Studies on the inhibition of sphingosine-1-phosphate lyase by stabilized reaction intermediates and stereodefined azido phosphates

Article abstract of DOI:10.1016/j.ejmech.2016.08.008

Two kinds of inhibitors of the PLP-dependent enzyme sphingosine-1-phosphate lyase have been designed and tested on the bacterial (StS1PL) and the human (hS1PL) enzymes. Amino phosphates 1, 12, and 32, mimicking the intermediate aldimines of the catalytic process, were weak inhibitors on both enzyme sources. On the other hand, a series of stereodefined azido phosphates, resulting from the replacement of the amino group of the natural substrates with an azido group, afforded competitive inhibitors in the low micromolar range on both enzyme sources. This similar behavior represents an experimental evidence of the reported structural similarities for both enzymes at their active site level. Interestingly, the anti-isomers of the non-natural enantiomeric series where the most potent inhibitors on hS1PL.

Full text of DOI:10.1016/j.ejmech.2016.08.008

European Journal of Medicinal Chemistry 123 (2016) 905e915
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Studies on the inhibition of sphingosine-1-phosphate lyase by
stabilized reaction intermediates and stereodefined azido phosphates
,
c
a
b
a
,
, c, *
Pol Sanllehí a , Jose-Luís Abad , Jordi Bujons , Josefina Casas **, Antonio Delgado a
ꢀ
a Spanish National Research Council (CSIC), Institute of Advanced Chemistry of Catalonia (IQAC-CSIC), Research Unit on Bioactive Molecules (RUBAM),
Department of Biomedicinal Chemistry, Jordi Girona 18-26, 08034, Barcelona, Spain
b Spanish National Research Council (CSIC), Institute of Advanced Chemistry of Catalonia (IQAC-CSIC), Department of Biological Chemistry and Molecular
Modelling, Jordi Girona 18-26, 08034, Barcelona, Spain
c University of Barcelona (UB), Faculty of Pharmacy, Department of Pharmacology, Toxicology and Medicinal Chemistry, Unit of Pharmaceutical Chemistry
(Associated Unit to CSIC), Avda. Joan XXIII s/n, 08028, Barcelona, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Two kinds of inhibitors of the PLP-dependent enzyme sphingosine-1-phosphate lyase have been
designed and tested on the bacterial (StS1PL) and the human (hS1PL) enzymes. Amino phosphates 1, 12,
and 32, mimicking the intermediate aldimines of the catalytic process, were weak inhibitors on both
enzyme sources. On the other hand, a series of stereodefined azido phosphates, resulting from the
replacement of the amino group of the natural substrates with an azido group, afforded competitive
inhibitors in the low micromolar range on both enzyme sources. This similar behavior represents an
experimental evidence of the reported structural similarities for both enzymes at their active site level.
Interestingly, the anti-isomers of the non-natural enantiomeric series where the most potent inhibitors
on hS1PL.
Received 6 July 2016
Received in revised form
4 August 2016
Accepted 5 August 2016
Available online 6 August 2016
Keywords:
Sphingolipids
Enzyme inhibitor
Sphingosine lyase
Synthesis
© 2016 Elsevier Masson SAS. All rights reserved.
Stereocontrolled
1. Introduction
for the discovery of potential neuro-restorative therapies with ap-
plications in traumatic brain injury, spinal cord injury, and stroke.
Phosphorylated sphingolipids are an important group of
sphingoid metabolites that present a terminal phosphate group.
Among them, sphingosine-1-phosphate (S1P) is a well-recognized
signaling molecule that regulates cell differentiation, survival,
inflammation, angiogenesis, calcium homeostasis and immunity,
among other functions [1]. Recent findings have associated high
S1P levels with neuroprotective effects that counteract many
noxious processes that follow CNS injury [2,3] (apoptotic cell death,
lipid hydrolysis, oxidative stress and tissue damage), while sup-
porting growth and trophic factor activities. In general, the roles of
phosphorylated sphingolipids are opposed to those of ceramides
(Cer), which are potent inducers of cell cycle arrest and apoptosis
[4]. Thus, the control of the S1P/Cer balance might be exploitable
These tissue protective properties make S1P a suitable cellular
survival signaling agent, in contraposition to Cer, which is a well-
recognized mediator of cellular stress responses. In addition to
the intracellular functions, paracrine functions of S1P through its
binding to specific lipid G-protein coupled S1P1-5 receptors are also
known. In this way, S1P becomes essential for vascular develop-
ment and endothelial integrity, control of cardiac rhythm, and
immunity responses [5e10].
Sphingosine-1-phosphate lyase (S1PL) is a pyridoxal 50-phos-
phate (PLP) dependent enzyme, localized in the endoplasmic re-
ticulum (ER), that catalyzes the irreversible degradation of S1P into
ethanolamine phosphate (EAP) and trans-2-hexadecenal (Scheme
1) [11]. According to the proposed mechanism of cleavage [11],
the incoming S1P replaces the catalytic Lys in internal aldimine A
(step 1, Scheme 1) to form the external aldimine B. Retro-aldol
cleavage takes place at the C3eOH group of S1P (step 2) to
release the corresponding aldehyde. Reprotonation of the transient
quinonoid intermediate C (step 3) leads to external aldimine D,
from which EAP is released after hydrolysis (step 4) and the internal
aldimine A is formed again for a second enzyme turnover. An
* Corresponding author. Spanish National Research Council (CSIC), Institute of
Advanced Chemistry of Catalonia (IQAC-CSIC), Research Unit on Bioactive Molecules
(RUBAM), Department of Biomedicinal Chemistry, Jordi Girona 18-26, 08034, Bar-
celona, Spain.
** Corresponding author.
E-mail address: delgado@rubam.net (A. Delgado).
http://dx.doi.org/10.1016/j.ejmech.2016.08.008
0223-5234/© 2016 Elsevier Masson SAS. All rights reserved.

906
P. Sanllehí et al. / European Journal of Medicinal Chemistry 123 (2016) 905e915
hydrolyzable analog of the putative intermediate D (Scheme 1).
Interestingly, dephosphorylated 32 had been reported in the liter-
ature as a potential S1PL inhibitor. However, its lack of activity in
cells was attributed to an inadequate phosphorylation in vivo [20].
As PLP analogs, compounds 1, 12, and 32 were expected to
displace the cofactor in the enzyme active site. In a second
approach, a series of configurationally defined azide analogs of both
enzyme substrates (S1P and dhS1P) were designed as non-reactive,
potential S1PL competitive inhibitors (compounds 89, 98, 103, and
114, Fig. 2). Since the C2-amino group in S1P is replaced by an azido
group, formation of the external aldimine B is expected to be pre-
cluded at the enzyme active site. Apart from the inherent lack of
reactivity towards imine formation and its remarkable stability in
cellulo [29], the azido group has also been used as a “pseudohalide”
in drug design [30]. In our case, the replacement of the amino group
present in S1P with an azido group is expected to cause a dramatic
effect in the electronic properties and on the interactions with the
enzyme active site, without disturbing the overall shape of the
molecule. Moreover, to gain insight into the enzyme stereo-
selectivity towards this type of analogs, all the configurations
around the C2 and C3 carbon atoms have been considered (ie.
compounds ent-89, ent-98, ent-103 and ent-114).
Scheme 1. Catalytic cycle of sphingosine-1-phosphate lyase (S1PL). 1) S1P or dhS1P
replaces Lys from A to form the external aldimine B; 2) Retro-aldol cleavage with
generation of 2-hexadecenal or hexadecanal and the quinonoid intermediate C; 3)
Formation of the aldimine system D by protonation of C; 4) Hydrolysis of D with
release of EAP and final restoration of A.
2. Results and discussion
2.1. Synthesis
identical mechanism is accepted for the alternative S1PL substrate
4,5-dihydro-S1P (dhS1P), giving rise to EAP and hexadecanal as
reaction products. The degradation of S1P by S1PL has been
considered as the “exit gate” of sphingolipid metabolism and the
natural connection with phospholipid metabolism through the
conversion of EAP into phosphatidylethanolamine, another
signaling molecule in lipid metabolism [12]. In combination with
S1P phosphatase and sphingosine kinases (SK) [13], the levels of
intracellular S1P can be efficiently regulated.
So far, the repertoire of available S1PL inhibitors structurally
related to the enzyme substrate is scarce. In this context, 1-
deoxysphinganine 1-phosphonate [14] and the 2-vinyl dihy-
drosphingosine 1-phosphate (2-vinyl dhS1P) (Fig. 1) are the only
ones reported in the literature [15]. The structurally related FTY720,
a well-known S1P receptor agonist after the enantioselective
phosphorylation to the (S) isomer, has also been reported as a
modest S1PL inhibitor in vitro [16,17]. On the other hand, the PLP
antagonist 4-deoxypyridoxine (DOP) has been described as a
functional S1PL inhibitor in vivo [18e20], as well as THI and related
analogs [20,21], whose mechanism of action is controversial
[22e24]. Finally, some heterocyclic compounds resulting from
massive HTS programs have been discovered as potent and selec-
tive S1PL inhibitors [18e20,25], some of them with IC50 values in
the nM range [26,27].
Compounds 1, 12 and 32 were obtained as outlined in Scheme 2
by reductive amination of PLP with either O-phosphorylethanol-
amine, dhS1P or S1P, respectively. Following an adaptation of re-
ported protocols [31,32], the corresponding dipotassium salts were
generated in situ with KOt-Bu in order to ensure their total solu-
bility. After the consumption of the starting amine was evidenced
by 1H NMR, reduction of the corresponding imines with NaBH4
afforded the desired diphosphates in acceptable overall yields,
together with pyridoxine-50-phosphate, which was also included in
our S1PL inhibition assays due to its high structural similarity with
PLP and DOP. On the other hand, crude diphosphates 12 and 32
presented a very low solubility in different organic solvents and in
aqueous mixtures. Nevertheless, preparation of the corresponding
triethylammonium salts afforded highly water-soluble species,
which could be properly characterized in terms of chemical identity
and biological activity.
The synthesis of the differently configured azidophosphates was
carried out from the corresponding stereodefined sphingosines,
whose synthesis from D- or L-Garner's aldehyde is depicted in
Scheme 3. For the 2S series, the anti-adducts resulted from the
diastereoselective addition of vinylmagnesium bromide to the
starting aldehyde, following a well-established reported protocol in
which the anti-configuration of the amino diol moiety was secured
by chemical correlation with a stereodefined reference compound
[33]. Cross-metathesis of 23 with 1-pentadecene, in the presence of
Grubbs catalyst (second generation) [34,35], followed by iso-
propylidene removal afforded the key sphingoid intermediate 25,
whose configuration was in agreement with the optical rotation
data reported for this compound in the literature [36]. On the other
As a result of our interest in the development of sphingolipid
modulators by targeting S1P metabolism [28], we undertook the
design of new S1PL inhibitors based on S1PL mechanistic consid-
erations. Thus, compounds 12 and 32 (Fig. 2) were designed as non-
reactive analogs of the intermediate aldimine B (Scheme 1),
whereas compound
1 (Fig. 2) can be regarded as a non-
Fig. 1. Reported S1PL inhibitors structurally related with the enzyme substrate.

P. Sanllehí et al. / European Journal of Medicinal Chemistry 123 (2016) 905e915
907
Fig. 2. Non-reactive S1PL reaction intermediates and substrates described in this work.
Scheme 2. Synthesis of PLP-derivatives. Reagents and conditions: (a) (i) selected amine (O- Phosphorylethanolamine for 1, S1P for 32 and dhS1P for 12), KOt-Bu, MeOH, reflux (ii)
NaBH4, MeOH, 0 ꢀC to rt, 79% for 1, 74% for 32, 71% for 12.
Scheme 3. Reagents and conditions: (a) vinylmagnesium bromide, THF, ꢁ78 ꢀC, 64%, dr ¼ 86:14; (b) 1-pentadecene, Grubbs catalyst 2nd gen., CH2Cl2, reflux, 78%, E/Z ¼ 94:6; (c) p-
TsOH, MeOH, rt, 94% for 25, 92% for 94; (d) H2, 5 wt% Rh on Al2O3, MeOH, rt, 91e97%; (e) AcCl, MeOH, 0 ꢀC to rt, 84%-quant. yield (f) (i) 1-pentadecyne, Cp2Zr(H)Cl, CH2Cl2, 0 ꢀC (ii)
Et2Zn, CH2Cl2, ꢁ40 to rt, 75%, dr ¼ 93:7, only E; (g) same sequence as from L-Garner's aldehyde.
hand, the syn-adducts were efficiently obtained from addition of 1-
pentadecenylethylzinc [37] to Garner's aldehyde. Again, the key
intermediate 94 proved to be spectroscopically and configura-
tionally in agreement with reported literature data [38]. Despite
minor amounts of the inseparable Z isomers were detected in these
initial steps, pure E isomers could be obtained after further purifi-
cation steps along the synthetic route. In an analogous manner, the
2R series was obtained from D-Garner's aldehyde.
The differently configured sphingosines were submitted to
diazotransfer reaction with imidazole-1-sulfonyl azide (ISA$HCl)
[39], a process that courses with retention of configuration at the
azide carbon [40,41] (Scheme 4). Gratifyingly, the resulting azides
were in agreement with the expected configuration, as evidenced
by comparison of their optical rotations with those reported in the
literature for azido diols 87 [42], 96 [43], and 112 [44]. In addition,
enantiomeric purity of these azido diols, together with that of the
unreported azido diol 101 was confirmed by chiral HPLC with ee
higher than 95% in all cases (see Supporting Material). Site-
selective phosphorylation with dimethylchlorophosphate and
methyl ester removal with Me3SiBr [45] afforded the required
azidophosphates in 15e25% overall yields from the starting Gar-
ner's aldehydes.
2.2. S1PL inhibition
The above compounds were tested against recombinant human

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P. Sanllehí et al. / European Journal of Medicinal Chemistry 123 (2016) 905e915
Scheme 4. Reagents and conditions: (a) 1H-imidazole-1-sulfonylazide hydrochloride, CuSO4$5H2O, K2CO3, MeOH, rt, 80e91%; (b) Dimethyl chlorophosphate, N-methylimidazole,
CH2Cl2, 0 ꢀC to rt, 64e85%; (c) (i) Me3SiBr, MeCN, 0 ꢀC to rt (ii) MeOH/H2O (95:5), rt, 56e71%.
(hS1PL) [26] and bacterial (Symbiobacterium thermophilum, StS1PL)
[11] enzyme sources, using our previously developed fluorogenic
substrate [46]. The results are collected in Table 1.
Compound 1, designed as a mimic of the S1PL reaction inter-
mediate D (see Scheme 1) behaved as a weak inhibitor (around 20%
compounds 89, ent-89 and ent-114 were the most active com-
pounds of this series, with IC50 values of 10.1, 5.2 and 10.8 M,
respectively. Interestingly, the naturally configured (2S,3R) azido-
M) was not as active as the above
derivatives. The inhibition constants (Ki) against hS1PL showed a
competitive inhibition pattern in all cases, with Ki values in the
m
phosphate 114 (IC50 ¼ 25.7
m
inhibition at 250 mM) in both hS1PL and StS1PL, while pyridoxine-
50-phosphate was practically devoid of activity. The presence of the
5e40 mM range (See Supporting) and a good correlation with the
sphingoid moiety in 12 and 32 led to more potent inhibitors, with
IC50 values in the range of 50e80 mM for both enzymes.
corresponding IC50 values. However, despite the similar inhibitory
trends, compounds ent-89 and ent-114, both with the non-natural
2R, 3S configuration at the sphingoid backbone, were the most
active stereoisomers of the series (Table 1 and Fig. 3). For the
naturally configured series, 89 was found to be about two-fold
more potent than its saturated analog 114.
Finally, in order to study the contribution of the phosphate
group on S1PL activity, the corresponding azidodiols (Scheme 4)
were also evaluated as hS1PL inhibitors. All compounds were
Despite comparable IC50 values for 12 and 32 on each of the
enzyme sources, a slight selectivity towards the bacterial enzyme
was observed. Furthermore, the presence of a double bond at the
C4eC5 position of the sphingoid backbone seemed not crucial for
S1PL inhibition. A putative unspecific inhibition due to the trie-
thylammonium counterion present in 12 and 32 was disregarded
since incubation of both enzymes at different concentrations of
triethylamine hydrochloride (500e0.5
mM range) did not affect the
inactive at 250 mM, thus suggesting that the presence of the
enzymatic activity (results not shown). The modest results ob-
tained with enzyme intermediate mimics 1, 12, and 32, initially
designed to occupy the enzyme PLP binding site, could be explained
by their inability to displace the cofactor from its Schiff base with
the postulated Lys residue at the active site (Scheme 1). In addition,
the bulkiness of 12 and 32 may prevent their access through the
channel linking the active site with the cytosol, as observed in the
X-ray structure reported for hS1PL [26].
Unlike the above compounds, all the azidophosphate analogs
synthesized in this work behaved as low mM inhibitors on the two
S1PL tested (Table 1). However, despite the subtle activity differ-
ences observed, some trends are worthy of mention. As StS1PL
inhibitors, unsaturated derivatives 89, ent-89, 98 and ent-98,
phosphate group, or a suitable surrogate, at C1 is critical to ensure a
strong enzyme binding.
Computer docking simulations allowed proposing similar
binding modes for the active azido phosphates in the active center
of StS1PL and hS1PL. Fig. 4 shows the bound poses of compounds
89 and ent-89 in the active center of both enzymes (PDB codes
3MAD and 4Q6R) [26,47]. In agreement with the above suggestion,
these poses show that the phosphate group can establish multiple
interactions with residues of a previously identified anion binding
site [11,48], i.e. residues Y105, N126, H129 and K317 of StS1PL, and
residues Y150, N171, H174 and K359 of hS1PL, thus confirming the
prominent role of the phosphate moiety.
The long aliphatic chain is always placed along the narrow hy-
drophobic access channel that communicates the active site of the
proteins with the surface, while the azido and hydroxyl groups of
both compounds are placed close to two aromatic residues (H201/
Y249 in StS1PL and H242/F290 in hS1PL), which are at short
showed IC50 values in the range of 10e16
active than their saturated analogs 114, ent-114, 103 and ent-103,
for which an IC50 value around 25 M was determined in all cases.
mM, around 2-fold more
m
On the other hand, as hS1PL inhibitors, the anti-configured
Table 1
Inhibitory activity of the synthesized compounds.
Compound
IC50
(
mM)
KI (m
M)a
StS1PL
hS1PL
hS1PL
12
32
89
ent-89
98
47.0
53.4
12.9
13.8
16.0
11.7
81.1
89.0
10.1
5.2
28.8
22.9
nd
nd
9.1
5.6
20.4
17.5
ent-98
103
ent-103
114
27.2
24.3
25.0
25.7
21.8
28.3
25.7
10.8
16.4
36.7
19.0
6.3
ent-114
Shaded rows correspond to dihydro analogs.
nd: not determined.
a
Competitive inhibitors.

P. Sanllehí et al. / European Journal of Medicinal Chemistry 123 (2016) 905e915
909
Fig. 3. Kinetics for the inhibition of hS1PL by ent-89 (A) and ent-114 (B). Double reciprocal plot of hS1PL incubated at different concentrations of substrate and compounds.
Regression lines arise from data obtained in two different experiments performed in triplicate.
Fig. 4. Best docked poses of azido phosphates 89 (A, B) and ent-89 (C, D) bound into the active site of StS1PL (A, C) and hS1PL (B, D), as determined by an Induced Fit Docking
protocol. Azido phosphates and the PLP prosthetic group are highlighted in orange and green, respectively. Protein residues interacting with the inhibitors are shown and labelled,
prime numbering indicates that residues belong to different subunits of the dimeric S1PL proteins. (For interpretation of the references to colour in this figure legend, the reader is
referred to the web version of this article.)
distance of the PLP prosthetic group. The high similarity between
the spatial arrangement of both compounds in the active sites of
both proteins and the interactions that they stablish correlates well
with their observed S1PL inhibitory activities. Furthermore, this
docking analysis revealed that the presence or absence of the C4-
double bond does not have a significant influence in the binding
mode of the inhibitors, as illustrated by comparing the docked
poses of azido phosphates 89 and ent-89 (Fig. 4) with those of the
corresponding saturated analogs 114 and ent-114 (Fig. S14).
The similar behavior of the above azidophosphates as StS1PL
and hS1PL inhibitors is also indicative of the similarities of both
enzymes at their active site level. Hence, the more easily available
StS1PL can be used as a reliable model of the human enzyme for
putative inhibitors targeting the active site. This is not the case for
inhibitors targeting the enzyme access channel, where the struc-
tural differences between both enzymes at that level may account

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P. Sanllehí et al. / European Journal of Medicinal Chemistry 123 (2016) 905e915
for the lack of activity on StS1PL observed for a recently reported
potent hS1PL inhibitor [48]. These observations justify the devel-
opment of an engineered StSPL as a model for the discovery of S1PL
inhibitors [25].
[49] and ISA$HCl [39] were synthesized in our laboratories
following previously described methods.
Bacterial S1PL from Symbiobacterium thermophilum (StS1PL)
was expressed and purified in our laboratory following a modified
procedure (see Supplementary Material) of a previously reported
protocol [11].
3. Conclusion
In summary, two kinds of S1PL inhibitors have been designed
and tested using bacterial (StS1PL) and human (hS1PL) enzyme
sources. Amino phosphates 1, 12, and 32, mimicking the interme-
diate aldimines of the catalytic process, were weak inhibitors on
both enzyme sources, probably due to the steric constraints
imposed by the PLP moiety. On the other hand, all the azido
phosphates behaved as competitive inhibitors in the low mM range,
being the anti-isomers with the non-natural enantiomeric config-
uration slightly more potent inhibitors on hS1PL.
4.2. Chemistry
4.2.1. General procedure 1: reductive amination between PLP and
selected amines
To an ice cooled solution of the selected amine (0.1 mmol) in dry
CH3OH (3 mL) was added KOtBu (2 equiv) and the mixture was
stirred at rt for 30 min (Solution A). Simultaneously, KOt-Bu
(2 equiv/mol of PLP) was added to a solution of PLP (1.3 eq.) in
CH3OH (3 mL) at 0 ꢀC and the mixture was stirred at rt for 30 min
(Solution B). Solution A was then added dropwise to solution B at
0 ꢀC and the mixture was refluxed in the dark for 3 h, cooled to 0 ꢀC
and treated with NaBH4 (1.3 equiv). After stirring at rt for 1 h, the
reaction mixture was acidified by dropwise addition of 6 M aq. HCl
and the solvent was evaporated to dryness to give a residue, which
was purified as indicated below.
4. Experimental section
4.1. General
Unless otherwise stated, reactions were carried out under argon
atmosphere. Dry solvents were obtained by passing through an
activated alumina column on a Solvent Purification System (SPS).
Methanol was dried over CaH2 and distilled prior to use.
Commercially available reagents and solvents were used with no
further purification. All reactions were monitored by TLC analysis
using ALUGRAM® SIL G/UV254 precoated aluminum sheets (Mach-
ery-Nagel). UV light was used as the visualizing agent and a 5% (w/
v) ethanolic solution of phosphomolybdic acid as the developing
agent. Flash column chromatography was carried out with the
4.2.1.1. Phosphopyridoxylethanolamine phosphate (1) and pyridox-
ine-50-phosphate. Compound 1 (pale yellow solid, 110 mg, 79%) was
obtained from solution A (O-phosphorylethanolamine (50 mg,
0.36 mmol), KOt-Bu (80 mg, 0.71 mmol)), solution B (PLP mono-
hydrate (122 mg, 0.46 mmol), KOt-Bu (103 mg, 0.92 mmol) and
NaBH4 (17 mg, 0.46 mmol), according to general procedure 1. The
crude material was dissolved in 10 mM aq. NH4HCO3 and applied to
a DEAE-Sephadex A-25-120 ion exchange column (10 g) previously
equilibrated with the same buffer. Elution with a linear gradient
from 10 to 300 mM aq. NH4HCO3 yielded the title compound.
Pyridoxine-50-phosphate (22 mg, white solid) was also isolated.
indicated solvents using flash-grade silica gel (37e70
mm). Pre-
parative reversed-phase purifications were performed on a Bio-
tage® Isolera™ One equipment with the indicated solvents using a
Biotage® SNAP cartridge (KP-C18-HS, 12 g) at a flow rate of 12 mL/
min. Yields refer to chromatographically and spectroscopically pure
compounds, unless otherwise stated.
For 1: 1H NMR (400 MHz, D2O)
2H), 4.45 (s, 2H), 4.10e4.01 (m, 2H), 3.38e3.30 (m, 2H), 2.48 (s, 3H).
13C NMR (101 MHz, D2O)
165.7, 147.4, 137.6 (d, JC-P ¼ 7.9 Hz),
d
7.68 (s, 1H), 4.86 (d, J ¼ 6.3 Hz,
d
NMR spectra were recorded at room temperature on a Varian
Mercury 400 instrument. The chemical shifts (d) are reported in
134.12, 126.1, 64.2 (d, JC-P ¼ 4.3 Hz), 62.3 (d, JC-P ¼ 4.4 Hz), 50.7 (d, JC-
¼ 6.9 Hz), 47.0, 17.6. 31P NMR (162 MHz, D2O)
d 2.93, 2.76. HRMS
P
ppm relative to the solvent signal, and coupling constants (J) are
reported in Hertz (Hz). 31P chemical shifts are relative to an 85%
H3PO4 external reference (0 ppm). For 13C NMR spectra recorded in
D2O, a 0.5% (w/v) solution of DSS (4,4-dimethyl-4-silapentane-1-
sulfonic acid) in D2O was used as external reference (0 ppm). The
following abbreviations are used to define the multiplicities in 1H
NMR spectra: s ¼ singlet, d ¼ doublet, t ¼ triplet, q ¼ quartet,
dd ¼ doublet of doublets, ddd ¼ doublet of doublet of doublets,
m ¼ multiplet, br ¼ broad signal and app ¼ apparent. High Reso-
lution Mass Spectrometry analyses were carried out on an Acquity
UPLC system coupled to a LCT Premier orthogonal accelerated time-
of-flight mass spectrometer (Waters) using electrospray ionization
(ESI) technique. Optical rotations were measured at room temper-
ature on a Perkin Elmer 341 polarimeter.
HPLC analyses were carried out on an Alliance HPLC system
consisting of a 2695 Separation Module (Waters) coupled to a 2996
PDA detector (Waters) and a light scattering ELS-1000 detector
(Polymer Laboratories). Column A corresponds to a Phenomenex®
Lux Amylose-2 (250 ꢂ 4.60 mm) and column B corresponds to a
Chiralpak® IA (250 ꢂ 4.60 mm). Hexane/isopropanol mixtures at a
flow rate of 1 mL/min were used as mobile phase and the moni-
calcd. for C10H19N2O9P2 ([MþH]þ): 373.0566, found: 373.0565.
Pyridoxine-5′-phosphate: 1H NMR (400 MHz, D2O)
d 7.78 (s,
1H), 4.96 (d, J ¼ 6.4 Hz, 2H), 4.85 (s, 2H), 2.49 (s, 3H). 13C NMR
(101 MHz, D2O)
d
172.7, 146.8, 141.9, 136.7 (d, JC-P ¼ 8.2 Hz), 128.1,
64.3 (d, JC-P ¼ 4.3 Hz), 58.8, 18.1. 31P NMR (162 MHz, D2O)
d 2.01.
HRMS calcd. for C8H13NO6P ([MþH]þ): 250.0481, found: 250.0486.
4.2.1.2. Phosphopyridoxylsphinganine-1-phosphate
Compound 12 (pale yellow solid, 40 mg, 71%) was obtained from
solution (4,5-dihydrosphingosine-1-phosphate, 30 mg,
(12).
A
0.08 mmol), KOt-Bu (18 mg, 0.16 mmol), solution B (PLP mono-
hydrate (27 mg, 0.10 mmol), KOt-Bu (23 mg, 0.21 mmol) and NaBH4
(4 mg, 0.10 mmol), according to general procedure 1. The title
compound was purified by preparative RP chromatography (from 5
to 100% CH3CN in 100 mM TEAA, pH 7.0 buffer).
20
[
a
]
¼ ꢁ4.5 (c 1.0, CH3OH). 1H NMR (400 MHz, CD3OD)
d 7.83 (s,
D
1H), 5.01e4.92 (m, 2H), 4.54 (d, J ¼ 13.8 Hz, 1H), 4.43 (d, J ¼ 13.8 Hz,
1H), 4.29e4.21 (m, 1H), 4.18e4.09 (m, 1H), 4.02e3.95 (m, 1H), 3.26
(dt, J ¼ 7.6, 3.7 Hz, 1H), 3.19 (q, J ¼ 7.3 Hz, 6H), 2.47 (s, 3H), 1.72e1.17
(m, 37H), 0.90 (t, J ¼ 6.9 Hz, 3H). 13C NMR (101 MHz, CD3OD)
d 157.9,
toring wavelength was set at 220 nm. Injection volume was 5
mL.
147.5, 134.5, 134.0 (d, JC-P ¼ 8.1 Hz), 130.9, 69.5, 64.0 (d, JC-
Retention times correspond to peaks observed using the PDA
detector.
¼ 6.5 Hz), 63.7 (d, JC-P ¼ 4.6 Hz), 62.0 (d, JC-P ¼ 4.9 Hz), 47.6, 44.6,
P
34.4, 33.1, 30.8, 30.8, 30.8, 30.7, 30.5, 27.1, 23.7, 17.7, 14.5, 9.2. 31P
All biochemical reagents were commercially available and were
used without further purification. RBM13 [46], Garner's aldehyde
NMR (162 MHz, CD3OD) d 2.78,1.92. HRMS calcd. for C26H51N2O10P2
([MþH]þ): 613.3019, found: 613.3024.

P. Sanllehí et al. / European Journal of Medicinal Chemistry 123 (2016) 905e915
911
4.2.1.3. Phosphopyridoxyl
sphingosine-1-phosphate
(32).
CDCl3) d 135.8, 128.4, 73.7, 67.7, 63.1, 32.4, 32.1, 29.8, 29.8, 29.7, 29.6,
Compound 32 (pale yellow solid, 47 mg, 74%) was obtained from
solution A (sphingosine-1-phosphate, 34 mg, 0.09 mmol), KOt-Bu
(20 mg, 0.18 mmol)), solution B (PLP monohydrate (31 mg,
0.12 mmol)), KOt-Bu (26 mg, 0.23 mmol) and NaBH4 (4 mg,
0.12 mmol), according to general procedure 1. The title compound
was purified by preparative RP chromatography (from 5 to 100%
29.5, 29.3, 29.1, 22.8, 14.3. HRMS calcd. for C18H35N3O2Na
([MþNa]þ): 348.2627, found: 348.2595. RT (Column A, hexane/
isopropanol (95:5)): 15.3 min ca. 97% ee.
4.2.2.4. (2R,3R,4E)-2-azidooctadec-4-ene-1,3-diol
(ent-96).
Compound ent-96 was obtained from ent-95 following the pro-
cedure described for the preparation of 96. Spectroscopic data were
identical to those indicated for 96.
CH3CN in 100 mM TEAA pH 7.0 buffer).
20
[
a]
¼ ꢁ14.1 (c 1.0, CH3OH). 1H NMR (400 MHz, CD3OD)
d 7.82
D
20
24
(s, 1H), 5.93e5.82 (m, 1H), 5.54 (dd, J ¼ 15.4, 6.6 Hz, 1H), 4.99e4.92
(m, 2H), 4.58 (d, J ¼ 13.9 Hz, 1H), 4.46 (app t, J ¼ 5.8 Hz, 1H), 4.41 (d,
J ¼ 13.9 Hz, 1H), 4.23 (ddd, J ¼ 11.7, 6.6, 3.4 Hz, 1H), 4.16e4.07 (m,
1H), 3.26 (ddd, J ¼ 8.2, 5.1, 3.5 Hz,1H), 3.19 (q, J ¼ 7.3 Hz, 6H), 2.46 (s,
3H), 2.11 (dd, J ¼ 14.1, 7.1 Hz, 2H), 1.56e1.17 (m, 31H), 0.90 (t,
[a
]
¼ ꢁ1.5 (c 1.0, CHCl3), [lit. [51] [
a]
¼ ꢁ2.4 (c 0.3, CHCl3)]. RT
D
D
(Column A, hexane/isopropanol (95:5)): 13.1 min ca. >99% ee.
4.2.2.5. (2S,3R)-2-azidooctadecane-1,3-diol (112). Compound 112
(white solid, 201 mg, 86%) was obtained from 62 (240 mg,
0.71 mmol), K2CO3 (265 mg, 1.92 mmol), CuSO4$5H2O (1.1 mg,
catalytic) and ISA$HCl (179 mg, 0.85 mmol), according to general
procedure 2.
J ¼ 6.9 Hz, 3H). 13C NMR (101 MHz, CD3OD)
d 157.2, 147.6, 135.9,
135.7, 133.2 (d, JC-P ¼ 8.7 Hz),131.4,129.7, 71.2, 63.8 (d, JC-P ¼ 6.9 Hz),
63.7 (d, JC-P ¼ 4.8 Hz), 62.5 (d, JC-P ¼ 4.8 Hz), 47.5, 45.5, 33.5, 33.1,
30.8, 30.8, 30.8, 30.8, 30.7, 30.5, 30.2, 23.7, 17.7, 14.5, 9.2. 31P NMR
20
24
[a
]
¼ þ7.9 (c 1.0, CHCl3) [lit. [44] [
a
]
¼ þ4.1 (c 0.5, CHCl3)]. 1H
D
D
(162 MHz, CD3OD)
d 2.64, 1.76. HRMS calcd. for C26H49N2O10P2
NMR (400 MHz, CDCl3) d 3.94e3.85 (m, 2H), 3.81e3.74 (m,1H), 3.43
([MþH]þ): 611.2862, found: 611.2859.
(dd, J ¼ 10.1, 5.1 Hz, 1H), 2.03 (br s, 2H), 1.66e1.17 (m, 28H), 0.88 (t,
J ¼ 6.8 Hz, 3H). 13C NMR (101 MHz, CDCl3)
d 72.8, 67.0, 62.7, 33.9,
4.2.2. General procedure 2: copper-catalyzed diazo-transfer
reaction
32.1, 29.8, 29.8, 29.8, 29.7, 29.7, 29.6, 29.5, 25.7, 22.8, 14.3. HRMS
calcd. for C18H37N3O2Na ([MþNa]þ): 350.2783, found: 350.2780. RT
(Column A, hexane/isopropanol (95:5)): 9.7 min ca. 98% ee.
To a stirred mixture of the corresponding amine hydrochloride
(0.70 mmol), K2CO3 (2.70 equiv) and CuSO4$5H2O (0.01 equiv) in
MeOH (7 mL) was added ISA$HCl (1.20 equiv) in one portion. After
stirring at rt overnight, the mixture was concentrated, diluted with
H2O (5 mL), acidified with 1 M aqueous HCl and extracted with
EtOAc (3 ꢂ 10 mL). The combined organic layers were washed with
brine (2 ꢂ 10 mL), dried over anhydrous MgSO4, filtered and
evaporated in vacuo. Purification of the residue by flash chroma-
tography (from 0 to 25% EtAcO in hexane) gave the required
compound.
4.2.2.6. (2R,3S)-2-azidooctadecane-1,3-diol
(ent-112).
Compound ent-112 was obtained from ent-62 following the pro-
cedure described for the preparation of 112. Spectroscopic data
were identical to those indicated for 112.
20
[a
]
¼ ꢁ10.6 (c 1.0, CHCl3). RT (Column A, hexane/isopropanol
D
(95:5)): 7.8 min ca. 98% ee.
4.2.2.7. (2S,3S)-2-azidooctadecane-1,3-diol (101). Compound 101
(white solid, 225 mg, 80%) was obtained from 100 (289 mg,
0.86 mmol), K2CO3 (319 mg, 2.31 mmol), CuSO4$5H2O (1.4 mg,
catalytic) and ISA$HCl (215 mg, 1.03 mmol), according to general
4.2.2.1. (2S,3R,4E)-2-azidooctadec-4-ene-1,3-diol
(87).
Compound 87 (white solid, 152 mg, 91%) was obtained from 86
(173 mg, 0.52 mmol), K2CO3 (192 mg, 1.39 mmol), CuSO4$5H2O
(0.8 mg, catalytic) and ISA$HCl (130 mg, 0.62 mmol), according to
general procedure 2.
procedure 2.
20
[a
]
¼ þ7.3 (c 1.0, CHCl3). 1H NMR (400 MHz, CDCl3)
d 3.97e3.82
D
20
25
(m, 2H), 3.79e3.70 (m, 1H), 3.44 (dt, J ¼ 6.1, 4.2 Hz, 1H), 2.07e2.00
[
a]
¼ ꢁ34.6 (c 1.0, CHCl3) [lit. [42] [
a
]
¼ ꢁ33.2 (c 2.0, CHCl3)].
D
D
1H NMR (400 MHz, CDCl3)
d 5.89e5.75 (m, 1H), 5.59e5.48 (m, 1H),
(m, 1H), 1.91e1.86 (m, 1H), 1.62e1.20 (m, 28H), 0.88 (t, J ¼ 6.8 Hz,
3H). 13C NMR (101 MHz, CDCl3)
d 72.5, 67.0, 63.8, 34.6, 32.1, 29.8,
4.28e4.21 (m, 1H), 3.84e3.73 (m, 2H), 3.51 (dd, J ¼ 10.4, 5.5 Hz, 1H),
2.12e1.99 (m, 4H), 1.46e1.18 (m, 22H), 0.88 (t, J ¼ 6.8 Hz, 3H). 13
C
29.8, 29.8, 29.8, 29.8, 29.7, 29.7, 29.6, 29.5, 25.7, 22.8, 14.3. HRMS
calcd. for C18H37N3O2Na ([MþNa]þ): 350.2783, found: 350.2755. RT
(Column A, hexane/isopropanol 96.5:3.5): 19.3 min ca. 95% ee.
NMR (101 MHz, CDCl3) d 136.1,128.1, 73.9, 66.9, 62.7, 32.5, 32.1, 29.8,
29.8, 29.8, 29.8, 29.7, 29.6, 29.5, 29.3, 29.1, 22.8, 14.2. HRMS calcd.
for C18H35N3O2Na ([MþNa]þ): 348.2627, found: 348.2637. RT (Col-
umn B, hexane/isopropanol (96.5:3.5)): 16.5 min ca. 99% ee.
4.2.2.8. (2R,3R)-2-azidooctadecane-1,3-diol
(ent-101).
Compound ent-101 was obtained from ent-100 following the pro-
cedure described for the preparation of 101. Spectroscopic data
4.2.2.2. (2R,3S,4E)-2-azidooctadec-4-ene-1,3-diol
(ent-87).
were identical to those indicated for 101.
Compound ent-87 was obtained from ent-86 following the pro-
cedure described for the preparation of 87. Spectroscopic data were
identical to those indicated for 87.
20
[a
]
¼ ꢁ8.3 (c 1.0, CHCl3). RT (Column A, hexane/isopropanol
D
(96.5:3.5)): 15.2 min ca. 99% ee.
20
24
[
a]
¼ þ32.4 (c 1.0, CHCl3) [lit. [50] [
a]
¼ þ39.3 (c 1.0, CHCl3)].
D
D
RT (Column B, hexane/isopropanol (96.5:3.5)): 15.4 min ca. 99% ee.
4.2.3. General procedure 3: site-selective phosphorylation of 1,3-
diols
4.2.2.3. (2S,3S,4E)-2-azidooctadec-4-ene-1,3-diol (96).
A solution of the starting diol (0.50 mmol) in CH2Cl2 (15 mL) at
0 ꢀC was treated successively with N-methylimidazole (1.50 equiv)
and dimethyl chlorophosphate (1.20 equiv). The reaction mixture
was stirred at rt for 1 h and cooled again to 0 ꢀC. The reaction was
then quenched by dropwise addition of saturated aqueous NH4Cl
and stirred for 5 min. The mixture was extracted with CH2Cl2
(3 ꢂ 15 mL) and the combined organic layers were washed with
brine (2 ꢂ 20 mL), dried over anhydrous MgSO4 and filtered.
Evaporation of the solvent afforded a crude mixture, which was
purified as indicated below.
Compound 96 (white solid, 217 mg, 82%) was obtained from 95
(272 mg, 0.81 mmol), K2CO3 (302 mg, 2.19 mmol), CuSO4$5H2O
(1.3 mg, catalytic) and ISA$HCl (204 mg, 0.97 mmol), according to
general procedure 2.
20
26
[
a]
¼ þ1.0 (c 1.0, CHCl3) [lit [43]. [
a]
¼ þ0.9 (c 1.0, CHCl3)]. 1H
D
D
NMR (400 MHz, CDCl3)
d
5.86e5.74 (m, 1H), 5.52 (dd, J ¼ 15.4,
7.1 Hz, 1H), 4.21 (app t, J ¼ 6.2 Hz, 1H), 3.82 (dd, J ¼ 11.4, 3.8 Hz, 1H),
3.70 (dd, J ¼ 11.4, 6.4 Hz, 1H), 3.52e3.42 (m, 1H), 2.12e1.95 (m, 4H),
1.48e1.10 (m, 22H), 0.88 (t, J ¼ 6.7 Hz, 3H). 13C NMR (101 MHz,

912
P. Sanllehí et al. / European Journal of Medicinal Chemistry 123 (2016) 905e915
4.2.3.1. (2S,3R,4E)-2-azido-3-hydroxyoctadec-4-en-1-yl
phosphate (88). Compound 88 (colorless oil, 171 mg, 85%) was
obtained from alcohol 87 (152 mg, 0.47 mmol), N-methylimidazole
dimethyl
Spectroscopic data were identical to those indicated for 113.
20
[
a
]
¼ ꢁ22.6 (c 1.0, CHCl3).
D
(56
mL, 0.70 mmol) and dimethyl chlorophosphate (60
mL,
4.2.3.7. (2S,3S)-2-azido-3-hydroxyoctadecyl dimethyl phosphate
0.56 mmol), according to general procedure 3. The title compound
(102). Compound 102 (colorless oil, 66 mg, 64%) was obtained from
was purified by flash chromatography on silica gel (from 0 to 100%
alcohol 101 (78 mg, 0.24 mmol), N-methylimidazole (29
mL,
Et2O in hexane).
0.36 mmol) and dimethyl chlorophosphate (31 L, 0.29 mmol),
according to general procedure 3. The title compound was purified
by flash chromatography on silica gel (from 0 to 1% CH3OH in
m
20
[a
]
¼
ꢁ7.4 (c 1.0, CHCl3). 1H NMR (400 MHz, CDCl3)
D
d
5.88e5.77 (m, 1H), 5.57e5.46 (m, 1H), 4.28e4.15 (m, 3H), 3.82 (d,
J ¼ 3.7 Hz, 3H), 3.80 (d, J ¼ 3.7 Hz, 3H), 3.57 (dd, J ¼ 10.3, 5.8 Hz,1H),
CH2Cl2).
20
2.11e2.02 (m, 2H), 1.45e1.19 (m, 22H), 0.88 (t, J ¼ 6.8 Hz, 3H). 13
C
[
a
]
¼
þ4.3 (c 1.0, CHCl3). 1H NMR (400 MHz, CDCl3)
D
NMR (101 MHz, CDCl3)
d
136.2, 127.7, 71.8, 67.2 (d, JC-P ¼ 5.4 Hz),
d 4.35e4.27 (m, 1H), 4.25e4.16 (m, 1H), 3.82 (s, 3H), 3.80 (s, 3H),
65.6 (d, JC-P ¼ 6.6 Hz), 54.7 (d, JC-P ¼ 6.1 Hz), 54.7 (d, JC-P ¼ 6.1 Hz),
3.71e3.64 (m, 1H), 3.57 (ddd, J ¼ 8.0, 5.1, 3.3 Hz, 1H), 1.68e1.17 (m,
32.4, 32.0, 29.8, 29.8, 29.8, 29.7, 29.6, 29.5, 29.3, 29.0, 22.8, 14.2. 31
P
28H), 0.88 (t, J ¼ 6.8 Hz, 3H). 13C NMR (101 MHz, CDCl3)
d 70.7, 67.6
NMR (162 MHz, CDCl3)
d
2.07. HRMS calcd. for C20H40N3O5PNa
(d, JC-P ¼ 5.5 Hz), 65.4 (d, JC-P ¼ 6.8 Hz), 54.8 (d, JC-P ¼ 6.1 Hz), 54.7 (d,
JC-P ¼ 6.1 Hz), 34.3, 32.1, 29.8, 29.8, 29.8, 29.8, 29.7, 29.7, 29.6, 29.5,
([MþNa]þ): 456.2603, found: 456.2603.
25.8, 22.8, 14.2. 31P NMR (162 MHz, CDCl3)
d 1.96. HRMS calcd. for
4.2.3.2. (2R,3S,4E)-2-azido-3-hydroxyoctadec-4-en-1-yl
dimethyl
C
20H42N3O5PNa ([MþNa]þ): 458.2760, found: 458.2764.
phosphate (ent-88). Compound ent-88 was obtained from ent-87
following the procedure described for the preparation of 88.
4.2.3.8. (2R,3R)-2-azido-3-hydroxyoctadecyl dimethyl phosphate
(ent-102). Compound ent-102 was obtained from ent-101
following the procedure described for the preparation of 102.
Spectroscopic data were identical to those indicated for 88.
20
[a
]
¼ þ6.8 (c 1.0, CHCl3).
D
Spectroscopic data were identical to those indicated for 102.
20
4.2.3.3. (2S,3S,4E)-2-azido-3-hydroxyoctadec-4-en-1-yl
dimethyl
[
a
]
¼ ꢁ3.4 (c 1.0, CHCl3).
D
phosphate (97). Compound 97 (colorless oil, 99 mg, 68%) was ob-
tained from alcohol 96 (110 mg, 0.34 mmol), N-methylimidazole
4.2.4. General procedure 4: deprotection of phosphate esters with
TMSBr
(40
mL, 0.51 mmol) and dimethyl chlorophosphate (44 mL,
0.41 mmol), according to general procedure 3. The title compound
was purified by flash chromatography on silica gel (from 0 to 100%
Et2O in hexane).
To an ice cooled solution of the starting dimethyl phosphate
(0.20 mmol) in dry CH3CN (8 mL), was added dropwise TMSBr (4.0
equiv). After stirring for 3 h at rt, the reaction mixture was
concentrated under reduced pressure. The residue was then taken
up in MeOH/H2O (95:5, same reaction volume), stirred for an
additional hour at rt and evaporated to dryness. The crude reaction
mixture was dissolved in methanol and loaded on an Amberlite
XAD-4 column (10 g), which had been washed thoroughly with
acetone and then equilibrated with water. Elution with a linear
gradient from 0 to 70% MeCN in H2O provided the required
compound.
20
[a
]
¼ ꢁ1.6 (c 1.0, CHCl3). 1H NMR (400 MHz, CDCl3)
d 5.86e5.72
D
(m, 1H), 5.49 (dd, J ¼ 15.4, 6.9 Hz, 1H), 4.29e4.20 (m, 1H), 4.18e4.12
(m, 1H), 4.12e4.02 (m, 1H), 3.80 (s, 3H), 3.77 (s, 3H), 3.63e3.55 (m,
1H), 2.10e1.99 (m, 2H), 1.47e1.12 (m, 22H), 0.86 (t, J ¼ 6.6 Hz, 3H).
13C NMR (101 MHz, CDCl3)
d
135.8, 127.8, 72.1, 67.2 (d, JC-P ¼ 5.4 Hz),
66.0 (d, JC-P ¼ 7.0 Hz), 54.7 (d, JC-P ¼ 5.9 Hz), 32.4, 32.0, 29.8, 29.8,
29.7, 29.6, 29.5, 29.3, 29.0, 22.8, 14.2. 31P NMR (162 MHz, CDCl3)
d
1.38. HRMS calcd. for C20H40N3O5PNa ([MþNa]þ): 456.2603,
found: 456.2591.
4.2.4.1. (2S,3R,4E)-2-azido-3-hydroxyoctadec-4-en-1-yl dihydrogen
phosphate (89). Compound 89 (pale yellow solid, 52 mg, 71%) was
obtained from dimethyl phosphate 88 (78 mg, 0.18 mmol) and
4.2.3.4. (2R,3R,4E)-2-azido-3-hydroxyoctadec-4-en-1-yl
phosphate (ent-97). Compound ent-97 was obtained from ent-96
following the procedure described for the preparation of 97.
dimethyl
TMSBr (95 mL, 0.72 mmol), according to general procedure 4.
20
Spectroscopic data were identical to those reported for 97.
[
a
]
¼ ꢁ18.5 (c 1.0, CH3OH). 1H NMR (400 MHz, CD3OD)
D
20
[a
]
¼ þ2.3 (c 1.0, CHCl3).
d
5.83e5.72 (m, 1H), 5.51 (dd, J ¼ 15.3, 7.4 Hz, 1H), 4.16e4.08 (m,
D
2H), 3.98e3.89 (m, 1H), 3.64e3.56 (m, 1H), 2.13e2.02 (m, 2H),
1.48e1.21 (m, 22H), 0.90 (t, J ¼ 6.7 Hz, 3H). 13C NMR (101 MHz,
4.2.3.5. (2S,3R)-2-azido-3-hydroxyoctadecyl dimethyl phosphate
(113). Compound 113 (colorless oil, 66 mg, 76%) was obtained from
alcohol 112 (65 mg, 0.20 mmol), N-methylimidazole (24
CD3OD)
d
136.0, 129.6, 73.1, 67.5 (d, JC-P ¼ 7.8 Hz), 67.1 (d, JC-
mL,
¼ 5.1 Hz), 33.4, 33.1, 30.8, 30.8, 30.8, 30.8, 30.7, 30.6, 30.5, 30.2,
P
0.30 mmol) and dimethyl chlorophosphate (26 L, 0.24 mmol),
m
30.2, 23.7, 14.4. 31P NMR (162 MHz, CD3OD)
d 1.22. HRMS calcd. for
according to general procedure 3. The title compound was purified
by flash chromatography on silica gel (from 0 to 100% Et2O in
C
18H36N3O5PNa ([MþNa]þ): 428.2290, found: 428.2287.
hexane).
4.2.4.2. (2R,3S,4E)-2-azido-3-hydroxyoctadec-4-en-1-yl dihydrogen
phosphate (ent-89). Compound ent-89 was obtained from ent-88
following the procedure described for the preparation of 89.
20
[a
]
¼ þ22.0 (c 1.0, CHCl3). 1H NMR (400 MHz, CDCl3)
D
d
4.43e4.26 (m, 2H), 3.83 (d, J ¼ 4.7 Hz, 3H), 3.80 (d, J ¼ 4.7 Hz, 3H),
3.74e3.67 (m, 1H), 3.43e3.36 (m, 1H), 1.67e1.20 (m, 28H), 0.88 (t,
Spectroscopic data were identical to those indicated for 89.
20
J ¼ 6.8 Hz, 3H). 13C NMR (101 MHz, CDCl3)
d
70.0, 67.3 (d, JC-
[
a
]
¼ þ16.8 (c 1.0, CH3OH).
D
¼ 5.4 Hz), 65.8 (d, JC-P ¼ 5.9 Hz), 54.9 (d, JC-P ¼ 6.1 Hz), 54.8 (d, JC-
P
¼ 6.1 Hz), 33.6, 32.1, 29.8, 29.8, 29.8, 29.7, 29.7, 29.7, 29.5, 25.6,
4.2.4.3. (2S,3S,4E)-2-azido-3-hydroxyoctadec-4-en-1-yl dihydrogen
phosphate (98). Compound 98 (pale yellow solid, 57 mg, 68%) was
obtained from dimethyl phosphate 97 (90 mg, 0.21 mmol) and
P
22.8, 14.3. 31P NMR (162 MHz, CDCl3)
d 2.44. HRMS calcd. for
C
20H42N3O5PNa ([MþNa]þ): 458.2760, found: 458.2765.
TMSBr (110 mL, 0.83 mmol), according to general procedure 4.
20
4.2.3.6. (2R,3S)-2-azido-3-hydroxyoctadecyl dimethyl phosphate
(ent-113). Compound ent-113 was obtained from ent-112
following the procedure described for the preparation of 113.
[
a
]
¼ ꢁ5.1 (c 0.7, CHCl3). 1H NMR (400 MHz, CD3OD) 5.85e5.72
D
(m, 1H), 5.53 (dd, J ¼ 15.4, 6.9 Hz, 1H), 4.20e4.04 (m, 2H), 3.96 (br s,
1H), 3.53 (br s, 1H), 2.14e2.01 (m, 2H), 1.62e1.19 (m, 22H), 0.90 (t,

P. Sanllehí et al. / European Journal of Medicinal Chemistry 123 (2016) 905e915
913
J ¼ 6.8 Hz, 3H). 13C NMR (101 MHz, CD3OD)
d
135.3, 130.1, 73.1, 67.7
by the addition of 50
m
L of MeOH. Finally, 100
m
L of a 200 mM
(d, JC-P ¼ 4.1 Hz), 66.9 (d, JC-P ¼ 4.2 Hz), 33.4, 33.1, 30.8, 30.8, 30.7,
glycine-NaOH buffer, pH 10.6, were added to the resulting solution
30.6, 30.5, 30.3, 30.2, 23.7, 14.4. 31P NMR (162 MHz, CD3OD)
d 3.05.
and the mixture was incubated for 20 additional min at 37 ꢀC in
HRMS calcd. for C18H36N3O5PNa ([MþNa]þ): 428.2290, found:
order to complete the b-elimination reaction. The amount of
umbelliferone formed was determined on either a SpectraMax M5
428.2287.
(Molecular Devices) or Synergy 2 (BioTek) microplate readers (lex/
4.2.4.4. (2R,3R,4E)-2-azido-3-hydroxyoctadec-4-en-1-yl dihydrogen
phosphate (ent-98). Compound ent-98 was obtained from ent-97
following the procedure described for the preparation of 98.
¼ 355/460 nm), using a calibration curve. IC50 values were
em
determined by plotting percent activity versus log [I] and fitting the
data to the log(inhibitor) vs. response equation in Prism 5
(GraphPad Software, La Jolla). Settings for curve adjustments were
kept with their default values. The type of inhibition and the Ki
values for active inhibitors were determined by Lineweaver-Burk or
Dixon plots of assays performed with different concentrations of
inhibitor and substrate.
Spectroscopic data were identical to those indicated for 98.
20
[
a]
¼ þ4.2 (c 0.7, CHCl3).
D
4.2.4.5. (2S,3R)-2-azido-3-hydroxyoctadecyl dihydrogen phosphate
(114). Compound 114 (pale yellow solid, 39 mg, 56%) was obtained
from dimethyl phosphate 113 (75 mg, 0.17 mmol) and TMSBr (91 mL,
0.69 mmol), according to general procedure 4.
4.4. Computational docking methods
¼ þ21.3 (c 0.7, CHCl3). 1H NMR (400 MHz, CD3OD)
20
[
a]
D
d
4.31e4.21 (m, 1H), 4.09e3.98 (m, 1H), 3.61e3.49 (m, 2H),
All the computational work was carried out with the
1.64e1.21 (m, 28H), 0.90 (t, J ¼ 6.7 Hz, 3H). 13C NMR (101 MHz,
€
Schrodinger Suite 2015 [52], through its graphical interface
CD3OD)
d
71.6, 67.7 (d, JC-P ¼ 6.6 Hz), 67.4 (d, JC-P ¼ 3.1 Hz), 34.4, 33.1,
Maestro [53]. Coordinates of StSPL and hSPL (PDB codes 3MAD and
4Q6R) [1,2] were obtained from the Protein Data Bank [54] at
Brookhaven National Laboratory. The protein X-ray structures were
prepared using the Protein Preparation Wizard [55e59] included in
Maestro to remove solvent molecules, ions and bound ligands,
adding hydrogens, setting protonation states [60] and minimizing
the energies. The 3-hydroxypyridine and imino groups of the Lys-
bound PLP prosthetic group were considered in their neutral
state. The azido phosphate structures were built within Maestro
and set up with the LigPrep module [61] to generate ionization
states, as well as for geometry optimization previous to docking.
Ligands were docked into the active site of S1PL using the Induced
Fit Docking Protocol [62] of the Schrodinger Suite, which uses Glide
XP [63e66] to perform the docking phase and takes into consid-
eration the flexibility of the protein residues within a given distance
(5 Å) from the bound ligands, in order to refine the geometries and
scores of the docked poses.
30.8, 30.8, 30.8, 30.7, 30.7, 30.7, 30.5, 26.6, 23.7, 14.5. 31P NMR
(162 MHz, CD3OD)
d 1.58. HRMS calcd. for C18H38N3O5PNa
([MþNa]þ): 430.2447, found: 430.2441.
4.2.4.6. (2R,3S)-2-azido-3-hydroxyoctadecyl dihydrogen phosphate
(ent-114). Compound ent-114 was obtained from ent-113
following the procedure described for the preparation of 114.
Spectroscopic data were identical to those indicated for 114.
20
[
a]
¼ ꢁ23.7 (c 0.7, CHCl3).
D
4.2.4.7. (2S,3S)-2-azido-3-hydroxyoctadecyl dihydrogen phosphate
(103). Compound 103 (pale yellow solid, 50 mg, 69%) was obtained
from dimethyl phosphate 102 (78 mg, 0.18 mmol) and TMSBr
(95
m
L, 0.72 mmol), according to general procedure 4.
20
[
a]
¼ þ8.4 (c 0.5, CHCl3). 1H NMR (400 MHz, CD3OD)
D
d
4.19e4.05 (m, 2H), 3.73e3.65 (m, 1H), 3.57e3.50 (m, 1H),
1.63e1.18 (m, 28H), 0.90 (t, J ¼ 6.8 Hz, 3H). 13C NMR (101 MHz,
CD3OD)
d
71.7, 67.4 (d, JC-P ¼ 5.1 Hz), 67.1 (d, JC-P ¼ 7.6 Hz), 34.9, 33.1,
Acknowledgments
30.8, 30.8, 30.7, 30.7, 30.7, 30.5, 26.8, 23.7, 14.5. 31P NMR (162 MHz,
CD3OD)
430.2447, found: 430.2445.
d
1.29. HRMS calcd. for C18H38N3O5PNa ([MþNa]þ):
We are grateful to Prof. Andreas Billich (Novartis) for a generous
gift of recombinant hS1PL. Partial financial support from the
ꢀ
“Ministerio de Ciencia e Innovacion”, Spain (Project CTQ2014-
54743-R is acknowledged). The authors also want to thank
4.2.4.8. (2R,3R)-2-azido-3-hydroxyoctadecyl dihydrogen phosphate
(ent-103). Compound ent-103 was obtained from ent-102
following the procedure described for the preparation of 103.
ꢀ
ꢀ
“Fundacio la Marato de TV3” for financial support through Projects
20112130 and 20112132.
Spectroscopic data were identical to those indicated for 103.
20
[
a]
¼ ꢁ8.1 (c 0.5, CHCl3).
D
Appendix A. Supplementary data
4.3. Fluorogenic assay of S1PL enzyme activity
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2016.08.008.
Recombinant bacterial or human S1PL (50
m
L from stock solu-
g/
g/mL for hS1PL) was added to a mixture of
RBM13 (added from a stock solution in 0.5 M potassium phosphate
buffer, pH 7.2; final concentration: 125 M) and the putative in-
tions in buffer A (StS1PL) or B (hS1PL), final concentration: 25
mL for StS1PL and 3
m
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