Synthesis of new ligands for targeting the S1P1 receptor

Article abstract of DOI:10.1016/j.bmc.2015.01.014

Sphingosine-1-phosphate (S1P) influences various fundamental biological processes by interacting with a family of five G protein-coupled receptors (S1P_1-5). FTY720, a sphingosine analogue, which was approved for treatment of relapsing forms of multiple sclerosis, is phosphorylated in vivo and acts as an agonist of four of the five S1P receptor subtypes. Starting from these lead structures we developed new agonists for the S1P₁ receptor. The biological activity was tested in vivo and promising ligands were fluorinated at different positions to identify candidates for positron emission tomography (PET) imaging after [¹⁸F]-labelling. The radioligands shall enable the imaging of S1P₁ receptor expression in vivo and thus may serve as novel imaging markers of S1P-related diseases.

Full text of DOI:10.1016/j.bmc.2015.01.014

Bioorganic & Medicinal Chemistry 23 (2015) 1011–1026
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of new ligands for targeting the S1P1 receptor
Stefanie S. Schilson a, , Petra Keul b, , Rizwan S. Shaikh a,c, Michael Schäfers d,e,f, Bodo Levkau b,
Günter Haufe a,e,f,
⇑
a Organic Chemistry Institute, University of Münster, Corrensstraße 40, D-48149 Münster, Germany
b Institute for Pathophysiology, Westdeutsches Herz- und Gefäßzentrum, University Hospital, Essen, University of Duisburg-Essen, Hufelandstraße 55, D-45122 Essen, Germany
c NRW Graduate School of Chemistry, University of Münster, Wilhelm-Klemm-Straße 10, D-48149 Münster, Germany
d Department of Nuclear Medicine, University of Münster, Albert-Schweizer-Campus 1, D-48149 Münster, Germany
e European Institute for Molecular Imaging, University of Münster, Waldeyerstraße 15, D-48149 Münster, Germany
f Cells-in-Motion, Centre of Excellence, University of Münster, Waldeyerstraße 15, D-48149 Münster, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Sphingosine-1-phosphate (S1P) influences various fundamental biological processes by interacting with a
family of five G protein-coupled receptors (S1P1–5). FTY720, a sphingosine analogue, which was approved
for treatment of relapsing forms of multiple sclerosis, is phosphorylated in vivo and acts as an agonist of
four of the five S1P receptor subtypes. Starting from these lead structures we developed new agonists for
the S1P1 receptor. The biological activity was tested in vivo and promising ligands were fluorinated at dif-
ferent positions to identify candidates for positron emission tomography (PET) imaging after [18F]-label-
ling. The radioligands shall enable the imaging of S1P1 receptor expression in vivo and thus may serve as
novel imaging markers of S1P-related diseases.
Received 20 November 2014
Revised 7 January 2015
Accepted 7 January 2015
Available online 16 January 2015
Keywords:
Fluorine
FTY720 analogues
G protein-coupled receptors
Sphingosine-1-phosphate (S1P)
S1P1 receptor agonists
Structure–activity relationship
Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction
NH2
OR
Sphingosine (1) and its phosphorylated form sphingosine-1-
phosphate (S1P, 2) (Fig. 1) influence various fundamental biologi-
cal processes such as cell growth, apoptosis, inflammation,
immune response and cardiovascular functions by interacting with
a family of five G protein-coupled receptors (S1P1–5) which are
expressed in different organ systems.1
In plasma, the majority of S1P is associated with high-density
lipoproteins (HDL) and is at least partially responsible for several
beneficial effects of HDL on cardiovascular risk.2 In patients with
coronary artery disease (CAD) and myocardial infarction, the S1P
levels in plasma are increased compared to healthy individuals
and could serve as potential biomarker.3
S1P is known to regulate lymphocyte trafficking out of second-
ary lymphatic organs. This function arises from the interaction of
S1P with the S1P1 receptor. There is a S1P concentration gradient
between lymph node (<pM), lymph (pM) and plasma (lM) along
which lymphocytes actively migrate to exit the lymph node. At
OH
1
(R = H)
2 (R = P(O)(OH)2
NH2
OH
OH
3
Figure 1. Structure of sphingosine (1), S1P (2) and FTY720 (3).
lymphocytes is down-regulated, which leads to their retention in
the lymph nodes and allows clonal expansion. Thereafter, the
S1P1 receptor is re-expressed and immunocompetent lymphocytes
exit the lymph nodes to battle the pathogen.4
FTY720 (3) is a sphingosine analogue which is phosphorylated
in vivo and functions as a partial agonist at four of the five S1P
receptor subtypes. The engagement of S1P1 by FTY720-phosphate
leads to its downregulation and results in the blockade of lympho-
cyte egress from lymph nodes.5 In 2010, FTY720 was approved by
the US Food and Drug Administration (FDA) and one year later by
the European Medicines Agency (EMA) for the treatment of
the beginning of an immune response, the expression of S1P1 on
⇑
Corresponding author. Tel.: +49 251 83 33281; fax: +49 251 83 39772.
E-mail address: haufe@uni-muenster.de (G. Haufe).
These two authors have contributed equally to the manuscript.
http://dx.doi.org/10.1016/j.bmc.2015.01.014
0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.

1012
S. S. Schilson et al. / Bioorg. Med. Chem. 23 (2015) 1011–1026
relapsing forms of multiple sclerosis (trade name GilenyaÒ) thus
becoming the first example of a new class of pharmaceuticals,
the S1P receptor modulators.6
2.1. Synthesis of 3-deoxysphingosine analogues
The synthesis of the 3-deoxysphingosine analogues (Scheme 1)
was based on preceding works by Marhold et al.15 The fluorinated
allylic alcohol 7 was prepared according to our well-established
three step sequence involving bromofluorination of hexadec-1-
ene, HBr-elimination and SeO2-mediated allylic oxidation.16 The
non-fluorinated allylic alcohol 6 was prepared by direct SeO2-med-
iated allylic oxidation of hexadec-1-ene (4). An enzyme-catalyzed
kinetic resolution of the racemic allylic alcohols using Candida ant-
arctica lipase provided the enantiomerically enriched (R)-alcohols.17
After esterification with Boc-glycine the chelate ester enolate Clais-
en rearrangement proceeded with complete chirality transfer from
C-3 of the allylic esters 8 and 9 to C-2 of the amino acids 10 and 11
due to the preferred lowest energy chair-like, six-membered transi-
tion state.15 Thus, starting from the (R)-alcohols, the amino function
is obtained in the correct configuration as compared to sphingosine.
FTY720, apart from sphingosine and S1P, is an interesting lead
structure for the development of fluorinated analogues, which
might deliver the non-radioactive parent compounds for later syn-
thesis of [18F]-labelled tracers for positron emission tomography
(PET) imaging.7 There is considerable evidence for the significant
effect of fluorine substituents on the physico-chemical proper-
ties,8,9 the chemical reactivity10,11 and the biological activity and
selectivity12,13 of particular compounds. Most recently, a fluori-
nated analogue of FTY720-phosphate has been synthesized and
its in vitro activity has been tested,14a and a [18F]-labelled analogue
of W146 has been prepared and tested as an S1P antagonist.14b
Here, we were interested in developing fluorinated S1P1 recep-
tor subtype-specific ligands, which—after [18F]-labelling and appli-
cation in PET imaging—may potentially serve to locate S1P1
expression in vivo.
The a-amino acids 10 and 11 were reduced to the correspond-
ing b-aminoalcohols using a one-pot two-step sequence reported
by Ho et al.18 First, using isobutyl chloroformate, the acids were
converted into mixed anhydrides which were subsequently
reduced to the alcohols with sodium borohydride. Cleavage of
the Boc-group with trifluoroacetic acid (TFA) gave the aminoalco-
hols 14 and 15 in high yields.
2. Results and discussion
For the synthesis of new ligands we focused on three structural
modifications of sphingosine (1) and FTY720 (3): (i) the synthesis
of 3-deoxy analogues of sphingosine, (ii) the combination of the
2-aminopropane-1,3-diol moiety with long aliphatic hydrocarbon
chains, and (iii) fluorinated analogues of these compounds. The tar-
get molecules are depicted in Figure 2.
The introduction ofthephosphatemoietywasplannedviaamixed
dimethylphosphateesterasintermediateslikethiscanbe easilypuri-
fied. In most publications concerning the synthesis of S1P the method
with trimethyl phosphate and tetrabromomethane described by Oza
and Corcoran is used for the synthesis of the phosphate ester
intermediate.19 But the reaction under these conditions gave insuffi-
cient and unreproducible yields of the esters 16 and 17. Therefore,
methods reported by Szulc et al. using phase transfer catalysis were
tested.20 But again the obtained yields were not reproducible. A
combination of both methods using dibromotetrachloroethane
(DBTCE) as halogen donor in absolute pyridine finally resulted in
reproducibly high yields of 16 (98%) and 17 (79%) (Scheme 2).
The deprotection of 16 and 17 using TMSBr proved to be diffi-
cult as the bromides 20 and 21 were obtained as main products
in a mixture with the desired phosphates 18 and 19. Unfortunately,
this reaction could not be improved. Therefore, the mixtures of 18
and 20 or 19 and 21, respectively, were separated by solid phase
extraction (SPE) to give the pure phosphates, which were tested
as S1P receptor agonists subsequently (see Section 2.5).
F
NH2
OH
OH
NH2
X
NH2
X
OH
OH
OH
O
P
11
11
O
X = H, F
X = H, F
X
NH2
X
NH2
OH
OH
11
X = H, F
11
X = H, F
OH
OH
NH2
F
NH2
OH
OH
X
11
n
2.2. Synthesis of aliphatic analogues of FTY720
OH
X = H, F; n = 10, 11
Based on preceding investigations by Fujita et al.21 we com-
bined the 2-aminopropane-1,3-diol moiety with long aliphatic
Figure 2. Target molecules.
X
X
X
i
iii
ii
11
11
11
OH
6 (X = H), 60%
O
NHBoc
8
(X = H), 86%
4 (X = H)
5 (X = F)
O
8
(R)- (X = H), 81%
6
(R)- (X = H), 84%
9
7
(X = F), 92%
(R)-9 (X = F), 86%
(X = F), 58%
(R)-7 (X = F), 80%
X
NHBoc
X
NHBoc
OH
X
NH2
vi
iv
v
OH
OH
11
11
11
O
10 (X = H), 92%
12 (X = H), 97%
14 (X = H), 98%
14
(X = F), 96%
(R)-15 (X = F), 90%
10
(R)- (X = H), 75%
(R)-12 (X = H), 64%
(R)- (X = H), 82%
15
11 (X = F), 86%
(R)-11 (X = F), 80%
13
(X = F), 68%
(R)-13 (X = F), 32%
Scheme 1. Synthesis of 3-deoxysphingosine analogues. Reagents and conditions: (i) SeO2, tBuOOH, HOAc, DCM, rt; (ii) lipase, vinyl acetate TBME, rt, (optional); (iii) Boc-
glycine, DCC, DMAP, DCM, rt; (iv) (a) LDA, Et2O, 0 °C to À78 °C (b) ZnCl2, Et2O, À78 °C to rt; (v) (a) iBuOCOCl, NEt3, THF, À10 °C, (b) NaBH4, water, THF, 0 °C; (vi) TFA, DCM, 0 °C
to rt.

S. S. Schilson et al. / Bioorg. Med. Chem. 23 (2015) 1011–1026
1013
X
NHBoc
OH
X
NHBoc
O
O
O
O
P
OMe
OMe
O
i
i
ii
Br
O
P
O
O
iv
O
11
11
11
32
2
O
12
16
(X = H), 98%
17 (X = F), 79%
(X = H)
13 (X = F)
30
31
, 90%
, 81%
NHAc
CO2Et
CO2Et
iii
v
X
NH2
X
NH2
Br
OH
OH
OH
O
O
Br
11
33, 69%
11
11
ii
+
P
11
11
34, 68%
35, 76%
18 (X = H), 5%
19
20
(X = H)
21 (X = F)
(X = F), 5%
NH2
OH
NHAc
vi
vii
OH
OH
Scheme 2. Synthesis of the 3-deoxy S1P analogues. Reagents and conditions: (i)
P(OMe)3, DBTCE, pyridine, molecular sieve, À10 °C to rt, 15 h; (ii) TMSBr, DCM, 0 °C
to rt, SPE with MeOH/H2O (60:40).
11
11
OH
36
, 68%
37
, 97%
Scheme 4. Synthesis of the unsaturated analogue. Reagents and conditions: (i)
P(OEt)3, 80 °C; (ii) NaH, tetradecanal, THF, 0 °C to rt; (iii) DIBAL-H, THF, 0 °C to rt;
(iv) NBS, PPh3, DCM, À20 °C to rt; (v) compound 23, Cs2CO3, MeCN, reflux; (vi) LiCl,
NaBH4, THF, EtOH, 0 °C to rt; (vii) NaOH, MeOH, reflux.
hydrocarbon chains applying their protocol with some minor mod-
ifications (Scheme 3). In addition we used the Boc-protected
amidomalonate 24 for the preparation of the corresponding Boc-
protected aminodiols. Inspired by the work of Deiters et al.,22 we
have chosen caesium carbonate as the base for the alkylation of
the amidomalonates 23 and 24. For small scale reactions (up to
3 mmol) the reaction time could be shortened from 4–5 h to 1 h
using microwave irradiation. The second modification was the
combination of lithium chloride and sodium borohydride for the
reduction of the diesters 25 and 26. An optimization of the reaction
conditions reported by Mori et al.23 gave the protected aminodiols
27 and 28 in high yields without side products.
NHBoc
NHBoc
O
PPh3Br
i
+
9
9
O
O
O
O
38
39
40
, 51%
NHBoc
NH2
ii
iii
OH
9
9
O
O
OH
For the synthesis of the unsaturated analogue 37 the preceding
work of Nikolova and Haufe24 was used. The allylic alcohol 33 was
prepared by Horner–Wadsworth–Emmons reaction of tetradecanal
with 31, followed by the reduction of the resulting ester 32 with
DIBAL-H (Scheme 4). The allylic alcohol was converted into the
bromide 34 by reaction of N-bromo succinimide (NBS) and tri-
phenylphosphine which was then used for the alkylation of 23.
An alternative synthetic route to 2-alkyl-2-aminopropane-1,3-
diols was offered by the aldehyde 39, which has already been used
by Kim et al.25 and Aidhen et al.26 for the synthesis of FTY720. 39
was prepared according to a two-step procedure by Ooi et al.27
starting from tris(hydroxymethyl) aminomethane. Wittig reaction
with the phosphonium bromide 38 gave the olefin 40, which was
subsequently reduced to 41 (Scheme 5). The protecting groups
were finally removed with TFA in analogy to a method by Kim
et al.25
Aminodiols 29, 37 and 42 were identified as potent immuno-
suppressants. In wild-type mice they showed an activity compara-
ble to that of FTY720 (see Section 2.5). Thus, 29 and 37 were
chosen as lead compounds. In the following we synthesized differ-
ent fluorinated analogues to identify the best position for labelling
with [18F] for PET imaging. The fluorine atom was introduced in
different positions: (i) in the head group, (ii) close to it and (iii)
41
42
, 58%
, 51%
Scheme 5. Alternative synthesis to 2-alkyl-2-aminopropane-1,3-diols. Reagents
and conditions: (i) K2CO3, 1,4-dioxane, reflux; (ii) H2, Pd/C, MeOH, rt; (iii), TFA,
DCM, water, rt.
compounds can be labelled at this alkyl chain. The syntheses of
the modified analogues are reported in the following sections.
2.3. Synthesis of monofluorinated and N-alkylated analogues
2.3.1. Synthesis of the fluorodeoxy analogue
The synthesis of the fluorodeoxy analogue 43 was accomplished
by two different strategies. The deoxyfluorination of alcohol 29
provided direct access to the desired product in one step. Alterna-
tively, 29 can be converted into an active intermediate like a cyclic
sulfite or a cyclic sulfate which has to be treated with a nucleo-
philic fluorine source in a second step. For the deoxyfluorination,
different fluorinating agents like DAST, XtalFluor-E and Fluolead™
were tested (Scheme 6, route A). Only the reaction with 1.0 equiv
DAST gave an isolatable amount of the desired product with a yield
of 11%, while there was no full conversion of 29. Following route B,
the aminodiol 29 was converted into the cyclic sulfite 44.28 The
originally planned synthesis involved a ring opening of a cyclic sul-
fate in order to avoid possible side products. The oxidation of 44 to
in the remote
x-position. Additionally, different N-alkylated
aminopropane-1,3-diols were synthesized to check whether the
O
O
O
O
NHR
CO2Et
ii
i
O
O
O
O
11
NH2.HCl
22
NHR
CO2Et
25 (R = Ac), 86%
(R = Boc), 79%
23 (R = Ac), 96%
(R = Boc), 91%
26
24
NHR
OH
(R = Ac), 75%
(R = Boc), 98%
NH2
OH
iv
iii
OH
OH
11
27
11
29, 97% (a), 93% (b)
28
Scheme 3. Synthesis of aminodiol 29. Reagents and conditions: (i) (a) NEt3, AcCl, DCM, 0 °C to rt, (b) Boc2O, NaOH, 1,4-dioxane, 5 °C to rt; (ii) 1-iodohexadecane, Cs2CO3,
MeCN, reflux; (iii) LiCl, NaBH4, THF, EtOH, 0 °C to rt; (iv) (a) NaOH, MeOH, reflux, (b) TFA, DCM 0 °C to rt.

1014
S. S. Schilson et al. / Bioorg. Med. Chem. 23 (2015) 1011–1026
NH2
OH
NH2
ferent leaving groups like bromide, iodide and tosylate were tested
in the substitution reaction. The reaction of 2-fluoro-1-iodo-hexa-
decane (46) gave the best result (47% yield) in a scale up to 3 mmol.
However, compound 46 has been incompletely consumed. After
column chromatographic purification of the fluorinated diester
48, unconverted iodide 46 has been recovered. Reduction of the
ester groups with lithium chloride and sodium borohydride and
the subsequent deprotection of the amino group yielded the final
product 50.
A
OH
OH
11
11
i
F
29
43, 11% (i), 28% (iii)
B
ii
NH2
iii
O
O
S
O
11
44, 71%
Scheme 6. Synthesis of the fluorodeoxy analogue. Reagents and conditions: (i)
DAST, DCM, À78 °C to rt; (ii) SOCl2, DCM, reflux, silica gel chromatography in the
presence of Et3N; (iii) Et3NÁ3HF, 100 °C, microwave.
For the synthesis of the unsaturated analogues preceding work
of our group was followed.24 The mixture of the diastereomeric flu-
oroallylic alcohols 54 was synthesized by Wittig reaction of
tetradecanal and the phosphoniumbromide 52 followed by the
reduction of the resulting unsaturated esters 53. The ratios of
E/Z-isomers were determined by GC or 19F NMR spectroscopy. After
tosylation of the alcohols and alkylation of 23, the diastereomeric
diesters 56 were separated by column chromatography and
reduced separately to the corresponding aminodiols. However,
the conversion was very slow. The reactions were stopped after
six days although there was still starting material remaining. The
aminoalcohols 58 were found as side products, which probably
result from traces of lithium hydroxide in the lithium chloride
leading to a hydrolysis of an ester function followed by decarbox-
ylation. While (Z)-57 was fully separated from the side product by
column chromatography, (E)-57 was isolated in a mixture with (E)-
58. Due to the small amount of substance this mixture was used in
the following deprotection reaction and the aminodiol (E)-59 was
separated from the corresponding aminoalcohol by column
chromatography.
the corresponding sulfate however was not successful. Hence, the
sulfite 44 was treated with different fluoride sources (TBAFÁ
(tBuOH)4, Et3NÁ3HF, KF). The best result was obtained by the reac-
tion with Et3NÁ3HF. Here a mixture of the starting material 44, the
aminodiol 29 (due to the nucleophilic attack at the sulfur atom)
and the fluorodeoxy analogue 43 was obtained. The desired com-
pound 43 was isolated by silica gel flash column chromatography
in 28% yield.
All reactions provided no full conversion and parts of the start-
ing material were reisolated. Under the reaction conditions with
the TBAF complex and KF only the aminodiol 29 was formed due
to the nucleophilic attack at the sulfur atom.29
2.3.2. Synthesis of the 4-fluoro aminodiols
The syntheses of the 4-fluoro analogues were realized by alkyl-
ation of 23 with already fluorinated building blocks like 46 and 55.
The reaction sequences are depicted in Schemes 7 and 8. For the
synthesis of the saturated analogue which also represents an
analogue of phytosphingosine with aminopropanediol moiety, dif-
2.3.3. Synthesis of
x
-substituted aminodiols
-modified compounds started from the
The synthesis of the
x
commercially available pentadecanolide (60) and hexadecanolide
(61) (Scheme 9). First, the lactones 60 and 61 were converted into
the bromoalcohols 62 and 63 according to a procedure by Luu
et al.30 The products 62 and 63 were contaminated with 17% and
5%, respectively, of the corresponding dibromides, which were eas-
ily removed by silica gel flash column chromatography. After the
substitution reaction, 64 and one portion of 65 were converted into
the corresponding fluorinated esters 66 and 67 by deoxyfluorina-
O
O
F
F
NHAc
CO2Et
CO2Et
X
i
EtO
OEt
+
11
11
NHAc
45 (X = Br)
46
23
48, 17% (from 45)
46
)
(X = I)
47 (X = OTs)
47% (from
3% (from 47)
tion with DAST at low temperature. The resulting
x-fluorinated
F
NHAc
OH
F
NH2
OH
ii
iii
diesters 66 and 67, as well as the other portion of 65, were reduced
to the aminodiols 68 to 70 by in situ prepared lithium borohydride.
The deprotection of the amino group gave the final compounds 71
to 73 in high yields. Surprisingly, it was difficult to dissolve 73 in
0.9% sodium chloride solution. Thus, the biological activity was
determined starting from the corresponding hydrochloride salt.
11
11
OH
OH
49
, 77%
50, 58%
Scheme 7. Synthesis of the saturated 4-fluoro aminodiol. Reagents and conditions:
(i) Cs2CO3, MeCN, reflux; (ii) LiCl, NaBH4, THF, EtOH, 0 °C to rt; (iii) NaOH, MeOH,
reflux.
O
O
F
i
ii
Br
PPh3Br
O
O
O
11
F
F
O
51
52, 94%
53, 77%, E/Z (GC): 34:66
F
F
F
NHAc
CO2Et
CO2Et
iii
iv
v
OH
OTs
11
11
11
54
55, 58%, E/Z (NMR): 15:85
, 61%, E/Z (GC): 39:61
(E)-56, 7%
(Z)-56, 53%
F
NHAc
OH
F
NH2
OH
F
NHAc
OH
vi
vii
OH
+
11
11
11
OH
(E)- , 50% (mixture with (E)-
57
57
(Z)- , 51%
58
)
58
58
(E)-59, 23%
(Z)-59, 87%
(E)-
(Z)-
Scheme 8. Synthesis of the unsaturated 4-fluoro analogues. Reagents and conditions: (i) PPh3; (ii) n-BuLi, tetradecanal, THF, 0 °C to rt; (iii) DIBAL-H, THF, 0 °C to rt; (iv) KOH,
TsCl, DCM, 0 °C to rt; (v) compound 23, Cs2CO3, MeCN, reflux; (vi) LiCl, NaBH4, THF, EtOH, 0 °C to rt; (vii) NaOH, MeOH, reflux.

S. S. Schilson et al. / Bioorg. Med. Chem. 23 (2015) 1011–1026
1015
Table 1
Immunosuppressive activity
NHAc
CO2Et
CO2Et
O
O
Br
ii
i
X
HO
n
n
Compound WBC
(106/mL)
CD4+
CD8+
B cells
Activity*
(%)
n
(106/mL)
(106/mL)
(106/mL)
60 (n = 10)
(n = 11)
62 (n = 10), 58%
63
64
(n = 10, X = OH), 80%
61
(n = 11), 97%
65 (n = 11, X = OH), 74%
66 (n = 10, X = F), 40%
Control**
92
(R)-14
18
8.94 0.59 0.73 0.07 0.48 0.05 4.72 0.53
—
0
28
43
30
iii
15.59
6.90
7.15
7.06
0.61
0.43
0.34
0.42
0.92
0.48
0.49
0.59
7.28
2.91
2.76
2.49
67
(n = 11, X= F), 25%
NHAc
OH
NH2
OH
iv
X
v
19
X
n
n
FTY720 (3) 3.69 0.63 0.02
27
29
37
42
43
50
(Z)-59
(E)-59
71
0
0.04 0.01 0.61 0.12 97
0.62 4.25 18
OH
(n = 10, X = F), 23%
69 (n = 11, X = F), 39%
70
OH
7.43
0.49
68
71 (n = 10, X = F), 90%
4.14 0.60 0.09 0.02 0.08 0.01 1.27 0.26 85
5.02 1.04 0.10 0.01 0.12 0.05 1.17 0.33 83
72
(n = 11, X = F), 90%
73 (n = 11, X = OH), 94%
(n = 11, X = OH), 90%
1.73
7.05
11.35
5.31
5.27
3.77
0.04
0.42
0.54
0.27
0.28
0.03
0.04
0.39
0.60
0.41
0.37
0.09
0.60
3.54
4.81
3.23
3.41
0.77
93
30
10
55
53
95
Scheme 9. Synthesis of the
x-substituted analogues. Reagents and conditions: (i)
(a) LiAlH4, THF, 0 °C to rt, (b) HBr, cyclohexane, reflux; (ii) 23, Cs2CO3, MeCN, reflux;
(iii) DAST, DCM, À78 °C to rt; (iv) LiCl, NaBH4, THF, EtOH, 0 °C to rt; (v) NaOH,
MeOH, reflux.
72
3.74 0.70 0.15 0.08 0.13 0.04 1.52 0.04 75
73
89
6.07
6.92
0.73
1.06
0.49
0.81
3.71
3.78
0
0
O
*
O
O
Activity: measured as potency (percent inhibition) of CD4+ blood cell counts
R
NH
R
NH
24 h after application.
EtO
OEt
R
i
ii
OH
**
CO2Et
CO2Et
Control: vehicle-untreated mice; SEM: standard error of the mean; mean: 6–8
HN
11
11
control mice, 2–3 experiments per compound.
OH
O
25
79
80
84
(R = CH3), 45%
(R = CH3)
(R = Pr), 71%
(R = C3H7), 74%
85 (R = iPr), 26%
i
i
74
75
76
(R = Pr)
-
2
3
72
84 87 88 90 91 85
86 (R = C3H7), 28%
87 (R = C11H23), 15%
(R = C3H7)
(R = C11H23
77 (R = C15H31
81 (R = C11H23), 74%
82 (R = C15H31), 85%
26 (R = OtBu)
)
)
Erk MAPK
88
89
90
(R = C15H31), 18%
(R = H), 15%
(R = Ph), 39%
83
78 (R = Ph)
(R = Ph), 83%
Figure 3. CHO cells overexpressing S1P1 were stimulated for 10 min. with S1P (2),
FTY720 (3) and selected compounds at M. Western blots for the dually
phosphorylated forms of Erk1 and Erk2 MAPK (Thr202/Tyr204) were performed
using a phosphor-specific antibody.
Scheme 10. Synthesis of the N-alkylated aminodiols. Reagents and conditions: (i)
Cs2CO3, MeCN, reflux; (ii) LiAlH4, THF, reflux.
1
l
O
O
R
NH
F
NH2
EtO
OEt
R
i
OH
OH
H
HN
OH
OH
91 (R = C15H31), 29%
O
92
77 (R = C15H31
)
Figure 4. 4-Fluorosphingosine (92).
Scheme 11. Synthesis of the N-alkylated aminodiol 91. Reagents and conditions: (i)
LiAlH4, THF, reflux.
addition, selected compounds were also tested for biological activ-
ity in vitro by monitoring phosphorylation of Erk MAPK in CHO
cells overexpressing the human S1P1 receptor (Fig. 3).
2.3.4. Synthesis of the N-alkylated analogues
The 3-deoxysphingosine analogues (R)-14, 18 and 19 were less
potent than FTY720 indicating that the 3-hydroxy group is impor-
tant for efficient binding to the receptor. The non-fluorinated
sphingosine analogue (R)-14 showed low potency. Therefore (R)-
15 was not tested. 4-Fluorosphingosine (92) (Fig. 4) which had
been previously synthesized in our group24 induced no lymphope-
nia. A possible reason is that the fluorine substituent increases the
acidity of the neighboring 3-hydroxy group and thus diminishes
the binding affinity. The non-fluorinated analogues 29, 37 and
42, bearing the 2-aminopropane-1,3-diol moiety, all induced
lymphopenia.
For the synthesis of the N-alkylated analogues 84 to 90 different
amidomalonates, prepared by the reaction of 2-aminomalonate
with the corresponding acid chlorides, were used as building
blocks for the alkylation with 1-iodohexadecane. After the alkyl-
ation, the resulting diesters 25, 26 and 79 to 83 were reduced with
lithium aluminumhydride to yield the N-alkylated aminodiols 84
to 90 (Scheme 10).
In addition the amidomalonate 77 was reduced with lithium
aluminiumhydride without previous alkylation to give the N-alkyl-
ated aminopropanediol 91 (Scheme 11).
The C2-ceramide analogue 27 was tested as a model compound
to learn whether an amide can function as a prodrug. However, 27
proved to be only a weak agonist. This indicates that either the
phosphorylated amide itself is a weak agonist or the conversion
to the 2-aminopropane-1,3-diol by a ceramidase is very slow. Thus,
using labelled amides of this type as prodrugs for S1P receptor
imaging is not adequate. The analogues 84–91 with an alkylated
amino group showed no activity, although the secondary amine
is more basic and should interact with the receptor. Therefore
direct alkylation of the amino group to attach a building block
2.4. Evaluation of the new ligands as immunosuppressants
The new ligands were evaluated for biological activity in vivo by
measuring their ability to induce lymphopenia as readout for their
effectiveness at the S1P1 receptor. Binding to the S1P1 receptor
requires phosphorylation of the synthesized ligands (unless
already phosphorylated) including FTY720, which physiologically
occurs in vivo. Therefore, all ligands were tested in wild-type mice
at a dose of 1.25 mg/kg/d and compared with the effect of FTY720
that was used here as the reference compound (Table 1). In

1016
S. S. Schilson et al. / Bioorg. Med. Chem. 23 (2015) 1011–1026
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
5
A
B
4.5
CD4+
CD8+
●
B cells
▲
4
3.5
3
■
2.5
2
1.5
1
0.5
0
0
24
48
72
96 120 144 168 192 216 240
0
24
48
72
96 120 144 168 192 216 240
time [h]
time [h]
Figure 5. FACS analysis of blood samples after intraperitoneal injection of a single dose (1.25 mg/kg) of compound 72. (A) CD4+ and CD8+ cells (106/mL). (B) B cells (106/mL).
which can be labelled with [18F] is not feasible. The reason why the
N-alkyl analogues are inactive, either because the phosphorylation
failed or because the phosphorylated compounds do not bind to
the receptor cannot be deduced from the current data.
(compound 30) led to a dramatic drop of potency. Similarly, the
potency of the N-acetyl derivative 27 was very low and all N-alkyl
derivatives 84 to 91 as well as the 18-hydroxy derivative 73 were
completely inactive showing that attachment of any tracer group
in such positions will not lead to suitable ligands. In contrast, the
Out of the fluorinated ligands only the
x-fluorinated com-
pounds 71 and 72 showed potency comparable to that of
FTY720. A reason for the low potency of the other fluorinated ana-
logues 43, 50, (E)-59 and (Z)-59 could be the decreased basicity of
the amino group caused by the nearby fluorine substituent. This
effect is generally known for fluorine substituents31 and was also
x-fluorinated 2-amino-17-fluoro-2-hydroxymethylheptadecanol
(71) was as potent as FTY720 (3) and its next higher homologue
72 was still a very good inhibitor. Thus, compounds with fluorine
in the
[
x-position are expected to be suitable as PET tracers after
18F]-labelling. Further results concerning the radiochemistry and
observed for
c-fluoro-
a
-amino acids.32
in vivo imaging will be reported in due course.
An additional polar substituent as OH in the
x
-position (73) did
not prove successful in retaining the potency of the unsubstituted
parent compound 29. The different effects of the modification of
4. Experimental section
4.1. General remarks
the
x-position can be explained by the S1P1 receptor crystal struc-
ture. The extracellular N-terminus forms a lid, which obstructs
direct access to the binding pocket. Thus, S1P (or other potential
ligands) enters the binding pocket by lateral diffusion.33 Polar
groups like OH prevent the inclusion into the membrane, averting
interaction with the receptor. Fluoro substituents however are
tolerated.
In addition to the potency, the biological half-life of selected
ligands was determined by flow cytometry (Fig. 5). Lymphopenia
lasted for at least 10 days after a single dose.
All reactions with moisture or air sensitive reagents were carried
out under an argon atmosphere (argon 4.8) in pre-dried reaction
vessels. Solvents and other reagents for these reactions were dried
according to standard procedures. For reactions under ambient con-
ditions all chemicals and reagents were of analytical grade and used
as delivered without further purification. Solvents were purified by
distillation. The synthesized compounds were characterized by
NMR spectra and HRMS, melting points were measured where pos-
sible. 1H NMR (300 MHz, 400 MHz and 600 MHz), 13C NMR (75 MHz,
The compounds 84 to 91 were inactive when tested in vitro in
CHO-S1P1 overexpressing cells using phosphorylation of Erk MAPK
as readout in comparison with S1P and FTY720.
101 MHz and 151 MHz), 19F NMR (282 MHz and 564 MHz) and 31
P
NMR (121 MHz) spectra were recorded using spectrometers by Bru-
ker or Agilent Technologies in CDCl3, CD3OD or in a mixture of both
3. Conclusion
solvents with TMS (1H NMR), the solvent (13C NMR) and CFCl3 (19
F
NMR) as internal standards. DEPT NMR was employed to assign
13C signals. Exact mass analyses were performed on a microTOF
spectrometer by Bruker Daltonics. Results are shown as the theoret-
ical and recorded m/z values of the calculated composition of the
pseudomolecular ion. NMR and MS investigations were performed
by the analytical service departments of the Organic Chemistry
Institute, University of Münster. Melting points were determined
on a Stuart SMP 10 apparatus by Stuart Scientific and are not cor-
rected. The specific rotation values were measured on a Perkin–
Elmer polarimeter 341 (k = 589 nm) at room temperature. For thin
layer chromatography (TLC) analyses silica coated aluminum foils
(silica gel 60 F254, 0.2 mm layer thickness, Merck) were used. The
TLC plates were developed with potassium permanganate or ceram-
monium molybdate reagent to show yellow or blue spots. Silica gel
(silica gel 60, Merck) was used for flash column chromatography.
Solid phase extractions (SPE) were performed using C18ec-cartridges
from Macherey–Nagel. The enantiomeric excess was determined by
chiral HPLC. Ultrapure hydrogen gas was generated with a Nitrox
UHP-40H hydrogen generator by Domnick Hunter. The lysing buffer
Different aliphatic structural analogues of sphingosine, S1P and
FTY720 have been synthesized as new ligands of the S1P1 receptor
and tested in wild-type mice for their ability to induce lymphope-
nia. 3-Deoxy- and 3-deoxy-4-fluoro-sphingosine phosphates 18
and 19 exhibited low agonist activity. (R)-3-Deoxysphingosine
(14), which is phosphorylated in vivo, was slightly less active than
the pre-phosphorylated racemic 18. 4-Fluorosphingosine (92) was
found to be inactive probably because of the electronic influence
of the fluorine in the b-position to this OH function. These results
illuminate the importance of this group for efficient binding to the
receptor. Saturated analogues of FTY720 such as 29 and its lower
homologue 42 were comparable in potency to FTY720 (3). A double
bond in 4-position (as in compound 37) did not influence the
potency, while fluorination of the vinylic 4-position led to
decreased, but still moderate potency independently of the stereo-
chemistry of the double bond (E/Z-isomers 59). In saturated deriva-
tives, fluorination of the 4-position (compound 50) or isosteric
replacement of the hydroxymethyl group by a fluoromethyl group

S. S. Schilson et al. / Bioorg. Med. Chem. 23 (2015) 1011–1026
1017
and the antibodies to CD4, CD8 and B220 for the FACS analysis were
purchased from Becton Dickinson. The labelled cells were analyzed
in a Gallios Beckman Coulter flow cytometer. All experiments to
determine the biological activity were performed at the Center for
Medical Biotechnology (ZMB), University of Duisburg-Essen.
Syntheses of the N-alkyl-2-aminopropane-1,3-diols 84 to 91 are
described in the Supporting information.
4.3.2. (4Z)-2-tert-Butoxycarbonylamino-4-fluorooctadec-4-ene
acid (11)
According to the general procedure described above the fluori-
nated glycine ester 915 (1.41 g, 3.39 mmol) was treated with LDA
and zinc chloride. The crude product was crystallized from pentane
to give a white solid. Yield: 1.20 g (86%).
21
O
22
23
4.2. Synthesis of the 3-deoxysphingosine analogues
20
19
1
F
HN
O
16
14
12
10
8
6
The fluorinated glycine allylic ester 9 has been synthesized from
18
4
2
OH
hexadec-1-ene as described by Marhold et al.15 The synthesis of
c-
17
15
13
11
9
7
5
3
fluorinated
c,d-unsaturated a-amino acids by chelate ester enolate
O
Claisen rearrangement is also described in the publication men-
tioned above. For the synthesis of the
c,d-unsaturated a-amino
acids 10 and 11 the published reaction conditions15 were changed
slightly as described below. The corresponding non-fluorinated
glycine allylic ester 8 and its precursors were prepared according
to the described procedures.15
Mp: 83–84 °C (pentane). 1H NMR (300 MHz, CDCl3): d 0.87 (m, 3H,
18-CH3), 1.21–1.34 (m, 22H, 7-CH2 to 17-CH2), 1.45 (s, 9H, 21-CH3
to 23-CH3), 2.04 (m, 2H, 6-CH2), 2.71 (m, 2H, 3-CH2), 4.35 (m,
3
0.3H, 2-CH), 4.46 (m, 0.7H, 2-CH), 4.64 (dt, JH,H = 7.5 Hz,
3JH,F = 37.3 Hz, 1H, 5-CH), 5.18 (d, JH,H = 7.9 Hz, 0.7H, 2-NH), 6.39
3
4.3. General procedure for the synthesis of
amino acids by chelate ester enolate Claisen rearrangement
c,d-unsaturated a-
3
(d, JH,H = 7.1 Hz, 0.3H, 2-NH), 10.83 (br s, 1H, 1-COOH). 13C NMR
(75 MHz, CDCl3): d 14.3 (q, C-18), 22.8 (t, C-17), 23.8 (dt, 3JC,F = 4.1 Hz,
C-6), 28.4 (q, C-21 to C-23), 29.3, 29.4, 29.5, 29.6, 29.8 (t, C-7 to
Similarly to the described procedure,15 dry diisopropylamine
(0.36 mL, 250 mg, 2.50 mmol, 2.5 equiv) was dissolved in abs
diethyl ether (4.0 mL). The solution was cooled to 0 °C and n-butyl-
lithium (1.6 M solution in hexane, 1.6 mL, 2.50 mmol, 2.5 equiv)
was added dropwise. The LDA solution was cooled to À78 °C and
a solution of the glycine allylic ester (1.00 mmol) in abs. diethyl
ether (5.0 mL) was administered using a syringe pump. After
10 min, the reaction mixture was treated with a 1.5 M solution of
anhydrous zinc chloride in abs. diethyl ether (1.0 mL, 1.50 mmol,
1.5 equiv) and then allowed to warm up to rt overnight. The reac-
tion was quenched with diethylether (20 mL) and the organic layer
was washed with 1 M HCl (2 Â 25 mL), water (2 Â 20 mL) and
brine (1 Â 20 mL). The organic layer was dried over MgSO4 and
the solvent was removed in vacuo.
2
C-15), 32.1 (t, C-16), 34.9 (dt, JC,F = 27.7 Hz, C-3), 51.3 (d, C-2),
80.5 (s, C-20), 110.0 (dd, 2JC,F = 14.7 Hz, C-5), 154.5 (d, 1JC,F = 251.6 Hz,
C-4), 155.5 (s, C-19), 176.4 (s, C-1). 19F NMR (282 MHz, CDCl3): d
3
3
À111.1 (dt, JH,F = 20.0 Hz, JH,F = 38.7 Hz, 1F, 4-CF). Exact mass
(ESI+): C23H42FNO4 + Na+: calcd 438.2990, found 438.2984.
The spectroscopic data agree with published ones.15
4.4. General procedure for the reduction of
amino alcohols
a-amino acids to b-
The Boc-protected amino acid (1.74 mmol) was dissolved in
abs. THF (8.0 mL), cooled to À10 °C and then treated with abs. tri-
ethylamine (0.24 mL, 176 mg, 1.74 mmol, 1.0 equiv) and isobutyl
chloroformate (0.23 mL, 237 mg, 1.74 mmol, 1.0 equiv). The reac-
tion mixture was stirred for 1 h at À5 °C. Then the precipitate
was filtered off and washed thoroughly with THF (45 mL). The fil-
trate was cooled to 0 °C and sodium borohydride (99.0 mg,
2.61 mmol, 1.5 equiv) in water (1.5 mL) was added. After 3 h at
0 °C, the reaction was quenched with water (12 mL) and the mix-
ture was adjusted to pH 3 with 1 M potassium hydrogensulfate
solution. The aqueous phase was extracted with ethyl acetate
(3 Â 35 mL). The combined organic phases were washed with
50% sodium bicarbonate solution (4 Â 5 mL) and brine (1 Â 9 mL)
and then dried over Na2SO4. The solvent was removed in vacuo
and the residue was purified by silica gel flash column
chromatography.
4.3.1. (4E)-2-(tert-Butoxycarbonylamino)octadec-4-ene acid
(10)
According to the general procedure described above the glycine
ester 815 (1.55 g, 3.90 mmol) was treated with LDA and zinc chlo-
ride. The crude product was crystallized from pentane to give a
white solid. Yield: 1.44 g (92%).
21
O
22
23
20
19
1
HN
O
16
14
12
10
8
6
4
18
2
OH
17
15
13
11
9
7
5
3
O
4.4.1. (4E)-2-(tert-Butoxycarbonylamino)octadec-4-ene-1-ol
(12)
The Boc-protected amino acid 10 (1.89 g, 4.75 mmol) was
reduced according to the general procedure described above. The
product was purified by flash column chromatography
(24 Â 5 cm, cyclohexane/ethyl acetate, 6:1 ? 3:1) and was
obtained as a white solid. Yield: 1.78 g (97%).
Mp: 75 °C (pentane). 1H NMR (400 MHz, CDCl3):
d
0.88 (t,
3JH,H = 6.8 Hz, 3H, 18-CH3), 1.16–1.37 (m, 22H, 7-CH2 to 17-CH2),
1.45 (s, 9H, 21-CH3 to 23-CH3), 2.00 (m, 2H, 6-CH2), 2.50 (m, 2H,
3-CH2), 4.12 (m, 0.3H, 2-CH), 4.30 (m, 0.7H, 2-CH), 5.03 (d,
3
3
3JH,H = 7.9 Hz, 0.7H, 2-NH), 5.31 (dt, JH,H = 6.6 Hz, JH,H = 14.2 Hz,
3
3
1H, 4-CH or 5-CH), 5.56 (dt, JH,H = 6.6 Hz, JH,H = 13.9 Hz, 1H, 4-
CH or 5-CH), 6.08 (br s, 0.3H, 2-NH), 11.28 (br s, 1H, 1-COOH). 13C
NMR (101 MHz, CDCl3): d 14.2 (q, C-18), 22.8 (t, C-17), 28.4 (q, C-
21 to C-23), 29.3, 29.4, 29.5, 29.6, 29.8, 32.1, 32.7 (t, C-6 to C-16),
35.4 (t, C-3), 53.1 (d, C-2), 80.2 (s, C-20), 123.2 (d, C-4 or C-5),
136.1 (d, C-4 or C-5), 155.6 (s, C-19), 177.3 (s, C-1). Exact mass
(ESI+): C23H43NO4 + Na+: calcd 420.3084, found 420.3080.
21
O
22
23
20
19
HN
O
16
14
12
10
8
6
4
18
2
OH
17
15
13
11
9
7
5
3
1

1018
S. S. Schilson et al. / Bioorg. Med. Chem. 23 (2015) 1011–1026
3
Mp: 52 °C. 1H NMR (300 MHz, CDCl3): d 0.88 (t, JH,H = 6.4 Hz, 3H,
18-CH3), 1.18–1.38 (m, 22H, 7-CH2 to 17-CH2), 1.44 (s, 9H, 21-CH3
to 23-CH3), 1.99 (m, 2H, 6-CH2), 2.19 (m, 2H, 3-CH2), 3.51–3.71
(14 Â 3 cm, cyclohexane/ethyl acetate, 4:1) and was obtained as
20
a white solid. Yield: 226 mg (32%). Specific rotation: [
a]
À2.89
D
(c 10.0 mg/mL, CHCl3); ee: 73% (chiral HPLC).
3
(m, 3H, 1-CH2, 2-CH), 4.79 (d, JH,H = 7.0 Hz, 1H, 2-NH), 5.36 (m,
1H, 4-CH or 5-CH), 5.52 (m, 1H, 4-CH or 5-CH). 13C NMR (75 MHz,
CDCl3): d 14.2 (q, C-18), 22.8 (t, C-17), 28.5 (q, C-21 to C-23), 29.3,
29.5, 29.6, 29.8, 32.0, 32.7 (t, C-6 to C-16), 34.8 (t, C-3), 52.6 (d,
C-2), 65.5 (t, C-1), 79.7 (s, C-20), 125.3 (d, C-4 or C-5), 134.6 (d,
C-4 or C-5), 156.6 (s, C-19). Exact mass (ESI+): C23H45NO3 + Na+:
calcd 406.3292, found 406.3295.
21
20
O
O
22
23
19
F
HN
16
14
12
10
8
6
18
4
OH
2
7
5
17
15
13
11
9
3
1
4.4.2. (2R)-(4E)-2-(tert-Butoxycarbonylamino)octadec-4-ene-1-
ol (R-12)
The spectroscopic data correspond to those of the racemate.
The amino acid (R)-1015 (497 mg, 1.25 mmol) was reduced
according to the general procedure described above. The product
was purified by flash column chromatography (9 Â 4 cm, cyclohex-
4.5. General procedure for the cleavage of the Boc-protecting
group
ane/ethyl acetate, 4:1) and was obtained as a white solid. Yield:
20
The protected aminoalcohol (0.50 mmol) was dissolved in abs.
dichloromethane (5.0 mL) and then treated with trifluoroacetic
acid (TFA, 5.0 mL) at 0 °C. The reaction mixture was stirred at
0 °C for 1 h and was then allowed to warm up to rt overnight. All
volatile components were removed in vacuo. To remove excess
TFA, the residue was dissolved in methanol (1.0 mL) and was again
concentrated in vacuo. This procedure was repeated three times.
The residue was dissolved in methanol (0.5 mL) and 1 M sodium
hydroxide solution (20 mL). The aqueous phase was extracted with
ethyl acetate or dichloromethane (3 Â 20 mL). The combined
organic layers were dried over Na2SO4 and the solvent was
removed under reduced pressure.
308 mg (64%). Specific rotation: [
ee: 80% (chiral HPLC).
a]
À4.61 (c 9.96 mg/mL, CHCl3);
D
21
O
22
23
20
19
HN
O
16
14
12
10
8
6
4
18
OH
2
7
5
17
15
13
11
9
3
1
The spectroscopic data correspond to those of the racemate.
4.4.3. (4Z)-2-tert-Butoxycarbonylamino-4-fluorooctadec-4-ene-
1-ol (13)
4.5.1. (4E)-2-Aminooctadec-4-ene-1-ol (14)
In accordance with the general procedure described above the
amino alcohol 12 (195 mg, 0.50 mmol) was treated with TFA. The
product was crystallized from ethyl acetate to yield a white solid.
Yield: 138 mg (98%).
The fluorinated amino acid 11 (3.17 g, 7.63 mmol) was reduced
according to the general procedure described above. The product
was purified by flash column chromatography (18 Â 5 cm, cyclo-
hexane/ethyl acetate, 6:1 ? 3:1) and was obtained as a white solid.
Yield: 2.08 g (68%).
NH2
16
14
12
10
8
6
4
18
2
OH
21
O
17
15
13
11
9
7
5
3
1
22
20
19
F
HN
O
23
16
14
12
10
8
6
18
4
2
OH
Mp: 90–91 °C (ethyl acetate). 1H NMR (300 MHz, CD3OD, CDCl3): d
0.89 (t, 3JH,H = 6.6 Hz, 3H, 18-CH3), 1.18–1.42 (m, 22H, 7-CH2 to 17-
CH2), 1.90–2.22 (m, 4H, 3-CH2, 6-CH2), 2.81 (m, 1H, 2-CH), 3.42 (m,
2H, 1-CH2), 5.39 (m, 1H, 4-CH or 5-CH), 5.54 (m, 1H, 4-CH or 5-CH).
13C NMR (75 MHz, CD3OD, CDCl3): d 14.4 (q, C-18), 23.4 (t, C-17),
29.9, 30.1, 30.2, 30.4 (t, C-7 to C-15), 32.7, 33.3 (t, C-6, C-16), 37.1
(t, C-3), 53.2 (d, C-2), 66.5 (t, C-1), 126.5 (d, C-4 or C-5), 134.9 (d,
C-4 or C-5). Exact mass (ESI+): C18H37NO + H+: calcd 284.2948,
found 284.2951, C18H37NO + Na+: calcd 306.2767, found 306.2767,
(C18H37NO)2 + Na+: calcd 589.5643, found 589.5631.
17
15
13
11
9
7
5
3
1
Mp: 68 °C. 1H NMR (300 MHz, CDCl3): d 0.87 (m, 3H, 18-CH3), 1.19–
1.37 (m, 22H, 7-CH2 to 17-CH2), 1.44 (s, 9H, 21-CH3 to 23-CH3), 2.05
(m, 2H, 6-CH2), 2.39 (m, 2H, 3-CH2), 3.67 (m, 2H, 1-CH2), 3.80 (m,
3
3
1H, 2-CH), 4.61 (dt, JH,H = 7.5 Hz, JH,F = 37.8 Hz, 1H, 5-CH), 4.95
(d, JH,H = 7.7 Hz, 1H, 2-NH). 13C NMR (75 MHz, CDCl3): d 14.2 (q,
C-18), 22.8 (t, C-17), 23.7 (dt, JC,F = 4.7 Hz, C-6), 28.4 (q, C-21 to
3
3
C-23), 29.3, 29.5, 29.8 (t, C-7 to C-15), 32.0 (t, C-16), 34.1 (dt,
2JC,F = 28.0 Hz, C-3), 50.5 (d, C-2), 64.7 (t, C-1), 79.9 (s, C-20),
4.5.2. (2R)-(4E)-2-Aminooctadec-4-ene-1-ol (R-14)
In accordance with the general procedure described above the
amino alcohol (R)-12 (150 mg, 0.39 mmol) was treated with TFA.
The product was crystallized from ethyl acetate to yield a white
solid. Yield: 91.0 mg (82%). Specific rotation:
2
1
108.7 (dd, JC,F = 15.3 Hz, C-5), 156.0 (d, JC,F = 251.8 Hz, C-4),
156.3 (s, C-19). 19F NMR (282 MHz, CDCl3):
d
À109.9 (dt,
3JH,F = 20.4 Hz, JH,F = 39.6 Hz, 1F, 4-CF). Exact mass (ESI+): C23H44
FNO3 + Na+: calcd 424.3197, found 424.3196; (C23H44FNO3)2 + Na+:
calcd 825.6503, found 825.6502.
3
20
[
a
]
À2.80 (c
D
2.5 mg/mL, CHCl3/CH3OH; 1:1); ee: 80% (chiral HPLC).
4.4.4. (2R)-(4Z)-2-tert-Butoxycarbonylamino-4-fluorooctadec-
4-ene-1-ol (R-13)
NH2
16
14
12
10
8
6
4
18
The fluorinated amino acid (R)-1115 (723 mg, 1.74 mmol) was
reduced according to the general procedure described above. The
product was purified by flash column chromatography
OH
2
5
17
15
13
11
9
7
3
1

S. S. Schilson et al. / Bioorg. Med. Chem. 23 (2015) 1011–1026
1019
The spectroscopic data correspond to those of the racemate.
17
15
13
11
9
7
5
3
1
24
O
O
O
2
P
O
O
18
16
14
12
10
8
6
4
25
4.5.3. (4Z)-2-Amino-4-fluorooctadec-4-ene-1-ol (15)
In accordance with the general procedure described above the
fluorinated amino alcohol 13 (207 mg, 0.50 mmol) was treated
with TFA. The product was crystallized from ethyl acetate to yield
a white solid. Yield: 144 mg (96%).
21
HN
19
20
22
O
23
Mp: 42–43 °C. 1H NMR (300 MHz, CDCl3): d 0.88 (t, JH,H = 6.6 Hz,
3H, 18-CH3), 1.21–1.34 (m, 22H, 7-CH2 to 17-CH2), 1.44 (s, 9H,
21-CH3 to 23-CH3), 1.99 (m, 2H, 6-CH2), 2.25 (m, 2H, 3-CH2), 3.76
(s, 3H, 24-CH3 or 25-CH3), 3.80 (s, 3H, 24-CH3 or 25-CH3), 3.99–
3
F
NH2
16
14
12
10
8
6
18
4
2
OH
17
15
13
11
9
7
5
3
1
3
4.07 (m, 3H, 1-CH2, 2-CH), 4.78 (d, JH,H = 7.8 Hz, 1H, 2-NH), 5.34
3
3
(dt, JH,H = 7.0 Hz, JH,H = 15.0 Hz, 1H, 4-CH or 5-CH), 5.53 (dt,
Mp: 71 °C (ethyl acetate). 1H NMR (300 MHz, CDCl3, CD3OD) d: 0.88
(t, 3JH,H = 6.5 Hz, 3H, 18-CH3), 1.18–1.39 (m, 22H, 7-CH2 to 17-CH2),
1.99–2.40 (m, 4H, 3-CH2, 6-CH2), 3.09 (m, 1H, 2-CH), 3.48 (m, 2H,
3JH,H = 6.6 Hz, JH,H = 13.6 Hz, 1H, 4-CH or 5-CH). 13C NMR
3
(75 MHz, CDCl3): d 14.2 (q, C-18), 22.8 (t, C-17), 28.5 (q, C-21 to
C-23), 29.3, 29.5, 29.6, 29.7, 29.8, 32.0, 32.7 (t, C-6 to C-16), 34.5
(t, C-3), 50.3 (m, C-2), 54.5 (2 dq, JC,P = 5.9 Hz, C-24, C-25), 68.5
3
3
1-CH2), 4.61 (dt, JH,H = 7.5 Hz, JH,F = 37.9 Hz, 1H, 5-CH). 13C NMR
(75 MHz, CDCl3, CD3OD): d 14.1 (q, C-18), 22.7 (t, C-17), 23.6 (dt,
3JC,F = 4.7 Hz, C-6), 29.3, 29.4, 29.5, 29.7 (t, C-7 to C-15), 32.0 (t, C-
2
2
(dt, JC,P = 6.2 Hz, C-1), 79.5 (s, C-20), 124.5 (d, C-4 or C-5), 135.1
(d, C-4 or C-5), 155.4 (s, C-19). 31P NMR (121 MHz, CDCl3): d 1.4
2
16), 36.8 (dt, JC,F = 26.9 Hz, C-3), 49.8 (d, C-2), 65.8 (t, C-1), 108.5
(s, 1P, C-OP). Exact mass (ESI+): C25H50NO6P + H+: calcd 492.3449,
2
1
(dt, JC,F = 15.6 Hz, C-5), 156.3 (d, JC,F = 252.0 Hz, C-4).19F NMR
(282 MHz, CDCl3, CD3OD): dÀ110.7 (ddd, 3JH,F = 17.0 Hz, 3JH,F = 24.9 Hz,
3JH,F = 39.9 Hz, 1F, 4-CF). Exact mass (ESI+): C18H37FNO + H+: calcd
302.2854, found 302.2843; C18H37FNO + Na+: calcd 324.2673, found
324.2662; (C18H37FNO)2 + H+: calcd 603.5635, found 603.5615;
(C18H37FNO)2 + Na+: calcd 625.5454, found 625.5433.
found 492.3443,
C
25H50NO6P + Na+: calcd 514.3268, found
514.3264, (C25H50NO6P)2 + Na+: calcd 1005.6644, found 1005.6627.
4.6.2. 2(4Z)-tert-Butyl{1-[(dimethoxyphosphoryl)oxy]-4-
fluorooctadec-4-ene-2-yl} carbamate (17)
According to the general procedure described above the Boc-
protected, fluorinated amino alcohol 13 (1.00 g, 2.49 mmol) was
treated with DBTCE and trimethyl phosphate. The product was
purified by flash column chromatography (20 Â 3 cm, cyclohex-
ane/ethyl acetate, 1:1) and was isolated as a white solid. Yield:
1.01 g (79%).
4.5.4. (2R)-(4Z)-2-Amino-4-fluorooctadec-4-ene-1-ol (R-15)
In accordance with the general procedure described above the
fluorinated amino alcohol (R)-13 (115 mg, 0.29 mmol) was treated
with TFA. The product was crystallized from ethyl acetate to yield a
20
white solid. Yield: 78.0 mg (90%). Specific rotation: [
a
]
+0.60 (c
D
10.1 mg/mL, CHCl3/CH3OH; 1:1); ee: 73% (chiral HPLC).
17
15
13
11
9
7
5
3
1
24
O
O
O
2
P
O
O
18
4
16
14
12
10
8
6
F
NH2
25
16
14
12
10
8
6
F
18
4
21
OH
HN
19
2
1
20
22
17
15
13
11
9
7
5
3
O
23
The spectroscopic data correspond to those of the racemate.
Mp: 38 °C. 1H NMR (300 MHz, CDCl3): d 0.87 (m, 3H, 18-CH3), 1.21–
1.35 (m, 22H, 7-CH2 to 17-CH2), 1.44 (s, 9H, 21-CH3 to 23-CH3),
2.04 (m, 2H, 6-CH2), 2.43 (m, 2H, 3-CH2), 3.77 (s, 3H, 24-CH3 or 25-
CH3), 3.81 (s, 3H, 24-CH3 or 25-CH3), 3.95–4.13 (m, 3H, 1-CH2, 2-
4.6. General procedure for the phosphorylation of amino
alcohols with trimethyl phosphite
3
3
CH), 4.63 (dt, JH,H = 7.5 Hz, JH,F = 37.7 Hz, 1H, 5-CH), 4.98 (d,
The aminoalcohol (2.49 mmol) was dissolved in abs. pyridine
(10 mL) in a predried schlenk tube containing a few beads of
molecular sieves. Dibromotetrachloroethane (DBTCE, 1.46 g,
4.48 mmol, 1.8 equiv) was added and the mixture was cooled to
À10 °C. Trimethyl phosphite (0.35 mL, 2.99 mmol, 1.2 equiv) was
slowly dripped into the mixture which was allowed to warm up
to rt afterwards and was stirred overnight. The reaction was
quenched with 2 M hydrochloric acid (20 mL) and the aqueous
phase was extracted with ethyl acetate (4 Â 20 mL). The combined
organic layers were washed with brine (1 Â 40 mL) and dried over
MgSO4. The solvent was removed in vacuo and the residue was
purified by silica gel flash column chromatography.
3JH,H = 8.4 Hz, 1H, 2-NH). 13C NMR (75 MHz, CDCl3): d 14.2 (q, C-18),
3
22.7 (t, C-17), 23.6 (dt, JC,F = 4.6 Hz, C-6), 28.3 (q, C-21 to C-23),
29.2, 29.4, 29.5, 29.7 (t, C-7 to C-15), 32.0 (t, C-16), 34.0 (dt,
3
2JC,F = 27.8 Hz, C-3), 48.3 (dd, JC,P = 7.1 Hz, C-2), 54.5 (2 dq,
2JC,P = 5.9 Hz, C-24, C-25), 68.3 (dt, 2JC,P = 6.0 Hz, C-1), 79.6 (s, C-20),
109.0 (dd, 2JC,F = 15.4 Hz, C-5), 155.2 (s, C-19), 155.3 (d, 1JC,F = 251.7 Hz,
3
C-4). 19F NMR (282 MHz, CDCl3): d À110.4 (dt, JH,F = 20.1 Hz,
3JH,F = 39.0 Hz, 1F, 4-CF). 31P NMR (121 MHz, CDCl3): d 1.3 (s, 1P,
1-COP). Exact mass (ESI+): C25H49FNO6P + Na+: calcd 532.3174, found
532.3170; (C25H49FNO6P)2 + Na+: calcd 1041.6455, found 1041.6457.
4.7. General procedure for the preparation of the free
phosphates
a-amino
4.6.1. (4E)-tert-Butyl{1-[(dimethoxyphosphoryl)oxy]octadec-4-
ene-2-yl} carbamate (16)
A
solution of the corresponding dimethyl phosphate
According to the general procedure described above the Boc-
protected amino alcohol 12 (191 mg, 0.50 mmol) was treated with
DBTCE and trimethyl phosphate. The product was purified by flash
column chromatography (23 Â 3 cm, cyclohexane/ethyl acetate,
3:1 ? 1:1) and was isolated as a white solid. Yield: 240 mg (98%).
(0.20 mmol) in dry dichloromethane (2.0 mL) was cooled to 0 °C
and treated with bromotrimethylsilane (0.13 mL, 1.00 mmol,
5.0 equiv). The reaction mixture was allowed to warm up to rt
and was stirred for 1 h. The reaction was quenched on a short silica
gel column (1.5 Â 0.5 cm) which was washed with methanol

1020
S. S. Schilson et al. / Bioorg. Med. Chem. 23 (2015) 1011–1026
(5.0 mL) afterwards. The solvent was removed under reduced pres-
sure and the crude product was stored in a freezer. The product
was separated from the corresponding bromide by solid phase
extraction (SPE, C18ec-phase, 500 mg-cartridge, methanol/water,
60:40) and was obtained as a white solid.
4.8.1. (E)-1-Bromohexadec-2-ene (34)
The allylic alcohol 3324,34 (7.22 g, 30.0 mmol) and triphenyl-
phosphine (7.87 g, 30.0 mmol, 1.0 equiv) were dissolved in dichlo-
romethane (60 mL) and cooled down to À20 °C. NBS (6.41 g,
36.0 mmol, 1.2 equiv) was added in portions and the mixture
was warmed to rt and stirred for additional 2 h. Then the volume
of the mixture was halved and the remainder was diluted with
pentane (200 mL). Triphenylphosphine oxide was filtered off and
the organic layer was washed with water (1 Â 80 mL) and dried
over MgSO4. The solvent was removed and the residue was
adsorbed on silica gel for purification. After column chromatogra-
phy (13 Â 5 cm, cyclohexane ? ethyl acetate) the bromide 34
was isolated as a viscous oil. Yield: 6.14 g (68%).
4.7.1. (4E)-2-Aminooctadec-4-ene-1-yl-dihydrogen phosphate
(18)
According to the general procedure dimethyl phosphate 16
(98 mg, 0.20 mmol) was converted to a mixture of the desired
product 18 and the bromide 20, which was separated by solid
phase extraction. Compound 18 was obtained as a white solid.
Yield: 3.48 mg (5%). Due to the small amount isolated after the
SPE, no melting point was measured and the spectroscopic data
were taken from the crude product.
14
12
10
8
6
4
2
16
Br
15
13
11
9
7
5
3
1
17
15
13
11
9
7
5
3
1
OH
OH
O
2
P
O
1H NMR (300 MHz, CDCl3): d 0.87 (m, 3H, 16-CH3), 1.23–1.34 (m,
18
16
14
12
10
8
6
4
3
NH2
22H, 5-CH2 to 15-CH2), 2.05 (q, JH,H = 6.6 Hz, 2H, 4-CH2), 3.95 (d,
3JH,H = 7.0 Hz, 2H, 1-CH2), 5.62–5.83 (m, 2H, 2-CH, 3-CH). 13C NMR
(75 MHz, CDCl3): d 14.3 (q, C-16), 22.9 (t, C-15), 29.0, 29.3, 29.5,
29.6, 29.7, 29.8 (t, C-5 to C-13), 32.1, 32.2 (t, C-4, C-14), 33.8 (t,
C-1), 126.4, 136.9 (d, C-2, C-3).
1H NMR (300 MHz, CD3OD): d 0.88 (m, 3H, 18-CH3), 1.20–1.46 (m,
22H, 7-CH2 to 17-CH2), 2.05 (m, 2H, 6-CH2), 2.42 (m, 2H, 3-CH2),
3
3.47 (m, 1H, 2-CH), 4.14 (m, 2H, 1-CH2), 5.42 (dt, JH,H = 7.3 Hz,
3JH,H = 14.8 Hz, 1H, 4-CH or 5-CH), 5.71 (dt, JH,H = 6.7 Hz,
3
3JH,H = 15.5 Hz, 1H, 4-CH or 5-CH). 13C NMR (75 MHz, CD3OD): d
14.5 (q, C-18), 23.7 (t, C-17), 30.3, 30.4, 30.5, 30.6, 30.7, 30.8 (t, C-
7 to C-15), 33.0, 33.4, 33.6 (t, C-3, C-6, C-16), 52.6 (dd, 3JC,P = 7.9 Hz,
C-2), 66.1 (dt, 2JC,P = 4.7 Hz, C-1), 123.6 (d, C-4 or C-5), 137.9 (d, C-4
or C-5).31P NMR (121 MHz, CD3OD): d À0.5 (s, 1P, 1-COP). Exact
mass (ESI+): C18H38NO4P + Na+: calcd 386.2431, found 386.2443.
Exact mass (ESIÀ): C18H37NO4P: calcd 362.2466, found 362.2491.
4.8.2. (E)-Diethyl-2-acetamido-2-(hexadec-2-en-1-yl)malonate
(35)
Diethyl 2-acetamidomalonate 23 (4.06 g, 18.7 mmol, 1.0 equiv),
bromide 34 (5.67 g, 18.7 mmol) and caesium carbonate (7.21 g,
22.2 mmol, 1.2 equiv) were suspended in acetonitrile (100 mL)
and refluxed for 8 h. After cooling to rt the precipitate was filtered
off and the solvent was removed in vacuo. The product was crystal-
lized from pentane and obtained as a white solid. Yield: 6.24 g (76%).
4.7.2. (4Z)-2-Amino-4-fluorooctadec-4-ene-1-yl-dihydrogen
phosphate (19)
According to the general procedure dimethyl phosphate 17
(102 mg, 0.20 mmol) was converted to a mixture of the desired
product 19 and the bromide 21, which was separated by solid
phase extraction. Compound 19 was obtained as a white solid.
Yield: 3.93 mg (5%). Due to the small amount isolated after the
SPE, no melting point was measured and the spectroscopic data
were taken from the crude product.
O
21
20
NH
17
15
13
11
9
7
5
19
2
O
1
4
O
18
16
14
12
10
8
6
3
22
23
O
O
24
25
3
Mp: 56 °C. 1H NMR (300 MHz, CDCl3): d 0.88 (t, JH,H = 6.7 Hz, 3H,
17
15
13
11
9
7
5
3
1
OH
OH
O
2
O
P
18
4
19-CH3), 1.21–1.33 (m, 28H, 8-CH2 to 18-CH2, 23-CH3, 25-CH3),
16
14
12
10
8
6
3
F
NH2
1.95 (q, JH,H = 6.8 Hz, 2H, 7-CH2), 2.03 (s, 3H, 21-CH3), 3.00 (d,
3JH,H = 7.4 Hz, 2H, 4-CH2), 4.23 (q, JH,H = 7.1 Hz, 4H, 22-CH2, 24-
3
3
3
CH2), 5.15 (dt, JH,H = 7.4 Hz, JH,H = 15.1 Hz, 1H, 5-CH), 5.49 (dt,
3JH,H = 6.9 Hz, JH,H = 15.1 Hz, 1H, 6-CH), 6.74 (s, 1H, 2-NH). 13C
3
1H NMR (300 MHz, CDCl3): d 0.88 (t, JH,H = 6.3 Hz, 3H, 18-CH3),
1.03–1.56 (m, 22H, 7-CH2 to 17-CH2), 2.07 (m, 2H, 6-CH2), 2.80
(m, 2H, 3-CH2), 3.92 (m, 1H, 2-CH), 4.36 (m, 2H, 1-CH2), 4.88 (m,
1H, 5-CH). 13C NMR (75 MHz, CDCl3): d 14.2 (q, C-18), 22.8 (t, C-
17), 24.1 (m, C-6), 29.3, 29.5, 29.6, 29.8, 29.9, 32.0, 32.4 (t, C-3, C-
3
NMR (75 MHz, CDCl3): d 14.1 (q, C-23, C-25), 14.2 (q, C-19), 22.8
(t, C-18), 23.1 (q, C-21), 29.2, 29.4, 29.5, 29.6, 29.7, 29.8 (t, C-8 to
C-16), 32.0, 32.7 (t, C-7, C-17), 35.9 (t, C-4), 62.5 (t, C-22, C-24),
66.6 (s, C-2), 122.3, 136.4 (d, C-5, C-6), 167.8 (s, C-1, C-3), 168.9
(s, C-20). Exact mass (ESI+): C25H45NO5 + Na+: calcd 462.3190, found
462.3190; (C25H45NO5)2 + Na+: calcd 901.6488, found 901.6476.
2
7 to C-16), 50.3 (m, C-2), 65.7 (m, C-1), 111.8 (dd, JC,F = 13.7 Hz,
C-5), 152.3 (d, JC,F = 251.4 Hz, C-4). 19F NMR (282 MHz, CDCl3): d
1
À112.6 (m, 1F, 4-CF). 31P NMR (121 MHz, CDCl3): d À0.6 (s, 1P, 1-
COP). Exact mass (ESI+): C18H37FNO4P + Na+: calcd 404.2336, found
404.2347. Exact mass (ESIÀ): C18H36FNO4P: calcd 380.2360, found
380.2385.
4.8.3. (E)-N-(1-Hydroxy-2-(hydroxymethyl)octadec-4-en-2-
yl)acetamide (36)
The ester 35 (2.86 g, 6.51 mmol) was dissolved in THF (40 mL)
and lithium chloride (1.38 g, 32.6 mmol, 5.0 equiv) and sodium
borohydride (1.23 g, 32.6 mmol, 5.0 equiv) were added. The mix-
ture was cooled to 0 °C and treated with ethanol (80 mL). After
30 min the mixture was warmed to rt and stirred for 4 d. The reac-
tion was quenched with 20% potassium sodium tartrate solution
(20 mL). The aqueous phase was extracted with dichloromethane
(3 Â 20 mL) and the combined organic layers were dried over
4.8. Synthesis of 2-Amino-2-(hexadec-2-en-1-yl)propane-1,3-
diol
The starting material, the allylic alcohol 33 was prepared as
described.24

S. S. Schilson et al. / Bioorg. Med. Chem. 23 (2015) 1011–1026
1021
Na2SO4. The solvent was removed under reduced pressure and the
residue was purified by column chromatography (11.5 Â 4 cm,
ethyl acetate). The product 36 was isolated as a white solid. Yield:
30:1 ? 6:1) and the product was isolated as a yellowish solid.
Yield: 36 mg (11%).
NH2
17
15
13
11
9
7
5
19
2
1.58 g (68%).
OH
1
O
3
18
16
14
12
10
8
6
4
F
21
20
HN
17
15
13
11
9
7
5
Mp: 52 °C. 1H NMR (300 MHz, CDCl3): d 0.87 (t, JH,H = 6.6 Hz, 3H,
3
19
2
OH
1
3
19-CH3), 1.21–1.34 (m, 28H, 5-CH2 to 18-CH2), 1.46 (m, 2H, 4-
18
16
14
12
10
8
6
4
CH2), 3.49 (m, 2H, 1-CH2), 4.32 (d, JH,F = 47.5 Hz, 2H, 3-CH2).13
C
2
OH
NMR (75 MHz, CDCl3): d 14.2 (q, C-18), 22.8 (q, C-19), 29.5,
Mp: 84 °C. 1H NMR (400 MHz, CDCl3): d 0.88 (t, JH,H = 6.9 Hz, 3H,
19-CH3), 1.13–1.38 (m, 22H, 8-CH2 to 18-CH2), 1.99–2.06 (m, 5H,
3
29.7,29.8 (t, C-5 to C-16), 30.4 (t, C-4), 32.1 (t, C-17), 56.2 (d,
2JC,F = 16.6 Hz, C-2), 65.2 (t, C-1), 86.3 (dt, JC,F = 174.3 Hz, C-3). 19F
1
3
7-CH2, 21-CH3), 2.30 (d, JH,H = 7.3 Hz, 2H, 4-CH2), 3.55 (d,
2
NMR (282 MHz, CDCl3): d À230.4 (t, JH,F = 47.3 Hz, 1F, 3-CH2F).
2JH,H = 11.6 Hz, 2H, 1-CH2, 3-CH2), 3.76 (d, JH,H = 11.6 Hz, 2H, 1-
2
Exact mass (ESI+): C19H40FNO + H+: calcd 318.3167, found
318.3167; C19H40FNO + Na+: calcd 340.2986, found 340.2991.
An alternative synthesis for this compound is described in the SI
(Section 4).
3
CH2, 3-CH2), 4.47 (br s, 2H, 1-OH, 3-OH), 5.41 (dt, JH,H = 7.4 Hz,
3JH,H = 15.1 Hz, 1H, 5-CH), 5.57 (dt, JH,H = 6.7 Hz, JH,H = 15.2 Hz,
1H, 6-CH), 6.09 (s, 1H, 2-NH). 13C NMR (101 MHz, CDCl3): d 14.2
(q, C-19), 22.8 (t, C-18), 24.0 (q, C-21), 29.3, 29.5, 29.6, 29.7, 29.8
(t, C-8 to C-16), 32.0, 32.8 (t, C-7, C-17), 36.1 (t, C-4), 61.0 (s, C-2),
65.4 (t, C-1, C-3), 123.4, 136.7 (d, C-5, C-6), 171.9 (s, C-20). Exact
mass (ESI+): C21H41NO3 + H+: calcd 356.3159, found 356.3161;
3
3
4.10. Synthesis of the saturated 4-fluoro compounds
4.10.1. 2-Fluoro-1-iodohexadecane (46)
C
21H41NO3 + Na+: calcd 378.2979, found 378.2978; (C21H41NO3)2 +
Similar to a known procedure,36 hexadec-1-ene (6.2 mL, 4.88 g,
20.0 mmol, 92 % purity) and triethylamine trishydrofluoride
(10 mL, 50.0 mmol, 2.5 equiv) were dissolved in dichloromethane
(40 mL) and N-iodosuccinimide (NIS) (4.95 g, 22.0 mmol,
1.1 equiv) in dichloromethane (40 mL) was added under stirring
at 0 °C. The mixture was stirred at rt overnight. Then the mixture
was poured into ice water (40 mL) and treated with conc. ammonia
solution until alkaline pH was reached. The phases were separated
and the aqueous phase was extracted with dichloromethane
(3 Â 50 mL). The combined organic layers were washed with 2 M
HCl (80 mL), 5% sodium bicarbonate solution and water. After dry-
ing over MgSO4 and evaporation of the solvent, the crude product
was purified by column chromatography (3 Â 6 cm, cyclohexane)
and recrystallized from ethyl acetate. The iodofluoride 46 was iso-
lated as a white, crystalline solid. Yield: 5.77 g (78%).
Na+: calcd 733.6065, found 733.6049.
4.8.4. (E)-2-Amino-2-(hexadec-2-en-1-yl)propane-1,3-diol (37)
The protected aminodiol 36 (186 mg, 0.52 mmol) was dissolved
in methanol (5 mL), treated with 1 M sodium hydroxide solution
(1.0 mL, 1.0 mmol, 1.9 equiv) and heated to reflux for 5 h. After
cooling to rt overnight the mixture was diluted with 1 M sodium
hydroxide solution (5 mL) and the aqueous phase was extracted
with dichloromethane (5 Â 10 mL). The combined organic layers
were dried over Na2SO4 and concentrated in vacuo. The product
was crystallized from ethyl acetate and isolated as a white solid.
Yield: 158 mg (97%).
NH2
17
15
13
11
9
7
5
19
2
OH
F
1
3
18
16
14
12
10
8
6
4
14
12
10
8
6
4
16
2
I
OH
15
13
11
9
7
5
3
1
Mp: 57 °C. 1H NMR (300 MHz, CD3OD): d 0.90 (t, JH,H = 6.7 Hz, 3H,
3
Mp: 50–52 °C. 1H NMR (400 MHz, CDCl3): d 0.87 (m, 3H, 16-CH3),
1.18–1.46 (m, 24H, 4-CH2 to 15-CH2), 1.71 (m, 2H, 3-CH2), 3.31
(m, 2H, 1-CH2), 4.44 (m, 1H, 2-CH). 13C NMR (101 MHz, CDCl3): d
19-CH2), 1.29 (m, 22H, 8-CH2 to 18-CH2), 2.03 (q, 3JH,H = 6.6 Hz, 2H,
3
2
7-CH2), 2.09 (d, JH,H = 6.7 Hz, 2H, 4-CH2), 3.38 (d, JH,H = 10.8 Hz,
2H, 1-CH2, 3-CH2), 3.41 (d, JH,H = 10.8 Hz, 2H, 1-CH2, 3-CH2), 5.49
2
2
7.2 (dt, JC,F = 24.6 Hz, C-1), 14.3 (q, C-16), 22.8 (t, C-15), 24.9 (dt,
(m, 2H, 5-CH, 6-CH). 13C NMR (75 MHz, CD3OD): d 14.5 (q, C-19),
23.8 (t, C-18), 30.3, 30.5, 30.7, 30.8 (t, C-8 to C-16), 33.1, 33.8 (t,
C-7, C-17), 38.2 (t, C-4), 56.8 (s, C-2), 66.5 (t, C-1, C-3), 125.5
136.0 (d, C-5, C-6). Exact mass (ESI+): C19H39NO2 + H+: calcd
314.3054, found 314.3071; C19H39NO2 + Na+: calcd 336.2873, found
336.2874; (C19H39NO2)2 + Na+: calcd 649.5854, found 649.5850.
The compound has been mentioned in a patent by Fujita but
was not characterized.35
3JC,F = 4.2 Hz, C-4), 29.4, 29.5, 29.6, 29.7, 29.8 (t, C-5 to C-13), 32.1
2
1
(t, C-14), 35.0 (dt, JC,F = 20.5 Hz, C-3), 92.3 (dd, JC,F = 174.6 Hz,
C-2). 19F NMR (282 MHz, CDCl3): d À171.3 (m, 1F, 2-CHF).
4.10.2. Diethyl 2-acetamido-2-(2-fluorohexadecyl)malonate
(48)
The iodofluoride 46 (370 mg, 1.00 mmol), the 2-acetamidomalo-
nate 23 (220 mg, 1.00 mmol, 1.0 equiv) and caesium carbonate
(1.10 g, 3.40 mmol, 3.4 equiv) were suspended in acetonitrile
(20 mL) and refluxed for 5 h. After cooling to rt and partial removal
of the solvent, the residue was adsorbed on silica gel (2 g) and puri-
fied by column chromatography (11 Â 4 cm, cyclohexane/ethyl ace-
tate, 4:1) to give the product as a white solid. Yield: 214 mg (47%).
4.9. Synthesis of 2-Amino-2-(fluoromethyl)octadecane-1-ol (43)
The aminodiol 29 (synthesized in analogy to Ref. 21, see SI)
(316 mg, 1.00 mmol) was suspended in dichloromethane (15 mL)
and cooled down to À78 °C. Diethylaminosulfur trifluoride (DAST)
(0.13 mL, 1.00 mmol, 1.0 equiv) was dripped slowly into the sus-
pension at À78 °C. The mixture was allowed to warm up to
À10 °C overnight and was neutralized with saturated sodium
bicarbonate solution (30 mL). The phases were separated and the
aqueous phase was extracted with dichloromethane (4 Â 25 mL).
The combined organic phases were dried over Na2SO4 and the sol-
vent was removed in vacuo. The residue was purified by column
O
21
20
F
NH
17
15
13
11
9
7
19
5
2
O
1
4
O
18
16
14
12
10
8
6
3
22
23
O
O
24
25
chromatography
(10 Â 4 cm,
dichloromethane/methanol,

1022
S. S. Schilson et al. / Bioorg. Med. Chem. 23 (2015) 1011–1026
(75 MHz, CDCl3, CD3OD): d 13.4 (q, C-19), 22.3 (t, C-18), 24.6 (dt,
3JC,F = 4.6 Hz, C-7), 29.0, 29.1, 29.2 (t, C-8 to C-16), 31.5 (t, C-17),
Mp: 71–73 °C. 1H NMR (300 MHz, CDCl3): d 0.88 (m, 3H, 19-CH3),
1.21–1.45 (m, 32H, 6-CH2 to 18-CH2, 23-CH3, 25-CH3), 2.06 (s, 3H,
21-CH3), 2.68 (m, 2H, 4-CH2), 4.23 (m, 4H, 22-CH2, 24-CH2), 4.56
(m, 1H, 5-CH), 7.07 (s, 1H, 2-NH). 13C NMR (75 MHz, CDCl3): d
13.8 (q, C-19), 14.0 (q, C-23 or C-25), 14.1 (q, C-23 or C-25), 22.7
2
2
36.0 (dt, JC,F = 20.8 Hz, C-6), 38.7 (dt, JC,F = 19.8 Hz, C-4), 55.1 (s,
1
C-2), 65.2 (t, C-1 or C-3), 65.6 (t, C-1 or C-3), 91.4 (dt, JC,F = 165.1
Hz, C-5). 19F NMR (282 MHz, CDCl3, CD3OD): d À178.3 (m, 1F,
5-CHF). Exact mass (ESI+): C19H40FNO2 + H+: calcd 334.3116, found
334.3110; C19H40FNO2 + Na+: calcd 356.2935, found 356.2936.
3
(t, C-18), 23.0 (q, C-21), 24.9 (dt, JC,F = 4.5 Hz, C-7), 29.4, 29.5,
2
29.6, 29.7 (t, C-8 to C-16), 31.9 (t, C-17), 35.2 (dt, JC,F = 20.2 Hz,
2
C-6), 38.0 (dt, JC,F = 19.1 Hz, C-4), 62.3 (t, C-22 or C-24), 62.9 (t,
1
4.11. Synthesis of the unsaturated 4-fluoro compounds
C-22 or C-24), 64.3 (s, C-2), 90.6 (dt, JC,F = 166.4 Hz, C-5), 167.4
(s, C-1 or C-3), 168.5 (s, C-1 or C-3), 169.4 (s, C-20). 19F NMR
(282 MHz, CDCl3): d À181.2 (m, 1F, 5-CHF). Exact mass (ESI+):
The allylic alcohol 54 was prepared as we described earlier (see
SI).24,34
C
25H46FNO5 + Na+: calcd 482.3252, found 482.3255; (C25H46FNO5)2 +
Na+: calcd 941.6612, found 941.6611.
4.11.1. 2-Fluorohexadec-2-ene-1-yl 4-methylbenzene sulfonate
(55)
4.10.3. N-(4-Fluoro-1-hydroxy-2-(hydroxymethyl)octadecan-2-
yl)acetamide (49)
The fluoroallylic alcohol 54 (2.36 g, 8.95 mmol, purity: 98% (GC))
was dissolved in dichloromethane (100 mL) and treated with potas-
sium hydroxide (1.33 g, 23.7 mmol, 2.6 equiv). The mixture was cooled
to 0 °C and tosyl chloride (3.83 g, 20.1 mmol, 2.2 equiv) was added.
After 1 h the mixture was warmed to rt and stirred for 2 days. The reac-
tion was stopped by addition of water (50 mL) and the phases were
separated. The aqueous layer was extracted with dichloromethane
(3 Â 30 mL) and the combined organic layers were dried over MgSO4.
The solvent was removed under reduced pressure and the tosylate 55
was purified by column chromatography (35 Â 4 cm, cyclohexane/
ethyl acetate, 40:1) and isolated as a colourless oil. Yield: 2.14 g
(58%). E/Z-ratio: 15:85 (NMR, changed due to purification).
The diester 48 (1.34 g, 2.90 mmol) was dissolved in THF (15 mL)
and reacted with lithium chloride (615 mg, 14.5 mmol, 5.0 equiv)
and sodium borohydride (549 mg, 14.5 mmol, 5.0 equiv). The mix-
ture was cooled to 0 °C and treated with ethanol (30 mL). After stir-
ring for one more hour at 0 °C the mixture was stirred at rt overnight.
After cooling to 0 °C 10% citric acid was added to adjust pH 4. THF
was removed and the residue was dissolved in water (50 mL). The
aqueous phase was extracted with dichloromethane (4 Â 30 mL).
The organic layer was washed with water and dried over Na2SO4.
The solvent was removed and the residue was purified by column
chromatography (18 Â 4 cm, cyclohexane/ethyl acetate, 1:3). The
product was isolated as a white solid. Yield: 840 mg (77%).
18
17
19
20
F
O
S
O
14
12
10
8
6
4
16
2
O
23
O
15
13
11
9
7
5
3
1
20
F
HN
22 21
21
OH
17
15
13
11
9
7
19
5
2
1
3
1H NMR (300 MHz, CDCl3): d 0.87 (m, 3H, 16-CH3), 1.18–1.34 (m, 22H,
5-CH2 to 15-CH2), 1.91 (m, 2H, (E)-4-CH2), 2.01 (m, 2H, (Z)-4-CH2),
2.45 (s, 3H, (Z)-23-CH3), 2.49 (s, 3H, (E)-23-CH3), 4.52 (d, 3JH,F = 17.9
18
16
14
12
10
8
6
4
OH
Mp: 95–96 °C. 1H NMR (300 MHz, CDCl3): d 0.88 (m, 3H, 19-CH3),
1.22–1.50 (m, 24H, 7-CH2 to 18-CH2), 1.73 (m, 2H, 6-CH2), 2.03 (s,
3H, 21-CH3), 2.21 (m, 2H, 4-CH2), 3.68 (m, 4H, 1-CH2, 3-CH2), 4.74
(m, 1H, 5-CH), 6.50 (s, 1H, 2-NH). 13C NMR (75 MHz, CDCl3): d 14.2
3
Hz, 2H, (Z)-1-CH2), 4.64 (d, JH,F = 20.9 Hz, 2H, (E)-1-CH2), 4.92 (dt,
3
3
3JH,H = 7.6 Hz, JH,F = 34.8 Hz, 1H, (Z)-3-CH), 5.33 (dt, JH,H = 8.2 Hz,
3JH,F = 19.6 Hz, 1H, (E)-3-CH), 7.34 (d, JH,H = 8.0 Hz, 2H, (Z)-19-CH,
(Z)-21-CH), 7.41 (d, JH,H = 8.3 Hz, 2H, (E)-19-CH, (E)-21-CH), 7.80
3
3
3
(q, C-19), 22.8 (q, C-21), 24.1 (t, C-18), 25.0 (dt, JC,F = 4.8 Hz, C-7),
3
3
(d, JH,H = 8.3 Hz, 2H, (Z)-18-CH, (Z)-22-CH), 7.92 (d, JH,H = 8.4 Hz,
29.5, 29.6, 29.7, 29.8 (t, C-8 to C-16), 32.0 (t, C-17), 36.2 (dt, 2JC,F = 21.1
Hz, C-6), 37.5 (dt, 2JC,F = 19.1 Hz, C-4), 60.9 (d, 3JC,F = 1.7 Hz, C-2), 65.0
(dt, 4JC,F = 1.7 Hz, C-1 or C-3), 66.2 (t, C-1 or C-3), 92.6 (dt, 1JC,F = 162.9
Hz, C-5), 172.0 (s, C-20). 19F NMR (282 MHz, CDCl3): d À176.67 (m, 1F,
5-CHF). Exact mass (ESI+): C21H42FNO3 + H+: calcd 376.3221, found
376.3220; C21H42FNO3 + Na+: calcd 398.3041, found 398.3040.
2H, (E)-18-CH, (E)-22-CH).13C NMR (75 MHz, CDCl3): d 14.3 (q, C-
3
16), 21.8 (q, C-23), 22.8 (t, C-15), 23.8 (dt, JC,F = 3.3 Hz, (Z)-C-4),
25.5 (dt, 3JC,F = 7.1 Hz, (E)-C-4), 28.8 (dt, 4JC,F = 1.7 Hz, (Z)-C-5), 29.2,
29.5, 29.7, 29.8 (t, C-6 to C-13), 32.1 (t, C-14), 63.6 (dt, 2JC,F = 31.6 Hz,
2
2
(E)-C-1), 68.0 (dt, JC,F = 31.8 Hz, (Z)-C-1), 114.4 (dd, JC,F = 13.5 Hz,
(Z)-C-3), 114.7 (dd, JC,F = 17.8 Hz, (E)-C-3), 127.2 (d, (E)-C-18, (E)-
2
C-22), 128.1 (d, (Z)-C-18, (Z)-C-22), 130.0 (d, (Z)-C-19, (Z)-C-21),
130.4 (d, (E)-C-19, (E)-C-21), 133.3 (s, C-17), 145.1 (s, C-20), 151.5
4.10.4. 2-Amino-2-(2-fluorohexadecyl)propane-1,3-diol (50)
The amidodiol 49 (400 mg, 1.06 mmol) was dissolved in meth-
anol (15 mL) and treated with 1 M NaOH (19 mL 1.90 mmol,
1.8 equiv). The reaction mixture was refluxed for 5 h. After cooling
to rt the mixture was extracted with dichloromethane (4 Â 15 mL).
The organic layer was washed with water and dried over Na2SO4
and the solvent was evaporated. The residue was recrystallized
from ethyl acetate to give a white solid. Yield: 206 mg (58%).
1
(d, JC,F = 253.4 Hz, (Z)-C-2). 19F NMR (282 MHz, CDCl3): d À120.22
3
(m, 1F, (Z)-2-CF), À112.74 (q, JH,F = 20.7 Hz, 1F, (E)-2-CF). Exact
mass (ESI+): C23H37FO3S + Na+: calcd 435.2340, found 435.2340;
(C23H37FO3S)2 + Na+: calcd 847.4787, found 847.4777.
4.11.2. Diethyl-2-acetamido-2-(2-fluorohexadec-2-ene-1-
yl)malonates (56)
F
NH2
The mixture of the tosylates 55 (1.90 g, 4.61 mmol), diethyl 2-
acetamidomalonate 23 (1.00 g, 4.61 mmol, 1.0 equiv) and caesium
carbonate (1.78 g, 5.48 mmol, 1.2 equiv) were suspended in aceto-
nitrile (30 mL) and heated to reflux for 6 h. After cooling to rt over-
night the mixture was filtrated, concentrated in vacuo and purified
by column chromatography (40 Â 3 cm, cyclohexane/ethyl acetate,
10:1). The diastereomers were separated and obtained as white
solids. Yield: 1.40 g (66%, both isomers and a mixed fraction).
17
15
13
11
9
7
19
5
2
OH
1
3
18
16
14
12
10
8
6
4
OH
Mp: 96–98 °C. 1H NMR (300 MHz, CDCl3, CD3OD): d 0.88 (t,
3JH,H = 6.4 Hz, 3H, 19-CH3), 1.21–1.79 (m, 28H, 4-CH2, 6-CH2 to
18-CH2), 3.50 (m, 4H, 1-CH2, 3-CH2), 4.79 (m, 1H, 5-CH). 13C NMR

S. S. Schilson et al. / Bioorg. Med. Chem. 23 (2015) 1011–1026
1023
4.11.2.1. (E)-Diethyl-2-acetamido-2-(2-fluorohexadec-2-ene-1-
O
yl)malonate ((E)-56).
21
O
20
F
HN
21
17
15
13
11
9
7
19
5
2
20
2
OH
F
NH
1
3
18
16
14
12
10
8
6
4
5
O
6
OH
1
O
4
O
17
15
13
11
9
3
19
22
23
7
O
18
16
14
12
10
8
24
Mp: 86 °C. 1H NMR (300 MHz, CDCl3): d 0.87 (m, 3H, 19-CH3), 1.24–
25
1.30 (m, 22H, 8-CH2 to 18-CH2), 2.00–2.12 (s, 5H, 7-CH2, 21-CH3),
3
2
2.54 (d, JH,F = 23.9 Hz, 2H, 4-CH2), 3.59 (d, JH,H = 11.7 Hz, 2H, 1-
2
CH2, 3-CH2), 3.81 (d, JH,H = 11.7 Hz, 2H, 1-CH2, 3-CH2), 4.72 (dt,
Yield: 148 mg (7%). Mp: 44 °C. 1H NMR (300 MHz, CDCl3): d 0.88 (t,
3JH,H = 6.8 Hz, 3H, 19-CH3), 1.21–1.30 (m, 28H, 8-CH2 to 18-CH2, 23-
CH3, 25-CH3), 1.83 (m, 2H, 7-CH2), 2.01 (s, 3H, 21-CH3), 3.37 (d,
3
3JH,H = 7.5, JH,F = 38.6 Hz, 1H, 6-CH), 6.20 (s, 1H, 2-NH). 13C NMR
(75 MHz, CDCl3): d 14.2 (q, C-19), 22.8 (t, C-18), 23.8 (dt, 3JC,F = 4.6 -
Hz, C-7), 24.1 (q, C-21), 29.3, 29.4, 29.5, 29.7, 29.8 (t, C-8 to C-16),
32.0 (t, C-17), 35.3 (dt, JC,F = 26.1 Hz, C-4), 60.8 (d, JC,F = 3.8 Hz,
3
3JH,F = 23.2 Hz, 2H, 4-CH2), 4.26 (q, JH,H = 7.1 Hz, 4H, 22-CH2, 24-
2
3
3
3
CH2), 5.18 (dt, JH,H = 8.0 Hz, JH,F = 22.8 Hz, 1H, 6-CH), 6.87 (s, 1H,
4
2
C-2), 65.6 (dt, JC,F = 1.8 Hz, C-1, C-3), 111.1 (dd, JC,F = 15.6 Hz, C-
2-NH). 13C NMR (75 MHz, CDCl3): d 14.0 (q, C-23, C-25), 14.2 (q,
C-19), 22.8 (t, C-18), 23.0 (q, C-21), 25.4 (d, JC,F = 8.5 Hz, C-7),
29.2, 29.5, 29.6, 29.7, 29.8 (t, C-9 to C-16), 30.1 (dt, JC,F = 2.1 Hz,
1
6), 155.3 (d, JC,F = 250.4 Hz, C-5), 172.0 (s, C-20). 19F NMR
3
3
3
(282 MHz, CDCl3): d À106.1 (dt, JH,F = 23.9 Hz, JH,F = 38.5 Hz, 1F,
4
5-CF). Exact mass (ESI+): C21H40FNO3 + H+: calcd 374.3065, found
2
374.3061;
C
21H40FNO3 + Na+: calcd 396.2884, found 396.2878;
C-8), 32.0 (t, C-17), 32.1 (dt, JC,F = 26.0 Hz, C-4), 62.9 (t, C-22,
3
2
C-24), 64.3 (d, JC,F = 3.5 Hz, C-2), 111.3 (dd, JC,F = 19.8 Hz, C-6),
(C21H40FNO3)2 + Na+: calcd 769.5877, found 769.5878. Exact mass
1
(ESI+):
C
20H38FNO2 + Na+: calcd 366.2779, found 366.2772;
154.6 (d, JC,F = 243.2 Hz, C-5), 167.6 (s, C-1, C-3), 169.2 (s, C-20).
19F NMR (282 MHz, CDCl3): d À104.3 (q, JH,F = 23.0 Hz, 1F, 5-CF).
3
(C20H38FNO2)2 + Na+: calcd 709.5665, found 709.5658.
Exact mass (ESI+): C25H44FNO5 + Na+: calcd 480.3096, found
480.3088; (C25H44FNO5)2 + Na+: calcd 937.6299, found 937.6317.
4.11.4. (E)-N-(4-Fluoro-1-hydroxy-2-(hydroxymethyl)octadec-
4-ene-2-yl)acetamide ((E)-57)
4.11.2.2. (Z)-Diethyl-2-acetamido-2-(2-fluorohexadec-2-ene-1-
yl)malonate ((Z)-56).
The diester (E)-56 (75 mg, 0.16 mmol) was dissolved in THF
(3 mL) and treated with lithium chloride (35 mg, 0.82 mmol,
5.0 equiv) and sodium borohydride (31 mg, 0.82 mmol, 5.0 equiv).
The mixture was cooled to 0 °C and ethanol (6 mL) was added.
After 35 min the mixture was warmed to rt and stirred for 6 days.
The reaction was quenched with 20% potassium sodium tartrate
solution (5 mL) and the aqueous layer was extracted with dichloro-
methane (6 Â 10 mL). The combined organic layers were dried over
Na2SO4 and concentrated under reduced pressure. Purification by
column chromatography (11.5 Â 2 cm, cyclohexane/ethyl acetate,
1:2) gave a mixture of the desired product (E)-57 and the side
product (E)-58. The mixture was used in the next reaction without
additional purification. Also starting material was reisolated. Yield:
30 mg (mixture, max. 50%).
O
21
20
F
NH
17
15
13
11
9
7
19
5
2
O
1
O
4
O
18
16
14
12
10
8
6
3
22
23
O
24
25
Yield: 1.11 g (53%). Mp: 80 °C. 1H NMR (300 MHz, CDCl3): d 0.88 (t,
3JH,H = 6.7 Hz, 3H, 19-CH3), 1.21–1.32 (m, 28H, 8-CH2 to 18-CH2, 23-
CH3, 25-CH3), 2.00 (m, 2H, 7-CH2), 2.03 (s, 3H, 21-CH3), 3.26 (d,
3
3JH,F = 21.6 Hz, 2H, 4-CH2), 4.26 (2q, JH,H = 7.1 Hz, 4H, 22-CH2, 24-
3
3
CH2), 4.57 (dt, JH,H = 7.6 Hz, JH,F = 37.5 Hz, 1H, 6-CH), 6.85 (s, 1H,
2-NH). 13C NMR (75 MHz, CDCl3): d 14.0 (q, C-23, C-25), 14.2 (q,
C-19), 22.8 (t, C-18), 23.0 (q, C-21), 23.7 (dt, JC,F = 4.5 Hz, C-7),
29.2, 29.5, 29.8 (t, C-8 to C-16), 32.0 (t, C-17), 35.9 (dt, JC,F = 25.6 -
O
3
3
21
20
F
HN
3
5
2
Hz, C-4), 62.9 (t, C-22, C-24), 64.7 (d, JC,F = 3.5 Hz, C-2), 110.7 (dd,
OH
6
2JC,F = 15.0 Hz, C-6), 154.4 (d, JC,F = 251.4 Hz, C-5), 167.5 (s, C-1,
1
1
3
4
17
15
13
11
9
19
C-3), 169.2 (s, C-20). 19F NMR (282 MHz, CDCl3): d À110.6 (dt,
7
OH
3
3JH,F = 21.6 Hz, JH,F = 37.5 Hz, 1F, 5-CF). Exact mass (ESI+): C25H44
18
16
14
12
10
8
FNO5 + Na+: calcd 480.3096, found 480.3095; (C25H44FNO5)2 + Na+:
calcd 937.6299, found 937.6321.
1H NMR (300 MHz, CDCl3): d 0.88 (t, JH,H = 6.4 Hz, 3H, 19-CH3),
3
4.11.3. (Z)-N-(4-Fluoro-1-hydroxy-2-(hydroxymethyl)octadec-
4-ene-2-yl)acetamide ((Z)-57)
1.23–1.35 (m, 22H, 8-CH2 to 18-CH3), 1.98 (m, 2H, 7-CH2), 2.03 (s,
3
3H, 21-CH3), 2.63 (d, JH,F = 25.9 Hz, 2H, 4-CH2), 3.56 (d,
2
2JH,H = 11.8 Hz, 2H, 1-CH2, 3-CH2), 3.81 (d, JH,H = 11.8 Hz, 2H, 1-
To a solution of the diester (Z)-56 (338 mg, 0.74 mmol) in THF
(6 mL) lithium chloride (157 mg, 3.70 mmol, 5.0 equiv) and sodium
borohydride (140 mg, 3.70 mg, 5.0 equiv) were added. The mixture
was cooled to 0 °C and treated with ethanol (12 mL). After 50 min
the reaction mixture was warmed to rt and stirred for 6 days. The
reaction was stopped by addition of 20% potassium sodium tartrate
solution (5 mL) and the aqueous phase was extracted with dichlo-
romethane (6 Â 10 mL). The combined organic phases were dried
over Na2SO4 and concentrated in vacuo. The residue was purified
by column chromatography (11 Â 3 cm, ethyl acetate) and the
product was obtained as a white solid. Yield: 142 mg (51%).
3
3
CH2, 3-CH2), 5.28 (dt, JH,H = 7.9 Hz, JH,F = 23.7 Hz, 1H, 6-CH), 6.18
(br s, 1H, 2-NH).13C NMR (75 MHz, CDCl3): d 14.3 (q, C-19), 22.8
3
(t, C-18), 24.2 (q, C-21), 25.6 (dt, JC,F = 8.8 Hz, C-7), 29.3, 29.4,
29.5, 29.6, 29.7, 29.8, 30.0, 30.1 (t, C-8 to C-16), 31.4 (dt, 3JC,F = 26.6 -
3
Hz, C-4), 32.1 (t, C-17), 61.1 (d, JC,F = 4.1 Hz, C-2), 65.7 (dt,
4JH,F = 1.2 Hz, C-1, C-3), 111.4 (dd, JC,F = 20.6 Hz, C-6),155.7 (d,
2
1JC,F = 241.5 Hz, C-5), 172.1 (s, C-20). 19F NMR (282 MHz, CDCl3): d
3
3
À99.4 (q, JH,F = 24.7 Hz, JH,F = 25.3 Hz, 1F, 5-CF). Exact mass
(ESI+): C21H40FNO3 + Na+: calcd 396.2884, found 396.2882;
(C21H40FNO3)2 + Na+: calcd 769.5877, found: 769.5880.

1024
S. S. Schilson et al. / Bioorg. Med. Chem. 23 (2015) 1011–1026
4.11.5. (Z)-2-Amino-2-(2-fluorohexadec-2-ene-1-yl)propane-
1,3-diol ((Z)-59)
4.12. Synthesis of the x-fluorinated analogues
The protected aminodiol (Z)-57 (61 mg, 0.16 mmol) was dis-
solved in methanol (5 mL) and treated with 1 M sodium hydroxide
solution (0.24 mL, 0.24 mmol, 1.5 equiv). The mixture was heated
to reflux for 6 h. After cooling to rt the mixture was diluted with
1 M sodium hydroxide solution (10 mL) and the aqueous phase
was extracted with dichloromethane (6 Â 10 mL). The combined
organic layers were dried over Na2SO4 and the solvent was
removed in vacuo. The product was crystallized from ethyl acetate
to give a white solid. Yield: 46 mg (87%).
4.12.1. Diethyl 2-acetamido-2-(15-hydroxypentadecyl)malonate
(64)
The bromide 62 was prepared in analogy to a procedure by Luu
et al.30,37 62 (966 mg, 3.1 mmol), diethyl 2-acetamidomalonate 23
(673 mg, 3.1 mmol, 1.0 equiv) and caesium carbonate (1.06 g,
3.3 mmol, 1.1 equiv) were suspended in acetonitrile (20 mL) and
the mixture was heated to reflux for 6 h. After cooling to rt, the
mixture was adsorbed on silica gel (1-2 g) and purified by column
chromatography (8 Â 4 cm, cyclohexane/ethyl acetate, 4:1 ? 100%
ethyl acetate). The product was obtained as a white solid. Yield:
1.11 g (80%).
F
NH2
17
15
13
11
9
7
19
5
2
OH
1
3
O
18
16
14
12
10
8
6
4
20
OH
19
NH
17
15
13
11
9
7
5
2
O
HO
1
O
4
O
18
16
14
12
10
8
6
3
21
22
Mp: 68–70 °C. 1H NMR (300 MHz, CDCl3): d 0.87 (m, 3H, 19-CH3),
1.20–1.37 (m, 22H, 8-CH2 to 18-CH2), 2.05 (m, 2H, 7-CH2), 2.29 (d,
3JH,F = 23.9 Hz, 2H, 4-CH2), 3.43–3.61 (m, 4H, 1-CH2, 3-CH2), 4.65
O
23
24
(dt, JH,H = 7.5 Hz, JH,F = 38.1 Hz, 1H, 6-CH). 13C NMR (75 MHz,
CD3OD, CDCl3): d 14.3 (q, C-19), 23.2 (t, C-18), 24.1 (dt, 3JC,F = 4.9 Hz,
C-7), 29.7, 29.9, 30.1, 30.2 (t, C-8 to C-16), 32.4 (t, C-17), 37.1 (dt,
3
3
Mp: 52 °C. 1H NMR (300 MHz, CDCl3): d 1.19–1.37 (m, 30H, 5-CH2 to
16-CH2, 22-CH3, 24-CH3), 1.56 (m, 2H, 17-CH2), 2.03 (s, 3H, 20-CH3),
2.30 (m, 2H, 4-CH2), 3.63 (t, JH,H = 6.6 Hz, 2H, 18-CH2), 4.24 (q,
2JC,F = 26.2 Hz, C-4), 56.1 (d, JC,F = 3.6 Hz, C-2), 65.9 (t, C-1,C-3),
3
110.5 (dd, JC,F = 15.6 Hz, C-6), 156.4 (d, JC,F = 252.2 Hz, C-5). 19F
2
1
3
3
NMR (282 MHz, CD3OD, CDCl3):
d
À104.6 (dt, JH,F = 23.9 Hz,
3JH,H = 7.2 Hz, 4H, 21-CH2, 23-CH2), 6.81 (s, 1H, 2-NH). 13C NMR
(75 MHz, CDCl3): d 14.1 (q, C-22, C-24), 23.1 (q, C-20), 23.7 (t, C-
5), 25.8 (t, C-16), 29.3, 29.4, 29.5, 29.7 (t, C-6 to C-15), 32.2 (t, C-
17), 32.9 (t, C-4), 62.5 (t, C-21, C-23), 63.0 (t, C-18), 66.7 (s, C-2),
168.3 (s, C-1, C-3), 169.0 (s, C-19). Exact mass (ESI+): C24H45NO6 +
Na+: calcd 466.3139, found 466.3138; (C24H45NO6)2 + Na+: calcd
909.6386, found 909.6394.
3JH,F = 37.8 Hz, 1F, 5-CF). Exact mass (ESI+): C19H38FNO2 + H+: calcd
332.2959, found 332.2955;
found 354.2776.
C
19H38FNO2 + Na+: calcd 354.2779,
4.11.6. (E)-2-Amino-2-(2-fluorohexadec-2-ene-1-yl)propane-
1,3-diol ((E)-59)
The mixture of the aminodiol (E)-57 and the aminoalcohol (E)-
58 (30 mg, max. 0.08 mmol) was dissolved in methanol (3 mL),
treated with 1 M sodium hydroxide solution (0.12 mL, 0.12 mmol,
max. 1.5 equiv) and heated to 100 °C for 7 h in a pressure vessel.
After cooling to rt the mixture was diluted with 1 M sodium
hydroxide solution (5 mL) and the aqueous phase was extracted
with dichloromethane (6 Â 10 mL). The combined organic layers
were dried over Na2SO4 and concentrated in vacuo. Purification
via column chromatography (20 Â 3 cm, dichloromethane/metha-
nol, 4:1) gave the product as a pale yellow solid. Yield: 6 mg (23%).
4.12.2. Diethyl 2-acetamido-2-(15-fluoropentadecyl)malonate
(66)
Under argon the alcohol 64 (443 mg, 1.0 mmol) was dissolved
in dry dichloromethane (8 mL), cooled down to À78 °C and care-
fully treated with DAST (0.18 mL, 1.3 mmol, 1.3 equiv). After 1 h,
the mixture was allowed to warm up to rt overnight. The reaction
was quenched with saturated sodium bicarbonate solution (20 mL)
at À10 °C. The phases were separated and the aqueous phase was
extracted with dichloromethane (4 Â 10 mL). The combined
organic layers were dried over Na2SO4 and the solvent was
removed in vacuo. The product was purified by column chroma-
tography (11.5 Â 3 cm, cyclohexane/ethyl acetate, 4:1) and was
obtained as a white and waxy solid. Yield: 179 mg (40%).
F
NH2
5
2
OH
6
1
3
4
17
15
13
11
9
19
7
OH
18
16
14
12
10
8
O
20
19
NH
3
17
15
13
11
9
7
5
Mp: 74 °C. 1H NMR (600 MHz, CDCl3): d 0.88 (t, JH,H = 7.0 Hz, 3H,
O
2
F
3
1
19-CH3), 1.24–1.39 (m, 22H, 8-CH2 to 18-CH2), 1.96 (q, JH,H = 7.5 -
4
O
18
16
14
12
10
8
6
3
21
22
3
O
Hz, 2H, 7-CH2), 2.40 (d, JH,F = 26.0 Hz, 2H, 4-CH2), 3.50 (d,
O
2JH,H = 11.1 Hz, 2H, 1-CH2, 3-CH2), 3.55 (d, JH,H = 10.9 Hz, 2H, 1-
2
23
CH2, 3-CH2), 5.23 (dt, JH,H = 7.9 Hz, JH,F = 23.4 Hz, 1H, 6-CH). 13C
NMR (151 MHz, CDCl3): d 14.3 (q, C-19), 22.8 (t, C-18), 25.9 (dt,
3JC,F = 8.8 Hz, C-7), 29.3, 29.5, 29.6, 29.7, 29.8, 30.0 (t, C-8 to C-16),
3
3
24
2
3
32.1 (t, C-17), 33.9 (dt, JC,F = 27.0 Hz, C-4), 56.3 (d, JC,F = 3.8 Hz,
Mp: 57 °C. 1H NMR (300 MHz, CDCl3): d 1.18–1.44 (m, 30H, 5-CH2 to
16-CH2, 22-CH3, 24-CH3), 1.68 (m, 2H, 17-CH2), 2.04 (s, 3H, 20-CH3),
2.31 (m, 2H, 4-CH2), 4.24 (q, JH,H = 7.1 Hz, 4H, 21-CH2. 23-CH2),
2
C-2), 67.5 (t, C-1, C-3), 110.3 (dd, JC,F = 20.7 Hz, C-6), 156.5 (d,
1JC,F = 243.7 Hz, C-5). 19F NMR (564 MHz, CDCl3): d À98.3 (q,
3
3JH,F = 25.7 Hz, 1F, 5-CF). Exact mass (ESI+): C19H38FNO2 + H+: calcd
3
2
332.2959, found 332.2959;
found 354.2779.
C
19H38FNO2 + Na+: calcd 354.2779,
4.43 (dt, JH,H = 6.2 Hz, JH,F = 47.4 Hz, 2H, 18-CH2), 6.83 (s, 1H,
2-NH). 13C NMR (75 MHz, CDCl3): d 14.0 (q, C-22, C-24), 23.0 (q,

S. S. Schilson et al. / Bioorg. Med. Chem. 23 (2015) 1011–1026
1025
3
2
C-20), 23.6 (t, C-5), 25.2 (dt, JC,F = 5.5 Hz, C-16), 29.3, 29.4, 29.6,
3JH,H = 6.1 Hz, JH,F = 47.5 Hz, 2H, 18-CH2). 13C NMR (75 MHz,
2
3
29.7 (t, C-6 to C-15), 30.4 (dt, JC,F = 19.3 Hz, C-17), 32.1 (t, C-4),
CD3OD, CDCl3): d 23.5 (t, C-5), 25.7 (dt, JC,F = 5.4 Hz, C-16), 29.8,
30.1, 30.2 (t, C-7 to C-15), 31.0 (dt, JC,F = 19.4 Hz, C-17), 31.0 (t,
1
2
62.4 (t, C-21, C-23), 66.6 (s, C-2), 84.2 (dt, JC,F = 163.9 Hz, C-18),
168.3 (s, C-1, C-3), 168.9 (s, C-19). 19F NMR (282 MHz, CDCl3): d
C-6), 34.8 (t, C-4), 56.3 (s, C-2), 66.3 (t, C-1, C-3), 84.6 (dt,
3
2
À218.4 (tt, JH,F = 24.8 Hz, JH,F = 47.4 Hz, 1F, 18-CH2F). Exact mass
(ESI+): C24H44FNO5 + H+: calcd 446.3276, found 446.3274; C24H44
FNO5 + Na+: calcd 468.3096, found 468.3093; (C24H44FNO5)2 + Na+:
calcd 913.6299, found 913.6286.
1JC,F = 163.7 Hz, C-18). 19F NMR (282 MHz, CD3OD, CDCl3):
d
3
2
À218.3 (tt, JH,F = 24.9 Hz, JH,F = 47.5 Hz, 1F, 18-CH2F). Exact mass
(ESI+): C18H38FNO2 + H+: calcd 320.2959, found 320.2961.
5. Determination of the immunosuppressive activity in vivo and
phosphorylation of Erk MAPK in vitro
4.12.3. N-[16-Fluoro-1,1-bis(hydroxymethyl)hexadecyl]
acetamide (68)
The diester 66 (170 mg, 0.38 mmol) was dissolved in THF
(6 mL). Lithium chloride (81 mg, 1.90 mmol, 5.0 equiv) and sodium
borohydride (72 mg, 1.90 mmol, 5.0 equiv) were added. The mix-
ture was cooled to 0 °C and treated with ethanol (12 mL). After
35 min, the mixture was allowed to warm up to rt and was stirred
overnight. After cooling to 0 °C potassium sodium tartrate solution
(20%, 5 mL) was added and THF was removed in vacuo. The residue
was diluted with water (5 mL) and extracted with dichlorometh-
ane (4 Â 15 mL). The organic layers were dried over Na2SO4 and
the solvent was removed in vacuo. The product was purified by
column chromatography (9.5 Â 3 cm, cyclohexane/ethyl acetate,
1:2) and was obtained as a white solid. Yield: 31 mg (23%).
The compounds were dissolved in a 0.9% NaCl-solution and
injected intraperitoneally at a dose of 1.25
lg/kg body weight of
wild-type mice in a total volume of 200 L. After 24 h, blood was
l
drawn from the retroorbital plexus in 17.8 mM EDTA as anti-coag-
ulant. The lymphocytes (T- and B-cells) were analyzed by flow
cytometry as follows: after lysis of the red blood cells by BD Pharm
Lyse in 50
100 L FACS buffer (1% BSA in PBS). The antibodies to CD45, CD4,
CD8 and B220 (all BD Biosciences) were added (1 L antibody
lL blood, the cells were washed and resuspended in
l
l
per sample each) and the mixture was incubated at room temper-
ature for 30 min. The cells were washed (3 times) and then ana-
lyzed by flow cytometry. Chinese hamster ovarian cells stably
transfected with the human S1P1 receptor (CHO-S1P1) were cul-
tured at 37 °C, 5% CO2, in selection media (aMEM+GlutaMAX, sup-
O
plemented with 10% FCS, 1%
streptomycin/amphotericin B, 50
mL G418; Gibco/Invitrogen). Confluent cells were incubated over-
night in serum-free medium prior to stimulation with 1 M of all
compounds for 10 min. Cell lysates (20 g of protein) were dena-
L
-glutamate, 1% penicillin/
20
19
NH
lg/mL Gentamicin, and 0.5 mg/
17
15
13
11
9
7
5
2
F
OH
1
3
18
16
14
12
10
8
6
4
l
OH
l
tured with sample buffer (0.5 M TrisHCl, pH 6.8, 10% glycerol,
10% SDS, 5% b-mercapto-ethanol, 0.1% bromphenol blue) for
10 min at 100 °C. Samples were separated on 10% SDS–polyacryl-
amide gels and transferred onto PVDF membranes. Western blot-
ting was performed with an antibody against pp 44/42 (Cell
signaling, rabbit polyclonal anti-phospho-p44/42, 1:1000) over-
night at 4 °C followed by incubation with a HRP-conjugated sec-
ondary antibody (Vector, Santa Cruz) at 1:5000 at room
temperature for 1 h. Blots were developed using ECL chemilumi-
nescence (Amersham ECL Western blotting detection reagents,
GE).
Mp: 101 °C. 1H NMR (300 MHz, CDCl3, CD3OD): d 1.20–1.44 (m,
24H, 5-CH2 to 16-CH2), 1.59–1.78 (m, 4H, 4-CH2, 17-CH2), 2.01 (s,
2
3H, 20-CH3), 3.62 (d, JH,H = 11.4 Hz, 2H, 1-CH2, 3-CH2), 3.69 (d,
2JH,H = 11.6 Hz, 2H, 1-CH2, 3-CH2), 4.43 (dt, 3JH,H = 6.2 Hz,
2JH,F = 47.4 Hz, 2H, 18-CH2). 13C NMR (101 MHz, CDCl3, CD3OD): d
3
23.5 (s, C-20), 23.6 (t, C-5), 25.7 (dt, JC,F = 5.3 Hz, C-16), 29.8,
2
30.1, 30.2, 30.7 (t, C-6 to C-15), 31.0 (dt, JC,F = 19.2 Hz, C-17), 32.5
1
(t, C-4), 62.1 (s, C-2), 64.9 (t, C-1, C-3), 84.6 (dt, JC,F = 163.6 Hz, C-
18), 173.4 (s, C-19). 19F NMR (282 MHz, CDCl3, CD3OD): d À218.3
3
2
(tt, JH,F = 23.8 Hz, JH,F = 47.5 Hz, 1F, 18-CH2F). Exact mass (ESI+):
C
20H40FNO3 + H+: calcd 362.3065, found 362.3068; C20H40FNO3 +
Acknowledgements
Na+: calcd 384.2884, found 384.2886.
This work was supported by the Deutsche Forschungsgemein-
schaft (Collaborative Research Center, SFB 656, Project A06).
4.12.4. 2-Amino-2-(15-fluoropentadecyl)propane-1,3-diol (71)
The aminodiol 68 (28 mg, 77 lmol) was dissolved in methanol
Supplementary data
(3 mL) and treated with 1 M NaOH (0.12 mL, 0.12 mmol, 1.6 equiv).
The reaction mixture was heated to 120 °C for 6 h in a pressure
vessel. After cooling to rt the mixture was diluted with 1 M NaOH
(3 mL) and extracted with dichloromethane (5 Â 10 mL). The com-
bined organic layers were dried over Na2SO4 and the solvent was
evaporated. The product was crystallized from ethyl acetate and
was isolated as a white solid. Yield: 23 mg (90%).
Supplementary data (copies of the NMR spectra of the described
compounds and spectroscopic data of starting materials and addi-
tional compounds) associated with this article can be found, in the
online version, at http://dx.doi.org/10.1016/j.bmc.2015.01.014.
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