routes of tracer
elimination. Furthermore, both [18F]34 and [18F]17 were
found to accumulate in lungs and the myocardium. After the
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Figure 6. (a) In vivo biodistribution of radioactivity in C57BL/6 WT mice after intravenous injection of [18F]34 (a, b) and [18F]17 (c, d). Time−
activity curves illustrate tracer dynamics in selected regions of interests (ROI) for tissues involved in tracer elimination (a, c) and S1P relevant
structures or reference tissues (b, d).
initial peak in the lungs early after injection, [18F]34’s activity
3. CONCLUSIONS
was decreased to 5% ID/mL at 20 min p.i. with a slight washout
Six fluorinated structural analogues of FTY720 (2) were
synthesized and tested for in vitro and in vivo activity. The goal
to 4% ID/mL until the end of the study. [18F]17’s kinetics in
the lungs showed similar characteristics as those of [18F]34,
with activity concentrations of 6.4% ID/mL at 20 min p.i. and
was to elaborate their application for PET imaging studies in
vivo after introducing 18F. The ω-fluorinated analogue 17 and
4.8% ID/mL at the end of the study. The myocardium
presented a biphasic time−activity curve, with an initial
perfusion peak and a smaller second maximum at 10 min p.i.
and activities of 8% ID/mL for [18F]34 and 8.6% ID/mL for
[18F]17. To evaluate organs typically hosting S1P receptor
positive immune cells, we analyzed the activity concentration in
the spleen and inguinal lymph nodes. In the spleen, an
accumulation of [18F]34 and [18F]17 was observed with a
maximum of 7% ID/mL at 10 min p.i. and 7.8% ID/mL at 30
min p.i., respectively.
In vivo defluorination of the radioligands (bone uptake of
[18F]fluoride ions, as observed in our earlier study with an 18F-
analogue of W146, a S1P1 selective antagonist19) was not
observed in these imaging studies.
In summary, biodistribution studies based on PET revealed
rapid clearance of radiotracers [18F]34 and [18F]17 from the
blood in wild-type mice within the first 10 min after injection.
Besides the predominantly hepatic elimination, an increased
uptake/retention of both [18F]34 and [18F]17 was observed in
S1P receptor positive tissues such as the lungs and the
myocardium as well as in the lymphocyte-containing spleen in
healthy mice.
its phosphorylated form 36 showed excellent biological activity
in vivo, suggesting that incorporation of a fluorine atom at the
end of the aliphatic chain, which is expected to increase
lipophilicity within the hydrophobic receptor pocket, preserves
interaction with the S1P receptor. Interestingly, shortening of
the chain length from eight to six carbons, as in 34, impaired
the activity. Moreover, we have synthesized two [18F]-
radiolabeled structural analogues of FTY720, [18F]34 and
[18F]17, in good radiochemical yields of 29 to 30% in 115 min
and >99% radiochemical purity. The in vivo biodistribution
studies of [18F]34 and [18F]17 using PET and CT showed
rapid clearance of the activity from the blood in C57BL/6 wild-
type mice within the first 10 min after injection mainly via the
liver. We observed a slightly increased uptake in S1P receptor
relevant tissues, namely, the lungs (mainly S1P1) and
myocardium (mainly S1P3), as well as in the lymphocyte-
containing spleen (mainly S1P4) in the healthy mouse. No
accumulation in bones and other organs was observed.
Although first imaging results showed positive uptake in
S1PR-rich tissues, the uptake, specificity (predosing, blocking
studies, S1PR knockout and overexpression models, etc.), and
metabolism of [18F]34 and [18F]17 have to be tested and
further optimized. This should finally result in a molecular
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2), 31.2, 31.1 (C-10, C-15), 29.5 (C-11), 29.4 (C-12), 29.2 (C-13),
25.8 (C-14). HRMS (ESI+): C16H23BrO2 + Na+: calcd, 349.0774;
found, 349.0775; (C16H23BrO2)2 + Na+: calcd, 677.1637; found,
677.1624.
Diethyl 2-Acetamido-2-[2-(4-octylphenyl)-2-oxoethyl]malonate
(10). Intermediate 8 (8.90 g, 19.7 mmol, 1.3 equiv), diethyl 2-
acetamidomalonate (3.21 g, 14.8 mmol), and cesium carbonate (5.15
g, 15.8 mmol, 1.1 equiv) were suspended in acetonitrile (125 mL) and
stirred under reflux for 4 h. After cooling to rt, the reaction mixture
was adsorbed on silica gel and purified by column chromatography
(10.5 × 8 cm, cyclohexane/ethyl acetate, 4:1 → 2:1) to give the
product as a pale yellow oil. Yield: 5.87 g (89%). 1H NMR (400 MHz,
imaging tracer with potential applications in cardiovascular,
neuroinflammatory, and immune system diseases.
4. EXPERIMENTAL SECTION
4.1. Materials and Methods. All substrates, reagents, and
solvents for the reactions were of analytical grade and were used
without further purification. Concerning reactions under dry
conditions, the glassware was heated under vacuum and flushed with
argon before use. The reactions were performed under an argon
atmosphere. Reactions using microwave irradiation were performed in
closed glass vessels using a CEM Discover microwave. To characterize
the synthesized compounds, the melting point (if the products were
solid at room temperature), NMR, and ESI-MS data were determined.
1H NMR (300, 400, and 600 MHz), 13C NMR (75, 100, and 150
MHz), and 19F NMR (282 and 564 MHz) spectra were recorded on
Bruker spectrometers in CDCl3 or MeOD for 13C NMR with TMS for
1H NMR, CDCl3 or MeOD, and CFCl3 for 19F NMR as the internal
standards. DEPT and two-dimensional NMR techniques (COSY,
HSQC, and HMBC) were recorded to assign the signals of some
complex structures. All chemical shifts are indicated in the δ scale in
parts per million. The numeration of the assigned nuclei is depicted in
the Supporting Information. Exact mass analyses were recorded with a
Bruker MicroTOF apparatus. All spectroscopic and analytical
investigations were performed by staff members of the Organic
CDCl3): δ 7.88 (m, 2H, 7-CH, 11-CH or 8-CH, 10-CH), 7.26 (m, 2H,
3
7-CH, 11-CH or 8-CH, 10-CH), 7.15 (s, 1H, 2-NH), 4.27 (q, JH,H
=
7.1 Hz, 4H, 22-CH2, 24-CH2), 4.25 (s, 2H, 4-CH2), 2.66 (m, 2H, 12-
CH2), 1.97 (s, 3H, 21-CH3), 1.61 (m, 2H, 13-CH2), 1.26−1.33 (m, 1
H, 14-CH2 to 18-CH2), 1.24 (t, 3JH,H = 7.1 Hz, 6H, 23-CH3, 25-CH3),
0.87 (t, 3JH,H = 6.7 Hz, 3H, 19-CH3). 13C NMR (101 MHz, CDCl3): δ
196.5 (C-5), 169.4 (C-20), 167.4 (C-1, C-3), 149.6 (C-6), 133.8 (C-
9), 128.7 (C-7, C-11), 128.4 (C-8, C-10), 64.0 (C-2), 62.8 (C-22, C-
24), 42.2 (C-4), 36.0 (C-12), 31.9 (C-17), 31.1 (C-14, C-15), 29.4 (C-
13), 29.2 (C-16), 22.9 (C-21), 22.7 (C-18), 14.1 (C-19), 13.9 (C-23,
C-25). HRMS (ESI+): C25H37NO6 + H+: calcd, 448.2694; found,
448.2689; C25H37NO6 + Na+: calcd, 470.2513; found, 470.2505;
(C25H37NO6)2 + Na+: calcd, 917.5134; found, 917.5132.
Diethyl 2-Acetamido-2-{2-[4-(8-hydroxyoctyl)phenyl]-2-
Chemistry Institute, University of Munster. Thin-layer chromatog-
̈
oxoethyl}malonate (11). Synthesis and purification were performed
raphy (TLC) analyses were performed on silica-coated aluminum foil
(silica gel 60 F254) with a 0.02 mm layer thickness. Silica gel (60−120
mesh) was used for column chromatography. The purity of all
compounds tested in vitro or in vivo was proved by HPLC to be >95%.
A Knauer HPLC system (RP-HPLC Nucleodur 100−10 C18ec column,
250 × 16 mm) was used for the purification of some intermediates.
4.2. Synthesis. 4-Octyl-α-bromoacetophenone (8). Aluminum
chloride (4.80 g, 32.0 mmol, 1.0 equiv) and 1-phenyloctane (6.84 g,
32.0 mmol) were dissolved in dry DCM (10 mL) and cooled to −10
°C. A solution of bromoacetyl bromide (3.14 mL, 32.0 mmol, 1.0
equiv) in DCM (12 mL) was added dropwise, and the mixture was
allowed to return to rt while stirring overnight. The mixture was slowly
poured into ice water (100 mL), and the aqueous phase was extracted
with DCM (3 × 50 mL). The combined organic layers were dried over
MgSO4 and concentrated under reduced pressure. The product was
obtained as a brown, highly viscous oil and was used in the next
reaction without purification. Yield: 8.90 g (62%). 1H NMR (300
MHz, CDCl3): δ 7.90 (m, 2 H, 5-CH, 7-CH), 7.29 (m, 2 H, 4-CH, 8-
CH), 4.44 (s, 2 H, 2-CH2), 2.67 (m, 2 H, 9-CH2), 1.63 (m, 2 H, 10-
CH2), 1.22−1.35 (m, 10 H, 11-CH2 to 15-CH2), 0.88 (m, 3 H, 16-
CH3). 13C NMR (75 MHz, CDCl3): δ 191.1 (C-1), 150.1 (C-3), 131.7
(C-6), 129.2 (C-4, C-8), 129.0 (C-5, C-7), 36.2 (C-9), 32.0 (C-2),
31.2 (C-10), 31.1 (C-14), 29.5 (C-12), 29.4 (C-13), 29.3 (C-11), 22.8
(C-15), 14.2 (C-16).
1
as described for compound 10. Yield: 221 mg (48%). H NMR (300
MHz, CDCl3): δ 7.88 (d, 3JH,H = 8.3 Hz, 2H, 7-CH, 11-CH or 8-CH,
10-CH), 7.26 (d, 3JH,H = 8.5 Hz, 2H, 7-CH, 11-CH or 8-CH, 10-CH),
7.15 (s, 1H, 2-NH), 4.22−4.32 (m, 6H, 4-CH2, 22-CH2, 24-CH2),
3
3
3.63 (t, JH,H = 6.6 Hz, 2H, 19-CH2), 2.65 (t, JH,H = 7.7 Hz, 2H, 12-
CH2), 1.97 (s, 3H, 21-CH3), 1.51−1.67 (m, 4H, CH2, 13-CH2, 18-
CH2), 1.28−1.36 (m, 8H, 14-CH2 to 17-CH2), 1.24 (t, 3JH,H = 7.1 Hz,
6H, 23-CH3, 25-CH3). 13C NMR (75 MHz, CDCl3): δ 196.6 (C-5),
169.6 (C-20), 167.4 (C-1, C-3), 149.7 (C-6), 133.9 (C-9), 128.8 (C-7,
C-11), 128.5 (C-8, C-10), 64.1 (C-2), 63.1, 63.0 (C-19, C-22, C-24),
42.3 (C-4), 36.1 (C-12), 32.8 (C-18), 31.1 (C-15), 29.5 (C-14), 29.4
(C-13), 29.2 (C-16), 25.8 (C-17), 23.1 (C-21), 14.0 (C-23, C-25).
HRMS (ESI+): C25H37NO7 + H+: calcd, 464.2643; found, 464.2639;
C25H37NO7 + Na+: calcd, 486.2462; found, 486.2447.
Diethyl 2-Acetamido-2-{2-[4-(8-fluorooctyl)phenyl]-2-oxoethyl}-
malonate (12). Intermediate 11 (166 mg, 0.35 mmol) was dissolved
in dry THF (3 mL) in a PTFE vessel. Perfluoro-1-butanesulfonyl
fluoride (PBSF, 0.19 mL, 1.06 mmol, 3.0 equiv), triethyl amine
trishydrofluoride (0.18 mL, 1.10 mmol, 3.1 equiv), and N,N-
diisopropyl ethyl amine (0.56 mL, 3.24 mmol, 9.0 equiv) were
added, and the mixture was stirred at rt for 2 days. The reaction was
quenched by addition of saturated sodium bicarbonate solution (5
mL), and the aqueous phase was extracted with DCM (3 × 15 mL).
The combined organic layers were dried over MgSO4 and
concentrated under reduced pressure. The residue was purified by
column chromatography (16.5 × 3 cm, cyclohexane/ethyl acetate, 2:1)
8-Hydroxyoctyl-α-bromoacetophenone (9). Aluminum chloride
(200 mg, 1.50 mmol, 3.0 equiv) was suspended in dry DCM (8 mL)
and cooled to 0 °C. 8-Phenyl-1-octanol (0.11 mL, 0.50 mmol) and α-
bromoacetyl bromide (53 μL, 0.60 mmol, 1.2 equiv) were added
dropwise. After 20 min, the mixture was warmed to rt and stirred
overnight. The reaction was stopped by pouring the solution into a
mixture of ice water (25 mL) and concentrated hydrochloric acid (10
mL). The phases were separated, and the aqueous layer was extracted
with DCM (2 × 10 mL). The combined organic phases were washed
with saturated sodium bicarbonate solution (1 × 10 mL) and brine (1
× 10 mL) and dried over MgSO4. The solvent was evaporated, and the
residue was purified by column chromatography (12 × 4 cm,
cyclohexane/ethyl acetate, 8:1) to give the product as a colorless oil.
1
to give the product as a colorless oil. Yield: 132 mg (80%). H NMR
(300 MHz, CDCl3): δ 7.88 (d, 3JH,H = 8.3 Hz, 2H, 7-CH, 11-CH or 8-
CH, 10-CH), 7.27 (d, 3JH,H = 8.2 Hz, 2H, 7-CH, 11-CH or 8-CH, 10-
CH), 7.13 (br s, 1H, 2-NH), 4.43 (dt, 3JH,H = 6.1 Hz, 2JH,F = 47.4 Hz,
3
2H, 19-CH2), 4.27 (q, JH,H = 7.2 Hz, 4H, 22-CH2, 24-CH2), 4.25 (s,
3
2H, 4-CH2), 2.66 (t, JH,H = 7.7 Hz, 2H, 12-CH2), 1.98 (s, 3H, 21-
CH3), 1.59−1.79 (m, 4H, 13-CH2, 18-CH2), 1.30−1.41 (m, 8H, 14-
3
CH2 to 17-CH2), 1.25 (t, JH,H = 7.1 Hz, 6H, 23-CH3, 25-CH3). 13C
NMR (75 MHz, CDCl3): δ 196.6 (C-5), 169.6 (C-20), 167.5 (C-1, C-
3), 149.6 (C-6), 133.9 (C-9), 128.9 (C-7, C-11), 128.5 (C-8, C-10),
1
Yield: 120 mg (73%). 1H NMR (300 MHz, CDCl3): δ 7.90 (d, 3JH,H
=
84.3 (d, JC,F = 164.1 Hz, C-19), 64.1 (C-2), 63.0 (C-22, C-24), 42.3
(C-4), 36.1 (C-12), 31.2 (C-13), 30.5 (d, 2JC,F = 19.4 Hz, C-18), 29.4
(C-14, C-16), 29.2 (C-15), 25.3 (d, 3JC,F = 5.5 Hz, C-17), 23.1 (C-21),
8.3 Hz, 2H, 4-CH/8-CH or 5-CH/7-CH), 7.29 (d, 3JH,H = 8.4 Hz, 2H,
4-CH/8-CH or 5-CH/7-CH), 4.45 (s, 2H, 2-CH2), 3.63 (t, 3JH,H = 6.6
Hz, 2H, 16-CH2), 2.67 (t, 3JH,H = 7.7 Hz, 2H, 9-CH2), 1.50−1.66 (m,
4H, 10-CH2, 15-CH2), 1.29−1.39 (m, 8H, 11-CH2 to 14-CH2). 13C
NMR (75 MHz, CDCl3): δ 191.1 (C-1), 150.0 (C-3), 131.7 (C-6),
129.2 (C-4, C-8), 129.0 (C-5, C-7), 63.0 (C-16), 36.1 (C-9), 32.8 (C-
14.0 (C-23, C-25). 19F NMR (282 MHz, CDCl3): δ −218.6 (tt, 3JH,F
=
2
25.0 Hz, JH,F = 47.4 Hz, 1F). HRMS (ESI+): C25H36FNO6 + H+:
calcd, 466.2599; found, 466.2605; C25H36FNO6 + Na+: calcd,
488.2419; found, 488.2422.
3477
DOI: 10.1021/jm502021d
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Journal of Medicinal Chemistry
Article
Diethyl 2-Acetamido-2-(4-octylphenethyl)malonate (13). To a
solution of triethyl silane (3.0 mL, 27.1 mmol, 5.4 equiv) in dry DCM
(25 mL) was added a solution of intermediate 10 (2.24 g, 5.0 mmol)
in dry DCM (6 mL) dropwise, followed by the addition of titanium
tetrachloride (2.0 mL, 19.1 mmol, 2.6 equiv). The reaction mixture
was stirred at rt overnight and was then slowly poured into ice water
(150 mL). After phase separation, the aqueous layer was extracted with
DCM (2 × 70 mL). The organic phases were collected, dried over
MgSO4, and concentrated in vacuo. The residue was purified by
column chromatography (14 × 4 cm, cyclohexane/ethyl acetate, 4:1)
to give the product as a white solid. Yield: 1.87 g, (86%). mp 55−57
(q, C-19), 65.5 (C-1, C-3), 61.4 (C-2), 35.5 (C-12), 34.3 (C-17), 31.9
(C-5), 31.6 (C-4), 29.5 (C-15), 29.4 (C-14), 29.3 (C-13), 29.2 (C-
16), 23.9 (C-21), 22.7 (C-18). HMRS (ESI+): C21H35NO3 + H+:
calcd, 350.2690; found, 350.2693; C21H35NO3 + Na+: calcd, 372.2509;
found, 372.2512; (C21H35NO3)2 + Na+: calcd, 721.5126; found,
721.5127.
N-{4-[4-(8-Fluorooctyl)phenyl]-1-hydroxy-2-(hydroxymethyl)-
butan-2-yl}acetamide (16). Diester 14 (48 mg, 106 μmol) was
dissolved in THF (3 mL) and cooled to 0 °C. Lithium borohydride (4
M solution in THF, 0.11 mL, 0.44 mmol, 4.2 equiv) was added to the
reaction mixture followed by ethanol (6 mL). The reaction mixture
was warmed to rt after 30 min and stirred overnight. The reaction was
diluted with 20% potassium sodium tartrate solution (4 mL), and the
aqueous phase was extracted with DCM (4 × 6 mL). The combined
organic layers were dried over Na2SO4, and the solvent was removed
under reduced pressure. Column chromatography (8 × 3 cm, DCM/
methanol, 20:1) gave the desired product as an oil. Yield: 13 mg
1
°C. H NMR (300 MHz, CDCl3): δ 7.06 (m, 4H, 7-CH, 8-CH, 10-
CH, 11-CH), 6.79 (s, 1H, 2-NH), 4.19 (m, 4H, 22-CH2, 24-CH2),
2.68 (m, 2H, 5-CH2), 2.55 (m, 2H, 12-CH2), 2.45 (m, 2H, 4-CH2),
1.97 (s, 3H, 21-CH3), 1.56 (m, 2H, 13-CH2), 1.23−1.33 (m, 10H, 14-
3
CH2 to 18-CH2), 1.24 (t, JH,H = 7.1 Hz, 6H, 23-CH3, 25-CH3), 0.86
(m, 3H, 19-CH3). 13C NMR (75 MHz, CDCl3): δ 169.0 (C-20), 168.1
(C-1, C-3), 140.7 (C-6), 137.7 (C-9), 128.4 (C-7, C-11), 128,3 (C-8,
C-10), 66.4 (C-2), 62.5 (C-22, C-24), 35.5 (C-12), 33.4 (C-17), 31.9
(C-5), 31.6 (C-4), 29.8 (C-15), 29.7 (C-14), 29.5 (C-13), 29.3(C-16),
23.0 (C-21), 22.7 (C-18), 14.1 (C-19), 14.0 (C-23, C-25). HRMS
(ESI+): C25H39NO5 + H+: calcd, 434.2901; found, 434.2904;
C25H39NO5 + Na+: calcd, 456.2720; found, 456.2725; (C25H39NO5)2
+ Na+: calcd, 889.5549; found, 889.5541.
(33%). 1H NMR (400 MHz, CD3OD, CDCl3): δ 7.02−7.18 (m, 4 H,
3
7-CH, 8-CH, 10-CH, 11-CH), 5.35 (s, 1H, 2-NH), 4.44 (dt, JH,H
=
6.2 Hz, 2JH,F = 47.4 Hz, 2H, 19-CH2), 3.76 (d, 2JH,H = 11.5 Hz, 2H, 1-
2
CH, 3-CH), 3.64 (d, JH,H = 11.5 Hz, 2H, 1-CH, 3-CH), 2.51−2.68
(m, 6H, 4-CH2, 5-CH2, 12-CH2), 1.99 (s, 3H, 21-CH3), 1.54−1.76 (m,
4H, 13-CH2, 18-CH2), 1.26−1.44 (m, 8H, 14-CH2 to 17-CH2). 13C
NMR (101 MHz, CD3OD, CDCl3): δ 172.4 (C-20), 140.2 (C-6),
1
138.9 (C-9), 128.0 (C-7, C-11), 128.2 (C-8, C-10), 84.1 (d, JC,F
=
Diethyl 2-Acetamido-2-[4-(8-fluorooctyl)phenethyl]malonate
(14). A solution of ketone 12 (63 mg, 136 μmol) in ethanol (4
mL) was treated with triethyl silane (89 μL, 544 μmol, 4.0 equiv) and
a catalytic amount of palladium chloride under an argon atmosphere.
The reaction mixture was stirred at rt overnight and was subsequently
heated at 98 °C for 3 h until completion of the reaction. After cooling
to rt, water (3 mL) was added, and the mixture was extracted with
DCM (5 × 6 mL). The organic layers were dried over Na2SO4 and
concentrated in vacuo. The residue was purified by column
chromatography (16 × 3 cm, cyclohexane/ethyl acetate, 2:1) to give
163.5 Hz, C-19), 64.4 (C-1, C-3), 61.2 (C-2), 35.3 (C-12), 33.7 (C-4),
2
31.3 (C-5), 30.2 (d, JC,F = 19.3 Hz, C-18), 29.2 (C-14, C-15), 29.0
(C-13, C-16), 24.9 (d, 3JC,F = 5.4 Hz, C-17), 23.0 (q, C-21). 19F NMR
(282 MHz, CDCl3): δ −218.4 (tt, 3JH,F = 25.0 Hz, 2JH,F = 47.4 Hz, 1F,
19-CH2F). HRMS (ESI+): C21H34FNO3 + Na+: calcd, 390.2415;
found, 390.2415.
2-Amino-2-(4-octylphenethyl)propane-1,3-diol (FTY720) (2). Pro-
tected aminodiol 15 (349 mg, 1.0 mmol) was dissolved in methanol
(20 mL) and treated with 1 M sodium hydroxide solution (1.2 mL, 1.2
mmol, 1.2 equiv). The reaction mixture was refluxed for 5 h. After
cooling to rt, the mixture was diluted with 1 M sodium hydroxide
solution (15 mL), and the aqueous phase was extracted with DCM (5
× 20 mL). The combined organic layers were dried over Na2SO4 and
concentrated in vacuo. The product was crystallized from ethyl acetate
to give a white solid. Yield: 252 mg (82%). mp 127 °C. 1H NMR (300
MHz, CD3OD, CDCl3): δ 7.09 (m, 4H, 7-CH, 8-CH, 10-CH, 11-CH),
3.54 (d, 2JH,H = 11.0 Hz, 2H, 1-CH2, 3-CH2), 3.48 (d, 2JH,H = 10.9 Hz,
2H, 1-CH2, 3-CH2), 2.51−2.65 (m, 4H, 5-CH2, 12-CH2), 1.69 (m, 2H,
4-CH2), 1.57 (m, 2H, 13-CH2), 1.22−1.37 (m, 10H, 14-CH2 to 18-
CH2), 0.87 (m, 3H, 19-CH3). 13C NMR (75 MHz, CD3OD, CDCl3):
δ 140.9 (C-6), 140.1 (C-9), 129.0 (C-7, C-11), 128.7 (C-8, C-10),
66.2 (C-1, C-3), 56.4 (C-2), 37.0 (C-12), 36.1 (C-17), 32.5 (C-4),
32.2 (C-5), 30.1 (C-13), 29.9 (C-15), 29.5 (C-14, C-16), 23.2 (C-18),
14.3 (C-19). HMRS (ESI+): C19H33NO2 + H+: calcd, 308.2584; found,
308.2585; C19H33NO2 + Na+: calcd, 330.2404; found, 330.2408;
(C19H33NO2)2 + Na+: calcd, 637.4915; found, 637.4917.
1
the product as a colorless oil. Yield: 35 mg (57%). H NMR (300
MHz, CDCl3): δ 7.04−7.09 (m, 4H, 7-CH, 8-CH, 10-CH, 11-CH),
3
2
6.76 (s, 1H, 2-NH), 4.43 (dt, JH,H = 6.2 Hz, JH,F = 47.4 Hz, 2H, 19-
CH2), 4.20 (m, 4H, 22-CH2, 24-CH2), 2.55 (m, 2H, 12-CH2), 2.41−
2.72 (m, 4H, 4-CH2, 5-CH2), 1.51−1.75 (m, 4H, 13-CH2, 18-CH2),
1.98 (s, 3H, 21-CH3), 1.29−1.35 (m, 8H, 14-CH2 to 17-CH2), 1.24 (t,
3JH,H = 7.1 Hz, 6H, 23-CH3, 25-CH2). 13C NMR (75 MHz, CDCl3): δ
169.1 (C-20), 168.2 (C-1, C-3), 140.7 (C-6), 137.8 (C-9), 128.5 (C-7,
C-11), 128.4 (C-8, C-10), 84.3 (d, 1JC,F = 164.0 Hz, C-19), 66.5 (C-2),
2
62.7 (C-22, C-24), 35.6 (C-12), 33.5 (C-4), 31.7 (C-5), 30.5 (d, JC,F
= 19.5 Hz, C-18), 29.8 (C-14, C-15), 29.5 (C-13), 29.3 (C-16), 25.3
3
(d, JC,F = 5.5 Hz, C-17), 23.1 (C-21), 14.1 (C-23, C-25). 19F NMR
(282 MHz, CDCl3): δ −218.5 (tt, 3JH,F = 24.9 Hz, 2JH,F = 47.4 Hz, 1F,
19-CH2F). Exact mass (ESI+): C25H38FNO5 + Na+: calcd, 474.2626;
found, 474.2621; (C25H38FNO5)2 + Na+: calcd, 925.5360; found,
925.5354.
N-[1-Hydroxy-2-(hydroxymethyl)-4-(4-octylphenyl)butan-2-yl]-
acetamide (15). To a solution of diester 13 (1.27 g, 2.93 mmol) in
THF (30 mL) were added lithium chloride (636 mg, 15.0 mmol, 5.1
equiv) and sodium borohydride (567 mg, 15.0 mmol, 5.1 equiv). The
mixture was cooled to 0 °C and treated with ethanol (60 mL). After 30
min at 0 °C, the mixture was allowed to warm to rt and was stirred for
3 days. The mixture was adjusted to pH 4 with 10% citric acid at 0 °C.
THF was removed in vacuo, and the residue was extracted with DCM
(4 × 30 mL). The combined organic phases were washed with brine
(1 × 80 mL), dried over Na2SO4, and concentrated under reduced
pressure. The product was purified by column chromatography (10 ×
4 cm, cyclohexane/ethyl acetate, 1:2) to give the product as a white
2-Amino-2-[4-(8-fluorooctyl)phenethyl]propane-1,3-diol (17).
Synthesis and purification were performed as described for compound
2. Yield: 5 mg (44%). 1H NMR (300 MHz, MeOD): δ 7.14 (m, 4H, 7-
CH to 10-CH), 4.41 (dt, 2JH,F = 47.6 Hz, 3JH,H = 6.1 Hz, 2H, 19-CH2),
3.70 (s, 4H, 3-CH2, 4-CH2), 2.62 (m, 4H, 5-CH2, 12-CH2), 1.94 (m,
2H, 1-CH2), 1.66 (m, 4H, 13-CH2, 18-CH2), 1.36 (m, 8H, 14-CH2 to
17-CH2). 13C NMR (75 MHz, MeOD): δ 141.9 (C-6), 139.5 (C-11),
129.6 (C-7, C-8), 129.1 (C-9, C-10), 82.8 (d, 1JC,F = 163.6 Hz, C-19),
62.6 (C-3, C-4), 61.9 (C-2), 36.4 (C-12), 34.8 (C-5), 32.7 (C-13),
31.5 (d, 2JC,F = 19.6 Hz, C-18), 30.5 (C-16), 30.3 (C-15), 30.2 (C-1),
3
29.6 (C-14), 26.3 (d, JC,F = 5.3 Hz, C-17). 19F NMR (282 MHz,
1
solid. Yield: 974 mg (95%). mp 87−88 °C. H NMR (400 MHz,
MeOD): δ −220.0 (tt, 2JH,F = 47.4 Hz, 3JH,F = 24.9 Hz, 19-F). HRMS
(ESI+): C19H32NO2F + Na+: calcd, 348.2315; found, 348.2310;
C19H32NO2F + H+: calcd, 326.2495; found, 326.2490.
CDCl3): δ 7.09 (m, 4H, 7-CH, 8-CH, 10-CH, 11-CH), 6.23 (s, 1H, 2-
2
NH), 4.61 (brs, 2H, 1-OH, 3-OH), 3.80 (d, JH,H = 11.5 Hz, 2H, 1-
2
CH, 3-CH), 3.61 (d, JH,H = 11.5 Hz, 2H, 1-CH, 3-CH), 2.52−2.60
2-Amino-2-(fluoromethyl)-4-(4-octylphenyl)butan-1-ol (18).
Compound 2 (310 mg, 1.00 mmol) was suspended in dry DCM
(10 mL) and cooled to −78 °C. Diethylaminosulfur trifluoride
(DAST, 0.13 mL, 1.00 mmol, 1.0 equiv) was slowly added to this
suspension, and the mixture was allowed to warm to rt and stirred
(m, 4H, 5-CH2, 12-CH2), 1.96 (m, 2H, 4-CH2), 1.93 (s, 3H, 21-CH3),
1.56 (m, 2H, 13-CH2), 1.20−1.33 (m, 10H, 14-CH2 to 18-CH2), 0.87
(m, 3 H, 19-CH3). 13C NMR (101 MHz, CDCl3): δ 172.0 (C-20),
140.7 (C-6), 138.7 (C-9), 128.6 (C-7, C-11), 128.2 (C-8, C-10), 14.1
3478
DOI: 10.1021/jm502021d
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Journal of Medicinal Chemistry
Article
overnight. The reaction was neutralized with saturated sodium
bicarbonate solution (30 mL) at −10 °C. The phases were separated,
and the aqueous phase was extracted with DCM (3 × 20 mL). The
combined organic layers were dried over MgSO4, and the solvent was
removed under reduced pressure. The residue was purified by column
chromatography (20 × 3 cm, DCM/methanol, 4:1) and subsequently
by gradient HPLC (RP-HPLC Nucleodur 100−10 C18ec column (250
× 16 mm), acetonitrile/water (0.1% TFA)). The obtained TFA salt
was dissolved in methanol (1 mL) and 1 M sodium hydroxide solution
(3 mL), and the mixture was extracted with DCM (5 × 5 mL). The
organic phases were dried over Na2SO4 and concentrated. The
product was dried in high vacuum and obtained as a highly viscous oil.
Yield: 5 mg (5%). 1H NMR (300 MHz, CDCl3): δ 7.01−7.11 (m, 4H,
rt. The aqueous phase was extracted with DCM (4 × 30 mL), and the
organic layers were dried over Na2SO4. The solvent was evaporated,
and the product was purified by column chromatography (DCM/
methanol, 4:1) to give the product as colorless, waxy solid. Yield: 503
1
mg (78%). mp 74−76 °C. H NMR (400 MHz, CDCl3): δ 7.15 (d,
3
3JH,H = 7.8 Hz, 2H, 7-CH/11-CH or 8-CH/10-CH), 7.04 (d, JH,H
=
7.7 Hz, 2H, 7-CH/11-CH or 8-CH/10-CH), 4.83 (d, 3JH,H = 10.3 Hz,
1H, 5-CH), 4.03 (br s, 5H, 1-OH, 3-OH, 5-OH, 2-NH2), 3.28−3.58
(m, 4H, 1-CH2, 3-CH2), 2.50 (m, 2H, 12-CH2), 1.47−1.70 (m, 4H, 4-
CH2, 13-CH2), 1.16−1.34 (m, 10H, 14-CH2 to 18-CH2), 0.87 (t, 3JH,H
= 6.7 Hz, 3H, 19-CH3). 13C NMR (101 MHz, CDCl3): δ 142.3 (C-6),
142.1 (C-9), 128.5 (C-7, C-11), 125.6 (C-8, C-10), 70.4 (C-5), 67.2
(C-1), 65.8 (C-3), 56.5 (C-2), 43.8 (C-4), 35.8 (C-12), 32.0 (C-16),
31.7 (C-15), 29.6 (C-13, C-17), 29.5 (C-14), 22.8 (C-18), 14.2 (C-
19). HRMS (ESI+): C19H33NO3 + H+: calcd, 324.2533; found,
324.2550; C19H33NO3 + Na+: calcd, 346.2353; found, 346.2355.
3-Amino-1-[4-(8-fluorooctyl)phenyl]-3-(hydroxymethyl)butane-
1,4-diol (22). Amide 20 (20 mg, 0.05 mmol) was dissolved in
methanol (3 mL), treated with 1 M sodium hydroxide solution (0.09
mL, 0.09 mmol, 1.7 equiv), and heated at 120 °C for 6 h in a pressure
vessel. The mixture was allowed to cool to rt and stirred overnight.
Then the reaction mixture was diluted with 1 M sodium hydroxide
solution (5 mL), and the aqueous phase was extracted with DCM (5 ×
8 mL). The combined organic layers were dried over Na2SO4 and
concentrated. The residue was purified by gradient HPLC (RP-HPLC
Nucleodur 100−10 C18ec column (250 × 16 mm), acetonitrile/water
(0.1% TFA)) with a Knauer HPLC system. Afterward, the obtained
TFA salt was dissolved in methanol (1 mL) and 1 M sodium
hydroxide solution (3 mL). The mixture was extracted with DCM (5
× 5 mL), and the organic phases were dried over Na2SO4 and
concentrated. The product was dried in high vacuum and obtained as a
highly viscous oil. Yield: 12 mg (68%). 1H NMR (300 MHz, CDCl3):
2
7-CH, 8-CH, 10-CH, 11-CH), 4.38 (d, JH,F = 47.3 Hz, 2H, 3-CH2),
3.52 (m, 2H, 1-CH2), 2.47−2.68 (m, 4H, 5-CH2, 12-CH2), 1.81 (m,
2H, 4-CH2), 1.56 (m, 2H, 13-CH2), 1.16−1.37 (m, 10H, 14-CH2 to
3
18-CH2), 0.87 (t, JH,H = 6.7 Hz, 3H, 19-CH3). 13C NMR (75 MHz,
CDCl3): δ 140.9 (C-6), 138.7 (C-9), 128.7 (C-7, C-11), 128.2 (C-8,
1
3
C-10), 86.1 (d, JC,F = 173.3 Hz, C-3), 65.2 (d, JC,F = 4.1 Hz, C-1),
2
56.1 (d, JC,F = 17.2 Hz, C-2), 35.7 (t, C-12), 34.8 (C-5), 32.0 (C-4),
31.7, 29.6 (C-15), 29.5 (C-16), 29.4 (C-13, C17), 29.3 (C-14), 23.5
(C-18), 14.2 (C-19). 19F NMR (282 MHz, CDCl3): δ −230.3 (t, 2JH,F
= 47.3 Hz, 1F, 3-CH2F). HRMS (ESI+): C19H32FNO + H+:
calcd,310.2541; found, 310.2542; C19H32FNO + Na+: calcd,
332.2360; found, 332.2379.
N-[1,4-Dihydroxy-2-(hydroxymethyl)-4-(4-octylphenyl)butan-2-
yl]acetamide (19). Lithium chloride (424 mg, 10.0 mmol, 5.0 equiv)
and sodium borohydride (378 mg, 10.0 mmol, 5.0 equiv) were added
to a solution of diester 10 (930 mg, 2.08 mmol) in THF (8 mL). The
mixture was cooled to 0 °C and treated with ethanol (16 mL). After 45
min, the reaction was warmed to rt and stirred overnight. The reaction
mixture was adjusted to pH 4 with 10% citric acid at 0 °C, and THF
was removed in vacuo. The residue was extracted with DCM (4 × 10
mL). The organic layers were washed with brine (1 × 15 mL), dried
over Na2SO4, and evaporated. The product was purified by column
chromatography (20 × 3 cm, DCM/methanol, 20:1) to give the
δ 7.23 (d, 3JH,H = 7.8 Hz, 2H, 7-CH, 11-CH or 8-CH, 10-CH), 7.11 (d,
3
3JH,H = 7.7 Hz, 2H, 7-CH, 11-CH or 8-CH, 10-CH), 4.94 (d, JH,H
=
10.2 Hz, 1H, 5-CH), 4.43 (dt, 3JH,H = 6.2 Hz, 2JH,F = 47.4 Hz, 2H, 19-
CH2), 3.42−3.79 (m, 4H, 1-CH2, 3-CH2), 2.55 (t, 3JH,H = 7.8 Hz, 2H,
12-CH2), 1.49−1.82, 1.21−1.46 (m, 14H, 4-CH2, 13-CH2 to 18-CH2).
13C NMR (101 MHz, CDCl3): δ 142.3 (C-6), 142.2 (C-9), 128.6 (C-
7, C-11), 125.6 (C-8, C-10), 84.4 (d, 1JC,F = 163.9 Hz, C-19), 70.7 (C-
5), 68.1(C-1), 66.4 (C-3), 56.6 (C-2), 43.8 (C-4), 35.7 (C-12), 30.5
(d, 2JC,F = 19.4 Hz, C-18), 31.6 (C-13), 29.8 (C-16), 29.5 (C-14), 29.3
(C-15), 25.3 (d, 3JC,F = 5.6 Hz, C-17). 19F NMR (282 MHz, CDCl3):
1
product as a white solid. Yield: 586 mg (77%). mp 123 °C. H NMR
(300 MHz, CDCl3): δ 7.26 (d, 3JH,H = 8.1 Hz, 2H, 7-CH/11-CH or 8-
CH/10-CH), 7.13 (d, 3JH,H = 8.1 Hz, 2H, 7-CH/11-CH or 8-CH/10-
CH), 4.85 (m, 1H, 5-CH), 3.59−3.91 (m, 4H, 1-CH2, 3-CH2), 2.58
(m, 2H, 12-CH2), 2.00 (m, 2H, 4-CH2), 2.00 (s, 3H, 21-CH3), 1.59
(m, 2H, 13-CH2), 1.21−1.36 (m, 10H, 14-CH2 to 18-CH2), 0.87 (m,
3H, 19-CH3). 13C NMR (101 MHz, CD3OD, CDCl3): δ 172.3 (C-
20), 141.9 (C-6), 141.8 (C-9), 128.1 (C-7, C-11), 125.2 (C-8, C-10),
69.7 (C-5), 65.0 (C-1), 63.7 (C-3), 61.1 (C-2), 41.2 (C-4), 35.3 (C-
12), 31.6 (C-16), 31.3 (C-15), 29.2 (C-13, C-17), 29.0 (C-14), 23.0
(C-21), 22.4 (C-18), 13.7 (C-19). HRMS (ESI+): C21H35NO4 + Na+:
calcd, 388.2458; found, 388.2463; (C21H35NO4)2 + Na+: calcd,
753.5024; found, 753.5032.
3
2
δ −218.5 (tt, JH,F = 24.9 Hz, JH,F = 47.4 Hz, 1F, 19-CH2F). HRMS
(ESI+): C19H32FNO3 + H+: calcd, 342.2439; found, 342.2437;
C19H32FNO3 + Na+: calcd, 364.2258; found, 364.2255.
tert-Butyl [5-(Hydroxymethyl)-2,2-dimethyl-1,3-dioxane-5-yl]-
carbamate (24).34 Compound 23 (7 g, 57 mmol, 1 equiv) was
suspended in DMF (100 mL), and Boc anhydride (13.87 g, 63 mmol,
1.1 equiv) was added. The reaction mixture was stirred at 25 °C for 2
h. Then, 2,2-dimethoxypropane (8.51 mL, 69 mmol, 1.2 equiv) and p-
toluenesulfonic acid (0.55 g, 2.9 mmol, 0.05 equiv) were added, and
the resulting mixture was stirred at 25 °C for 12 h. The reaction was
quenched by addition of diethyl ether (100 mL). The organic phase
was washed with saturated sodium carbonate solution (1 × 50 mL)
and brine (1 × 50 mL). The separated organic layer was dried over
MgSO4, and solvent was removed under reduced pressure. The
product was obtained as a white solid and used without further
N-{4-[4-(8-Fluorooctyl)phenyl]-1,4-dihydroxy-2-(hydroxymethyl)-
butan-2-yl}-acetamide (20). Synthesis and purification were
1
performed as described for compound 19. Yield: 21 mg (48%). H
NMR (300 MHz, CDCl3): δ 7.09−7.27 (m, 4H, 7-CH, 8-CH, 10-CH,
11-CH), 5.30 (1 H, 2-NH), 4.86 (d, 3JH,H = 10.5 Hz, 1H, 5-CH), 4.43
3
2
(dt, JH,H = 6.2 Hz, JH,F = 47.4 Hz, 2H, 19-CH2), 3.42−3.80 (m, 4H,
1-CH2, 3-CH2), 2.58 (m, 2H, 12-CH2), 2.31 (m, 2H, 4-CH2), 2.02 (s,
3H, 21-CH3), 1.53−1.85 and 1.24−1.43 (m, 12H, 13-CH2 to 18-
CH2). 13C NMR (75 MHz, CDCl3): δ 172.0 (C-20), 142.9 (C-6),
1
purification. Yield: 13.0 g (87%). mp 100 °C. H NMR (400 MHz,
1
3
141.6 (C-9), 128.8 (C-7, C-11), 125.6 (C-8, C-10), 84.4 (d, JC,F
=
CDCl3): δ 5.33 (s, 1H, NH), 4.33 (s, 1H, OH), 3.83 (q, JH,H = 11.7
Hz, 4H, 3-CH2, 4-CH2), 3.73 (d, 3JH,H = 6.5 Hz, 2H, 1-CH2), 1.46 (m,
15H, 5-CH3, 6-CH3, 8-CH3, 9-CH3, 10-CH3). 13C NMR (101 MHz,
CDCl3): δ 156.4 (C-11), 98.7 (C-7), 80.4 (C-12), 64.6 (C-1), 64.3 (C-
3, C-4), 53.2 (C-2), 28.3 (C-8, C-9, C-10), 26.7 (C-6), 20.2 (C-5).
HRMS (ESI+): C12H23NO5 + Na+: calcd, 284.1468; found,: 284.1468.
tert-Butyl (5-Formyl-2.2-dimethyl-1,3-dioxan-5-yl)carbamate
(25).34 A solution of oxalyl chloride (1.31 mL, 15.3 mmol, 2 equiv)
in DCM (25 mL) was cooled to −78 °C, treated with a solution of
DMSO (1.63 mL, 22.9 mmol, 1.1 equiv) in DCM (10 mL), and
allowed to stir for 15 min. After that, a solution of 24 (2 g, 7.66 mmol)
in DCM (20 mL) was added slowly dropwise, and stirring was
163.9 Hz, C-19), 71.2 (C-5), 66.4 (C-1), 65.1 (C-3), 61.5 (C-2), 41.4
(C-4), 35.7 (C-12), 30.5 (d, 2JC,F = 19.3 Hz, C-18), 31.6 (C-16), 29.5
(C-13), 29.3 (C-14, C-15), 25.3 (d, 3JC,F = 5.4 Hz, C-17), 24.0 (C-21).
3
2
19F NMR (282 MHz, CDCl3): δ −218.5 (tt, JH,F = 25.0 Hz, JH,F
=
47.4 Hz, 1F, 19-CH2F). HRMS (ESI+): C21H34FNO4 + Na+: calcd,
406.2364; found, 406.2358.
3-Amino-3-(hydroxymethyl)-1-(4-octylphenyl)butane-1,4-diol
(21). Amide 19 (1.37 g, 2.00 mmol) was dissolved in methanol (30
mL) and treated with 1 M sodium hydroxide solution (2.4 mL, 2.40
mmol, 1.2 equiv). The reaction mixture was refluxed for 6 h and then
diluted with 1 M sodium hydroxide solution (20 mL) after cooling to
3479
DOI: 10.1021/jm502021d
J. Med. Chem. 2015, 58, 3471−3484

Journal of Medicinal Chemistry
Article
continued for a further 15 min. Triethylamine (5.37 mL, 38.3 mmol, 5
equiv) was added to the reaction mixture, and stirring was continued
for a further 15 min. The mixture was allowed to warm to rt and
stirred for 30 min. The reaction was quenched with water and
extracted with DCM (2 × 20 mL). Then, the organic layer was washed
with 1% H2SO4 (1 × 30 mL) and brine (2 × 20 mL), dried over
MgSO4, and concentrated in vacuo to obtain the crude product, which
was further purified by column chromatography (cyclohexane/ethyl
acetate, 4:1) to obtain the desired pure product as a white solid. Yield:
Hz, 3JH,H = 0.2 Hz, 2H, 28-CH2), 2.42 (t, 3JH,H = 6.9 Hz, 2H, 23-CH2),
1.66−1.57 (m, 4H, 24-CH2, 27-CH2), 1.50−1.42 (m, 4H, 25-CH2, 26-
CH2), 1.38 (s, 12H, 5-CH3, 8-CH3, 9-CH3, 10-CH3), 1.32 (s, 3H, 6-
CH3). 13C NMR (75 MHz, CDCl3): δ 154.4 (C-11), 136.7 (C-14),
131.6 (C-15), 131.0 (C-18, C-19), 129.8 (C-1), 128.5 (C-16, C-17),
126.2 (C-20), 98.1 (C-7), 90.6 (C-21), 87.3 (C-22), 80.4 (C-12), 65.8
(C-28), 62.9 (C-3, C-4), 52.4 (C-2), 32.6 (C-27), 28.6 (single peak
with high intensity, C-24, C-25), 28.34 (C-8, C-9), 28.38 (C-10), 25.2
(single peak with high intensity, C-5, C-6), 19.3 (C-26), 18.8 (C-23).
1
3
1
Minor E-Isomer. H NMR (300 MHz, CDCl3): δ 7.32 (dd, JH,H
=
1.71 g (86%). mp 118 °C. H NMR (300 MHz, CDCl3): δ 9.63 (s,
3
8.3 Hz, 1.9 Hz, 2H, 18-CH, 19-CH), 7.18 (d, JH,H = 8.1 Hz, 2H, 16-
1H, 1-CHO), 5.58 (s, 1H, NH), 4.04 (m, 4H, 3-CH2, 4-CH2), 1.47 (s,
15H, 5-CH3, 6-CH3, 8-CH3, 9-CH3, 10-CH3). 13C NMR (75 MHz,
CDCl3): δ 199.2 (C-1), 155.4 (C-11), 98.7 (C-7), 80.9 (C-12), 62.6
(C-3, C-4), 59.8 (C-2), 28.2 (C-8, C-9, C-10), 27.3 (C-6), 19.5 (C-5).
HRMS (ESI+): C12H21NO5 + Na+: calcd, 282.1317; found,: 282.1312.
tert-Butyl [5-(4-Iodostyryl)-2,2-dimethyl-1,3-dioxan-5-yl]-
carbamate (27). Potassium carbonate (1.26 g, 9.2 mmol, 4 equiv),
was flame-dried in a pressure tube. After cooling to rt, 25 (0.6 g, 2.3
mmol, 1 equiv) dissolved in toluene (7 mL) and 26 (2.58 g, 4.6 mmol,
2 equiv) were added, and the mixture was heated at 120 °C for 12 h.
Then, the reaction mixture was cooled to rt, absorbed on silica gel, and
purified by column chromatography (12 × 2 cm, cyclohexane/ethyl
acetate, 4:1) to give the Z/E-isomeric products (dr 3:1) as a white
solid. Yield: 1.02 g (96%). mp 107 °C. Z-isomer: 1H NMR (300 MHz,
CDCl3): δ 7.66−7.57 (m, 2H, 18-CH, 19-CH), 7.05−6.94 (m, 2H, 16-
3
3
CH, 17-CH), 6.49 (d, JH,H = 16.4 Hz, 1H, 1-CH), 6.20 (d, JH,H
=
16.4 Hz, 1H, 14-CH), 5.18 (s, 1H, NH), 3.93−3.83 (m, 2H, 3-CH, 4-
CH), 3.73 (d, 2JH,H = 12.2 Hz, 4JH,H = 1.4 Hz, 2H, 3-CH, 4-CH), 3.66
3
3
3
(td, JH,H = 6.6 Hz, JH,H = 0.2 Hz, 2H, 28-CH2), 2.42 (t, JH,H = 6.9
Hz, 2H, 23-CH2), 1.66−1.57 (m, 4H, 24-CH2, 27-CH2), 1.50−1.42
(m, 4H, 25-CH2, 26-CH2), 1.38 (s, 12H, 5-CH3, 8-CH3, 9-CH3, 10-
CH3), 1.32 (s, 3H, 6-CH3). 13C NMR (75 MHz, CDCl3): δ 154.8 (C-
11), 136.7 (C-14), 135.6 (C-20), 132.6 (C-15), 131.8 (single peak, C-
18, C-19), 129.8 (C-1), 122.7 (single peak, C-16, C-17), 98.2 (C-7),
91.0 (C-21), 87.3 (C-22), 79.4 (C-12), 65.8 (C-28), 62.9 (C-3, C-4),
52.9 (C-2), 32.6 (C-27), 28.6 (C-25), 28.38 (C-10), 28.34 (C-8, C-9),
27.8 (C-24), 25.2 (single peak with high intensity, C-5, C-6), 19.4 (C-
+
26), 19.2 (C-23). HRMS (ESI+): C27H39NO5 Na+: calcd, 480.2726;
found, 480.2720.
3
3
CH, 17-CH), 6.58 (d, JH,H = 12.7 Hz, 1H, 14-CH), 5.58 (d, JH,H
=
tert-Butyl {5-[4-(6-Hydroxyhexyl)phenethyl]-2,2-dimethyl-1,3-di-
oxan-5-yl}carbamate (30). Ultrapure hydrogen gas was generated
with a Nitrox UHP-40H hydrogen generator (Domnick Hunter,
England). Alkyne 28 (310 mg, 0.65 mmol) was dissolved in ethyl
acetate (20 mL) in a pressure vessel, to which Pd/C (31 mg, 10 mol
%) was added. Then, the flask was flushed with hydrogen (2 atm
pressure), and the reaction mixture was stirred vigorously at rt for 2 h.
The reaction was stopped by releasing hydrogen gas, and the mixture
was filtered over Celite. The solvent was removed under reduced
pressure, and the residue was purified by column chromatography (4.5
× 3 cm, cyclohexane/ethyl acetate, 4:1) to give the product as a white
solid. Yield: 240 mg (76%). mp 57 °C. 1H NMR (300 MHz, CDCl3):
δ 7.12−7.04 (m, 4H, 16-CH, 17-CH, 18-CH, 19-CH), 4.98 (bs, 1H,
12.6 Hz, 1H, 1-CH), 5.14 (s, 1H, 13-NH), 3.87 (d, 3JH,H = 5.2 Hz, 2H,
3
3-CH, 4-CH), 3.74 (dt, JH,H = 12.2 Hz, 1.4 Hz, 2H, 3-CH, 4-CH),
1.40−1.32 (m, 15H, 5-CH3, 6-CH3, 8-CH3, 9-CH3, 10-CH3). 13C
NMR (75 MHz, CDCl3): δ 154.3 (C-11), 137.5 (C-14), 136.9 (C-18,
C-19), 131.2 (C-1), 130.5 (C-16, C-17), 128.1 (C-15), 98.3 (C-20),
98.1 (C-7), 79.4 (C-12), 65.8 (C-3, C-4), 52.4 (C-2), 28.3 (C-8, C-9,
C-10), 18.7 (C-5, C-6). HRMS (ESI+): C19H26INO4+Na+: calcd,
482.0804; found, 482.0799.
tert-Butyl {5-[4-(6-Hydroxyhex-1-yn-1-yl)styryl]-2,2-dimethyl-1,3-
dioxan-5-yl}carbamate (28). Intermediate 27 (0.15 g, 0.27 mmol, 1
equiv) was dissolved in acetonitrile (2 mL) and treated with
triphenyphosphine (15 mg, 10 mol %), Pd/C (15 mg, 10 mol %),
copper iodide (7.5 mg, 5 mol %), and triethylamine (0.14 mL, 0.98
mmol, 3 equiv). The mixture was stirred for 20 min, hex-5-yn-1-ol (53
μL, 0.49 mmol, 1.5 equiv) was added, and the mixture was stirred at 25
°C for 1 h. Finally, the reaction mixture was heated under microwave
irradiation at 80 °C for 90 min. After cooling to rt, the reaction
mixture was diluted with ethyl acetate (2 mL) and filtered through
Celite. The organic layer was concentrated, and the crude compound
was purified by column chromatography (13.5 × 3 cm, cyclohexane/
ethyl acetate, 3:2) to get the Z/E-isomeric products (dr 3:1) as a
2
2
13-NH), 3.89 (d, JH,H = 11.8 Hz, 2H, 3-CH, 4-CH), 3.67 (d, JH,H
=
3
12.1 Hz, 1.1 Hz, 2H, 3-CH, 4-CH), 3.63 (t, JH,H = 6.6 Hz, 2H, 26-
CH2), 2.60−2.49 (m, 4H, 14-CH2, 21-CH2), 1.97 (t, JH,H = 8.5 Hz,
3
2H, 1-CH2), 1.70−1.50 (m, 6H, 22-CH2, 23-CH2, 25-CH2), 1.47 (s,
9H, 8-CH3, 9-CH3, 10-CH3), 1.45−1.40 (m, 6H, 5-CH3, 6-CH3), 1.36
(dt, 3JH,H = 6.9 Hz, 4.5 Hz, 3H, 24-CH2, −OH). 13C NMR (75 MHz,
CDCl3): δ 154.8 (C-11), 140.2 (C-15), 139.1 (C-20), 128.4 (single
peak with high intensity, C-16, C-17), 128.2 (single peak with high
intensity, C-18, C-19), 98.3 (C-7), 80.1 (C-12), 66.3 (C-26), 62.9
(single peak with high intensity C-3, C-4), 51.7 (C-2), 35.4 (C-21),
32.6 (C-25), 31.4 (C-22), 29.0 (single peak C-14, C-23), 28.6 (C-1),
28.4 (single peak with high intensity C-8, C-9, C-10), 27.4 (C-6), 25.5
1
slightly yellowish liquid. Yield: 120 mg, (85%). Z-isomer: H NMR
(300 MHz, CDCl3): δ 7.29−7.35 (m, 2H, 18-CH, 19-CH), 7.15−7.21
3
(m, 2H, 16-CH, 17-CH), 6.64 (d, JH,H = 12.6 Hz, 1H, 14-CH), 5.58
3
2
+
(C-24), 19.6 (C-5). HRMS (ESI+): C25H41NO5 Na+: calcd, 458.2882;
(d, JH,H = 12.7 Hz, 1H, 1-CH), 5.18 (brs, 1H, NH), 3.88 (d, JH,H
=
11.7 Hz, 2H, 3-CH2), 3.67−3.79 (m, 4H, 4-CH2, 26-CH2), 2.47 (t,
3JH,H = 6.5 Hz, 2H, 23-CH2), 1.65−1.82 (m, 4H, 24-CH2, 25-CH2),
1.43 (s, 6H, 5-CH3, 6-CH3), 1.38 (s, 9H, 8-CH3, 9-CH3, 10-CH3),
1.32 (bs, 1H, OH). 13C NMR (75 MHz, CDCl3): δ 164.7 (C-11),
136.8 (C-14), 131.8 (C-1), 131.7 (C-15), 131.0 (single peak with high
intensity, C-18, C-19), 128.5 (single peak with high intensity, C-16, C-
17), 125.2 (C-20), 98.1 (C-7), 90.2 (C-21), 80.7 (C-22), 77.2 (C-12),
65.8 (C-26), 62.4 (C-3, C-4), 52.4 (C-2), 31.8 (C-25), 28.39 (C-10),
28.35 (C-8, C-9), 26.9 (C-24), 24.9 (C-23), 19.2 (C-6), 18.7 (C-5).
found, 458.2877.
tert-Butyl {5-[4-(8-Hydroxyoctyl)phenethyl]-2,2-dimethyl-1,3-di-
oxan-5-yl}carbamate (31). Synthesis and purification of product 31
were performed as described for compound 30. Yield: 100 mg (65%).
1
3
mp 61 °C. H NMR (300 MHz, CDCl3): δ 7.09 (d, JH,H = 3.9 Hz,
4H, 16-CH, 17-CH, 18-CH, 19-CH), 5.00 (brs, 1H, NH), 3.89 (m,
2H, 3-CH, 4-CH), 3.64 (m, 4H, 28-CH2, 3-CH, 4-CH), 2.55 (m, 4H,
14-CH2, 21-CH2), 1.93 (m, 2H, 1-CH2), 1.51−1.65 (m, 6H, 22-CH2,
26-CH2, 27-CH2), 1.47 (s, 6H, 5-CH3, 6-CH3), 1.41−1.46 (m, 7H, 23-
CH2, 24-CH2, 25-CH2, OH), 1.31 (s, 9H, 8-CH3, 9-CH3, 10-CH3).
13C NMR (75 MHz, CDCl3): δ 140.4 (C-15), 139.0 (C-20), 128.4
(single peak high intensity, C-16, C-17), 128.1 (single peak high
intensity, C-18, C-19), 98.3 (C-7), 77.2 (C-12), 66.3 (C-28), 63.0
(single peak high intensity, C-3, C-4), 51.6 (C-2), 35.4 (C-21), 32.7
(C-27), 31.5 (C-22), 30.9 (C-25), 29.4 (C-14), 29.3 (C-1), 29.2 (C-
24), 28.6 (C-23), 28.4 (single peak high intensity, C-8, C-9), 28.3 (C-
10), 25.6 (single peak high intensity, C-26, C-6), 19.6 (C-5). HRMS
(ESI+): C27H45NO5 + H+: calcd, 464.3376; found, 464.3371,
C27H45NO5 + Na+: calcd, 486.3195; found, 486.3190.
+
HRMS (ESI+): C25H35NO5 Na+: calcd, 452.2413; found, 452.2407
tert-Butyl {5-[4-(8-Hydroxyoct-1-yn-1-yl)styryl]-2,2-dimethyl-1,3-
dioxan-5-yl}carbamate (29). Synthesis and purification of com-
pounds 29 (dr 3:1) were performed as described for compound 28.
Yield: 95 mg (85%).
1
3
Major Z-Isomer. H NMR (300 MHz, CDCl3): δ 7.32 (dd, JH,H
=
3
8.3 Hz, 1.9 Hz, 2H, 18-CH, 19-CH), 7.18 (d, JH,H = 8.1 Hz, 2H, 16-
CH, 17-CH), 6.64 (d, JH,H = 12.7 Hz, 1H, 1-CH), 5.58 (d, JH,H
3
3
=
12.7 Hz, 1H, 14-CH), 5.18 (s, 1H, NH), 3.93−3.83 (m, 2H, 3-CH, 4-
CH), 3.73 (d, 2JH,H = 12.2 Hz, 2H, 3-CH, 4-CH), 3.66 (td, 3JH,H = 6.6
3480
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Journal of Medicinal Chemistry
Article
tert-Butyl N-{5-[4-(6-Fluorohexyl)phenethyl]-2,2-dimethyl-1,3-di-
oxan-5-yl}-carbamate (32). Alcohol 30 (161 mg, 0.37 mmol) was
dissolved in dry THF (5 mL) and treated with PBSF (0.14 mL, 0.78
mmol, 2.1 equiv), triethylamine trishydrofluoride (0.12 mL, 0.73
mmol, 2.0 equiv), and N,N-diisopropylethyl amine (0.39 mL, 2.22
mmol, 6.0 equiv). The reaction mixture was stirred at rt for 2 days and
quenched by addition of saturated sodium bicarbonate solution (5
mL). The aqueous phase was extracted with DCM (3 × 15 mL), and
the combined organic layer was dried over MgSO4. The solvent was
removed under reduced pressure, and the residue was purified by
column chromatography (9.5 × 3 cm, cyclohexane/ethyl acetate, 2:1)
to give the product as a white solid. Yield: 72 mg (45%). mp 88−90
°C. 1H NMR (300 MHz, CDCl3): δ 7.08 (s, 4H, 7-CH, 8-CH, 10-CH,
10H, 4-CH2, 13-CH2 to 16-CH2). 13C NMR (75 MHz, CDCl3,
CD3OD): δ 140.1, 139.0 (C-6, C-9), 128.3 (C-7, C-11), 128.0 (C-8,
1
C-10), 84.1 (d, JC,F = 163.5 Hz, C-17), 65.5 (single peak, C-1, C-3),
56.1 (C-2), 35.2 (C-12), 31.2 (C-13), 30.2 (d, 2JC,F = 19.3 Hz, C-16),
3
28.7 (single peak, C-4, C-5, C-14), 24.9 (d, JC,F = 5.4 Hz, C-15). 19F
NMR (282 MHz, CDCl3, CD3OD): δ −218.5 (tt, 3JH,F = 25.1 Hz, 2JH,F
= 47.4 Hz, 1F, 17-CH2F). HRMS (ESI+): C17H28FNO2 + H+: calcd,
298.2177; found, 298.2182; C17H28FNO2 + Na+: calcd, 320.1996;
found, 320.2001.
{4-[4-(8-Fluorooctyl)phenethyl]-2-methyl-4,5-dihydro-oxazol-4-
yl}methanol (35). A solution of diol 17 (100 mg, 0.307 mmol, 1
equiv), N,N-diisopropylethyl amine (0.080 mL, 0.461 mmol, 1.5
equiv), and triethyl orthoacetate (0.084 mL, 0.461 mmol, 1.5 equiv) in
DMF (2 mL) was stirred at 120 °C for 2 h. The solution was extracted
with ethyl acetate (2 × 5 mL). The combined organic layer was
washed with brine (5 mL), dried over MgSO4, and concentrated. The
crude product was purified by column chromatography (12 × 2 cm,
cyclohexane/ethyl acetate, 5:1) to give the product as a yellowish oil.
3
2
11-CH), 4.99 (brs, 1H, 2-NH), 4.42 (dt, JH,H = 6.1 Hz, JH,F = 47.4
Hz, 2H, 17-CH2), 3.89 (d, 2JH,H = 11.7 Hz, 2H, 1-CH, 3-CH), 3.68 (d,
2JH,H = 11.7 Hz, 2H, 1-CH, 3-CH), 2.49−2.60 (m, 4H, 5-CH2, 12-
CH2), 1.97 (m, 2H, 4-CH2), 1.47 (s, 9H, 23-CH3 to 25-CH3), 1.35−
1.44, 1.56−1.76 (m, 14H, 13-CH2 to 16-CH2, 19-CH3, 20-CH3). 13C
NMR (75 MHz, CDCl3): δ 155.0 (C-21), 140.3, 139.3 (C-6, C-9),
128.5 (single peak, C-7, C-11), 128.3 (single peak, C-8, C-10), 98.5
(C-18), 84.2 (d, 1JC,F = 164.1 Hz, C-17), 79.4 (C-22), 66.4 (C-1, C-3),
51.8 (C-2), 35.5 (C-12), 31.5 (C-13), 30.4 (d, 2JC,F = 19.4 Hz, C-16),
29.0 (C-4, C-14), 28.8 (C-5), 28.6 (single peak, C-23, C-24, C-25),
1
Yield: 70 mg (58%). H NMR (400 MHz, CDCl3): δ 7.09 (s, 4H, 7-
CH to 10-CH), 4.43 (dt, 2JH,F = 47.4 Hz, 3JH,H = 6.2 Hz, 2H, 19-CH2),
2
2
4.26 (d, JH,H = 11.4 Hz, 1H, 3-CH), 4.09 (d, JH,H = 11.4 Hz, 1H, 3-
CH), 3.71 (d, 2JH,H = 8.5 Hz, 1H, 4-CH), 3.45 (d, 2JH,H = 8.5 Hz, 1H,
4-CH), 2.56 (m, 4H, 5-CH2, 12-CH2), 2.05 (s, 3H, 21-CH3), 1.89
(ddd, 3JH,H = 13.7 Hz, 11.5 Hz, 5.8 Hz, 1H, 1-CH), 1.50−1.81 (m, 5H,
13-CH2, 18-CH2, 1-CH), 1.33 (m, 8H, 14-CH2 to 17-CH2). 13C NMR
(101 MHz, CDCl3): δ 140.4 (C-6), 138.7 (C-11), 128.4 (single peak
with high intensity, C-7, C-8), 128.1 (single peak with high intensity,
C-9, C-10), 109.9 (C-20), 84.2 (d, 1JC,F = 163.5 Hz, C-19), 77.2 (C-2),
67.1 (C-3, C-4), 38.0 (C-12), 35.4 (C-5), 31.4 (C-13), 29.36 (C-15),
3
25.2 (d, JC,F = 5.4 Hz, C-15), 19.8 (single peak, C-19, C-20). 19F
3
2
NMR (282 MHz, CDCl3): δ −218.6 (tt, JH,F = 23.7 Hz, JH,F = 47.6
Hz, 1F, 17-CH2F). HRMS (ESI+): C25H40FNO4 + H+: calcd,
438.3014; found, 438.3014; C25H40FNO4 + Na+: calcd, 460.2834;
found, 460.2832.
tert-Butyl {5-[4-(8-Fluorooctyl)phenethyl]-2,2-dimethyl-1,3-diox-
an-5-yl}carbamate (33). A stirred solution of alcohol 31 (800 mg,
1.72 mmol, 1 equiv) in dry DCM (10 mL) was cooled to −78 °C,
under nitrogen, and treated with DAST (0.456 mL, 3.45 mmol, 2
equiv). The flask was stoppered, and the mixture was kept at rt for 8 h.
Then, the reaction was quenched with ice, neutralized with 10%
aqueous NaHCO3, and extracted with DCM (2 × 5 mL). The extract
was washed with water and brine, dried over MgSO4, and
concentrated. The product was purified by column chromatography
(15 × 3.5 cm, cyclohexane/ethyl acetate, 5:1) to give the product as a
29.29 (C-18), 29.2 (C-1), 29.16 (single peak, C-17, C-16), 25.1 (C-
2
14), 13.9 (C-21). 19F NMR (282 MHz, CDCl3): δ −218.0 (tt, JH,F
=
47.3 Hz, 3JH, F = 24.9 Hz). HRMS (ESI+): C21H32NO2F + Na+: calcd,
372.2315; found, 372.2309; C19H32NO2F + H+: calcd, 350.2495;
found, 350.2490.
2-Amino-4-[4-(8-fluorooctyl)phenyl]-2-(hydroxymethyl)butyl di-
hydrogen phosphate (36). Alcohol 35 (300 mg, 0.859 mmol) was
dissolved in DCM (7 mL), and 0.45 M tetrazole in MeCN (0.6 mL,
2.57 mmol, 3 equiv) was added. The reaction was stirred for 15 min at
rt, and then N,N-diethyl-di-tert-butyl-phosphoramidite (0.48 mL, 1.71
mmol, 2 equiv) was added. The mixture was stirred overnight, a 30%
solution of hydrogen peroxide (2 mL) was added, and the mixture was
further stirred for 4 h. The organic solution was washed with Na2S2O3
solution (5 mL) and saturated NaHCO3 solution (5 mL), dried over
Na2SO4, filtered, and concentrated in vacuo. The crude material was
passed through a short column (DCM/methanol/acetic acid, 79:20:1)
to remove some impurities. After concentration, the residue was
dissolved in conc. HCl/ethanol, 1:10 (2 mL), and the solution was
stirred at 50 °C for 6 h. Water (1 mL) was added, and the mixture was
stirred at rt for 1 h to form a precipitate. The precipitate was collected
by filtration and washed with ethyl acetate to obtain the fluffy off-white
product. Yield: 36 mg (10%). mp 219 °C. 1H NMR (600 MHz,
1
white solid. Yield: 700 mg (87%). mp 63 °C. H NMR (300 MHz,
CDCl3): δ 7.04−7.13 (m, 4H, 16-CH to 19-CH), 4.97 (brs, 1H, NH),
4.43 (dt, 2JH,F = 47.4 Hz, 3JH,H = 6.2 Hz, 2H, 28-CH2), 3.90 (d, 2JH,H
=
2
11.8 Hz, 2H, 3-CH, 4-CH), 3.67 (d, JH,H = 11.8 Hz, 2H, 3-CH, 4-
CH), 2.47−2.62 (ddd, 3JH,H = 8.8 Hz, 7.1 Hz, 5.0 Hz, 4H, 14-CH2, 21-
CH2), 1.91−2.03 (m, 2H, 1-CH2), 1.52−1.67 (m, 4H, 22-CH2, 27-
CH2), 1.47 (s, 9H, 8-CH3, 9-CH3, 10-CH3), 1.40−1.45 (m, 8H, 23-
CH2 to 26-CH2), 1.32 (s, 6H, 5-CH3, 6-CH3). 13C NMR (75 MHz,
CDCl3): δ 156.4 (C-11), 140.5 (C-15), 138.7 (C-20), 128.52 (C-17),
128.42 (C-16), 128.19 (C-19), 128.13 (C-18), 98.3 (C-7), 84.2 (d,
1JC,F = 163.9 Hz, C-28), 80.1 (C-12), 66.6 (C-3), 59.3 (C-4), 51.7 (C-
2
2), 35.5 (C-21), 31.5 (C-22), 30.3 (d, JC,F = 9.4 Hz, C-27), 29.3 (C-
24, C-14), 29.19 (C- 23), 29.16 (C-1), 29.09 (C-26), 28.4 (C-8), 28.3
(single peak high intensity, C-9, C-10, C-25), 25.17 (C-6), 25.10 (C-
MeOD): δ 7.14 (d, 3JH,H = 7.9 Hz, 2H, 9-CH, 10-CH), 7.09 (d, 3JH,H
8.0 Hz, 2H, 7-CH, 8-CH), 4.39 (dt, JH,F = 47.6 Hz, JH,H = 6.1 Hz,
=
5). 19F NMR (282 MHz, CDCl3): δ −218.0 (tt, 2JH,F = 47.4 Hz, 3JH,F
=
2
3
24.9 Hz, 28-F). HRMS (ESI+): C27H44NO4F + Na+: calcd, 488.31;
found, 488.30.
2H, 19-CH2), 4.00 (m, 2H, 3-CH, 4-CH), 3.69 (m, 2H, 3-CH, 4-CH),
3
2.63 (m, 2H, 5-CH2), 2.56 (t, JH,H = 7.7 Hz, 2H, 12-CH2), 1.96 (m,
2-Amino-2-[4-(6-fluorohexyl)phenethyl]propane-1,3-diol (34).
According to ref 25, intermediate 32 (72 mg, 0.16 mmol) was
dissolved in a mixture of DCM, TFA, and water (v/v 2:2:1; 2.5 mL).
The reaction mixture was allowed to warm to rt and stirred further for
12 h. Then, the solvent was removed under reduced pressure, and the
residue was diluted with water (2 mL) and neutralized with a saturated
NaHCO3 solution. The aqueous phase was extracted with DCM (4 ×
8 mL). The combined organic phase was washed with brine (1 × 8
mL) and dried over Na2SO4. The solvent was evaporated, and the
residue was crystallized from ethyl acetate to yield white crystals. Yield:
38 mg (81%). mp 108 °C (ethyl acetate). 1H NMR (300 MHz,
CDCl3, CD3OD): δ 6.97−7.10 (m, 4H, 7-CH, 8-CH, 10-CH, 11-CH),
2H, 1-CH2), 1.55−1.71 (m, 4H, 13-CH2, 18-CH2), 1.35 (m, 8H, 14-
CH2 to 17-CH2). 13C NMR (151 MHz, MeOD): δ 140.4 (C-6), 138.0
(C-11), 128.1 (single peak with high intensity C-7, C-8), 127.7 (single
1
peak with high intensity C-9, C-10), 83.4 (d, JC,F = 163.7 Hz, C-19),
64.3 (C-4), 61.0 (C-3), 59.9 (C-2), 35.0 (C-12), 33.4 (C-5), 31.2 (13),
30.1 (d, 2JC,F = 19.6 Hz, C-18), 29.0 (C-16), 28.9 (C-15), 28.7 (C-14),
3
28.1 (C-1), 24.8 (d, JC,F = 5.3 Hz, C-17). 19F NMR (564 MHz,
MeOD): δ −220.0 (tt, 2JH,F = 47.5 Hz, 3JH,F = 24.9 Hz, 1F, 19-F). 31
P
NMR (243 MHz, MeOD): δ 0.859 (s). HRMS (ESI+): C19H33NO5FP
+ Na+: calcd, 428.1978; found, 428.1973.
7-[4-(2-{5-[(tert-Butoxycarbonyl)amino]-2,2-dimethyl-1,3-diox-
an-5-yl}ethyl)heptyl]-4-methylbenzenesulfonate (37). Alcohol 30
(100 mg, 0.22 mmol, 1 equiv) was dissolved in dry DCM (10 mL).
The solution was cooled to 0 °C, and then triethylamine (64 μL, 0.445
mmol, 2 equiv) was added dropwise followed by the addition of tosyl
4.34 (dt, 3JH,H = 6.1 Hz, 2JH,F = 47.4 Hz, 2H, 17-CH2), 3.48 (d, 2JH,H
=
2
10.9 Hz, 2H, 1-CH, 3-CH), 3.40 (d, JH,H = 11.1 Hz, 2H, 1-CH, 3-
CH), 2.43−2.60 (m, 4H, 5-CH2, 12-CH2), 1.12−1.40, 1.46−1.70 (m,
3481
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Journal of Medicinal Chemistry
Article
chloride (63 mg, 0.334 mmol, 1.5 equiv) in one portion slowly. The
reaction mixture was allowed to warm to rt and was stirred overnight.
After completion of the reaction, the mixture was diluted with water
(10 mL) and extracted with DCM (2 × 10 mL). The combined
organic layer was washed with brine (1 × 10 mL) and water (2 × 5
mL), dried over MgSO4, and concentrated under reduced pressure.
The residue was purified by column chromatography (15 × 2 cm,
cyclohexane/ethyl acetate, 5:1) to get the product as a pure white
solid. Yield: 75 mg (55%). mp 75 °C. 1H NMR (400 MHz, CDCl3): δ
7.83−7.73 (m, 2H, 28-CH, 29-CH), 7.38−7.30 (m, 2H, 30-CH,31-
CH), 7.12−7.01 (m, 4H, 16-CH, 17-CH, 18-CH, 19-CH), 4.98 (s, 1H,
Siemens) by irradiation of a 2.8 mL water target using 10 MeV proton
beams on 97.0% enriched [18O]H2O by the 18O(p,n)18F nuclear
reaction.
4.3.2. Synthesis of [18F]34 and [18F]17. The same synthetic
protocol was used for both compounds. In a computer controlled
TRACERLab FxFDG Synthesizer, a batch of aqueous [18F]fluoride ions
(213−2918 MBq) from the cyclotron target was passed through anion
exchange resin (Sep-Pak Light Waters Accell Plus QMA cartridge,
preconditioned with 1 M K2CO3 (5 mL) and water for injection (10
mL)). [18F]Fluoride ions were eluted from the resin with a mixture of
40 μL of 1 M K2CO3, 200 μL of water for injection, and 800 μL of
DNA-grade CH3CN containing 20 mg (53 μmol) of Kryptofix 222
(K222) in the reactor. Subsequently, the aqueous K(K222) [18F]F
solution was carefully evaporated to dryness in vacuo. Then, precursor
(5.0−5.5 mg, 8.1−9.3 μmol) in dry acetonitrile (1 mL) was added, and
the reaction mixture was heated at 84 °C for 10−12 min. Then, the
solvent was evaporated, and a solution of 1.1 mL TFA/H2O (10:1)
was added at 40 °C and allowed to stir for 5 min. After that, the
solvent was evaporated to dryness, water (10 mL) was added, and the
mixture was passed through a Waters Sep-Pak C18 Light cartridge
(preconditioned with 10 mL of ethanol and 10 mL of water). The
cartridge was washed with water (10 mL) and eluted with MeOH (0.5
mL). After addition of water (0.5 mL), the raw product solution was
purified by gradient-radio-HPLC system A (method A). The product
fraction of compound [18F]34 (retention time tR = 16.63 min) and
[18F]17 (tR = 18.15 min) was evaporated to dryness in vacuo and
formulated in 1.0 mL of water for injection/EtOH (9:1, (v/v)).
Products [18F]34 and [18F]17 were obtained in radiochemical yields
(rcy) of 29.2 3.3% (n = 5) and 30.2 4.2% (n = 3) (decay corrected
3
3
13-NH), 4.01 (t, JH,H = 6.5 Hz, 2H, 26-CH2), 3.90 (d, JH,H = 11.8
2
Hz, 2H, 3-CH, 4-CH), 3.68 (d, JH,H = 11.9 Hz, 2H, 3-CH, 4-CH),
2.57−2.48 (m, 4H, 14-CH2, 21-CH2), 2.44 (s, 3H, 33-CH3), 1.96 (t,
3JH,H = 8.5 Hz, 2H, 1-CH2), 1.60−1.67 (m, 2H, 25-CH2), 1.50−1.57
(m, 2H, 22-CH2), 1.47 (s, 9H, 8-CH3, 9-CH3, 10-CH3), 1.42 (m, 6H,
5-CH3, 6-CH3), 1.38−1.20 (m, 4H, 23-CH2, 24-CH2). 13C NMR (101
MHz, CDCl3): δ 154.8 (C-11), 144.6 (C-32), 140.0 (C-15), 139.2 (C-
20), 133.2 (C-27), 129.7 (single peak with high intensity C-30, C-31),
128.3 (single peak with high intensity C-16, C-17), 128.2 (single peak
with high intensity C-18, C-19), 127.8 (single peak with high intensity
C-28, C-29), 98.3 (C-7), 79.3 (C-12), 70.6 (C-26), 66.3 (single peak,
C-3, C-4), 51.6 (C-2), 35.3 (C-21), 33.6 (C-25), 31.2 (C-22), 28.7 (C-
14), 28.6 (C-1), 28.5 (C-24), 28.4 (single peak with high intensity, C-
23, C-9, C-10), 27.4 (C-8), 25.2 (C-6), 21.6 (C-33), 19.6 (C-5).
HRMS (ESI+): C32H47NO7S+Na+: calcd, 612.2971; found,. 612.2965.
8-[4-(2-{5-[(tert-Butoxycarbonyl)amino]-2,2-dimethyl-1,3-diox-
an-5-yl}ethyl)phenyl]octyl-4-methylbenzenesulfonate (38). Synthe-
sis and purification of 38 were performed as described for compound
1
37. Yield: 69 mg (59%). H NMR (300 MHz, CDCl3): δ 7.83−7.74
based on cyclotron-derived [18F]fluoride ions) in 115
12 min
(m, 2H, 32-CH, 33-CH), 7.37−7.30 (m, 2H, 30-CH, 31-CH), 7.13−
7.03 (m, 4H, 16-CH, 17-CH, 18-CH, 19-CH), 4.98 (s, 1H, NH), 4.01
(t, 3JH,H = 6.5 Hz, 2H, 28-CH2), 3.90 (d, 3JH,H = 11.8 Hz, 2H, 3-CH, 4-
([18F]34) and 115 16 min ([18F]17) from the end of the
radionuclide production. The target compounds were isolated in >99%
radiochemical purity (rcp). Radiochemical purities of [18F]34 (tR
([18F]34) = 10.82 min) and [18F]17 (tR ([18F]17) = 11.15 min)
were determined by analytical radio-HPLC B (method B).
4.4. Biological Studies. 4.4.1. In Vivo Testing. The compounds
were dissolved to a concentration of 0.2 mg/mL in 0.9% NaCl and
injected intraperitoneally at 1.25 mg/kg body weight. Twenty four
hours later, blood was drawn from the retroorbital plexus in 17.8 mM
EDTA as an anticoagulant, and lymphocytes were analyzed by flow
cytometry (Gallios, Beckman Coulter)35 using antibodies to CD4,
CD8, CD45, and B220 (Becton Dickinson) after lysis of red blood
cells with BD PharmLyse (Becton Dickinson) according to the
manufacturer’s instructions.
4.4.2. In Vitro Testing. Chinese hamster ovary (CHO) cells
overexpressing the human S1P1 and S1P3 receptors were serum-
starved overnight and stimulated with compounds and S1P side-by-
side in the same experiment for the indicated times. Cells were then
lysed, and samples were separated on a 12% SDS-PAGE gel. After
transfer to PVDF membranes, western blotting was performed with an
antibody to the dually phosphorylated p44/42 MAP kinase (Cell
Signaling), and signals were visualized by ECL.
3
CH), 3.68 (d, JH,H = 11.7 Hz, 2H, 3-CH, 4-CH), 2.60−2.48 (m, 4H,
14-CH2, 20-CH2), 2.44 (s, 3H, 35-CH3), 2.02−1.91 (m, 2H, 1-CH2),
1.65−1.59 (m, 2H, 22-CH2), 1.47 (s, 9H, 8-CH3, 9-CH3, 10-CH3),
1.42 (m, 8H, 5-CH3, 6-CH3, 27-CH2), 1.26 (m, 8H, 23-CH2, 24-CH2,
25-CH2, 26-CH2). 13C NMR (75 MHz, CDCl3): δ 154.8 (C-11),
144.6 (C-34), 140.3 (C-15), 139.1 (C-20), 133.1 (C-29), 129.7 (single
peak with high intensity, C-32, C-33), 128.4 (single peak with high
intensity, C-16, C-17), 128.2 (single peak with high intensity, C-18, C-
19), 127.8 (single peak with high intensity, C-30, C-31), 98.3 (C-7),
77.2 (C-12), 70.6 (C-28), 66.3 (C-3, C-4), 51.6 (C-2), 35.4 (C-21),
31.4 (C-22), 29.2 (C-24), 29.1 (C-25), 28.8 (C-14), 28.7 (C-27), 28.6
(C-1), 28.4 (single peak with high intensity, C-8, C-9, C-10), 27.4 (C-
23), 26.9 (C-26), 25.3 (C-5), 21.6 (C-6), 19.7 (C-35). HRMS (ESI+):
C34H51NO7S + Na+: calcd, 640.3284; found, 640.3278.
4.3. Radiolabeling Experiments. 4.3.1. General Methods. The
radiosynthesis was carried out on a modified PET tracer radio-
synthesiser (TRACERLab FxFDG, GE Healthcare). The recorded data
were processed by TRACERLab Fx software (GE Healthcare).
Separation and purification of the radiosynthesized compounds were
performed on the following semipreparative radio-HPLC system A: K-
500 and K-501 pump, K-2000 UV detector (Herbert Knauer GmbH),
NaI(TI) Scintibloc 51 SP51 γ-detector (Crismatec), and an ACE 5 AQ
column (250 mm × 10 mm). Method A started with a linear gradient
from 10 to 90% CH3CN in water (0.1% TFA) over 30 min, holding
for 5 min, and followed by a linear gradient from 90 to 10% CH3CN in
water (0.1% TFA) over 5 min, with λ = 254 nm and a flow rate of 5.5
mL min−1. Radiochemical purities were determined using the
analytical radio-HPLC system B: two Smartline 1000 pumps and a
Smartline UV detector 2500 (Herbert Knauer GmbH), a GabiStar γ-
4.4.3. In Vitro Stability. An aliquot of formulated [18F]17 (20 μL,
8.7 MBq) was added to a sample of human serum (200 μL), and the
mixture was incubated at 37 °C. Samples of 20 μL each were taken
after periods of 10, 30, 60, and 90 min and quenched in MeOH/
CH2Cl2 (1:1 (v/v), 100 μL) followed by centrifugation for ≥5 min.
The supernatant was analyzed by analytical radio-HPLC B [method B,
tR ([18F]17) = 11.15 min].
4.4.4. Animals. Adult C57BL/6 mice (age 13 weeks, 21−26 g body
weight, male) were anaesthetised by isoflurane/O2, and one lateral tail
vein was cannulated using a 27 G needle connected to 15 cm
polyethylene catheter tubing. [18F]34 or [18F]17 (400 kBq/g
bodyweight) was injected as a bolus (100 μL of compound flushed
with 100 μL of saline; syringe pump speed, 300 μL/min) via the tail
vein, and subsequent PET scanning was performed. All experiments
performed in the study were in accordance with the German Law on
the Care and Use of Laboratory Animals and approved by the local
authorizing agency of North Rhine-Westphalia.
detector (Raytest Isotopenmessgerate GmbH), and a Nucleosil 100-5
̈
C-18 column (250 mm × 4 mm). Method B started with a linear
gradient from 10 to 90% CH3CN in water (0.1% TFA) over 15 min,
followed by a linear gradient from 90 to 10% CH3CN in water (0.1%
TFA) over 3 min, with λ = 254 nm and a flow rate of 1.0 mL min−1.
The recorded data of both HPLC systems were processed by GINA
Star software (Raytest Isotopenmessgerate GmbH). No-carrier-added
̈
aqueous [18F]fluoride was produced on a RDS 111e cyclotron (CTI-
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4.4.5. Small Animal PET Scanning. PET experiments were carried
out using a submillimeter high-resolution (0.7 mm full width at half-
maximum) small animal scanner (32 module quadHIDAC, Oxford
Positron Systems Ltd., Oxford, UK) with uniform spatial resolution
(<1 mm) over a large cylindrical field (165 mm diameter, 280 mm
axial length).39 List-mode data were acquired for 120 min and
reconstructed into dynamic time frames using an iterative
reconstruction algorithm. Subsequently, the scanning bed was
transferred to the computed tomography (CT) scanner (Inveon,
Siemens Medical Solutions, USA), and a CT acquisition with a spatial
resolution of 80 μm was performed for each mouse. Reconstructed
image data sets were coregistered based on extrinsic markers attached
to the multimodal scanning bed and the image analysis software
(Inveon Research Workplace 3.0, Siemens Medical Solutions, USA).
Three-dimensional volumes of interest (VOIs) were defined over the
respective organs in CT data sets, transferred to the coregistered PET
data, and analyzed quantitatively. Regional uptake was calculated as the
percentage of injected dose by dividing counts per milliliter in the VOI
by total counts in the mouse multiplied by 100 (%ID/mL).
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ASSOCIATED CONTENT
■
S
* Supporting Information
1
Copies of the H, 13C, and 19F NMR spectra of synthesized
compounds and Radio-HPLC chromatograms. In vitro stability
of [18F]17 after incubation in human blood serum at 37 °C for
30 min. This material is available free of charge via the Internet
at http://pubs.acs.org.
(10) Brinkmann, V.; Davis, M. D.; Heise, C. F.; Albert, R.; Cottens,
S.; Hof, R.; Bruns, C.; Prieschl, E.; Baumruker, T.; Hiestland, 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.
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Fingolimod (FTY720), the sphingosine-1-phosphate receptor modu-
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AUTHOR INFORMATION
■
Corresponding Author
*Phone: +49 251 83 33281. Fax: +49 251 83 39772. E-mail:
haufe@uni-muenster.de.
Author Contributions
◆R.S.S. and S.S.S. contributed equally to this work.
Notes
The authors declare no competing financial interest.
(13) Mehling, M.; Brinkmann, V.; Antel, J.; Bar-Or, A.; Goebels, N.;
Vedrine, C.; Kuhle, J.; Lindberg, R. L. P.; Kappos, L. FTY720 therapy
exerts differential effects on T cell subsets in multiple sclerosis.
Neurology 2008, 71, 1261−1267.
(14) Urbano, M.; Guerrero, M.; Rosen, H.; Robert, E. Modulators of
the sphingosine-1-phosphate receptor 1. Bioorg. Med. Chem. Lett. 2013,
23, 6377−6389.
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subjects: seeing fundamental biological processes in a new light. Genes
Dev. 2003, 17, 545−580.
(16) Foster, C. A.; Howard, L. M.; Schweitzer, A.; Persohn, E.;
Hiestand, P. C.; Balatoni, B.; Reuschel, R.; Beerli, C.; Schwartz, M.;
Billich, A. Brain penetration of the oral immunomodulatory drug
FTY720 and its phosphorylation in the central nervous system during
experimental autoimmune encephalomyelitis: consequences for mode
of action in multiple sclerosis. J. Pharmacol. Exp. Ther. 2007, 323,
469−475.
ACKNOWLEDGMENTS
■
This work was supported in part by the Deutsche
Forschungsgemeinschaft (Collaborative Research Center, SFB
656, projects A06, C06, and Z05). R.S.S. is grateful to the NRW
International Graduate School of Chemistry, University of
Munster (GSC-MS), for a scholarship.
̈
ABBREVIATIONS USED
■
AS, specific activity; CNS, central nervous system; CVS,
cardiovascular system; CT, computed tomography; DAST,
diethylamino sulfurtrifluoride; DIPEA, N,N-diisopropylethyl-
amine; DMP, 2,2-dimethoxypropane; GPCR, G-protein
coupled receptor; MS, multiple sclerosis; NKs, natural killer
cells; PTSA, p-toluenesulfonic acid; PBSF, perfluoro-1-
butanesulfonyl fluoride; ROI, region of interest; SEM, standard
error of the mean; SphK, sphingosine kinase; S1P, sphingosine-
1-phosphate; S1PR, sphingosine-1-phosphate receptor;
TBDPA, di-tert-butyl N,N-diethyl-phosphoramidite; VOI, vol-
ume of interest; WBC, white blood cells
(17) Meno-Tetang, G. M. L.; Li, H.; Mis, S.; Pyszczynski, N.;
Heining, P.; Lowe, P.; Jusko, W. J. Physiologically based
pharmacokinetic modeling of FTY720 (2-amino-2[2-(4-octylphenyl)-
ethyl]propane-1,3-diol hydrochloride) in rats after oral and intra-
venous doses. Drug Metab. Dispos. 2006, 34, 1480−1487.
(18) Briard, E.; Orain, D.; Beerli, C.; Billich, A.; Streiff, M.; Bigaud,
M.; Auberson, Y. P. BZM055, an iodinated radiotracer candidate for
PET and SPECT imaging of myelin and FTY720 brain distribution.
ChemMedChem 2011, 6, 667−677.
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