Synthesis of fluorinated agonist of sphingosine-1-phosphate receptor 1

Article abstract of DOI:10.1016/j.bmc.2014.06.038

The bioactive metabolite sphingosine-1-phosphate (S1P), a product of sphingosine kinases (SphKs), mediates diverse biological processes such as cell differentiation, proliferation, survival and angiogenesis. A fluorinated analogue of S1P receptor agonist has been synthesized by utilizing a ring opening reaction of oxacycles by a lithiated difluoromethylphosphonate anion as the key reaction. In vitro activity of this S1P analogue is also reported.

Full text of DOI:10.1016/j.bmc.2014.06.038

Accepted Manuscript
Synthesis of fluorinated agonist of sphingosine-1-phosphate receptor 1
Lucie Aliouane, Sovy Chao, Leyre Brizuela, Emmanuel Pfund, Olivier
Cuvillier, Ludovic Jean, Pierre-Yves Renard, Thierry Lequeux
PII:
S0968-0896(14)00484-2
DOI:
http://dx.doi.org/10.1016/j.bmc.2014.06.038
BMC 11675
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
21 January 2014
16 June 2014
18 June 2014
Please cite this article as: Aliouane, L., Chao, S., Brizuela, L., Pfund, E., Cuvillier, O., Jean, L., Renard, P-Y.,
Lequeux, T., Synthesis of fluorinated agonist of sphingosine-1-phosphate receptor 1, Bioorganic & Medicinal
Chemistry (2014), doi: http://dx.doi.org/10.1016/j.bmc.2014.06.038
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Lucie Aliouane, Sovy Chao, Leyre Brizuela, Emmanuel Pfund, Olivier Cuvillier, Ludovic Jean, Pierre-Yves
Renard*, Thierry Lequeux*

Bioorganic & Medicinal Chemistry
j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m
Synthesis of fluorinated agonist of sphingosine-1-phosphate receptor 1
Lucie Aliouanea,b, Sovy Chaob, Leyre Brizuelac,d, Emmanuel Pfunda, Olivier Cuvillierc,d, Ludovic Jeanb,
Pierre-Yves Renardb∗, Thierry Lequeuxa∗
aLaboratoire de Chimie Moléculaire et Thioorganique, UMR CNRS 6507 & FR3038, ENSICAEN, Normandie Université, Université de Caen Basse-Normandie,
6 Bd du Maréchal Juin, Caen 14050, France
b Normandie Université, COBRA, UMR 6014 & FR 3038; Université de Rouen; INSA Rouen; CNRS, 1 rue Tesnière 76821 Mont-Saint-Aignan, Cedex, France
c CNRS, Institut de Pharmacologie et de Biologie Structurale, 205 Route de Narbonne, 31077 Toulouse, France
d Université de Toulouse, UPS, IPBS, 205 Route de Narbonne, 31077 Toulouse, France
ARTICLE INFO
ABSTRACT
Article history:
Received
The bioactive metabolite Sphingosine-1-phosphate (S1P), a product of sphingosine kinases
(SphKs), mediates diverse biological processes such as cell differentiation, proliferation,
survival and angiogenesis. A fluorinated analogue of S1P receptor agonist has been synthesized
by utilizing a ring opening reaction of oxacycles by a lithiated difluoromethylphosphonate anion
as the key reaction. In vitro activity of this S1P analogue is also reported.
Received in revised form
Accepted
Available online
2009 Elsevier Ltd. All rights reserved.
Keywords:
Agonist
Sphingosine-1-phosphate
Difluorophosphonate
Fluorine
their movement to the central nervous system.9 This is turn would
limit the autoimmune responses in multiple sclerosis and was
actually proposed as an antirejection medication after
transplantation. With regards to mechanism of action underlying
lymphopenia, it is mostly suggested that FTY720-P causes
receptor internalization and proteosomal degradation, leading to a
S1P1-null state.10
1. Introduction
Sphingosine-1-phosphate (S1P) is a phospholipid regulating
pleiotropic biological activities including proliferation, survival,
inflammation and angiogenesis.1 The bioactive sphingolipid
metabolite, sphingosine-1-phosphate (S1P) is a high affinity
ligand for five G-protein coupled receptors (S1P1-5)2 that
functions as a key regulator in many biological processes
including blood vessel development, hearth rhythm, blood
pressure, and immune regulationS.3-6
NH2
HO
H2N
X
R
O
HO
P
HO
P
11
O
OH
HO
In the 1990s, Fujita’s group isolated the natural compound,
myriocin, from the fungus Isaria sinclairii, which showed
impressive immunosuppressive activity.7 Various structural
modifications of myriocine led to the development of the
O
Sphingosine-1-phosphate (S1P)
7
FTY720-P
(X = O, R = CH2OH)
1
2
(X = CH2, R = CH2OH)
(X = O, R = H)
immunomodulator FTY720 (FingolimodTM). FTY720 is
a
NH2
F2
C
HO
prodrug requiring metabolization in vivo by sphingosine kinase-2
isoform into its bioactive S-isomeric monophosphate ester
FTY720-phosphate (FTY720-P),8 which interacts with four S1P
receptors (S1P1, S1P3-5); however, the majority of biological
effects of FTY720-P are attributed to its interaction with S1P1.
The interaction of FTY720-P with S1P1 is thought to induce
sequestration of lymphocytes in lymph nodes thereby preventing
P
HO
O
3
7
Scheme 1. Structures of S1P and S1P1 receptor agonists
Therefore, the discovery of subtype selective S1P1 modulators
12
are highly desirable as potential therapeutics.11, The success
———
∗ Corresponding authors. Fax: (+) 33 2 35 52 29 71; e-mail: pierre-yves.renard@univ-rouen.fr (P.-Y. Renard) and fax: (+) 33 2 31 45 28 65; e-
mail: thierry.lequeux@ensicaen.fr (T. Lequeux).

Scheme 2. Two retrosynthetic pathways to prepare α,α-difluorophosphonate derivative 3
of FTY720 in human clinical investigations has prompted
synthetic efforts to provide both pharmacological tools to study
S1P signaling and therapeutics.13 Since FTY720-P is not stable in
physiological medium, there remains a tremendous need to
develop a nonhydrolysable methylene-phosphonate 1 analogue as
a S1P receptor agonist.14, 15 Previous work has demonstrated that
deletion of hydroxymethyl group (compound 2) have no effect on
S1P receptor affinity.14 Other results demonstrated that
lithiation of 4 in the presence of t-BuLi and subsequent capture
with a Lewis acid can effectively control the reactivity of the
corresponding carbanion species, which in turn, enables rapid
access to γ, δ and ε-hydroxyl α,α-difluoromethylphosphonates
after addition to strained heterocycles such as epoxides,
trimethylene oxide and even THF.27
To expand the scope of this ring opening reaction with other
heterocycles, we applied this methodology towards the
generation of γ-amino-α,α-difluoromethylphosphonates in an
effort to synthesize fluorinated analogues of 1. As shown in
scheme 3, sulfamidates 8a-h, prepared from the corresponding
aminoalcohols,28 were subjected to the in situ derived
phosphonodifluoromethyl lithium anion to readily generate
various γ-amino-α,α-difluoromethylphosphonates 9a, 9b, 9d and
9e after acidic workup. However, the methodology failed in the
presence of longer carbon chain sulfamide derivatives 9f-h.
Despite the various tested conditions (Lewis acid addition, higher
temperature …), none of sulfamidates 8f-h did successfully react
with the anion 4.
enzymatically
and
hydrolytically
stable
α,α-
difluoromethylphosphonates mimics are better isopolar and
isosteric analogues than methylene-phosphonate ones.16,17 The
combination of these reports has generated significant interest in
our group to develop
a
physiologically stable α,α-
difluorophosphonate derivative 3 of FTY720-P. Herein, we
report the synthesis and biological evaluation of a fluorinated
agonist for the sphingosine-1-phosphate receptor through a novel
ring opening of oxacycles with α,α-difluoromethylphosphonate
anion. Two pathways have been explored to obtain the key γ-
amino-α,α-difluoromethylphosphonates moiety : the first one
based on ring opening reaction of 1,2 cyclic sulfamidates, and the
second one based on an epoxide ring opening (scheme 2).
2. Results and Discussion
2.1. Chemistry
Preparation of the α,α-difluoromethyl phosphonates is
commonly conducted through lithiation of commercially
available diethyl difluoromethylphosphonate.18-25 However, the
strict use of HCFC and CFC from Montreal’s protocol has
severely limited access to these starting materials. To circumvent
this limitation, our developed an alternative approach utilizing
readily available phosphonodifluoromethyl sulfide 4, prepared
from nucleophilic fluoride sources.26 In contrast to the previously
described methods using diethyl α,α-difluoromethylphosphonate,
Scheme 3. Ring opening reaction of 1,2-cyclic sulfamidates 8a-h

Scheme 4. Synthesis of fluorinated S1P agonist 3
In accordance with our desired goal to synthesize a fluorinated
analogue of 1, we altered our original strategy to incorporate an
epoxide, instead of a sulfamidate as the electrophile, which after
ring opening, the alcohol would be transformed into the
necessary amine group. As shown in scheme 4, preparation of the
pivotal epoxide 15 commenced with the Friedel Crafts acylation
of bromobenzene with octanoyl chloride29 followed by
Sonogashira coupling of bromide 10 to furnish the desired ketone
11. Simultaneous reduction of the alkyne and ketone moieties
with H2 afforded alcohol 12 which was subsequently transformed
into the alkyl iodide 13 with I2 and PPh3. After extensive
optimization, only the combination of the 13 and tBuOK30 could
the elimination product 14 be obtained in high yields which was
then converted to the key epoxide 15 under standard epoxidation
conditions with m-CPBA.31
TMSBr and methanol quench generated the phosphonic acid 3 in
50% yield.
2.2. Biological tests
We compared the proliferative effects of fluorinated S1P
agonist 3 (C3) with S1P the active (S)-enantiomer of FTY720-P,
which is the in vivo phosphorylated product of FTY720 in
representative PC-3 and C4-2B prostate cancer cell lines.33, 34 As
shown in Figure 1, whereas treatment with S1P and FTY720-P
resulted in a robust proliferative effect peaking at 100 nM,
compound 3 did not exhibit any proliferative effect after 24 h of
treatment in both PC-3 and C4-2B cell lines. On the contrary,
compound 3 showed an inhibitory effect at concentrations above
1 µM with a marked effect at 10 µM (IC50 ≈ 3 and 20 µM for C4-
2B and PC-3, respectively) somehow similar to the inhibition of
proliferation observed with high doses of FTY720-P. A similar
finding was observed using an MTT-based cell viability (data not
shown).
With the necessary epoxide in hand, we next turned our
attention towards the incorporation of the difluorophosphonate
moiety. Following our previous protocols and under optimized
conditions, lithiation of phosphonate 4 with t-BuLi in the
.
presence of BF3 Et2O generated the necessary nucleophilic
equivalents to react with epoxide 15 to rapidly gain access to y-
hydroxy-a,a-difluoromethylphosphonate 16 in high yields. To
obtain the desired y-aminophosphonate analogue 3, the alcohol
was converted to the necessary amine functionality in a three-step
process. Accordingly, azide formation under Mitsunobu
conditions with diphenylphosphoryl azide DPPA32 as the azide
source followed by acidic reduction with H2 afforded amine 18.
Gratifyingly, careful hydrolysis of the phosphonate ester 18 with
3. Conclusion
Two synthetic pathways were assayed to obtained the targeted
γ-amino-α,α-difluoromethylphosphonic acid 3 stable FTY720-P
analogue. If the ring opening of cyclic sulfamidates route was

successfully achieved on sulfamidate substituted with short alkyl
or aryl chains (25% and 71% yields), this strategy failed on
sulfamidates bearing longer alkyl chains. Stable FTY720-P
analogue 3 was obtained through the epoxide ring opening route
in ten steps in 9% overall yield.
type, receptor expression, and receptor availability, FTY720-P
could modify S1P1 coupling to specific intracellular signals such
as Ca2+, cAMP or MEK/ERK 1/2 signalling36 and therefore
altering proliferative signaling in cells. This last characteristic
could explain the inhibitory effects of fluorinated FTY720-P
analogue 3 on cell proliferation and viability of prostate cancer
cells.
Interaction of S1P to its cognate receptors mostly results in
internalization and consequent recycling of the receptors back to
the membrane, yet the binding of FTY720-P to S1P1 is associated
with S1P1 receptor internalization and degradation,10 making
FTY720-P a “functional antagonist”.35 Depending on the cell
PC-3 cells
C4-2B cells
150
***
**
150
***
*
***
*
**
**
**
100
100
50
*
*
**
**
***
***
50
Figure 1. Evaluation of cell proliferation in PC-3 and C4-2B prostate cancer cell lines treated by S1P, FTY720-P and FTY720-P analogue 3 (C3).
and concentrated under reduced pressure The resulting residue
was purified by flash chromatography (pentane/EtOAc, 100/0,
90/10, 80/20) to afford 10 as a yellow solid (243 mg, 86%). H
4. Experimental
4.1. Chemistry
1
NMR (400 MHz, CDCl3) δ 0.85 (t, 3JHH = 7.4 Hz, 3H), 1.25-1.32
(m, 8H), 1.32-1.35 (m, 2H), 1.57-1.71 (m, 2H), 2.89 (t, 3JHH = 7.4
Hz, 2H), 7.57 (d, 3JHH = 7.4 Hz, 2H), 7.80 (d, 3JHH = 7.4 Hz, 2H);
13C NMR (100 MHz, CDCl3) δ 14.2, 22.8, 24.4, 29.3, 29.4, 31.8,
38.7, 128.1, 129.7, 132.0, 135.9, 198.8; HMRS (ESI) m/z calcd
for C14H19BrO 282.0619 [M+H+], found 282.0620.
All solvents were dried following standard procedures
(dichloromethane: distillation over CaH2, THF: distillation over
Na/benzophenone).
Column chromatography purifications were performed on
Geduran® Si 60 silica gel (40-63 µm) from Merck. TLC were
carried out on Merck DC Kieselgel 60 F-254 aluminium sheets.
Compounds were visualised by one or more of the following
methods: (1) illumination with a short wavelength UV lamp (i.e.,
λ = 254 nm), (2) spray with a 0.2% (w/v) ninhydrin solution in
absolute ethanol, (3) spray with a 3.5% (w/v) phosphomolybdic
acid solution in absolute ethanol.
4.1.2. 1-(4-(4-hydroxybut-1-ynyl)phenyl)octan-1-
one 11
A solution of butyn-1-ol (50 mg, 0.71 mmol, 1.0 equiv) in a
mixture CH2Cl2/triethylamine (v/v: 2/1) were added to a solution
of bromobenzene 10 (200 mg, 0.71 mmol, 1.0 equiv) in CH2Cl2
(2 mL) at room temperature. The reaction mixture was degassed
by softly bubbling argon during 30 min and then CuI (6 mg, 0.07
mmol, 0.1 equiv) and Pd(PPh3)4 (40 mg, 0.35 µmol, 0.05 equiv)
were successively added. The reaction mixture was further
degassed by argon bubbling and stirred overnight at room
temperature under an argon atmosphere. After filtration on celite
followed by removal of the solvent under reduced pressure, the
residue was purified by flash chromatography (pentane/EtOAc,
50/50) to give 11 as a white solid (178 mg, 92%). 1H NMR (400
Instruments and methods. 1H, 13C (C13APT or C13CPD
experiments), 31P and 19F NMR spectra were recorded either on
a Bruker AC 200, on a Bruker DPX 300 or on a Bruker avance
III400 spectrometers working at 200, 300 and 400 MHz
respectively. Chemical shifts are expressed in parts per million
(ppm) from the residual non-deuterated solvent signal CDCl3 (δΗ
= 7.26, δC = 77.00) or DMSO-d6 (δΗ = 2.50, δC = 39.43). J values
are in hertz (Hz). High-resolution mass spectra were recorded on
a LCT Premier XE benchtop orthogonal acceleration time-of-
flight (oa-TOF) mass spectrometer (Waters Micromass) equipped
with an ESI source and in the positive mode.
3
MHz, CDCl3) δ 0.87 (t, JHH = 7.4 Hz, 3H), 1.28-1.34 (m, 8H),
1.69-1.74 (m, 2H), 2.72 (t, 3JHH = 6.4 Hz, 2H), 2.93 (t, 3JHH = 9.6
Hz, 2H), 3.84 (t, 3JHH = 8.4 Hz, 2H), 7.48 (d, 3JHH = 8.8 Hz, 2H),
7.89 (d, 3JHH = 8.6 Hz, 2H); 13C NMR (100 MHz, CDCl3) δ 14.2
(s), 22.7, 22.8, 24.4 (s), 29.3, 29.4, 31.8, 38.7, 61.3, 83.4, 98.7,
128.1, 129.7, 131.9, 135.9, 198.8.
4.1.1. 4-Bromo-octanoylbenzene 10
Octanoyl chloride (0.17 mL, 1.0 mmol, 1.0 equiv) was added
dropwise to a mixture of bromobenzene (0.21 mL, 2.0 mmol, 2.0
equiv) and aluminium chloride (160 mg, 1.2 mmol, 1.2 equiv).
The reaction mixture was then stirred at 50 °C for 3 h, poured
into ice water, and extracted with CH2Cl2. The organic phase was
washed with aq solution HCl (1 N) and brine, dried over MgSO4
4.1.3. 3-(4-octylphenyl)propan-1-ol 12
Pd/C (30 mg, 10 wt. %) was added to a solution of 11 (200
mg, 0.73 mmol, 1.0 equiv) in MeOH (2 mL). The reaction
mixture was degassed by using argon bubbling during 30 min

4.1.7. (3-hydroxy-1,1-difluoro-5-phenyloctyl)-
phosphonic acid diisopropyl ester 16
followed by hydrogen and stirred overnight at room temperature
under hydrogen atmosphere (1 bar). After filtration on celite
followed by concentration under reduced pressure, the residue
was purified by flash chromatography (pentane/EtOAc, 80/20) to
A
solution
of
diisopropyl
methylsulfanyl
difluoromethylphosphonate 4 (262 mg, 1.0 mmol, 1.0 equiv) in
dry Et2O (1 mL) was added dropwise to a cold solution of t-
butylithium (1.3 M, 1 mL, 1.3 mmol, 1.3 equiv) in dry Et2O (5
mL) at - 80 °C. After 10 min of stirring, a solution of 15 (348 mg,
1.3 mmol, 1.3 equiv) in dry Et2O (1 mL) was added slowly at -
80 °C, followed by BF3.OEt2 (0.26 mL, 2.0 mmol, 2.0 equiv) at
the same temperature. After 15 min, the reaction mixture was
quenched at - 80 °C by addition of an aqueous saturated NH4Cl
and the mixture was slowly warmed up to room temperature. The
aqueous layer was extracted with Et2O (twice) and combined
organic layers were washed with aqueous solution of NaHCO3
and dried over MgSO4. Solvents were evaporated under reduced
pressure and crude product was purified by flash column
chromatography (pentane/EtOAc, 90/10) to give 16 as a
1
afford 12 as a yellow solid (115 mg, 60%). H NMR (400 MHz,
3
CDCl3) δ 0.90 (t, JHH = 7.4 Hz, 3H), 1.27-1.30 (m, 10H), 1.61-
1.72 (m, 6H), 2.54-2.64 (m, 4H), 3.66 (t, 3JHH = 8.4 Hz, 2H), 7.10
(s, Ar, 5H); 13C NMR (100 MHz, CDCl3) δ 14.1, 22.8, 25.4, 27.6,
29.2, 29.4, 29.5, 31.6, 32.3, 35.2, 35.2, 62.8, 125.9, 128.2, 128.3,
128.4; HMRS (ESI) m/z calcd for C18H30O 262.2297 [M+H+],
found 262.2299.
4.1.4. 1-(4-iodobutyl)-4-octylbenzene 13
Iodine (223, 0.88 mmol, 1.2 equiv) and imidazole (90 mg,
1.32 mmol, 1.8 equiv) were successively added to a stirred
solution of triphenylphosphine (300 mg, 1.1 mmol, 1.5 equiv) in
CH2Cl2 (5 mL) at room temperature. The resulting solution was
stirred for 10 min before addition of a solution of the alcohol 12
(191 mg, 1.0 mmol, 1.0 equiv) in CH2Cl2. The reaction was
stirred 4 h at room temperature, and then was quenched with
addition of solution of Na2S2O3, extracted with dichloromethane,
washed with brine, dried over MgSO4 and concentrated under
reduced pressure. The crude product was purified by flash
chromatography (pentane) to give 13 as a colourless oil (346 mg,
86%). 1H NMR (400 MHz, CDCl3) δ 0.86 (t, 3JHH = 7.4 Hz, 3H),
1.24-1.27 (m, 10H), 1.67-1.79 (m, 4H), 1.81-1.86 (m, 2H), 2.52-
2.60 (m, 4H), 3.18 (t, 3JHH = 9.2 Hz, 2H), 7.07 (s, Ar, 5H); 13C
NMR (100 MHz, CDCl3) δ 6.8, 14.1, 22.8, 25.4, 27.6, 29.2, 29.4,
29.5, 31.6, 32.3, 35.2, 35.2, 128.2, 128.4, 138.9, 140.5; HMRS
(ESI) m/z calcd for C18H29I 372.1314 [M+H+], found 372.1307.
1
colourless oil (357 mg, 75%). H NMR: (400 MHz, CDCl3) δ
0.89 (t, JHH = 7.5 Hz, 3H), 1.03-1.33 (m, 24H), 1.48-1.52 (m,
3
2H), 1.97-2.02 (m, 4H), 2.46-2.48 (m, 2H), 2.50-2.53 (m, 2H),
3.75-3.76 (m, 1H), 4.04 (s, OH, 1H), 4.77-4.81 (m, 2H) 7.07 (s,
Ar, 5H); 19F NMR: (377 MHz, CDCl3) δ - 111.05 (dddd, JFH
=
3
2
19.4 Hz, 3JFH = 24.3 Hz, JFP = 111.3 Hz, 2JFF = 263.1 Hz, 1F), –
3
105.80 (dddd, JFH= 19.4 Hz, , JFH = 24.3 Hz, JFP = 108.3 Hz,
3
2
2JFF = 296.1 Hz, 1F); 31P NMR: (162 MHz, CDCl3) δ 6.12 (dd,
2
2JPF = 111.3 Hz, JPF = 108.3, 1P); 13C NMR: (CDCl3, 100 MHz)
δ 14.1, 22.7, 23.6, 23.7, 23.8, 24.0 (d, 3JCP = 4.8 Hz), 24.1 (d, 3JCP
= 3.3 Hz), 29.2, 29.5, 31.4, 31.5, 35.7, 39.2, 51.2, 74.1 (d, 2JCP
=
7.0 Hz), CF2 not detected, 128.3, 128.4, 138.9, 140.4; HMRS
(ESI) m/z calcd for C25H43F2O4P 476.2867 [M+H+], found
476.2925.
4.1.5. 1-(but-3-enyl)-4-octylbenzene 14
A solution of 13 (372 mg, 1.0 mmol, 1.0 equiv) in dry THF (2
mL) was added dropwise to a solution of t-BuOK (111 mg, 2.7
mmol, 2.7 equiv) in dry THF (8 mL) under argon. Precipitate was
observed even before the addition was complete and TLC control
indicated the reaction was completed after 30 min. The reaction
mixture was quenched using an aqueous saturated solution of
NaHCO3. The organic layer was separated and concentrated
under reduced pressure and the resulting residue was purified by
flash chromatography (pentane) to furnish 14 as a colourless oil
4.1.8. (3-azido-1,1-difluoro-5-phenyloctyl)-
phosphonic acid diisopropyl ester 17
Triphenylphosphine (1.31 g, 5.0 mmol, 5.0 equiv) was added
to a solution of alcohol 16 (476 mg, 1.0 mmol, 1.0 equiv) in THF
(3 mL) at 0 °C. The mixture was stirred for a minute. Diisopropyl
azodicarboxylate (0.95 mL, 5.0 mmol, 5.0 equiv) was then added
dropwise, and a white precipitate appeared. Diphenyl phosphoryl
azide (DPPA) (1.04 mL, 5.0 mmol, 5.0 equiv) was added
dropwise at 0 °C, and the reaction mixture was allowed to warm
to room temperature and stirred for an additional 24 h. After
concentration, the residue was directly purified by silica gel
chromatography (pentane/EtOAc, 100/0, 98/2, 95/5, 90/10) to
give 17 as a colorless oil (482 mg, 95%).1H NMR: (400 MHz,
1
(212 mg, 86%). H NMR (400 MHz, CDCl3) δ 0.80 (t, 3JHH = 7.5
Hz, 3H), 1.24-1.27 (m, 10H), 1.47-15.4 (m, 2H), 2.25-2.32 (m,
2H), 2.49 (t, 3JHH = 10.4 Hz, 2H), 2.59 (t, 3JHH = 9.6 Hz, 2H), 4.91
(dd, 2JHHgem = 2.0 Hz, 3JHHcis = 9.2 Hz, 1H), 4.91 (dd, 2JHHgem = 2.0
3
Hz, JHHtrans = 21.6 Hz, 1H), 5.80 (m, 1H), 7.07 (s, Ar, 5H); 13C
3
CDCl3) δ 0.86 (t, JHH = 7.4 Hz, 3H), 1.09-1.33 (m, 24H), 1.54-
NMR (100 MHz, CDCl3) δ 14.1, 22.7, 29.3, 29.4, 29.6, 31.6,
31.9, 35.0, 35.2, 35.2, 35.2, 114.8, 128.3, 128.4, 138.3, 139.0,
140.5; HMRS (ESI) m/z calcd for C18H28 244.2191 [M+H+],
found 244.2201.
1.59 (m, 2H), 1.83-1.86 (m, 2H), 2.13-2.25 (m, 2H), 2.52-2.57
(m, 2H), 2.67-2.90 (m, 2H), 3.77-3.81 (m, 1H), 4.80-4.86 (m,
2H) 7.07 (s, Ar, 5H); 19F NMR: (377 MHz, CDCl3) δ –112.33
3
2
(td, JFH = 20.4 Hz, JFP = 106.1 Hz, 2F), 31P NMR: (162 MHz,
CDCl3) δ 4.55 (t, JPF = 106.1 Hz, 1P); 13C NMR: (CDCl3, 100
2
4.1.6. 2-(4-octylphenethyl)oxirane 15
3
MHz) δ 14.1, 22.7, 23.6, 23.7, 23.8, 24.0 (d, JCP = 4.8 Hz), 24.1
A solution of m-CPBA (332 mg, 1.5 mmol, 1.5 equiv) in dry
CH2Cl2 (2 mL) was added dropwise to a solution of alkene 14
(244 mg, 1.0 mmol, 1.0 equiv) in dry CH2Cl2 (5 mL). The
reaction mixture was stirred overnight at room temperature and
then quenched using an aqueous saturated solution of NaHCO3.
The organic layer was separated and concentrated under reduced
pressure. Purification by flash chromatography (pentane/EtOAc,
(d, 3JCP = 3.3 Hz), 29.2, 29.5, 31.4, 31.5, 35.7, 39.2, 51.2, 74.1 (d,
2JCP = 7.0 Hz), CF2 not detected, 128.3, 128.4, 138.9, 140.4;
HMRS (ESI) m/z calcd for C25H42F2N3O3P 501.2932 [M+H+],
found 501.3009.
4.1.9. (3-amino-1, 1-difluoro-5-phenyloctyl)-
phosphonic acid diisopropyl ester 18
1
100/0 to 95/5) to give 15 as a colourless oil (192 mg, 74%). H
To a solution of azide 17 (508 mg, 1.0 mmol, 1.0 equiv) in
MeOH (2 mL) was added Pd/C (51 mg, 10 wt. %) and aq
solution HCl 1 M (1 mL). The reaction mixture was degassed by
using argon bubbling during 30 min followed by hydrogen and
stirred 48 h at room temperature under hydrogen atmosphere (1
bar). After filtration on celite followed by removal of the solvent
under reduced pressure, the residue was purified by flash
chromatography (CH2Cl2/EtOAc, 90/10, 80/20, 70/30, 50/50) to
NMR (400 MHz, CDCl3) δ 0.89 (t, 3JHH = 7.5 Hz, 3H), 1.26-1.30
(m, 10H), 1.81-1.83 (m, 2H), 1.86-1.89 (m, 2H), 2.48-2.50 (m,
3H), 2.70-2.78 (m, 3H), 2.95-2.97 (m, 1H), 7.07 (s, Ar, 5H); 13C
NMR (100 MHz, CDCl3) δ 14.1, 22.7, 29.3, 29.4, 29.6, 31.6,
31.9, 35.0, 35.2, 35.2, 35.2, 47.3, 51.9, 128.2, 128.4, 138.4,
140.6; HMRS (ESI) m/z calcd for C18H28O 260.2140 [M+H+],
found 260.2124.

afford 18 as a yellow solid (427 mg, 90%). 1H NMR: (400 MHz,
Acknowledgments
3
CDCl3) δ 0.86 (t, JHH = 7.4 Hz, 3H), 1.26-1.38 (m, 24H), 1.68-
1.78 (m, 2H), 2.02-2.31 (m, 2H), 2.36-2.60 (m, 4H), 3.35-3.64
This work was supported by the Régions Haute and Basse
Normandie (the CRUNCh program, and a PhD fellowship for
LA). We thank Dr. Anthony Romieu (Université de Rouen) for
MS analyses.
(m, 1H), 4.80-4.86 (m, 2H) 7.07 (s, Ar, 5H); 19F NMR: (377
3
MHz, CDCl3) δ δ –111.85 (dddd, JFH = 14.8 Hz, 3JFH = 28.1 Hz,
2
2JFP = 107.6 Hz, JFF = 296.6 Hz, 1F), –109.61 (dddd, 3JFH= 14.8
3
2
2
Hz, , JFH = 28.1 Hz, JFP = 108.4 Hz, JFF = 296.6 Hz, 1F); 31
P
=
2
2
NMR: (162 MHz, CDCl3) δ 5.12 (dd, JPF = 107.6 Hz, JPF
References and notes
108.4, 1P); 13C NMR: (CDCl3, 100 MHz) δ 14.1, 22.7, 23.6,
3
3
23.7, 23.8, 24.0 (d, JCP = 4.8 Hz), 24.1 (d, JCP = 3.3 Hz), 29.2,
1.
2.
3.
Maceyka, M.; Harikumar, K. B.; Milstien, S.; Spiegel, S.
Trends Cell Biol. 2012, 22, 50.
Rosen, H.; Stevens, R. C.; Hanson, M.; Roberts, E.; Oldstone,
M. B. A. Annu. Rev. Biochem 2013, 82, 637.
2
29.5, 31.4, 31.5, 35.7, 39.2, 51.2, 74.1 (d, JCP = 7.0 Hz), CF2 not
detected, 128.3, 128.4, 138.9, 140.4; HMRS (ESI) m/z calcd for
C25H42F2NO3P 475.3027 [M+H+], found 475.3102.
Forrest, M.; Sun, S. Y.; Hajdu, R.; Bergstrom, J.; Card, D.;
Doherty, G.; Hale, J.; Keohane, C.; Meyers, C.; Milligan, J.;
Mills, S.; Nomura, N.; Rosen, H.; Rosenbach, M.; Shei, G. J.;
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4.1.10. (3-amino-1,1-difluoro-5-phenyloctyl)-
pentylphosphonic acid 3
TMSBr (0.6 mL, 4.2 mmol, 5.0 equiv.) was added to a
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0.8 mmol, 1.0 equiv) in CH2Cl2 (1 mL). The reaction mixture
was stirred 48 h at room temperature under argon atmosphere.
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4.
5.
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7.
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1
3
50%). H NMR: (300 MHz, DMSO+DCl) δ 0.75 (t, JHH = 6.9
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3
3
2H), 2.29-2.58 (m, 5H), 7.04 (dd, JHH = 8.1 Hz, JHH = 8.1 Hz,
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3
2
2
3JFH = 14.1 Hz, JFH = 39.4 Hz, JFP = 95.8 Hz, JFF = 304.6 Hz,
3
1F), –109.21 (dddd, JFH = 14.8 Hz, JFH = 36.6 Hz, JFP = 95.8
3
2
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3
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Supplementary Material
General procedure for the preparation and characterizations of
compounds 8a-h. General procedure for the ring opening
reactions of sulfamidates and characterizations of compounds 9a-
b and 9d-e.