Novel S1P1 receptor agonists - Part 4: Alkylaminomethyl substituted aryl head groups

Article abstract of DOI:10.1016/j.ejmech.2016.03.048

In a previous communication we reported on the discovery of alkylamino pyridine derivatives (e.g. 1) as a new class of potent, selective and efficacious S1P₁ receptor (S1PR₁) agonists. However, more detailed profiling revealed that this compound class is phototoxic in vitro. Here we describe a new class of potent S1PR₁ agonists wherein the exocyclic nitrogen was moved away from the pyridine ring (e.g. 11c). Further structural modifications led to the identification of novel alkylaminomethyl substituted phenyl and thienyl derivatives as potent S1PR₁ agonists. These new alkylaminomethyl aryl compounds showed no phototoxic potential. Based on their in vivo efficacy and ability to penetrate the brain, the 5-alkyl-aminomethyl thiophenes appeared to be the most interesting class. Potent and selective S1PR₁ agonist 20e, for instance, maximally reduced the blood lymphocyte count (LC) for 24 h after oral administration of 10 mg/kg to rat and its brain concentrations reached >500 ng/g over 24 h.

Full text of DOI:10.1016/j.ejmech.2016.03.048

European Journal of Medicinal Chemistry 116 (2016) 222e238
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Novel S1P1 receptor agonists e Part 4: Alkylaminomethyl substituted
aryl head groups
Cyrille Lescop*, Claus Müller, Boris Mathys, Magdalena Birker, Ruben de Kanter,
Christopher Kohl, Patrick Hess, Oliver Nayler, Markus Rey, Patrick Sieber, Beat Steiner,
Thomas Weller, Martin H. Bolli
Drug Discovery Chemistry, Actelion Pharmaceuticals Ltd., Gewerbestrasse 16, CH-4123 Allschwil, Switzerland
a r t i c l e i n f o
a b s t r a c t
Article history:
In a previous communication we reported on the discovery of alkylamino pyridine derivatives (e.g. 1) as a
new class of potent, selective and efficacious S1P1 receptor (S1PR1) agonists. However, more detailed
profiling revealed that this compound class is phototoxic in vitro. Here we describe a new class of potent
S1PR1 agonists wherein the exocyclic nitrogen was moved away from the pyridine ring (e.g. 11c). Further
structural modifications led to the identification of novel alkylaminomethyl substituted phenyl and
thienyl derivatives as potent S1PR1 agonists. These new alkylaminomethyl aryl compounds showed no
phototoxic potential. Based on their in vivo efficacy and ability to penetrate the brain, the 5-alkyl-
aminomethyl thiophenes appeared to be the most interesting class. Potent and selective S1PR1 agonist
20e, for instance, maximally reduced the blood lymphocyte count (LC) for 24 h after oral administration
of 10 mg/kg to rat and its brain concentrations reached >500 ng/g over 24 h.
Received 26 August 2015
Received in revised form
17 December 2015
Accepted 18 March 2016
Available online 23 March 2016
Keywords:
S1P1 receptor agonist
Immunomodulator
Lymphocyte count
Aminomethyl pyridine
Aminomethyl benzene
Aminomethyl thiophene
Phototoxicity
© 2016 Elsevier Masson SAS. All rights reserved.
1. Introduction
[10e12] as well as tissue and organ function such as cell growth
[13], vascular development [14], immune cell trafficking [15e18],
Sphingosine-1 phosphate (S1P, Fig.1) is a bioactive phospholipid
produced by sphingosine kinase-mediated phosphorylation of
sphingosine, a metabolite of ceramide. S1P is involved in a number
of pathological conditions such as autoimmune diseases, cancer,
fibrosis, atherosclerosis, diabetes and osteoporosis [1e9]. These
observations confirm the critical role of S1P in cellular processes
cardiac function and inflammation [5] to name a few. Regulation of
these functions is primarily orchestrated by the interaction of S1P
with a family of five G-protein coupled receptors known as S1PR1-5
[10,19e21]. The S1PR1 e 3 are widely expressed in almost all organs,
while S1PR4 is predominantly expressed on lymphoid and he-
matopoietic tissues and S1PR5 is primarily located in the spleen and
in the white matter of the central nervous system. Over the last
decade, the role of the S1PeS1P1 receptor signaling axis in
immune-mediated diseases has been studied in detail [4,22,23].
The S1P1 receptor subtype plays a central role in the control of
lymphocyte migration. Upon activation in the lymph node by an
antigen presenting cell, lymphocytes up-regulate S1P1 receptor
expression and are thus able to sense the concentration gradient of
S1P that exists between lymph and blood. By migrating along this
gradient, lymphocytes exit the lymph nodes and secondary
lymphoid organs, reach the systemic circulation and finally the
intruder and target tissue. Understanding of the mechanism of
action of S1PR1 agonists has grown rapidly with the discovery that
fingolimod (FTY720, Fig. 1) e the first-in-class oral drug approved
for the treatment of relapsing remitting multiple sclerosis [24] - is
Abbreviations used: BuLi, n-butyl lithium; CC, column chromatography; DCM,
dichloromethane; DIPEA, diisopropylethylamine; DEAD, diethyl azodicarboxylate;
DMF,
dimethylformamide;
EA,
ethyl
acetate;
EDC,
1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide; fumic
,
unbound fraction in microsomal in-
cubations; fup, unbound fraction in plasma; HOBt, N-hydroxybenzotriazole; LC,
lymphocyte count; LC-MS, (high pressure) liquid chromatography combined with
mass spectrometry; MeCN, acetonitrile; MeOH, methanol; NMP, N-methyl-
pyrrolidine; PK, pharmacokinetics; PPh3, triphenylphosphine; RLM, rat liver mi-
crosomes; SAR, structure-activity relationship; S1P, sphingosine 1-phosphate;
S1PR1, sphingosine 1-phosphate 1 receptor; S1PR3, sphingosine 1-phosphate 3 re-
ceptor;
TBTU,
O-(benzotriazol-1-yl)-N,N,N0,N0-tetramethyluronium
tetra-
fluoroborate; TFA, trifluoroacetic acid; THF, tetrahydrofuran.
* Corresponding author.
E-mail address: cyrille.lescop@actelion.com (C. Lescop).
http://dx.doi.org/10.1016/j.ejmech.2016.03.048
0223-5234/© 2016 Elsevier Masson SAS. All rights reserved.

C. Lescop et al. / European Journal of Medicinal Chemistry 116 (2016) 222e238
223
Fig. 1. Structures of sphingosine 1-phosphate, FTY720 (fingolimod), p-FTY720, ponesimod and siponimod
phosphorylated in vivo by sphingosine kinases to become a potent,
non-selective S1PR agonist, p-FTY720 (Fig. 1) [25e27]. Activation of
the S1P1 receptor by a synthetic S1PR1 agonist leads to receptor
internalization and thus desensitization of the lymphocytes toward
the S1P gradient. As a consequence lymphocytes lose their ability to
exit the lymph node and can no longer reach sites of inflammation
in the tissue. Mechanistically, synthetic S1PR1 agonists therefore
act as functional antagonists [28e31]. Disrupted trafficking of
activated lymphocytes by down-regulation of S1P1 receptors using
synthetic S1PR1 modulators represents an attractive concept to
treat a variety of lymphocyte dependent immune diseases such as
multiple sclerosis, Crohn's disease, systemic lupus erythematosus,
or psoriasis [1,15,32]. Over the last few years, numerous synthetic
S1PR1 agonists have been identified as a result of dedicated drug
discovery efforts [33e35].
In addition to the peripheral immunomodulatory action of
S1PR1 agonists, more recent studies identified activation of the
S1P1, S1P3 or S1P5 receptors on neural cells like astrocytes and ol-
igodendrocytes to lead to beneficial effects [19]. Following these
arguments, brain penetration of a synthetic S1PR1 agonist may be
contributing to the compound's efficacy in CNS related diseases
such as multiple sclerosis [36e39].
Several publications showed that activation of the S1P3 receptor
is associated with cardiovascular liabilities in rodents such as heart
rate reduction, hypertension and vaso-constriction [40e44]. On the
other hand, in the absence of data with selective S1PR2 and S1PR4
modulators, the benefit of modulating S1PR2 and S1PR4 remains
unclear [45]. Therefore, more recent research focused on the
identification and exploration of selective S1PR1 agonists in order
to improve the safety profile of pan-S1PR agonist which induces
transient heart rate reduction [35,46e51]. Lately, some of these
more selective compounds such as ponesimod [52], siponimod
[53], and RPC1063 [54] (structure not published) (Fig. 1) success-
fully completed phase II clinical trials in relapsing remitting mul-
tiple sclerosis [55]. However, transient heart rate reduction was not
totally eliminated implying that the S1P1 receptor is involved in the
regulation of the heart rate in humans [56e58].
Uptake (NRU) Phototoxicity assay. This assay measures cell viability
by comparing the concentration-dependent reduction in NRU by
normal BALB/c 3T3 mouse fibroblasts after exposure to a test
compound in the presence and absence of UVA light [60,61]. The
cytotoxicity IC50 values obtained in the presence and absence of
light are then used to calculate a photo-irritancy factor (PIF). A PIF
value greater than 2.5 is indicative of phototoxic potential [62]. All
tested alkylamino pyridine derivatives shown in Fig. 2 were above
the cut-off value of 2.5, with compounds 1e3 exhibiting a PIF > 200.
Planar and conjugated polycyclic structures containing hetero-
atoms are considered a structural alert for drug-induced photo-
toxicity [63,64], as they can absorb UV light and generate an
electronically excited species that could cause biological damage.
We therefore systematically evaluated other related pyridine series
in the 3T3 NRU phototoxicity assay. We discovered that the dialkyl
pyridine series was devoid of this property as illustrated by
example 4 [59] and we hypothesized that the alkylamino pyridine
moiety is the major driver of this unwanted effect (Fig. 2). We then
speculated that introducing a methylene linker between the pyri-
dine and the amino group would reproduce the clean profile of the
dialkyl pyridine series while keeping clogP ꢀ3.0. Indeed, first ex-
amples such as compounds 5 and 6 were clean in the cellular
photoxicity assay (PIF < 2.5). Even though compounds 5 and 6 were
less potent with EC50 values on S1PR1 of 45 nM and 110 nM,
respectively, they were still attractive because of their reduced
lipophilicity compared to compound 4 (clogP of 2.15, 3.06 and 3.71
respectively). These results prompted us to investigate the scope of
various alkylaminomethyl substituted aryl series in more detail. In
this article we describe the discovery of potent alkylaminomethyl
pyridine, benzene and thiophene based S1PR1 agonists with the
following profile: the compound is very potent on S1PR1
(EC50 < 5 nM) with a high selectivity against S1PR3 (ꢁ100-fold), has
a clogP ꢀ 3, is devoid of phototoxicity potential (PIF < 2.5), and
shows maximal lymphocyte count reduction for at least 24 h at an
oral dose of 10 mg/kg in the Wistar rat.
2. Results and discussion
In a previous communication we reported on the discovery of
alkyl-amino pyridine derivatives such as 1 as a new class of selec-
tive and efficacious S1PR1 agonists (Fig. 2) [59]. However, more
detailed profiling of this alkylamino pyridine-based series revealed
that this compound class is phototoxic in vitro. The potential for
phototoxicity was assessed in vitro using the 3T3 Neutral Red
2.1. Synthesis
Alkylaminomethyl substituted pyridine derivatives 5 and 11a-e
were prepared as outlined in Scheme 1. Like in previous series
[59,65,66] several side chains at position 4 of the phenyl ring have

224
C. Lescop et al. / European Journal of Medicinal Chemistry 116 (2016) 222e238
Fig. 2. Photo-irritancy factor (PIF) and EC50 S1PR1 (GTPgS) values in six related pyridine series.
been studied and many led to potent compounds (data not shown).
The glycolamide moiety shown in 5 appeared to be one of the most
interesting side chains in terms of affinity and selectivity for the
S1P1 receptor and was selected to discuss the structure activity
relationship of the present series. The key intermediate of the
synthesis is the 2-(hydroxymethyl)-6-methylisonicotinic acid
methyl ester 8. First, commercially available 2-methylpyridine-4-
carboxylic acid 7 was converted to its methyl ester which was
aReagents and conditions: (a) (1) H2SO4, MeOH, 65 °C, 24 h ; (2) (NH4)S2O8, H2O,
65 °C, 10 h, 62%; (b) MsCl, DIPEA, DCM, rt, 1 h, 96% crude; (c) RR’NH, MeCN, 40
°C, 3-16 h then 2N LiOH, 1 h, 20-52%; (d) (1) TBTU or EDC.HCl/HOBt, DIPEA,
DMF, rt, 1-2 h; (2) dioxane or DMF 80-110 °C, 2-48 h; 1-48%.
Scheme 1. Preparation of 2-alkylaminomethyl substituted pyridine derivativesa.

C. Lescop et al. / European Journal of Medicinal Chemistry 116 (2016) 222e238
225
then reacted with ammonium persulfate under Minisci conditions
[67] to afford 2-(hydroxymethyl)pyridine derivative 8. The
hydroxymethyl pyridine 8 was reacted with methanesulfonyl
chloride to provide the corresponding mesylate intermediate
which, after nucleophilic substitution with various amines
and subsequent saponification, gave the corresponding 2-
aminomethyl-6-methylisonicotinic acids 9a-f. These acids were
coupled with (S)-N-(3-(2-ethyl-4-(N-hydroxycarbamimidoyl)-6-
methylphenoxy)-2-hydroxypropyl)-2-hydroxyacetamide 10 [59].
Typical coupling conditions involved TBTU or EDC.HCl/HOBt as
activating agents. Subsequent cyclization of the hydroxyamidine
ester intermediate at 80e100 ꢂC furnished the desired oxadiazole
compounds 5 and 11a-e in moderate yield. We usually obtained
better yields in the cyclization step when all reagents were added
at once rather than in a sequential manner allowing the activation
of the pyridine carboxylic acids 9a-f. However, we did not further
optimize this procedure as this synthesis delivered sufficient ma-
terial for a first evaluation of the desired target compounds.
Related alkylaminomethyl aryl series were prepared from the
corresponding formyl aryl carboxylic acids. Thus, the synthesis of
the 3-alkylaminomethyl benzene series (14a-s) was carried out as
depicted in Scheme 2. As in the pyridine series, the alkylamino-
methyl group is in meta position to the oxadiazole linker. 3-Formyl
benzoic acids 12a-g were prepared according to literature pro-
cedures (see Supplementary Material), and were then coupled with
(S)-N-(3-(2-ethyl-4-(N-hydroxycarbamimidoyl)-6-
with respect to the oxadiazole. As outlined in Scheme 3, derivatives
17a-o were synthesized from the corresponding 4-formyl benzoic
acids 15a-e following a similar “coupling-cyclization-reductive
amination” sequence. The 4-formyl benzoic acids 15a-e were
commercially available or synthesized according to literature pro-
cedures [68,69]. For details on the synthesis of 15a-e see Supple-
mentary Material.
The same strategy was applied to the synthesis of the 5-alkyl-
aminomethyl thiophene derivatives 20a-s (Scheme 4). 5-Formyl-
thiophene-2-carboxylic acids 18a-d [70,71] were coupled with (S)-
N-(3-(2-ethyl-4-(N-hydroxycarbamimidoyl)-6-methylphenoxy)-2-
hydroxypropyl)-2-hydroxyacetamide 10 using EDC.HCl and HOBt
as activating agents and the resulting hydroxyamidine ester in-
termediates were then cyclized at 80e100 ꢂC to the oxadiazole
derivatives 19a-d. Reductive amination with the appropriate amine
in the presence of sodium cyanoborohydride or sodium triacetox-
yborohydride furnished the desired target compounds 20a-s usu-
ally in good yield.
In previous work we demonstrated that the chiral integrity is
maintained during the above described coupling-cyclization pro-
cedure [59]. This was verified exemplarily by subjecting the (S)-
enantiomer 20e and its (R)-enantiomer 20e′ (prepared from (R)-N-
(3-(2-ethyl-4-(N-hydroxycarbamimidoyl)-6-methylphenoxy)-2-
hydroxypropyl)-2-hydroxyacetamide 10′, see Supplementary Ma-
terial) to HPLC using a chiral stationary phase (Chiralpak AD-H
250 ꢃ 4.6 mm ID, 5
mM). Compounds 20e and 20e′ each dis-
played a single peak with a retention time of 16.8 and 12.4 min,
respectively, and showed no sign of racemization.
methylphenoxy)-2-hydroxypropyl)-2-hydroxyacetamide 10 using
EDC.HCl and HOBt, and cyclized at 80e110 ꢂC in DMF or dioxane to
give the oxadiazole intermediates 13a-g. A final reductive amina-
tion of benzaldehydes 13a-g using either sodium cyanoborohydride
or sodium triacetoxyborohydride as the reducing agent provided
the corresponding 3-aminomethyl benzene derivatives 14a-s in
moderate yield.
2.2. In vitro SAR discussion
For the different alkylaminomethyl substituted aryl series dis-
cussed here, affinity and selectivity for the S1P1 receptor were
In the benzene series, we also investigated compounds 17a-o
wherein the aminomethyl residue was moved to the para position
determined using a GTP
taining the S1P1 or the S1P3 receptor. In the 2-alkylaminomethyl
gS binding assay with membranes con-
aReagents and conditions: (a) (1) (S)-N-(3-(2-ethyl-4-(N-hydroxycarbamimidoyl)-6-
methylphenoxy)-2-hydroxypropyl)-2-hydroxyacetamide 10, EDC.HCl, HOBt, DMF, rt,
2-18 h; (2) dioxane or DMF, 80-110 °C, 2-48 h, 25-61%; (b) primary or secondary
amine, NaBH3CN, MeOH/NMP or NaBH(OAc)3, HOAc, DCM, NMP, rt, 18 h, 23-67%.
For the structure of compounds 12a-g, 13a-g, and 14a-s see Table 2 and
Supplementary Material.
Scheme 2. Preparation of 3-alkylaminomethyl benzene derivativesa.

226
C. Lescop et al. / European Journal of Medicinal Chemistry 116 (2016) 222e238
aReagents and conditions: (a)
(S)-N-(3-(2-ethyl-4-(N-hydroxycarbamimidoyl)-6-
methylphenoxy)-2-hydroxypropyl)-2-hydroxyacetamide 10, EDC.HCl, HOBt, DMF, rt,
1-2 h; (2) dioxane or DMF, 80-110 °C, 2-48 h, 26-67% ; (b) primary or secondary
amine, NaBH3CN or NaBH(OAc)3, HOAc, DCM/NMP, rt, 18 h, 17-93%. For the
structure of compounds 15a-e, 16a-e, and 17a-o see Table 3 and Supplementary
Material.
Scheme 3. Preparation of 4-alkylaminomethyl benzene derivativesa.
substituted pyridine series a selection of representative compounds
(5,11a-e) is compiled in Table 1. Compound 11a displayed an EC50 at
S1PR1 of 40 nM demonstrating that the S1P1 receptor can accom-
modate a basic moiety in the head group. In this compound class,
the potency on S1PR1 could be improved by increasing the size of
the dialkylamino residue. For example replacing one methyl group
of the dimethylamino derivative 11a by an n-propyl, an n-butyl or
an iso-butyl group furnished compounds 11b,11d and 11c with very
high affinity for the S1P1 receptor (EC50 S1PR1 ¼8.5, 4.3 and 3.2 nM,
respectively). Interestingly, increasing the size of the second alkyl
group attached to the nitrogen led to clearly less potent com-
pounds. This is illustrated with the diethylaminomethyl pyridine 5
aReagents and conditions: (a) (1)
(S)-N-(3-(2-ethyl-4-(N-hydroxycarbamimidoyl)-6-
methylphenoxy)-2-hydroxypropyl)-2-hydroxyacetamide 10, EDC.HCl, HOBt, DMF, rt
1-2 h; (2) dioxane or DMF, 80-110 °C, 2-48 h, 46-84%; (b) primary or secondary
amine, NaBH3CN or NaBH(OAc)3, DCM, NMP, HOAc, rt, 18-72 h, 4-88%. For the
structure of compounds 19a-d and 20a-s see Table 4 and Supplementary Material.
Scheme 4. Preparation of 5-alkylaminomethyl substituted thiophene derivativesa.

C. Lescop et al. / European Journal of Medicinal Chemistry 116 (2016) 222e238
227
Table 1
SAR of the 2-aminomethyl pyridine series.
a
a
Compd
R1
clogP
1.33
EC50 S1PR1 [nM]
40
EC50 S1PR3 [nM]
Selectivityb
11a
>10000
>250
102
175
349
180
11b
11c
11d
5
2.20
2.41
2.65
2.15
8.5
3.2
4.3
45
870
560
1500
8100
11e
2.13
95
>10000
>105
a
EC50 values measured in a GTPgS assay using membranes of CHO cells expressing either S1PR1 or S1PR3; EC50 values are the geometric mean of at least three independent
measurements in duplicate.
b
Selectivity against S1PR3 calculated as the ratio EC50 S1PR3/EC50 S1PR1.
and the pyrrolidine-methyl derivative 11e. Usually, the 2-
alkylaminomethyl substituted pyridine series exhibited a more
than 100-fold selectivity against the S1P3 receptor.
with the dimethylamino compound 17a, also this substitution
pattern furnished compounds with low nanomolar affinity for
S1PR1 and good selectivity against S1PR3. Increasing the size of the
alkyl group attached to the benzylic nitrogen (compounds 17a-f)
had little effect on the compound's potency on S1PR1 and produced
a slight trend towards higher affinity for S1PR3. As illustrated with
the two pairs 17d/17g, and 17h/17i, replacing the tertiary amine by
a secondary amine resulted in a significant potency loss on both
S1PR1 and S1PR3. Similarly, removing the methyl group attached as
R1 to the benzene head (17h vs 17a) led to a clear reduction in
potency. Adding a methyl group in the second ortho position to the
aminomethyl substituent (R3, compound 17j) reduced the com-
pound's affinity for S1PR1 as well. A methyl group in meta to the
aminomethyl group (R4) was also detrimental (compound 17k). As
illustrated with the pairs 17a/17l, 17b/17m, 17e/17n, and 17f/17o,
increasing the size of the alkyl group in R1 improved the com-
pound's affinity for S1PR1 and S1PR3 about 10-fold, while providing
a 100-fold selectivity against S1PR3.
In a last series we replaced the benzene ring by a thiophene. The
SAR of this class is illustrated with the compounds listed in Table 4.
In general the thiophene derivatives were often significantly more
potent on S1PR1 when compared to the corresponding analogs of
the 3- and 4-aminomethyl benzene series. For instance, thiophenes
20a, 20b, 20c, 20d, and 20e, are more than 150 times more potent
on S1PR1 when compared to the corresponding 3-aminomethyl
benzenes 14m, 14n, 14o, 14p, and 14q, respectively. In compari-
son to the 4-aminomethyl benzenes 17a, 17b, 17c, and 17d the
thiophenes 20a, 20b, 20d, and 20e are about 10 times more potent.
As already observed in the two benzene series, the affinity for
S1PR1 did not change in going from the dimethylamino derivative
20a to the much bulkier isobutyl-methylamino analog 20e, and
there was no clear SAR for compounds 20a to 20e with respect to
potency on S1PR3. Interestingly, while an alcohol function attached
Next, we extended our studies to compounds wherein the pyr-
idine was replaced by a benzene ring to evaluate the influence of a
more lipophilic ring on the receptor affinity. As shown with the
examples listed in Table 2, the aminomethyl residue alone is suf-
ficient to keep a reasonable logP value. While the benzene deriv-
ative 14a was more potent on S1PR1 when compared to its pyridine
analog 11a, the benzene derivative 14e showed a similar affinity for
S1PR1 as its pyridine analog 11c. Increasing the size of the alkyl
chain attached to the amino group (compounds 14a-e) left the af-
finity for S1PR1 largely unchanged but clearly increased the com-
pound's potency on S1PR3. On the other hand, increasing the size of
the R3 residue resulted in potent dual S1PR1/3 agonists (14f-h) e an
observation we already made in a related series [59].
Replacing the tertiary amine by a secondary amine (14i)
increased the selectivity against the S1P3 receptor (140-fold) but at
the cost of a 7-fold decrease in potency on the S1P1 receptor.
Compounds missing an additional alkyl residue at the phenyl ring
(compare 14j to 14a, 14k to 14c and 14l to 14e) were clearly less
potent on S1PR1 and showed no measurable affinity for S1PR3.
Moving the methyl substituent from the meta (R3) to the ortho
position (R2) with respect to the aminomethyl substituent led to a
significant loss in affinity for the S1PR1 but affected the potency on
the S1PR3 only moderately (compare e.g. 14n vs 14b, 14o vs 14c, 14p
vs 14d, etc.). A similar effect was observed when the R3 methyl
group was moved to the para (R4) position. Compound 14s was
more than 200 times less potent on S1PR1 when compared to its
regio-isomer 14e.
Table 3 compiles some representatives of the 4-aminomethyl
benzene series. In general, the SAR of the 4-aminomethyl series is
similar to the one of the 3-aminomethyl derivatives. As illustrated

228
C. Lescop et al. / European Journal of Medicinal Chemistry 116 (2016) 222e238
Table 2
SAR of the 3-aminomethyl benzene series.
a
a
Compd
R1
R2
H
R3
R4
H
clogP
2.23
EC50 S1PR1 [nM]
8.0
EC50 S1PR3 [nM]
Selectivityb
250
14a
Me
2000
1250
380
425
100
8.0
14b
14c
14d
14e
14f
14g
14h
14i
H
H
H
H
H
H
H
H
Me
Me
Me
Me
Et
H
H
H
H
H
H
H
H
2.63
3.09
3.54
3.31
3.72
4.18
4.15
3.04
3.6
4.1
2.6
2.1
1.1
2.8
1.1
15
347
93
163
48
7
n-Pr
i-Pr
Me
3.5
<2
<2
140
1.4
2100
14j
14k
14l
H
H
H
H
H
H
H
H
H
1.62
2.47
2.69
190
305
230
>10000
>10000
>10000
>53
>33
>43
14m
14n
14o
14p
14q
14r
Me
Me
Me
Me
Me
Me
H
H
H
H
H
H
H
H
H
2.22
2.63
3.09
3.54
3.31
3.01
3.31
385
285
305
310
97
>10000
9000
>26
32
H
H
4300
14
H
2900
9
H
1200
12
H
2350
490
>10000
>10000
>4
>20
14s
Me
a
EC50 values measured in a GTPgS assay using membranes of CHO cells expressing either S1PR1 or S1PR3; EC50 values are the geometric mean of at least three independent
measurements in duplicate.
b
Selectivity against S1PR3 calculated as the ratio EC50 S1PR3/EC50 S1PR1.
to the alkylamino moiety was tolerated by S1PR1 (20f), an addi-
tional dimethylamino group as in compound 20g led to a significant
potency loss. As in the benzene series, secondary amines showed a
reduced activity when compared to the corresponding tertiary
amine analogs (e.g. 20h vs 20a, 20i vs 20e). Removing the methyl
group in the 4 position (R2) of the thiophene reduced the com-
pound's affinity for both receptors (20j-n). Nevertheless, the thio-
phenes 20j and 20m, for instance, were still significantly more
potent than their corresponding 4-aminomethyl benzenes 17i and
17h, respectively. Elongation of R2 to an ethyl group improved the

C. Lescop et al. / European Journal of Medicinal Chemistry 116 (2016) 222e238
229
compound's potency, in particular on S1PR3 and the two ethyl de-
rivatives 20o and 20p were the more potent dual S1PR1/3 agonists
of this series. Compound 20o was also less selective for S1PR1 when
compared to its 4-aminomethyl benzene analog 17l. Finally,
attaching a methyl group to position 3 (R3) of thiophenes 20a, 20c,
and 20e gave analogs 20q, 20r, and 20s, respectively, which were all
less potent on S1PR1 and S1PR3. The affinity loss appears to be more
pronounced for compounds bearing larger alkylaminomethyl
groups (e.g. 20s).
Overall, the SAR analysis indicated that all four series behave
very similar in terms of selectivity against the S1P3 receptor. On the
other hand the 5-aminomethyl thiophene series appeared superior
with respect to the affinity to the S1P1 receptor. We observed in the
2-aminomethyl pyridine series that increasing the lipophilicity of
the compounds gave a gain in potency (compare 11a with 11c and
11d in Table 1) whereas in the other three series this gain was
minimal (compare within subgroup 14a-e (Table 2), or 17a-d (Ta-
ble 3), or 20a-e (Table 4)).
10 mg/kg to male Wistar rats. Blood lymphocyte count (LC) was
measured shortly before and 3, 6, and 24 h after compound
administration. Later sampling time points were added for com-
pounds 14e and 20e to confirm complete recovery of the LC.
Because of significant inter-individual variability and the circadian
rhythm of the absolute number of circulating lymphocytes, a LC
change of 15% was considered to be within baseline. On the other
hand, a LC reduction of ꢁ60% was interpreted as the maximal effect
that is achieved under the experimental conditions chosen [47].
The duration of action (DA) was defined as the latest time point at
which maximal LC reduction was observed.
We first looked at the plasma exposure of compounds 11c, 14e,
20a, and 20e to understand how in vitro clearance translated into
in vivo clearance. We corrected the apparent intrinsic in vitro
clearance of the four compounds for fumic and scaled to total body
weight (CLint. scaled, Table 6). The two thiophenes 20a and 20e show
similarly high scaled up CLint values of 14600 and 25200 mL/min/
kg, respectively. The lower oral plasma clearance of 20e compared
to 20a (19 vs 275 mL/min/kg respectively) can be attributed to the
significantly higher plasma protein binding of 20e (Table 5) [72,75].
The high extent of plasma protein binding therefore protects 20e
from in vivo metabolism. Similarly, despite showing comparable
values for CLint. scaled, the pyridine 11c and the benzene 14e clearly
show different oral plasma clearance (80 vs 8 mL/min/kg respec-
tively). This can again be attributed to the significantly higher
plasma protein binding of compound 14e, which protects the
compound from being metabolized in vivo.
2.3. Pharmacokinetics and pharmacodynamics
The metabolic stability (Table 5) of a few potent representatives
from each subseries was studied using rat liver microsomes (RLM).
There are clear differences in the apparent in vitro intrinsic clear-
ance (CLint, app.) not only between, but also within, the subseries.
The pyridines appear to be less stable than the corresponding
benzene and thiophene analogs. This is illustrated with the isobutyl
derivative 11c, which shows a 3e10 times higher CLint,
as
With respect to efficacy, all compounds tested showed a rapid
onset of action and the largest LC reduction was usually observed at
3 h after compound administration. LC reduction followed largely
plasma exposure and usually returned to baseline 24 h after com-
pound administration. Specifically, agonist 11c produced maximal
LC reduction (ꢄ80%) 3 h post dose and was of short duration only.
Plasma concentrations of 11c determined in the LC experiment
reached a maximum of 385 ng/mL at 3 h after compound admin-
istration and were below 2 ng/mL at 24 h. The short duration of
action reflected therefore the rapid decline in plasma exposure
(oral plasma clearance CL/F ¼ 80 mL/min/kg). When compared to
pyridine 11c, the 3-aminomethyl benzene analog 14e, displayed a
more sustained LC reduction for 24 h in line with its lower oral
plasma clearance of 8 mL/min/kg. Interestingly, despite high
exposure of 14e at 3 and 6 h no maximal reduction of the circu-
lating lymphocytes was observed. The reason for the submaximal
app.
compared to its benzene (14e and 17d) and thiophene (20e) ana-
logs. On the other hand, the size of the alkyl chain attached to the
amino group influences the CLint, app. as well. As illustrated with the
four benzene compounds 14a, 14b, 14d and 14e the metabolic
stability appears to improve with increasing size of the alkyl chain.
While the dimethylamine derivative 14a showed a high CLint, app. of
>1250
appeared more stable with an apparent in vitro intrinsic clearance
of 20 L/min/mg. A similarly marked difference was observed be-
mL/min/mg, the corresponding isobutylmethyl amine 14e
m
tween the dimethylamines 17l and 20a, and their corresponding
isobutylmethylamine analogs 17d and 20e, respectively. It is well
established that reliance on apparent in vitro intrinsic clearance
(CLint, app.) values for assessment of metabolic stability may be
misleading due to nonspecific microsomal binding (fumic), in
particular for a series of lipophilic basic compounds like ours [72].
Table 5 demonstrates that CLint, app. in RLM and fumic decrease in
parallel. This suggests that fumic is a major determinant of
“apparent” metabolic stability of those examples. Hence, the
efficacy remains unclear. For instance, neither in the GTPgS assay
nor in receptor internalization assays did compound 14e behave as
a partial agonist (data not shown). Another explanation could be
that the fup of 14e is much higher than 99.9% while, for instance, for
the fully efficacious compound 20e fup could be close to 99.9%. In
addition, differences in tissue distribution (e.g. plasma versus
lymph) may account for the distinct efficacies [66,76]. A rather
short DA (<6 h) was observed with analogs 14a,b,d and 17l. The two
thiophenes 20a and 20e showed a clear difference in duration of
action. While the number of circulating lymphocytes was back to
baseline levels at 24 h after administration of dimethylamine 20a,
the LC was still maximally reduced at 24 h with low-clearance
isobutylmethylamine 20e. Calculating the free compound concen-
tration available for receptor binding in plasma (free drug
concentration ¼ total plasma concentration x fup) suggested that
reaching concentrations of around EC50 is sufficient to observe
significant LC reduction.
apparent gain in metabolic stability when looking at CLint,
app.
values appears to be driven by the increase of microsomal binding
rather than rendering the compounds worse substrates of the
microsomal enzymes (compare 14a,b with 14d, and 17l with 17d).
The increase in microsomal binding is known to follow lipophilicity
as reflected in Table 5 (logD) [73,74]. Interestingly, when correcting
the CLint
,
for unbound fraction (CLint), compounds 11c and 14e
app.
clearly stand out from the remainder of the S1PR1 agonists exem-
plified suggesting that branched alkyl chains are less susceptible to
metabolism compared to the straight chains.
In vivo efficacy of S1PR1 agonists with an EC50 below 10 nM on
S1PR1 was evaluated by measuring their ability to reduce the
number of lymphocytes in blood. For a few compounds plasma
exposure was measured at the same time points as LC was deter-
mined. Table 6 summarizes the pharmacodynamic profiles of the 2-
alkylaminomethyl pyridine 11c, the 3-alkylaminomethyl benzenes
14a,b,d,e, the 4-alkylaminomethyl benzene 17l and the 5-
alkylaminomethyl thiophenes 20a and 20e, and the plasma con-
centrations of 11c, 14e, 20a and 20e after oral administration of
Of the compounds discussed above, thiophene 20e displayed
the best in vivo efficacy along with high affinity and a more than
200-fold selectivity for S1PR1 vs. S1PR3 (EC50 > 1000, 145, >1000,
and 753 nM for S1PR2, S1PR3, S1PR4 and S1PR5, respectively). We
therefore decided to characterize this compound in more detail.

230
C. Lescop et al. / European Journal of Medicinal Chemistry 116 (2016) 222e238
Table 3
SAR of the 4-aminomethyl benzene series.
a
a
Compd
R1
R2
R3
H
R4
H
clogP
2.23
EC50 S1PR1 [nM]
7.1
EC50 S1PR3 [nM]
Selectivityb
451
17a
Me
3200
910
350
570
560
17b
17c
17d
17e
Me
Me
Me
Me
H
H
H
H
H
H
H
H
2.63
3.54
3.31
3.04
14
5.3
13
10
65
66
44
56
17f
Me
H
H
3.01
50
3100
62
17g
17h
Me
H
H
H
H
H
3.04
1.88
270
160
>10000
>10000
>37
>62
17i
17j
H
H
H
H
1.62
2.57
2090
69
>10000
>4.8
>80
Me
Me
5500
17k
17l
H
H
H
H
H
Me
H
2.22
2.64
3.05
3.46
230
1.3
1.0
1.0
>10000
480
>43
369
100
210
Et
Et
Et
17m
17n
H
100
H
210
17o
Et
H
H
3.43
3.5
4300
130
a
EC50 values measured in a GTPgS assay using membranes of CHO cells expressing either S1PR1 or S1PR3; EC50 values are the geometric mean of at least three independent
measurements in duplicate.
b
Selectivity against S1PR3 calculated as the ratio EC50 S1PR3/EC50 S1PR1.
LC and plasma exposure were measured in a dose response
experiment. The results listed in Table 7 demonstrate that plasma
concentrations were dose proportional over the tested dose range
and the whole time course. In addition, the reduction of the LC was
clearly dose dependent. A single oral dose of 1 mg/kg reduced the
LC by 46%, while a dose of 3 mg/kg induced already maximal LC
reduction at 6 h. At 24 h, no effect on the number of circulating
lymphocytes was observed anymore at the 1 mg/kg dose, while the
3 mg/kg dose still resulted in a LC reduction of 25%. As mentioned
earlier, maximal LC reduction for at least 24 h was achieved with an
oral dose of 10 mg/kg and the LC returned to baseline levels 96 h
after dosing.
2.4. Brain penetration
In view of recent reports on CNS related autoimmune diseases,
which suggest beneficial, direct effects of S1PR1 agonists on cells of
the central nervous system [36,38,39], we studied the potential of
thiophene 20e to penetrate the brain in Wistar rats (Table 8). Based
on parameters used to assess the CNS penetration potential of drug
candidates (e.g. molecular weight, pKa, polar surface area, number
of hydrogen bond donors) [77e80], compound 20e was predicted
to have low permeability and thus a low probability to cross the
blood brain barrier. As evident from the data in Table 8, thiophene
20e displayed a surprisingly good brain penetration, albeit brain

C. Lescop et al. / European Journal of Medicinal Chemistry 116 (2016) 222e238
231
Table 4
SAR of the 5-aminomethyl thiophene series.
a
a
Compd
R1
R2
R3
H
clogP
2.33
EC50 S1PR1 [nM]
0.9
EC50 S1PR3 [nM]
Selectivityb
20a
Me
660
733
115
200
171
290
607
20b
20c
20d
20e
20f
Me
Me
Me
Me
Me
H
H
H
H
H
2.74
3.19
3.65
3.41
1.81
1.3
0.6
0.7
0.5
2.8
150
120
120
145
1700
20g
Me
H
2.03
155
>10000
>64
20h
20i
Me
Me
H
H
2.07
3.15
14
20
>10000
>714
1400
70
20j
20k
20l
H
H
H
H
H
H
1.72
2.12
2.49
189
293
203
>10000
>10000
>10000
>53
>34
>49
20m
20n
20o
20p
20q
20r
H
H
1.99
2.40
2.75
3.61
2.68
3.54
3.76
5.4
6.8
0.3
0.5
6.4
14
>10000
4600
45
>1852
676
150
40
H
H
Et
H
Et
H
20
Me
Me
Me
Me
Me
Me
4400
370
687
26
20s
55
2300
42
a
EC50 values measured in a GTPgS assay using membranes of CHO cells expressing either S1PR1 or S1PR3; EC50 values are the geometric mean of at least three independent
measurements in duplicate.
b
Selectivity against S1PR3 calculated as the ratio EC50 S1PR3/EC50 S1PR1.

232
C. Lescop et al. / European Journal of Medicinal Chemistry 116 (2016) 222e238
Table 5
LogD, fumic, fup, apparent and corrected intrinsic clearance in rat liver microsomes.
CLint, app. RLMa
[
mL/min/mg]
logDb
fumic
fup ratd
CLint. RLMe
[mL/min/mg]
c
Compd
11c
14a
14b
14d
14e
17d
17l
197
>1250
>1250
183
20
74
1011
1215
28
3.8
2.8
3.4
>4.1
>4.1
>4.1
3.8
0.18*
0.26
0.004
0.029
0.012
<0.001
<0.001
<0.001
0.005
1090
>4800
>5000
7300
740
74000
9200
0.25*
0.025*
0.027
0.001*
0.11*
0.15
20a
20e
3.2
>4.1
0.012
<0.001
8100
14000
0.002
* Recovery < 80%.
a
Apparent intrinsic clearance in the presence of rat liver microsomes at 1
Distribution coefficient between 1-octanol and phosphate buffer pH ¼ 7.
Unbound fraction in microsomal incubations.
Unbound fraction in rat plasma.
Apparent intrinsic clearance corrected for unbound fraction, CLint ¼ CLint,app/fumic
m
M compound starting concentration.
b
c
d
e
.
Table 6
Lymphocyte count, plasma concentrations, oral plasma clearance in male Wistar rats and scaled up in vitro CLint
.
e
Compd
LC reduction [%]a
Plasma concentration [ng/mL]b
3 h 6 h
385 176
CL/Fd
CLint. scaled
3 h
6 h
24 h
24 h
72 h
n.d.
96 h
[mL/min/kg]
80
11c
14a
14b
14d
14e
17l
ꢄ80
ꢄ44
ꢄ57
ꢄ60
ꢄ52
ꢄ56
ꢄ64
ꢄ69
ꢄ75
ꢄ37
ꢄ58
ꢄ57
ꢄ53
ꢄ61
ꢄ56
ꢄ77
þ1
þ21
þ24
ꢄ6
<2
n.d.
1970
ꢄ44
þ27
þ5
1377
1121
342
3.7
n.d.
0.5
8
1330
20a
20e
106
722
31
541
<4
92
275
19
14600
25200
ꢄ70c
n.d.
n.d. ¼ not determined.
a
Lymphocyte count reduction at indicated time point post compound administration as compared to vehicle treated group.
After oral administration of 10 mg/kg of aminomethyl aryl derivatives (n ¼ 6).
þ4% at 96 h.
b
c
d
e
Oral plasma clearance as estimated from the plasma concentration data.
In vitro clearance scaled up to full body weight according to CLint ¼ CLint; appꢅSF, wherein the scaling factor (SF) is the product of 45 mg microsomal protein/g liver and 40 g
liver/kg body weight ¼ 1.8 including the change in units from
m
L/min/mgfu(CmLicint, app.) to mL/min/kg (CLint. scaled) [72].
exposure is clearly delayed as compared with plasma exposure. At
2 h after compound administration total brain concentration
reached 522 ng/g, while total plasma concentration was somewhat
higher with 783 ng/mL. Interestingly, 6 h after dosing brain con-
centration further increased to 1028 ng/g and exceeded that in
plasma by a factor of 1.7. While the concentration in brain was still
high at 24 h, the plasma concentration had dropped significantly.
The observed shift between total plasma and brain concentration
supports a rather slow passage of compound 20e through the blood
brain barrier. In a multiple dose experiment using 10 mg/kg, the
brain concentration is expected to be >1000 ng/mL at steady state.
Therefore, compound 20e is potentially an interesting molecule to
treat immune-mediated diseases, where activity in the brain is
deemed beneficial or even required.
>10000 nM). This decomposition is more pronounced under UV
conditions as indicated by LCMS analysis of aliquots exposed to the
assay conditions (data not shown). The mechanism of the pre-
sumed air-oxidation is thought to involve a hydrogen abstraction
by singlet oxygen followed by an electron transfer that generates
the hydrolyzable iminium [81,82]. As aldehyde 19b is clearly
phototoxic (PIF ¼ 31), it is not possible to accurately measure the
PIF value of 20e, but we can assume that the PIF value of 20e is
below 6. Close inspection of the chemical instability of other 5-
aminomethyl thiophenes showed that they also partially decom-
posed but to a lesser extent (<1%).
Thus, we have reached our goal to find potent, selective and
in vivo efficacious aminomethyl aryl S1PR1 agonists that are devoid
of phototoxic potential. However, as observed for the 5-
aminomethyl thiophene 20e, we now face chemical instability via
air-oxidation to a phototoxic aldehyde which represents a strong
obstacle to further develop this compound class.
2.5. In vitro phototoxicity measurement
Representatives of the different amino-methyl aryl series were
measured for their phototoxicity potential in the in vitro mouse 3T3
fibroblast (NRU) phototoxicity assay [60,61]. The obtained PIF
values are summarized in Table 9. In contrast to aminopyridine 1
none of new compounds showed any sign of phototoxicity
(PIF < 2.5). The only exception was the 5-aminomethyl thiophene
20e for which the measured PIF value was 6. Further investigation
showed that 20e in particular displays some chemical instability
and partially decomposes (ꢀ6%) to its corresponding aldehyde 19b
(Scheme 4 with R2 ¼ Me and R3 ¼ H, EC50 S1PR1 ¼ 26 nM, S1PR3
3. Conclusions
With the aim to abolish the phototoxic potential of amino-
pyridine based S1PR1 agonists such as 1, we discovered a new class
of potent, selective and in vivo active aminomethyl substituted
pyridine derivatives. Low nanomolar affinity of compound 11c
illustrated that the S1P1 receptor can accommodate the basic
aminomethyl moiety. However, these aminomethyl pyridine based
S1PR1 agonists suffered from metabolic instability which translated

C. Lescop et al. / European Journal of Medicinal Chemistry 116 (2016) 222e238
233
Table 7
Table 9
Lymphocyte count and plasma concentrations after p.o administration of 20e to
Photo-irritancy factor (PIF) values of some representatives of the four alkyl-ami-
nomethyl substituted aryl series.
male Wistar rats.a
Dose [mg/kg] LC reduction [%]
3 h 6 h 24 h 96 h 3 h
þ4 67
Plasma concentration [ng/mL]
Compd
PIF valuea
Comment
6 h
49
24 h 72 h 96 h
5.2 BLQ
1
5
>200
1
phototoxic
clean
clean
clean
clean
clean
clean
clean
1
3
ꢄ33 ꢄ46
11a
11c
14e
17a
17i
20a
20c
20e
19b
1
1
1
1
1
1
1
ꢄ59 ꢄ70 ꢄ25
ꢄ69 ꢄ76 ꢄ70
210 170 25
722 540 92
BLQ
n.d.
10
þ4
0.5
a
n ¼ 6; n.d.: not determined; BLQ: below limit of quantification.
clean
into low exposure and a short duration of action in our in vivo
model. Further structural modifications replacing the pyridine ring
by a benzene or a thiophene ring led to new series of potent S1PR1
agonists with higher in vivo exposure and in vivo efficacy. The 5-
isobutylmethyl aminomethyl thiophene 20e, a representative of
the most interesting series, displayed low nanomolar affinity for
S1PR1 and a more than 200-fold selectivity vs. the S1PR3. In vivo 20e
showed a dose dependent reduction of the number of circulating
lymphocytes. At a dose of 10 mg/kg, maximal LC reduction was
observed for 24 h and the effect was fully reversible within 96 h.
Interestingly, the basic S1PR1 agonist 20e penetrated well into
brain, making 20e an interesting candidate for immune-mediated
diseases for which activity in the brain is beneficial or even
required. In addition, all representatives showed a PIF value far
below the one measured for the original aminopyridine series.
However, as the alkyl-aminomethyl thiophenes suffer from slow
oxidation in the air, we decided to explore other chemical modifi-
cations to abolish the phototoxic potential observed in the
aminopyridine-based S1PR1 agonist series. The results of these
investigations will be discussed in a follow-up publication [83].
ꢀ6
partial oxidation into 19b
phototoxic
31
a
Values measured in the in vitro mouse 3T3 fibroblast neutral red uptake (NRU)
phototoxicity assay (see Supplementary Material for details); mean of n ¼ 2.
spectroscopy. LC-HRMS: Analytical pump ¼ Waters Acquity Binary,
Solvent Manager, MS: SYNAPT G2 MS, source temperature: 150 ꢂC,
desolvatation temperature: 400 ꢂC, desolvatation gas flow: 400 L/
hr; cone gas flow: 10 L/hr, extraction cone: 4 RF; lens: 0.1 V; sam-
pling cone: 30; capillary:1.5 kV; high resolution mode; gain: 1.0, MS
function: 0.2 s per scan, 120e1000 amu in full scan, centroid mode.
Lock spray: Leucine enkephalin 2 ng/mL (556.2771 Da) scan time
0.2 s with interval of 10 s and average of 5 scans; DAD: Acquity UPLC
PDA Detector. Column: Acquity UPLC BEH C18 1.7
mm 2.1 ꢃ 50 mm
from Waters, thermostated in the Acquity UPLC Column Manager at
60 ꢂC. Eluents: water þ0.05% formic acid; B: acetonitrile þ0.05%
formic acid. Gradient: 2e98% B over 3.0 min. Flow: 0.6 mL/min.
Detection: UV 214 nm and MS, tR is given in min. NMR spectros-
copy: Bruker Avance II, 400 MHz UltraShield™, 400 MHz (1H),
100 MHz (13C); or Bruker Avance III HD, Ascend 500 MHz (1H),
125 MHz (13C) magnet equiped with DCH cryoprobe, chemical
shifts are reported in parts per million (ppm) relative to tetrame-
4. Experimental section
4.1. Chemistry
thylsilane (TMS), and multiplicities are given as
s (singlet),
d (doublet), t (triplet), q (quartet), quint (quintuplet), h (hextet),
hept (heptuplet) or m (multiplet), br ¼ broad, coupling constants
are given in Hz. Several compounds have been prepared on a
15e50 mmol scale. For those compounds 1H NMR spectra were
acquired using non-deuterated 10 mM DMSO stock solution sub-
mitted for biological testing [84]. The solvent and water signals
were suppressed by irradiation at 2.54 and 3.54 ppm, respectively.
As a consequence, signal integrals close to those frequencies are not
always accurate or visible. Compounds were purified by either flash
column chromatography on silica gel 60 (Fluka SigmaeAldrich,
All reagents and solvents were used as purchased from com-
mercial sources (SigmaeAldrich Switzerland, Lancaster Synthesis
GmbH, Germany, Acros Organics USA). Moisture sensitive reactions
were carried out under an argon atmosphere. Progress of the re-
actions was followed either by thin-layer chromatography (TLC)
analysis (Merck, 0.2 mm silica gel 60 F254 on glass plates) or by LC-
MS. LC-MS parameters: Finnigan MSQ™ plus or MSQ™ surveyor
(Dionex, Switzerland), with HP 1100 Binary Pump and DAD (Agi-
lent, Switzerland), column: Zorbax SB-AQ,
5 mm, 120 Å,
4.6 ꢃ 50 mm (Agilent), gradient: 5e95% acetonitrile in water con-
taining 0.04% of trifluoroacetic acid, within 1 min, flow: 4.5 mL/
min; tR is given in min. LC-MS marked with * refers to LC run on
Switzerland), or by prep. HPLC (Waters XBridge™ Prep C18, 5
m
m
m,
m,
OBD, 19 ꢃ 50 mm, or Waters X-terraTM RP18, 19 ꢃ 50 mm, 5
gradient of acetonitrile in water containing 0.4% of formic acid, flow
75 mL/min), or by MPLC (Labomatic MD-80-100 pump, Linear
UVIS-201 detector, column: 350 ꢃ 18 mm, Labogel-RP-18-5s-100,
gradient: 10% methanol in water to 100% methanol).
Zorbax SB-AQ, 1.8
LC run on Waters X-Bridge C18, 5
conditions, i.e. eluting with a gradient of MeCN in water containing
13 mM of ammonium hydroxide.
m
m, 4.6 ꢃ 20 mm. LC-MS marked with ** refers to
m
m, 4.6 ꢃ 50 mm under basic
According to the LC-MS analyses, final compounds showed a
purity >95% (UV at 230 nm and 214 nm) except for compounds 14a,
14k, 17i, 17k, 20k and 20p with purities of 90e93%. Purity and
identity was further confirmed by LC-HRMS and NMR
4.1.1. 2-(Hydroxymethyl)-6-methylisonicotinic acid methyl ester (8)
A suspension of 2-methylpyridine-4-carboxylic acid 7 (5.0 g,
36.5 mmol) in methanol (500 mL) was treated with concentrated
Table 8
Total brain and plasma concentrations of 20e after single oral dose of 10 mg/kg in Wistar rats.
Time (h)
Total plasma concentrationa [ng/mL]
Total brain concentrationa [ng/g]
522 212
Brain/plasma ratio
2
6
24
783 149
622 86
66.5 15.6
0.64 0.15
1.69 0.24
11.49 0.75
1028
6
776 230
a
Values are the mean of n ¼ 2 animals.

234
C. Lescop et al. / European Journal of Medicinal Chemistry 116 (2016) 222e238
sulfuric acid (2.5 mL). The white suspension is refluxed for 24 h to
give a clear solution of the corresponding ester. A solution of
ammomium persulfate (16.6 g, 72.9 mmol) in water (40 mL) was
then added and the reaction mixture was stirred at reflux for 4 h.
Another 20 mL of ammomium persulfate aq. solution (8.3 g,
36.5 mmol) were added. After another 6 h at reflux, the reaction
mixture was concentrated and partionized between EA (300 mL)
and NaHCO3 (6 ꢃ 100 mL). The organic phase was dried over
MgSO4, filtered and evaporated to give a residue which was purified
by prep. HPLC (XBridge Prep C18) to afford 2-hydroxymethyl-6-
methyl-isonicotinic acid methyl ester as a white crystalline solid
(4.07 g, 62%); LC-MS**: tR ¼ 0.61 min, [Mþ1]þ ¼ 182.25; 1H NMR
J ¼ 7.5 Hz, 2 H), 2.60 (s, 3 H), 2.35 (s, 3 H), 2.23 (s, 3 H), 2.16 (d,
J ¼ 7.3 Hz, 2 H), 1.89e1.78 (m, 1 H), 1.22 (t, J ¼ 7.5 Hz, 3 H), 0.91 (d,
J ¼ 6.5 Hz, 6 H); 13C NMR (D6-DMSO):
d 174.5, 172.4, 168.7, 161.7,
159.5, 158.4, 138.2, 132.4, 131.4, 128.2, 126.4, 121.8, 119,1, 117.2, 75.7,
69.1, 66.0, 63.9, 61.9, 43.1, 41.9, 26.1, 24.4, 22.6, 21.1, 16.6, 15.2.
4.1.4. 3-Formyl-5-methyl-benzoic acid (12a) [68].
a) A solution of 2,5-dimethyl benzoic acid (42.0 g, 282 mmol),
2,20-azo-bis(2-methylpropionitrile) (4.73 g, 28.2 mmol) and N-
bromo-succinimide (52.7 g, 296 mmol) in DCM (1.1 L) was refluxed
for 18 h. The mixture was extracted with 1 N aq. NaOH and water.
The combined aqueous extracts were acidified by adding 1 N aq.
HCl and extracted with twice EA. The combined org. extracts were
washed with water, dried over Na2SO4, filtered and concentrated.
The crude product was crystallized from hot EtOH (25 mL) by
adding cold water (50 mL). The mixture was cooled to rt, the
crystallized material was collected and dried to give 3-
bromomethyl-5-methyl-benzoic acid (32.0 g) as a white solid; LC-
MS*: tR ¼ 0.95 min, [Mþ1]þ ¼ not detectable.
(D6-DMSO):
d
7.74 (s, 1 H), 7.57 (s, 1 H), 5.54 (t, J ¼ 5.8 Hz, 1 H), 4.59
(d, J ¼ 5.8 Hz, 2 H), 3.90 (s, 3 H), 2.53 (s, 3 H).
4.1.2. 2-((Isobutyl(methyl)amino)methyl)-6-methylisonicotinic acid
(9c)
a) To a solution of 2-(hydroxymethyl)-6-methylisonicotinic acid
methyl ester (2.83 g, 15.6 mmol) and DIPEA (8.0 mL, 46.9 mmol) in
DCM (250 mL) was added methanesulfonyl chloride (1.46 mL,
18.7 mmol). The reaction mixture was stirred at rt for 1 h and was
then washed with 1N Na2CO3 (50 mL), followed by 1N HCl (50 mL),
water (25 mL) and brine (25 mL). The organic phase was dried over
MgSO4, filtered and evaporated to give the crude 2-methyl-6-
(((methylsulfonyl)oxy)methyl)isonicotinic acid methyl ester (4.57 g
crude containing about 15% of methyl 2-(chloromethyl)-6-
methylisonicotinate as side product, 96%) which was used
without purification. LC-MS**: tR ¼ 0.73 min, [Mþ1]þ ¼ 259.84.
b) To a solution of 2-methyl-6-(((methylsulfonyl)oxy)methyl)
isonicotinic acid methyl ester (557 mg, 2.15 mmol) in acetonitrile
(3 mL) was added a N-methylisobutylamine (562 mg, 6.44 mmol).
The reaction mixture was stirred at 45 ꢂC for 3 h. The reaction
mixture was concentrated. Crude methyl 2-((isobutyl(methyl)
amino)-methyl)-6-methylisonicotinate (482 mg) was then dis-
solved in 10 mL MeOH/THF (4:1), treated with 2N LiOH (3.0 mL) and
stirred for 1 h to hydrolyze the ester to the corresponding acid. The
reaction mixture was then concentrated to afford the title com-
pound 9c as a beige wax (490 mg crude, 96%); LC-MS **:
b) To a solution of 3-bromomethyl-5-methyl-benzoic acid
(3.90 g, 17 mmol) in water (30 mL) and MeCN (30 mL), Cu(NO3)2
hemipentahydrate (12.77 g, 68 mmol) followed by water (50 mL)
was added. The turquoise mixture was refluxed for 2 h before it was
concentrated to about half of the original volume. The dark green
solution was extracted three times with EA (150 mL). The org. ex-
tracts were washed twice with water (2 ꢃ 50 mL), combined, dried
over Na2SO4, filtered and concentrated. The white residue was
separated by prep. HPLC (XBridge Prep C18) to give 3-
hydroxymethyl-5-methyl-benzoic acid (897 mg) as a white solid,
LC-MS: tR ¼ 0.64 min ([Mþ1]þ ¼ not detected), along with the title
compound (160 mg) as a white solid. To a solution of the above 3-
hydroxymethyl-5-methyl-benzoic acid (947 mg, 5.70 mmol) in
MeCN (40 mL), MnO2 (1.49 g, 17.1 mmol) was added and the
resulting mixture was stirred at 80 ꢂC for 16 h before another
portion of MnO2 (1.21 g, 13.9 mmol) and acetonitrile (40 mL) were
added and stirring was continued at 80 ꢂC for 4 h. The mixture was
filtered over a glass fibre filter, the colourless filtrate was concen-
trated and purified by prep. HPLC (as above) to give the title com-
pound 12a (504 mg) as a white solid; LC-MS: tR ¼ 0.64 min,
tR ¼ 0.50 min, [Mþ1]þ ¼ 237.20. 1H NMR (CDCl3):
d 7.55 (s,1 H), 7.34
(s, 1 H), 3.40 (s, 2 H), 2.19 (s, 3 H), 2.08e2.01 (m, 2 H), 1.98 (s, 3H),
1.68e1.56 (m, 1 H), 0.69 (d, J ¼ 6.4 Hz, 6 H).
[Mþ1]þ ¼ not detectable; 1H NMR (D6-DMSO):
d 13.29 (s, 1 H),
10.06 (s, 1 H), 8.26 (s, 1 H), 8.07 (s, 1 H), 7.96 (s, 1 H), 2.47 (s, 3 H).
4.1.3. (S)-N-(3-(2-ethyl-4-(5-(2-((isobutyl(methyl)amino)methyl)-
6-methylpyridin-4-yl)-1,2,4-oxadiazol-3-yl)-6-methylphenoxy)-2-
hydroxypropyl)-2-hydroxyacetamide (11c)
4.1.5. (S)-N-(3-(2-ethyl-4-(5-(3-((isobutyl(methyl)amino)methyl)-
5-methylphenyl)-1,2,4-oxadiazol-3-yl)-6-methylphenoxy)-2-
hydroxypropyl)-2-hydroxyacetamide (14e)
To
a
solution of 2-((isobutyl(methyl)amino)methyl)-6-
a) A solution of 3-formyl-5-methyl-benzoic acid 12a (340 mg,
2.1 mmol), HOBt (336 mg, 2.5 mmol) and EDC (437 mg, 2.3 mmol)
in DMF (40 mL) was stirred at rt for 30 min before adding N-((S)-3-
[2-ethyl-4-(N-hydroxycarbamimidoyl)-6-methyl-phenoxy]-2-
hydroxy-propyl)-2-hydroxy-acetamide 10 [59] (674 mg, 2.1 mmol).
The mixture was stirred at rt for 18 h and was then diluted with EA
(50 mL), washed with sat. NaHCO3 (25 mL), followed by brine
(25 mL). The organic phase was dried over MgSO4, filtered and
concentrated. The orange residue was dissolved in dioxane
(120 mL) and heated at 100 ꢂC for 2 h. The reaction mixture was
then evaporated and the crude compound was purified by CC
(eluent DCM/MeOH 9:1) to afford (S)-N-(3-(2-ethyl-4-(5-(3-
formyl-5-methylphenyl)-1,2,4-oxadiazol-3-yl)-6-methyl-phe-
noxy)-2-hydroxypropyl)-2-hydroxyacetamide 13a (407 mg, 43%);
LC-MS: tR ¼ 0.85 min, [Mþ1]þ ¼ 454.11; 1H NMR (D6-DMSO):
methylisonicotinic acid 9c (385 mg, 1.63 mmol) and (S)-N-(3-(2-
ethyl-4-(N-hydroxycarbamimidoyl)-6-methylphenoxy)-2-hydrox-
ypropyl)-2-hydroxyacetamide 10 [59] (562 mg, 1.73 mmol) in DMF
(3.0 mL), HOBt (264 mg, 1.96 mmol) and EDC (375 mg, 1.96 mmol)
were added. The reaction mixture was stirred at rt overnight and
was then diluted with EA (150 mL), washed with sat. NaHCO3
(25 mL) followed by brine (25 mL). The organic phase was dried
over MgSO4, filtered and evaporated to give an orange residue
(886 mg) which was dissolved in dioxane (120 mL) and heated at
100 ꢂC for 18 h. The reaction mixture was concentrated and the
residue was purified by prep. HPLC (Waters X-Bridge C18, gradient
of acetonitrile in water containing 0.5% NH4OH) to give 11c (415 mg,
48%) as a beige solid; LC-MS**: tR ¼ 0.97 min, [Mþ1]þ ¼ 526.12;
HRMS (ESIþ) m/z calculated for C28H39N5O5 [Mþ1]þ 526.3029,
found 526.3033; 1H NMR (D6-DMSO):
d
7.97 (s, 1 H), 7.85 (s, 1 H),
d 10.15 (s, 1 H), 8.50 (s, 1 H), 8.34 (s, 1 H), 8.06 (s, 1 H), 7.82 (s, 2 H),
7.79 (s, 2 H), 7.72 (t, J ¼ 5.7 Hz, 1 H), 5.57 (t, J ¼ 5.7 Hz, 1 H), 5.33 (d,
J ¼ 5.1 Hz, 1 H), 4.01e3.92 (m, 1 H), 3.84 (d, J ¼ 5.7 Hz, 2 H), 3.77 (dd,
J1 ¼ 9.5 Hz, J2 ¼ 4.6 Hz, 1 H), 3.72 (dd, J1 ¼ 9.5 Hz, J2 ¼ 6.1 Hz, 1 H),
3.67 (s, 2 H), 3.48e3.39 (m, 1 H), 3.30e3.20 (m, 1 H), 2.73 (q,
7.71 (t, J ¼ 5.0 Hz, 1 H), 5.56 (t, J ¼ 5.5 Hz, 1 H), 5.32 (d, J ¼ 4.6 Hz,
1 H), 4.01e3.93 (m, 1 H), 3.84 (d, J ¼ 4.7 Hz, 2 H), 3.81e3.71 (m, 2 H),
3.39e3.48 (m, 1 H), 3.30e3.19 (m, 1 H), 2.74 (q, J ¼ 7.3 Hz, 2 H), 2.55
(s, 3 H), 2.36 (s, 3 H), 1.23 (t, J ¼ 7.4 Hz, 3 H).

C. Lescop et al. / European Journal of Medicinal Chemistry 116 (2016) 222e238
235
b) N-methylisobutylamine (78 uL, 0.65 mmol) was added to a
suspension of (S)-N-(3-(2-ethyl-4-(5-(3-formylphenyl)-1,2,4-
oxadiazol-3-yl)-6-methylphenoxy)-2-hydroxypropyl)-2-
methyl-2-thiophencarboxylic acid according to the procedures
described in the literature [46,70]; LC-MS: tR
¼
0.70 min,
[Mþ1]þ ¼ not detectable, 1H NMR (D6-DMSO):
d 13.67 (s br, 1 H),
hydroxyacetamide 13a (103 mg, 0.24 mmol) in DCM (10 mL) and
NMP (2.0 mL). The reaction mixture was stirred at rt for 1 h and was
then cooled to 0 ꢂC and NaBH(OAc)3 (250 mg,1.18 mmol) was added
followed by acetic acid (0.4 mL). The reaction mixture was warmed
to rt and stirred for 18 h. The reaction was then quenched with
MeOH (3 mL) and concentrated to give an orange wax which was
purified by prep-HPLC (XBridge Prep C18, gradient of acetonitrile in
water containing 0.5% NH4OH) to afford the title compound 14e
(55.4 mg, 48%); LC-MS*: tR ¼ 0.58 min, [Mþ1]þ ¼ 525.38; HRMS
(ESIþ) m/z calculated for C29H40N4O5 [Mþ1]þ 525.3077, found
10.09 (s, 1 H), 7.67 (s, 1 H), 2.57 (s, 3 H).
4.1.9. (S)-N-(3-(2-ethyl-4-(5-(5-((isobutyl(methyl)amino)methyl)-
4-methylthiophen-2-yl)-1,2,4-oxadiazol-3-yl)-6-methylphenoxy)-
2-hydroxypropyl)-2-hydroxy-acetamide (20e)
The title compound was prepared in analogy to compound 14e
starting from 18b and using N-isobutylmethylamine for the
reductive amination (82 mg); LC-MS: tR
¼
0.77 min;
[Mþ1]þ ¼ 531.32; HRMS (ESIþ) m/z calculated for C27H38N4O5S
[Mþ1]þ 531.2681, found 531.2645; 1H NMR (D6-DMSO):
d 7.82 (s,
525.3080; 1H NMR (D6-DMSO):
d
7.94 (s, 1 H), 7.90 (s, 1 H), 7.79 (s,
1 H), 7.73 (s, 2 H), 7.69 (t, J ¼ 5.8 Hz,1 H), 5.55 (t, J ¼ 5.7 Hz,1 H), 5.30
(d, J ¼ 5.2 Hz, 1 H), 4.00e3.91 (m, 1 H), 3.84 (d, J ¼ 5.7 Hz, 2 H), 3.77
(dd, J1 ¼ 9.7 Hz, J2 ¼ 4.5 Hz, 1 H), 3.72 (dd, J1 ¼ 9.7 Hz, J2 ¼ 6.1 Hz,
1 H), 3.67 (s, 2 H), 3.48e3.39 (m, 1 H), 3.29e3.20 (m, 1 H), 2.72 (q,
J ¼ 7.5 Hz, 2 H), 2.34 (s, 3 H), 2.23 (s, 6 H), 2.21 (d, J ¼ 7.4 Hz, 2 H),
1.87e1.75 (m, 1 H), 1.22 (t, J ¼ 7.5 Hz, 3 H), 0.91 (d, J ¼ 6.6 Hz, 6 H).
2 H), 7.69 (t, J ¼ 5.3 Hz,1 H), 7.47 (s, 1 H), 5.55 (t, J ¼ 5.7 Hz,1 H), 5.31
(d, J ¼ 5.2 Hz, 1 H), 3.96e3.93 (m, 1 H), 3.84 (d, J ¼ 5.7 Hz, 2 H),
3.81e3.70 (m, 2 H), 3.54 (s, 2 H), 3.48e3.40 (m, 1 H), 3.29e3.21 (m,
1 H), 2.73 (q, J ¼ 7.8 Hz, 2 H), 2.44 (s, 3 H), 2.35 (s, 3 H), 2.15 (s, 3 H),
2.12 (d, J ¼ 7.3 Hz, 2 H), 1.88e1.77 (m, 1 H), 1.23 (t, J ¼ 7.5 Hz, 3 H),
0.89 (d, J ¼ 6.5 Hz, 6 H); 13C NMR (CDCl3):
d
176.0,172.6,168.6,157.6,
13C NMR (CD3OD):
d 174.1, 171.6, 168.3, 157.8, 146.4, 137.8, 135.6,
141.0, 138.9, 137.5, 134.1, 131.5, 128.4, 127.3, 126.7, 125.7, 124.0, 123.1,
74.0, 70.3, 66.2, 62.2, 42.7, 42.2, 26.2, 22.9, 21.3, 20.9, 16.5, 14.8.
134.5,131.8, 127.8, 126.1,122.3,122.2, 74.6, 69.1, 65.9, 61.2, 54.9, 41.8,
41.6, 26.2, 22.5, 19.8, 15.3, 14.0, 12.5. Despite purification by prep.
HPLC the product always contained a few % of aldehyde 19b ac-
cording to the LCMS and NMR analysis.
4.1.6. 3-Ethyl-4-formyl-benzoic acid (15e) [68].
a) 4-Formyl-3-hydroxy-benzoic acid (1.5 g, 9.0 mmol) and pyr-
idine (2.9 mL, 36.1 mmol) were dissolved in DCM (50 mL). The
solution was cooled to 0 ꢂC before adding triflic anhydride (3.1 mL,
19 mmol) and was then stirred for 1 h. The reaction mixture was
quenched with 1 mL NH4OH, evaporated and the crude compound
was purified by prep. HPLC (XBridge Prep C18) to give 4-formyl-3-
(((trifluoromethyl)sulfonyl)oxy)benzoic acid (413 mg, 15%). LC-
MS*: tR ¼ 0.52 min, [Mþ1]þ ¼ not detectable; 1H NMR (D8-THF):
In vitro 3T3 Neutral Red Uptake (NRU) Phototoxicity Test.
[60,61] The 3T3 NRU phototoxicity assay utilizes normal Balb/c 3T3
mouse fibroblasts to measure the concentration-dependent
reduction in neutral red uptake by the cells one day after treat-
ment with the chemical either in the presence or in the absence of a
non-cytotoxic dose of UVA radiation. Balb/c 3T3 cells (clone 31,
ECACC) were cultured overnight in duplicate 96-well microtiter
ml in Dulbecco's modified
Eagle's medium (DMEM) containing 10% new born calf serum
(NBCS, PAA B15-102), 4 mM L-glutamine, 100 U/ml penicillin and
plates at a density of 1 ꢃ 104 cells/100
d
9.03 (s, 1 H), 8.40 (s, 1 H), 6.40 (d, J ¼ 8.0 Hz, 1 H), 6.30 (d,
J ¼ 8.0 Hz, 1 H), 6.24 (s, 1 H).
b) To a solution of 4-formyl-3-(((trifluoromethyl)sulfonyl)oxy)
benzoic acid (200 mg, 0.67 mmol) in dioxane (5 mL) was added
Pd(dppf)Cl2 (4.5 mg, 0.01 eq.) under nitrogen atmosphere. Diethyl
zinc (15wt % in toluene, 0.9 mL,1.0 mmol) was then added dropwise
and the reaction mixture was stirred at 75 ꢂC for 1 h. After evap-
oration, the crude compound was purified by prep. HPLC (XBridge
Prep C18) to give the title compound (66 mg, 55%). LC-MS*:
tR ¼ 0.62 min, [Mþ1]þ ¼ not detectable; 1H NMR (D6-DMSO):
100 mg/mL streptomycin. The next day the cells were washed with
phosphate buffered saline containing CaCl2 and MgCl2 (PBSþþ). 3T3
fibroblasts were exposed to eight different concentrations of
chemical, ranging from 0.05 to 100 mM, with a pre-incubation time
of 1 h (at 37 ꢂC, 5% CO2). One of the duplicate plates was exposed to
4 J/cm2 UVA (1.7 mW/cm2) for 40 min while the other plate was
kept in the dark. By using the UV-sun simulator UVACUBE 400
equipped with a SOL500 lamp and an H1-Filter (H1 Filter and
€
d
13.36 (s br, 1 H), 10.34 (s, 1 H), 7.94 (s, 2 H), 7.93 (s, 1 H), 3.10 (q,
frame: Cat. Number 4730, Dr. Honle UV Technology) a UV-spectrum
J ¼ 7.5 Hz, 2 H), 1.21 (t, J ¼ 7.5 Hz, 3 H).
almost devoid of UVB is obtained. After UV exposure, PBSþþ was
replaced with culture medium and cell viability was assessed after
24 h. The NRU by cells was measured at 540 nm and the absorption
of wells exposed to the test chemical in the presence of UVA
exposure (þUVA) was compared to their NRU in the absence of UVA
exposure (-UVA). Dose-response curves were established for each
experiment. EC50 (þUVA) and EC50 (-UVA) values, i.e. the effective
concentration inhibiting cell viability by 50% of untreated controls
in the presence and absence of UVA exposure, were calculated by
using Phototox software version 2.0 [61]. The software was ob-
tained at http://www.oecd.org/document/55/0,3343,en_2649_
4.1.7. (S)-N-(3-(4-(5-(4-((dimethylamino)methyl)-3-ethylphenyl)-
1,2,4-oxadiazol-3-yl)-2-ethyl-6-methylphenoxy)-2-hydroxypropyl)-
2-hydroxyacetamide (17l)
The title compound was prepared in analogy to compound 14e
starting from 3-ethyl 4-formyl benzoic acid 15e and using 2N
dimethylamine in THF and NaBH(OAc)3 for the reductive amination
(48 mg); LC-MS*: tR ¼ 0.53 min, [Mþ1]þ ¼ 497.80; HRMS (ESIþ) m/
z calculated for C27H36N4O5 [Mþ1]þ 497.2764, found 497.2758; 1H
NMR (CDCl3):
d
8.06 (s, 1 H), 8.02 (dd, J1 ¼ 1.6 Hz, J2 ¼ 7.9 Hz, 1 H),
7.88 (s, 1 H), 7.86 (s, 1 H), 7.54 (d, J ¼ 8.0 Hz, 1 H), 7.08 (t, J ¼ 5.8 Hz,
1 H), 4.24e4.16 (m, 3 H), 3.89 (dd, J1 ¼ 4.7 Hz, J2 ¼ 9.6 Hz, 1 H), 3.84
(dd, J1 ¼ 6.2 Hz, J2 ¼ 9.5 Hz, 1 H), 3.81e3.75 (m, 1 H), 3.56e3.46 (m,
3 H), 2.85 (q, J ¼ 7.5 Hz, 2 H), 2.74 (q, J ¼ 7.5 Hz, 2 H), 2.38 (s, 3 H),
34377_2349687_1_1_1_1,00.html.
Chlorpromazine
(Sigma-
eAldrich C0982-5G) served as the positive control. A PIF was
calculated according to the formula PIF ¼ EC50 (-UVA)/EC50 (þUVA).
PIF values > 2.5 were considered to be predictive for phototoxic
potential.
2.31 (s, 6 H), 1.32 (t, J ¼ 7.5 Hz, 6 H); 13C NMR (CDCl3):
d 175.8, 172.8,
168.6, 157.0, 144.2, 141.8, 137.5, 131.5, 130.5, 128.4, 128.2, 126.7,
125.4, 123.1, 123.0, 74.0, 70.3, 62.2, 61.1, 45.6, 42.2, 25.3, 22.9, 16.5,
15.0, 14.8.
In vitro potency assessment using GTP
GTP S binding assays were performed in 96 well polypropylene
microtiter plates in a final volume of 200 L. Membrane prepara-
gS binding assays.
g
m
tions of CHO cells expressing recombinant human S1P1 or S1P3
receptors were used. Assay conditions were 20 mM Hepes, pH 7.4,
4.1.8. 5-Formyl-4-methyl-thiophene-2-carboxylic acid (18b)
The title compound is prepared from commercially available 3-
100 mM NaCl, 5 mM MgCl2, 0.1% fatty acid free BSA, 1 or 3 mM GDP

236
C. Lescop et al. / European Journal of Medicinal Chemistry 116 (2016) 222e238
(for S1P1 or S1P3, respectively), 2.5% DMSO and 50 pM 35S-GTP
Test compounds were dissolved, diluted and pre-incubated with
the membranes, in the absence of 35S-GTP
S, in 150 L assay buffer
at rt for 30 min. After addition of 50 S in assay buffer,
L of 35S-GTP
the reaction mixture was incubated for 1 h at rt. The assay was
terminated by filtration of the reaction mixture through a Multi-
screen GF/C plate, pre-wetted with ice-cold 50 mM Hepes pH 7.4,
100 mM NaCl, 5 mM MgCl2, 0.4% fatty acid free BSA, using a Cell
Harvester. The filterplates were then washed with ice-cold 10 mM
Na2HPO4/NaH2PO4 (70%/30%, w/w) containing 0.1% fatty acid free
gS.
Pharmacokinetics in the Rat. Blood samples were obtained
from the above in vivo efficacy experiments in vials containing
EDTA as anticoagulant and plasma was prepared by centrifugation
at 3000 g for 10 min at 4 ꢂC. Plasma samples were analyzed using
liquid chromatography coupled to mass spectrometry (LC-MS/MS)
after protein precipitation.
In vivo intrinsic clearances were derived from measured plasma
concentration-time data and from the well-stirred liver model [86]
suggesting that unbound oral clearance equates to intrinsic clear-
ance under the assumption that the compound is completely
absorbed.
For brain penetration in the rat at 2, 6 and 24 h after dosing,
male Wistar rats (n ¼ 2) were anaesthetised with 5% isoflurane and
sacrificed by opening the diaphragm. A blood sample was taken,
plasma was prepared and the brain was slowly perfused with 10 mL
0.9% NaCl solution in water through the carotid. The whole brain
was then removed and homogenized in an equal volume of ice-cold
0.1 M Na-phosphate buffer, pH 7.4, using a IKA-WERKE ultra-turrax
T25 tissue homogeniser for 10 s, and the brain homogenate was
snap frozen in liquid nitrogen. Drug concentrations were then
determined as described for plasma, using a calibration curve in
blanco brain homogenate.
g
m
m
g
BSA. Then the plates were dried, sealed, 25
added, and membrane-bound 35S-GTP
S was determined on the
TopCount. Specific 35S-GTP
S binding was determined by sub-
mL MicroScint20 was
g
g
tracting non-specific binding (the signal obtained in the absence of
agonist) from maximal binding (the signal obtained with
10 mM S1P). The EC50 of a test compound is the concentration of a
compound inducing 50% of specific binding. Data (EC50) are given
as geometric means. Our assay sensitivity allows the accurate
measurement of EC50 as low as 0.1 nM.
The In Vivo Efficacy of the target compounds was assessed by
measuring the circulating lymphocytes after oral administration of
1e10 mg/kg of a target compound to male Wistar rats. Male Wistar
rats (RccHan:WIST) were obtained from Harlan Laboratories
(Venray, the Netherlands). This study was conducted in accordance
with Swiss Animal Protection Laws and with local guidelines
(Basel-Landschaft Cantonal Veterinary Office). The animals were
housed in climate-controlled conditions with a 12 h-light/dark
cycle, and had free access to normal rat chow and drinking water.
Blood was collected under isoflurane anaesthezia by sublingual
vein puncture shortly before and 3, 6 and 24 h after drug admin-
istration. For several compounds later sampling time points were
added to confirm complete recovery of the LC. Full blood was
subjected to hematology using Beckman Coulter Ac.T 5diff CP
(Beckman Coulter International SA, Nyon, Switzerland). The effect
on lymphocyte count (%LC) was calculated for each animal as the
difference between LC at a given time point and the pre-dose value
(¼ 100%). All data are presented as mean of 6 animals. A lympho-
cyte count (LC) reduction of ꢁ - 60% was interpreted as the maximal
effect that is achieved under this experimental conditions. For
formulation, the compounds were dissolved in DMSO. This solution
was added to a stirred solution of succinylated gelatine (7.5%) in
water. The resulting milky suspension containing a final concen-
tration of 5% of DMSO was administered to the animals by gavage.
In vitro CLint determinations. Intrinsic metabolic clearance
(CLint) was determined by substrate depletion experiments, with a
default starting concentration of 1 uM in the presence of 0.5 mg/mL
rat liver microsomes (RLM) in 100 mM sodium phosphate buffer, at
pH 7.4. Incubations were initiated by the addition of an NADPH
regenerating system. All incubations were conducted by shaking
reaction mixtures under air, at 37 ꢂC. Aliquots (0.1 ml) were
removed at 0, 2.5, 5, 10 and 15 min and 0.1 mL of ice-cold methanol
was added. After protein precipitation, by centrifugation, the
remaining concentrations were analyzed by liquid chromatography
coupled to mass spectrometry (LC-MS/MS), CLint was calculated
from the concentration remaining versus time, fitted to a first order
decay constant versus time [85].
Notes
The authors declare no competing financial interest.
Acknowledgments
ꢀ
The authors gratefully acknowledge Lucy Baumann, Celine
ꢀ
Bortolamiol, Maxime Boucher, Roberto Bravo, Stephane Delahaye,
Alexandre Flock, Elvire Fournier, Hakim Hadana, Julie Hoerner,
Benedikt Hofstetter, Niklaus Kuratli, François Le Goff, Claire
€
Maciejasz, Matthias Merrettig, Michel Rauser, Manuela Schlapfer,
ꢀ
Virginie Sippel, Daniel Strasser, Gaby von Aesch, Rene Vogelsanger,
Daniel Wanner, Aude Weigel, Rolf Wuest and Nancy Yoganathan for
the excellent work and Martine Clozel for support.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2016.03.048.
References
[1] M. Maceyka, K.B. Harikumar, S. Milstien, S. Spiegel, Sphingosine-1-phosphate
signaling and its role in disease, Trends Cell Biol. 22 (2012) 50e60.
[2] T. Mutoh, R. Rivera, J. Chun, Insights into the pharmacological relevance of
lysophospholipid receptors, Br. J. Pharmacol. 165 (2012) 829e844.
[3] K. Takabe, S.W. Paugh, S. Milstien, S. Spiegel, “Inside-Out” signaling of
sphingosine-1-phosphate: therapeutic targets, Pharmacol. Rev. 60 (2008)
181e195.
[4] J. Rivera, R.L. Proia, A. Olivera, The alliance of sphingosine-1-phosphate and its
receptors in immunity, Nat. Rev. Immunol. 8 (2008) 753e763.
[5] C.A. Oskeritzian, S. Milstien, S. Spiegel, Sphingosine-1-phosphate in allergic
responses, asthma and anaphylaxis, Pharmacol.Ther. 115 (2007) 390e399.
[6] N.J. Pyne, S. Pyne, Sphingosine 1-phosphate and cancer, Nat. Rev. Cancer 10
(2010) 489e503.
[7] N.J. Pyne, G. Dubois, S. Pyne, Role of sphingosine 1-phosphate and lysophos-
phatidic acid in fibrosis, Biochim. Biophys. Acta 1831 (2012) 228e238.
[8] Y. Okamoto, F. Wang, K. Yoshioka, N. Takuwa, Y. Takuwa, Sphingosine-1-
phosphate-specific G protein-coupled receptors as novel therapeutic targets
for atherosclerosis, Pharmaceuticals 4 (2011) 117e137.
[9] M. Ishii, J.G. Egen, F. Klauschen, M. Meier-Schellersheim, Y. Saeki, J. Vacher,
R.L. Proia, R.N. Germain, Sphingosine-1-phosphate mobilizes osteoclast pre-
cursors and regulates bone homeostasis, Nature 458 (2009) 524e528.
[10] T. Baumruker, A. Billich, Sphingolipids in cell signalling: their function as
receptor ligands, second messengers, and raft constituents, Curr. Immunol.
Rev. 2 (2006) 101e118.
Free fraction in plasma microsomal incubations. The free
fraction in rat plasma and in rat liver microsomes were determined
by equilibrium analysis using a Pierce rapid equilibrium dialysis
(RED) device (Thermo Fisher Scientific, Lausanne, Switzerland) by
incubating on a shaker at 37 ꢂC for 6 h. The donor compartment was
either rat plasma (pH 7.2e7.6) or 1 mg/mL liver microsomes in
phosphate buffer (pH 7.4). Donor and receiver (containing phos-
phate buffer, pH 7.4) were analyzed by LC-MS/MS and the free
fraction and recovery were calculated.
[11] S.E. Alvarez, S. Milstien, S. Spiegel, Autocrine and paracrine roles of sphin-
gosine-1-phosphate, Trends Endocrinol. Metab. 18 (2007) 300e307.

C. Lescop et al. / European Journal of Medicinal Chemistry 116 (2016) 222e238
237
[12] S. Spiegel, S. Milstien, Sphingosine-1-phosphate: an enigmatic signalling lipid,
Nat. Rev. Mol. Cell. Biol. 4 (2003) 397e407.
Immune cell regulation and cardiovascular effects of sphingosine 1-phosphate
receptor agonists in rodents are mediated via distinct receptor subtypes, JPET
309 (2004) 758e768.
[13] H. Zhang, N.N. Desai, A. Olivera, T. Seki, G. Brooker, S. Spiegel, Sphingosine 1-
phosphate, a novel lipid, involved in cellular proliferation, J. Cell Biol. 114
(1991) 155e167.
[41] M.G. Sanna, J. Liao, E. Jo, C. Alfonso, M.-Y. Ahn, M.S. Peterson, B. Webb,
S. Lefebvre, J. Chun, N. Gray, H. Rosen, Sphingosine 1-phosphate (S1P) re-
ceptor subtypes S1P1 and S1P3, respectively, regulate lymphocyte recircula-
tion and heart rate, J. Biol. Chem. 279 (2004) 13839e13848.
[42] S. Salomone, E. Potts, S. Tyndall, P. Ip, J. Chun, V. Brinkmann, C. Waeber,
Analysis of sphingosine 1-phosphate receptors involved in constriction of
isolated cerebral arteries with receptor null mice and pharmacological tools,
Br. J. Pharmacol. 153 (2007) 1e8.
[43] A. Murakami, H. Takasugi, S. Ohnuma, Y. Koide, A. Sakurai, S. Takeda,
T. Hasegawa, J. Sasamori, T. Konno, K. Hayashi, Y. Watanabe, Y.S. Koji Mori,
A. Takahashi, N. Mochizuki, N. Takakura, Sphingosine 1-phosphate (S1P)
regulates vascular contraction via S1P3 receptor: investigation based on a
new S1P3 receptor antagonist, Mol. Pharmacol. 77 (2010) 704e713.
[44] R.M. Fryer, A. Muthukumarana, P.C. Harrison, S.N. Mazurek, R.R. Chen,
K.E. Harrington, R.M. Dinallo, J.C. Horan, L. Patnaude, L.K. Modis, G.A. Reinhart,
The clinically-tested S1P receptor agonists, FTY720 and BAF312, demonstrate
subtype-specific bradycardia (S1P1) and hypertension (S1P3) in rat, PLoS One
7 (2012) e52985.
€
[14] M. Schuchardt, M. Tolle, J. Prüfer, M. Giet, Pharmacological relevance and
potential of sphingosine 1-phosphate in the vascular system, Br. J. Pharmacol.
163 (2011) 1140e1162.
[15] S. Spiegel, S. Milstien, The outs and the ins of sphingosine-1-phosphate in
immunity, Nat. Rev. Immunol. 11 (2011) 403e415.
[16] H. Chi, Sphingosine-1-phosphate and immune regulation: trafficking and
beyond, Trends Pharmacol. Sci. 32 (2011) 16e24.
[17] H. Rosen, E.J. Goetzl, Sphingosine 1-phosphate and its Receptors: an autocrine
and paracrine network, Nat. Rev. Immunol. 5 (2005) 560e570.
[18] J.G. Cyster, Chemokines, sphingosine-1-phosphate and cell migration in sec-
ondary lymphoid organs, Annu. Rev. Immunol. 23 (2005) 127e159.
[19] T. Hla, V. Brinkmann, Sphingosine 1-phosphate (S1P) Physiology and the ef-
fects of S1P receptor modulation, Neurology 76 (2011) S3eS8.
[20] T. Hla, Physiological and pathological actions of sphingosine 1-phosphate,
Semin. Cell. Dev. Biol. 15 (2004) 513e520.
[21] J. Chun, E.J. Goetzl, T. Hla, Y. Garashi, K.R. Lynch, W. Moolenaar, S. Pyne,
G.O. Tigyi, International union of pharmacology. XXXIV. Lysophospholipid
receptor nomenclature, Pharmacol. Rev. 54 (2002) 265e269.
[45] L.M. Healy, J.P. Antel, Sphingosine-1-phosphate receptors in the central ner-
vous and immune systems, Curr. Drug Targets 17 (2016) 1e10.
[22] J.G. Cyster, S.R. Schwab, Sphingosine-1-phosphate and lymphocyte egress
from lymphoid organs, Annu. Rev. Immunol. 30 (2012) 69e94.
[23] C.S. Garris, V.A. Blaho, T. Hla, H. M., T. Haaf, Sphingosine-1-phosphate receptor
[46] S. Crosignani, A. Bombrun, D. Covini, M. Maio, D. Marin, A. Quattropani,
D. Swinnen, D. Simpson, W. Sauer, B. Françon, T. Martin, Y. Cambet, A. Nichols,
I. Martinou, F. Burgat-Charvillon, D. Rivron, C. Donini, O. Schott, V. Eligert,
L. Novo-Perez, P.-A. Vitte, J.-F. Arrighi, Discovery of a novel series of potent
S1P1 agonists, Bioorg. Med. Chem. Lett. 20 (2010) 1516e1519.
1
signalling in
T cells: trafficking and beyond, Immunology 142 (2014)
347e353.
[24] O.J. David, J.M. Kovarik, R.L. Schmouder, Clinical pharmacokinetics of fingoli-
mod, Clin. Pharmacokinet. 51 (2012) 15e28.
[47] M.H. Bolli, S. Abele, C. Binkert, R. Bravo, S. Buchmann, D. Bur, J. Gatfield,
P. Hess, C. Kohl, C. Mangold, B. Mathys, K. Menyhart, C. Mueller, O. Nayler,
M. Scherz, G. Schmidt, V. Sippel, B. Steiner, D. Strasser, A. Treiber, T. Weller, 2-
Imino-thiazolidin-4-one derivatives as potent, orally active S1P1 receptor
agonists, J. Med. Chem. 53 (2010) 4198e4211.
[48] E.H. Demont, B.I. Andrews, R.A. Bit, C.A. Campbell, J.W.B. Cooke, N. Deeks,
S. Desai, S.J. Dowell, P. Gaskin, J.R.J. Gray, A. Haynes, D.S. Holmes, U. Kumar,
M.A. Morse, G.J. Osborne, T. Panchal, B. Patel, A. Perboni, S. Taylor, R. Watson,
J. Witherington, R. Willis, Discovery of a selective S1P1 receptor agonist effi-
cacious at low oral dose and devoid of effects on heart rate, Med. Chem. Lett. 2
(2011) 444e449.
[49] H. Kurata, K. Kusumi, K. Otsuki, R. Suzuki, M. Kurono, N. Tokuda, Y. Takada,
H. Shioya, H. Mizuno, T. Komiya, T. Ono, H. Hagiya, M. Minami, S. Nakade,
H. Habashita, Structureeactivity relationship studies of S1P agonists with a
dihydronaphthalene scaffold, Bioorg. Med. Chem. Lett. 22 (2012) 144e148.
[50] D.J. Buzard, S. Han, L. Lopez, A. Kawasaki, J. Moody, L. Thoresen, B. Ullman,
J. Lehmann, I. Calderon, X. Zhu, T. Gharbaoui, D. Sengupta, A. Krishnan, Y. Gao,
J. Edwards, J. Barden, M. Morgan, K. Usmani, C. Chen, A. Sadeque, J. Thatte,
M. Solomon, L. Fu, K. Whelan, L. Liu, H. Al-Shamma, J. Gatlin, M. Le, C. Xing,
S. Espinola, R.M. Jones, Fused tricyclic indoles as S1P1 agonists with robust
efficacy in animal models of autoimmune disease, Bioorg. Med. Chem. Lett. 22
(2012) 4404e4409.
[25] S. Mandala, R. Hajdu, J. Bergstrom, E. Quackenbush, J. Xie, J. Milligan,
R. Thornton, G.-J. Shei, D. Card, C. Keohane, M. Rosenbach, J. Hale, C.L. Lynch,
K. Rupprecht, W. Parsons, H. Rosen, Alteration of lymphocyte trafficking by
sphingosine-1-phosphate receptor agonists, Science 296 (2002) 346e349.
[26] V. Brinkmann, M.D. Davis, C.E. Heise, R. Albert, S. Cottens, R. Hof, C. Bruns,
E. Prieschl, T. Baumruker, P. Hiestand, C.A. Foster, M. Zollinger, K.R. Lynch, The
immune modulator FTY720 targets sphingosine 1-phosphate receptors, J. Biol.
Chem. 277 (2002) 21453e21457.
[27] S.R. Schwab, J.G. Cyster, Finding a way out: lymphocyte egress from lymphoid
organs, Nat. Immunol. 8 (2007) 1295e1301.
[28] J. Chun, V. Brinkmann, A mechanistically novel, first oral therapy for multiple
sclerosis: the development of fingolimod (FTY720, Gilenya), Discov. Med. 64
(2011) 213e228.
[29] M. Matloubian, C.G. Lo, G. Cinamon, M.J. Lesneski, Y. Xu, V. Brinkmann,
M.L. Allende, R.L. Proia, J.G. Cyster, Lymphocyte egress from thymus and pe-
ripheral lymphoid organs is dependent on S1P receptor 1, Nature 427 (2004)
355e360.
[30] J.J. Hale, G. Doherty, L. Toth, S.G. Mills, R. Hajdu, C.A. Keohane, M. Rosenbach,
J. Milligan, G.-J. Shei, G. Chrebet, J. Bergstrom, D. Card, M. Forrest, S.-Y. Sun,
S. West, H. Xie, N. Nomura, H. Rosen, S. Mandalab, Selecting against S1P3
enhances the acute cardiovascular tolerability of 3-(N-benzyl)amino-
propylphosphonic acid S1P receptor agonists, Bioorg. Med. Chem. Lett. 14
(2004) 3501e3505.
[31] V. Brinkmann, J.G. Cyster, T. Hla, FTY720: sphingosine 1-phosphate receptor-1
in the control of lymphocyte egress and endothelial barrier function, Am. J.
Transpl. 4 (2004) 1019e1025.
[32] K. Chiba, Sphingosine 1-phosphate receptor type 1 as a novel target for the
therapy of autoimmune diseases, Inflamm. Regen. 30 (2010) 160e168.
[33] D.J. Buzard, J. Thatte, M. Lerner, J. Edwards, R.M. Jones, Recent progress in the
development of selective S1P1 receptor agonists for the treatment of in-
flammatory and autoimmune disorders, Expert Opin. Ther. Pat. 18 (2008)
1141e1159.
[51] T. Nakamura, M. Asano, Y. Sekiguchi, Y. Mizuno, K. Tamaki, T. Kimura, F. Nara,
Y. Kawase, T. Shimozato, H. Doi, T. Kagari, W. Tomisato, R. Inoue, M. Nagasaki,
H. Yuita, K. Oguchi-Oshima, R. Kaneko, N. Watanabe, Y. Abe, T. Nishi, Dis-
covery of CS-2100, a potent, orally active and S1P3-sparing S1P1 agonist,
Bioorg. Med. Chem. Lett. 22 (2012) 1788e1792.
ꢀ
ꢀ
[52] M.S. Freedman, T. Olsson, M. Melanson, O. Fernandez, A. Boster, D. Bach,
O. Berkani, M. Mueller, T. Sidorenko, C. Pozzilli, Dose-dependent effect of
ponesimod, an oral, selective sphingosine 1-phosphate receptor-1 modulator,
on magnetic resonance imaging outcomes in patients with relapsingeremit-
ting multiple sclerosis, in: 28th European Committee for the Treatment and
Research in Multiple Sclerosis (ECTRIMS), 2012. P923.
[53] S. Pan, N.S. Gray, W. Gao, Y. Mi, Y. Fan, X. Wang, T. Tuntland, J. Che, S. Lefebvre,
Y. Chen, A. Chu, K. Hinterding, A. Gardin, P. End, P. Heining, C. Bruns,
N.G. Cooke, B. Nuesslein-Hildesheim, Discovery of BAF312 (siponimod), a
potent and selective S1P receptor modulator, Med. Chem. Lett. 4 (2013)
333e337.
[54] Receptos, Receptos Reports Positive Phase 2 Results for RPC1063 in Relapsing
Multiple Sclerosis, 2014. RCPT_News_2014_6_9_General_Releases.
[55] M. Bigaud, D. Guerini, A. Billich, F. Bassilana, V. Brinkmann, Second generation
S1P pathway modulators: research strategies and clinical developments,
Biochim. Biophys. Acta 1841 (2013) 745e758.
[34] M.H. Bolli, C. Lescop, O. Nayler, Synthetic sphingosine 1-phosphate receptor
modulators - opportunities and potential pitfalls, Curr. Top. Med. Chem. 11
(2011) 726e757.
[35] E. Roberts, M. Guerrero, M. Urbano, H. Rosen, Sphingosine 1-phosphate re-
ceptor agonists: a patent review (2010-2012), Expert Opin. Ther. Pat. 23
(2013) 817e841.
[36] V. Brinkmann, FTY720 (fingolimod) in Multiple Sclerosis: therapeutic effects
in the immune and the central nervous system, Br. J. Pharmacol. 158 (2009)
1173e1182.
[37] R.P. Coelho, H.S. Saini, C. Sato-Bigbee, Sphingosine-1-phosphate and oligo-
dendrocytes: from cell development to the treatment of multiple sclerosis,
Prostagl. Other Lipid Mediat. 91 (2010) 139e144.
[38] J.W. Choi, S.E. Gardell, D.R. Herr, R. Rivera, C.-W. Lee, K. Noguchi, S.T. Teo,
Y.C. Yung, M. Lu, G. Kennedy, J. Chun, FTY720 (fingolimod) efficacy in an
animal model of multiple sclerosis requires astrocyte sphingosine 1-
phosphate receptor 1 (S1P1) modulation, Proc. Natl. Acad. Sci. U.S.A. 108
(2011) 751e756.
[39] A. Groves, Y. Kihara, J. Chun, Fingolimod: direct CNS effects of sphingosine 1-
phosphate (S1P) receptor modulation and implications in multiple sclerosis
therapy, J. Neurol. Sci. 328 (2013) 9e18.
[40] M. Forrest, S.-Y. Sun, R. Hajdu, J. Bergstrom, D. Card, G. Doherty, J. Hale,
C. Keohane, C. Meyers, J. Milligan, S. Mills, N. Nomura, H. Rosen, M. Rosenbach,
G.-J. Shei, I.I. Singer, M. Tian, S. West, V. White, J. Xie, R.L. Proia, S. Mandala,
[56] P. Brossard, H. Derendorf, J. Xu, H. Maatouk, A. Halabi, J. Dingemanse, Phar-
macokinetics and pharmacodynamics of ponesimod, a selective S1P receptor
modulator, in the first-in-human study, Br. J. Clin. Pharmacol. 76 (2013)
888e896.
[57] P. Gergely, E. Wallstrom, B. Nuesslein-Hildesheim, C. Bruns, F. Zecri, N. Cooke,
M. Traebert, T. Tuntland, M. Rosenberg, M. Saltzman, Phase I study with the
selective S1P1/S1P5 receptor modulator BAF312 indicates that S1P1 rather
than S1P3 mediates transient heart rate reduction in humans, in: 25th
Congress of the European Committee for the Treatment and Research in
Multiple Sclerosis (ECTRIMS), 2009. P437.
€
ꢀ
[58] P. Gergely, B. Nuesslein-Hildesheim, D. Guerini, V. Brinkmann, M. Traebert,
C. Bruns, S. Pan, N.S. Gray, K. Hinterding, N.G. Cooke, A. Groenewegen,
A. Vitaliti, T. Sing, O. Luttringer, J. Yang, A. Gardin, N. Wang, W.J. Crumb Jr.,
M. Saltzman, M. Rosenberg, E. Wallstrom, The selective sphingosine 1-

238
C. Lescop et al. / European Journal of Medicinal Chemistry 116 (2016) 222e238
phosphate receptor modulator BAF312 redirects lymphocyte distribution and
has species-specific effects on heart rate, Br. J. Pharmacol. 167 (2012)
1035e1047.
[72] R.S. Obach, Prediction of human clearance of twenty-nine drugs from hepatic
microsomal intrinsic clearance data: an examination of the in vitro half-life
approach and nonspecific binding to micrososmes, Drug Metab. Dispos. 27
(1999) 1350e1359.
[73] R.P. Austin, P. Barton, S.L. Cockroft, M.C. Wenlock, R.J. Riley, The influence of
nonspecific microsomal binding on apparent intrinsic clearance, and its pre-
diction from physicochemical properties, Drug Metab. Dispos. 33 (2002)
1497e1503.
[59] M.H. Bolli, S. Abele, M. Birker, R. Bravo, D. Bur, R. De Kanter, C. Kohl, J. Grimont,
P. Hess, C. Lescop, B. Mathys, C. Müller, O. Nayler, L. Piali, M. Rey, M. Scherz,
G. Schmidt, J. Seifert, B. Steiner, J. Velker, T. Weller, Novel S1P1 receptor
agonists ꢄ part 3: from thiophenes to pyridines, J. Med. Chem. 57 (2014)
110e130.
[60] H. Spielmann, M. Balls, J. Dupuis, W.J. Pape, G. Pechovitch, O. de Silva, H.-
G. Holzhütter, R. Clothier, P. Desolle, F. Gerberick, M. Liebsch, W.W. Lovell,
T. Maurer, U. Pfannenbecker, J.M. Potthast, M. Csato, D. Sladowski, W. Steiling,
P. Brantom, The international EU/COLIPA in vitro phototoxicity validation
study: results of the phase II (blind trial). Part1: the 3T3 NRU phototoxicity
test, Toxicol. In Vitro 12 (1998) 305e327.
[61] B. Peters, H.-G. Holzhütter, In vitro phototoxicity testing: development and
validation of a new concentration response analysis software and biostatis-
tical analyses related to the use of various prediction models, ATLA 30 (2002)
415e432.
[74] M.J. Sykes, M.J. Sorich, J.O. Miners, Molecular modeling approaches for the
prediction of the nonspecific binding of drugs to hepatic microsomes, J. Chem.
Inf. Model. 46 (2006) 2661e2673.
[75] X. Liu, M. Wright, C.E.C.A. Hop, Rational use of plasma protein and tissue
binding data in drug design, J. Med. Chem. 57 (2014) 8238e8248.
[76] J. Wagner, P.v. Matt, B. Faller, N.G. Cooke, R. Albert, R. Sedrani, H. Wiegand,
C. Jean, C. Beerli, G. Weckbecker, J.-P. Evenou, G. Zenke, S. Cottens, Structur-
eeactivity relationship and pharmacokinetic studies of sotrastaurin (AEB071),
a promising novel medicine for prevention of graft rejection and treatment of
psoriasis, J. Med. Chem. 54 (2011) 6028e6039.
[62] A.M. Lynch, P. Wilcox, Review of the performance of the 3T3 NRU invitro
phototoxicity assay in the pharmaceutical industry, Exp.Toxicol. Pathol. 63
(2011) 209e214.
[77] N.J. Abbott, D.E.M. Dolman, A.K. Patabendige, Assays to predict drug perme-
ation across the blood-brain barrier, and distribution to brain, Curr. Drug
Metab. 9 (2008) 901e910.
[63] S. Onoue, N. Igarashi, Y. Yamauchi, T. Kojima, N. Murase, Y. Zhou, S. Yamada,
Y. Tsuda, In vitro phototoxic potential and photochemical properties of imi-
dazopyridine derivative: a novel 5-HT4 partial agonist, J. Pharm. Sci. 97 (2008)
4307e4318.
[78] T.T. Wager, A. Villalobos, P.R. Verhoest, X. Hou, C.L. Shaffer, Strategies to
optimize the brain availability of central nervous system drug candidates, Exp.
Op. Drug Disc. 6 (2011) 371e381.
[79] T.T. Wager, R.Y. Chandrasekaran, X. Hou, M.D. Troutman, P.R. Verhoest,
A. Villalobos, Y. Will, Defining desirable central nervous system drug space
through the alignment of molecular properties, in vitro ADME, and safety
attributes, ACS Chem. Neurosci. 1 (2010) 420e434.
[80] T.T. Wager, X. Hou, P.R. Verhoest, A. Villalobos, Moving beyond rules: the
development of a central nervous system multiparameter optimization (CNS
MPO) approach to enable alignment of druglike properties, ACS Chem. Neu-
rosci. 1 (2010) 435e449.
[81] E. Baciocchi, T.D. Giacco, A. Lapi, Oxygenation of benzyldimethylamine by
singlet oxygen. Products and mechanism, Org. Lett. 6 (2004) 4791e4794.
[82] E. Baciocchi, T.D. Giacco, O. Lanzalunga, A. Lapi, Singlet oxygen promoted
carbon-heteroatom bond cleavage in dibenzyl sulfides and tertiary diben-
zylamines. Structural effects and the role of exciplexes, J. Org. Chem. 72 (2007)
9582e9589.
[64] M.D. Barratt, Structure-activity relationship and prediction of the phototox-
icity and phototoxic potential of new drugs, ATLA 32 (2004) 511e524.
[65] M.H. Bolli, C. Müller, B. Mathys, S. Abele, M. Birker, R. Bravo, D. Bur, P. Hess,
C. Kohl, D. Lehmann, O. Nayler, M. Rey, S. Meyer, M. Scherz, G. Schmidt,
B. Steiner, A. Treiber, J. Velker, T. Weller, Novel S1P1 receptor agonists ꢄ part
1: from pyrazoles to thiophenes, J. Med. Chem. 56 (2013) 9737e9755.
[66] M.H. Bolli, J. Velker, C. Müller, B. Mathys, M. Birker, R. Bravo, D. Bur, R.
d. Kanter, P. Hess, C. Kohl, D. Lehmann, S. Meyer, O. Nayler, M. Rey, M. Scherz,
B. Steiner, Novel S1P1 receptor agonists e part 2: from bicyclo[3.1.0]hexane-
fused thiophenes to isobutyl substituted thiophenes, J. Med. Chem. 57 (2014)
78e97.
[67] W. Buratti, G.P. Gardini, F. Minisci, Nucleophilic character of alkyl radicals-V
Selective homolytic alpha-oxyalkylation of heteroaromatic bases, Tetrahe-
dron 27 (1971) 3655e3688.
[83] M.H. Bolli, C. Lescop, M. Birker, R.d. Kanter, P. Hess, C. Kohl, O. Nayler, M. Rey,
P. Sieber, J. Velker, T. Weller, B. Steiner, Novel S1P1 receptor agonists e Part 5:
from amino-to alkoxy-pyridines, Eur. J. Med. Chem. 115 (2016) 326e341.
[84] J. Grimont, F. Le Goff, G. Bourquin, J. Silva, Quality assessment and concen-
tration determination of Actelion's compound solutions by automated LCMS
and quantitative NMR, in: MipTec 2010-The Leading European Event for Drug
Discovery, Basel, Switzerland, Sep 20ꢄ24, 2010.
[68] M.H. Bolli, C. Lescop, B. Mathys, C. Mueller, O. Nayler, B. Steiner, Novel Ami-
nomethyl Benzene Derivatives. WO2009109904, Sep. 11, 2009.
[69] Mihara, J.; Murata, T.; Yamazaki, D.; Yoneta, Y.; Shibuya, K.; Shimojo, E.;
€
Gorgens, U.; Turberg, A.; Bach, T. Insecticidal aryl isoxazoline derivatives.
US2010/0179194 A1, Jul 15, 2010.
[70] G. Costantino, M. Marinozzi, E. Camaioni, N. Natalini, I. Sarichelou, F. Micheli,
P. Cavanni, S. Faedo, C. Noe, F. Moroni, R. Pellicciari, Stereoselective synthesis
and preliminary evaluation of (þ)- and (-)-3-methyl-5-carboxy-thien-2-
ylglycine (3-MATIDA): identification of (þ)-3-MATIDA as a novel mGluR1
competitive antagonist, Il Farm. 59 (2004) 93e99.
[85] R.S. Obach, A.E. Reed-Hagen, Measurement of Michaelis constants for cyto-
chrome P450-mediated biotransformation reactions using a substrate deple-
tion approach, Drug Metab. Dispos. 30 (2002) 831e837.
[86] M. Rowland, L.Z. Benet, G.G. Graham, Clearance concepts in pharmacokinetics,
J. Pharmacokinet. Pharmacodyn. 1 (1973) 123e136.
[71] M.H. Bolli, C. Lescop, B. Mathys, C. Mueller, O. Nayler, B. Steiner, Novel Thio-
phene Derivatives. WO2009074950, June 18, 2009.