Novel S1P1 receptor agonists - Part 1: From pyrazoles to thiophenes

Article abstract of DOI:10.1021/jm4014373

From a high-throughput screening campaign aiming at the identification of novel S1P₁ receptor agonists, the pyrazole derivative 2 emerged as a hit structure. Medicinal chemistry efforts focused not only on improving the potency of the compound but in particular also on resolving its inherent instability issue. This led to the discovery of novel bicyclo[3.1.0]hexane fused thiophene derivatives. Compounds with high affinity and selectivity for S1P₁ efficiently reducing the blood lymphocyte count in the rat were identified. For instance, compound 85 showed EC₅₀ values of 7 and 2880 nM on S1P₁ and S1P₃, respectively, had favorable pharmacokinetic properties in rat and dog, distributed well into brain tissue, and efficiently and dose dependently reduced the blood lymphocyte count in the rat. After oral administration to spontaneously hypertensive rats, the S1P ₁ selective compound 85 showed no effect on mean arterial blood pressure and affected the heart rate during the wake phase of the animals only.

Full text of DOI:10.1021/jm4014373

Article
pubs.acs.org/jmc
Novel S1P1 Receptor Agonists − Part 1: From Pyrazoles to
Thiophenes
Martin H. Bolli,* Claus Muller, Boris Mathys, Stefan Abele, Magdalena Birker, Roberto Bravo, Daniel Bur,
̈
Patrick Hess, Christopher Kohl, David Lehmann, Oliver Nayler, Markus Rey, Solange Meyer,
Michael Scherz, Gunther Schmidt, Beat Steiner, Alexander Treiber, Jorg Velker, and Thomas Weller
̈
Drug Discovery Chemistry, Actelion Pharmaceuticals Ltd., Gewerbestrasse 16, Allschwil CH-4123, Switzerland
S
* Supporting Information
ABSTRACT: From a high-throughput screening campaign aiming at the
identification of novel S1P1 receptor agonists, the pyrazole derivative 2
emerged as a hit structure. Medicinal chemistry efforts focused not only on
improving the potency of the compound but in particular also on resolving its
inherent instability issue. This led to the discovery of novel bicyclo[3.1.0]-
hexane fused thiophene derivatives. Compounds with high affinity and
selectivity for S1P1 efficiently reducing the blood lymphocyte count in the rat
were identified. For instance, compound 85 showed EC50 values of 7 and 2880
nM on S1P1 and S1P3, respectively, had favorable pharmacokinetic properties
in rat and dog, distributed well into brain tissue, and efficiently and dose
dependently reduced the blood lymphocyte count in the rat. After oral
administration to spontaneously hypertensive rats, the S1P1 selective compound 85 showed no effect on mean arterial blood
pressure and affected the heart rate during the wake phase of the animals only.
junctions was postulated to be the main reason for the inability
of the lymphocytes to leave the lymphoid tissue.5,12−16 In a
second hypothesis, the S1P concentration gradient17 that exists
between blood plasma and lymph was proposed as a driving
force for lymphocytes to exit the lymph nodes and re-enter
systemic circulation.18−21 The activation of the S1P1 receptor
by synthetic agonists is thought to lead to receptor internal-
ization and thus desensitization of the cell toward the external
S1P gradient. According to this hypothesis, the S1P1 receptor
agonists therefore behave as functional antagonists. Indeed,
S1P1 receptor antagonists have recently been shown to also
induce lymphocyte sequestration in rodents.22−25
INTRODUCTION
With the recent approval of FTY720 (1, fingolimod, Figure 1)
for the treatment of relapsing remitting multiple sclerosis (MS),
■
In addition to the benefit of lymphocyte sequestration,
several reports also proposed direct effects of S1P receptor
agonism in brain26−33 and other tissues.34 While agonism of the
S1P1, and possibly the S1P5 receptor,30,35−37 are beneficial in
the context of treating MS, activation of the S1P3 receptor is
deemed an undesired property. Several reports showed that
activation of the S1P3 receptor leads to heart rate
reduction,38−43 vaso- and bronchoconstriction,42,44 blood
pressure increase,45 pulmonary epithelial leakage,46 and
fibrosis7 in rodents. More recently, however, Hamada et al.
have shown that, in the rat, selectivity against S1P3 is necessary
but not sufficient for a compound to be devoid of effects on
heart rate.47 Furthermore, Fryer et al.45 carefully compared
heart rate and arterial blood pressure recordings of Sprague−
Dawley rats after iv infusion of the nonselective compound 1
Figure 1. Structures of sphingosine-1-phosphate, FTY720 (fingolimod,
1), and p-FTY720 (p-1).
a first sphingosine-1-phosphate (S1P, Figure 1) receptor
agonist is now available to patients suffering from this
debilitating disease.1−3 After oral administration, the prodrug
1 is phosphorylated to p-FTY720 (p-1) and is thus rendered a
nonselective agonist of the five known S1P receptors.4−7 It has
been shown that sphingosine-1-phosphate receptor 1 (S1P1)
agonism leads to sequestration of lymphocytes in lymph nodes
and hereby prevents these immune cells from causing
inflammation and tissue damage.8−11 Two hypotheses have
been proposed to explain the observed effects. In a first
hypothesis, the tightening of the lymphatic endothelial cell
Received: September 18, 2013
© XXXX American Chemical Society
A
dx.doi.org/10.1021/jm4014373 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
with those obtained after treatment with the S1P3 receptor
sparing compound BAF312 (siponimod).48 These analyses
suggest that, in the rat, the short-lasting heart rate decrease seen
upon iv infusion of the two agonists is caused by S1P1 receptor
agonism while the arterial blood pressure increase observed
with 1 is associated with S1P3 activation. However, it is
noteworthy that Fryer et al. observed no heart rate reduction
after oral administration of 1 and siponimod to Sprague−
Dawley rats. There is also a growing body of evidence
suggesting that S1P1 receptor activation triggers a transient
heart rate reduction in humans.49−54
cyclopentanone derivative 13, which was then chlorinated
under Appel conditions59 to yield the desired chlorovinyl
ketone 14.
Treating diketone 13 with hydrazine hydrate in a mixture of
methanol and acetic acid furnished the desired pyrazole scaffold
1560 that was then reacted with either a cinnamic acid
derivative, a dihydrocinnamic acid, or 2-methoxybenzylamine in
the presence of carbonyl di-imidazole (CDI) to give the
corresponding amides 2, 3, and 4, or urea 5, respectively
(Scheme 2).
The studies summarized above defined the key properties we
deemed desirable for S1P1 agonists suitable for clinical
development. Hence, a potential drug candidate needs to be
selective against the S1P3 receptor. In the rat, the
pharmacokinetic (PK) properties of the compound should
allow oral dosing in the low milligram range to obtain sustained
24 h maximal lymphocyte count (LC) reduction. The ability of
an S1P1 agonist to penetrate brain tissue was seen as a potential
benefit when treating central nervous system related auto-
immune diseases. Furthermore, we selected the spontaneously
hypertensive rat to assess the cardiovascular safety profile of the
compound. In the following paragraphs, we describe our efforts
aiming at identifying compounds fulfilling the above-mentioned
requirements. Compound 85, which emerged from these
efforts, showed EC50 values of 7 and 2880 nM on the S1P1
and S1P3 receptors, respectively, had favorable PK properties in
rat and dog, distributed well into brain tissue, and efficiently
and dose dependently reduced the blood LC in the rat. After
oral administration to spontaneously hypertensive rats, the
S1P1 selective compound 85 showed no effect on mean arterial
blood pressure and affected the heart rate during the wake
phase of the animals only.
Scheme 2. Preparation of Pyrazole, Pyrrole, and Thiophene
Derivatives
a
RESULTS AND DISCUSSION
■
Synthesis. The chlorovinyl ketone 14 represents a key
intermediate in the syntheses of the compounds discussed in
this account, and its preparation55−58 started from (+)-3-carene
10 (Scheme 1). Ozonolysis followed by oxidative workup
furnished the heptanoic acid derivative 11. Its corresponding
ester 12 was cyclized in a Dieckmann reaction to form the
Scheme 1. Preparation of the Key Intermediate Chlorovinyl
a
Ketone 14
a
Reagents and conditions: (a) NH2NH2·H2O, AcOH, MeOH, rt, 1 h,
67%; (b) (dihydro-) cinnamic acid, EDC or TBTU, THF or DMF,
45−57% (compounds 2−4); (c) 2-methoxy-benzyl amine, CDI, THF,
rt, 1 h, then 15, 50 °C, 1 h, 32% (compound 5); (d) diethyl oxalate,
KOtBu, THF, rt, 1 h; (e) MeOH, HOAc, NH2NH2 H2O, rt, 30 min,
14% over two steps; (f) MeI, Cs2CO3, DMF, 0 °C to rt, 2 h; (g) 2 N
aq LiOH, H2O, CH3CN, 45 °C, 1 h, 25% (over two steps, + 27% of
N2-methyl isomer); (h) (1) appropriate benzylamine, Hunig’s base,
̈
TBTU, DMF, rt, 4 h, 12−75%; for 27, 28: (2) 2-bromo-ethanol, 2 N
NaOH, MeOH 85 °C, 24 h, 21−54%; (i) diethyl 2-aminomalonate
HCl, NaOEt, EtOH, rt, 1 h; (j) 2 N aq LiOH, EtOH, 75 °C, 16 h; (k)
TFA, DCM, rt, 15 min, 31−62% (over 3 steps); (l) NaHMDS,
pentafluorophenyl 3-(2-methoxyphenyl)propanoate, THF, rt, 15 min,
34%; (m) glycine ethyl ester HCl, Et3N, NaHCO3, EtOH, 70 °C, 15 h;
(n) NaOEt, EtOH, microwave 130 °C, 10 min, 25%; (o) 2 N aq
LiOH, EtOH, 65 °C, 18 h, 96%; (p) NaOEt, HSCH2COOEt, EtOH,
rt, 1 h; (q) NaOEt, EtOH, 75 °C, 1 h; (r) 2 N aq LiOH, EtOH, 75 °C,
2 h, rt, 16 h, 67% over 3 steps.
a
Reagents and conditions: (a) (i) O3, MeOH, −65 °C, (ii) H2O2, rt to
35 °C, 2 h, 45−65%; (b) SOCl2, MeOH, 0 °C, 30 min, 70%; (c)
NaOMe, MeOH, reflux 1 h, 90% crude; (d) PPh3, CCl4, CHCl3, 65 °C
1−3 h, 46−68%.
B
dx.doi.org/10.1021/jm4014373 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
The preparation of the isomeric pyrazole scaffold 17 started
from ketone 16.61,62 Hence, Claisen condensation of 16 with
diethyl oxalate under basic conditions gave the corresponding
pyruvate intermediate that, upon treatment with hydrazine
hydrate in methanol and acetic acid, cyclized to the desired
pyrazole scaffold.63,64 Reacting the pyrazole intermediate with
MeI in the presence of Cs2CO3 furnished the N-alkylated
pyrazole scaffold as an approximately 1:1 mixture of the two
regioisomers. The mixture was separated by preparative high-
performance liquid chromatography (HPLC; X-terra column),
and structural assignment was confirmed by X-ray crystal
structure analysis of one of the two isomers (see the Supporting
Information). Ester hydrolysis under basic conditions gave the
pyrazole carboxylic acid 17 that was coupled with 2-
methoxybenzylamine to produce amide 7.
Reacting chlorovinyl ketone 14 with diethyl 2-amino-
malonate65 in the presence of sodium ethoxide followed by
LiOH-mediated ester hydrolysis produced the 2-carboxylic acid
derivative of pyrrole 18, which upon exposure to TFA in DCM,
rapidly decarboxylated to furnish pyrrole 18.66 N-acylation to
give 6 was achieved with moderate yield by reacting
deprotonated pyrrole 18 with the appropriate carboxylic acid
as its pentafluorophenyl ester. Pyrrole carboxylic acid 19 was
prepared by reacting chlorovinyl ketone 14 with ethyl
glycinate67,68 in the presence of Et3N and NaHCO3 at 70 °C.
Cyclization was achieved under microwave irradiation at 130
°C in the presence of sodium ethoxide. Lithium hydroxide
mediated ester hydrolysis furnished the desired pyrrole
carboxylic acid 19. As this compound has a tendency to slowly
decarboxylate, it is best purified at the ester stage. Amide
coupling was achieved under standard conditions using TBTU
as the coupling reagent and gave the target compound 8 in 12%
yield. Finally, by treating chlorovinyl ketone 14 with methyl
mercaptoacetate in the presence of sodium methoxide first at
room temperature then at 75 °C followed by hydrolyzing the
ester intermediate using aqueous LiOH produced the
thiophene carboxylic acid 20 in good yield.69 As before,
amide formation to obtain compounds 9 and 21−29 was
achieved under standard coupling conditions using TBTU as
the activating agent.70,71
Starting from carboxylic acid 20, the preparation of
thiophene derivatives incorporating a propanone linker
involved three steps (Scheme 3). First, the carboxylic acid
was transformed into the corresponding methylketone by
treating 20 with MeLi. Progress of the reaction had to be
carefully monitored to avoid overreaction to the tertiary
alcohol. Then, condensation of methylketone 30 with an
appropriately substituted benzaldehyde and subsequent reduc-
tion of the thus formed propenone using Pd/C and hydrogen
gas furnished the propanone derivative of general structure 31
in usually good yield. As indicated in step (d) in Scheme 3,
further transformation of the phenol furnished the target
compounds 32−65 and 67−70. The carboxylic acid derivative
66 was obtained in a similar fashion by condensing 3-(4-formyl-
2,6-dimethylphenyl)propenoic acid71 to ketone 30 followed by
reducing the corresponding propenone intermediate.
The preparation of thiophene derivatives incorporating a
five-membered heteroaromatic ring as linker between the
thiophene and the phenyl ring is outlined in Schemes 4 and 5.
The 5-thiophen-2-yl-1,2,4-oxadiazole derivative 78 was pre-
pared by coupling thiophene-2-carboxylic acid 20 with N,4-
dihydroxy-3,5-dimethylbenzamidine using TBTU as the activat-
ing agent. Cyclization of the hydroxyamidine ester intermediate
Scheme 3. Preparation of 1-Thien-2-yl-propan-1-one
Derivatives (for R1 and R2, see Tables 2 and 3)
a
a
Reagents and conditions: (a) MeLi, diethyl ether, 35 °C, 1−2 h, 44−
77%; (b) substituted benzaldehyde, KOH, EtOH, 60−80 °C, 4−6 h;
31%; or substituted benzaldehyde, ∼6 N HCl in 2-propanol, EtOH, rt,
20 h, 42−90%; or 3-(4-formyl-2,6-dimethyl-phenyl)-acrylic acid, 10%
w/v NaOH in MeOH, rt, 3 d, 40% (for 66); (c) H2, Pd/C, EtOH,
THF, DIPEA (for 66), rt, 1−18 h, 31−89%; (d) 2-bromoethanol
(33−45), 3-bromopropanol (46) or (R)- or (S)-3-chloro-propane-1,2-
diol (47 and 48), 2 N aq NaOH, NaI, 2-propanol, 70−90 °C, 4−24 h,
34−87%; or (2,2-dimethyl-1,3-dioxan-5-yl)methanol, Ph3P, DEAD,
THF, rt, 24 h, 67%, then 6 N HCl in 2-propanol, H2O, THF, 1 h, 40%
(49), or 41 or 46, methanesulfonyl chloride, Hunig’s base, DCM, rt, 1
̈
h, 84−95%, then appropriate amine or amino acid, Hunig’s base,
̈
DMF, 70−85 °C, 7−22 h, 46−73% (50−56 and 58−60), or
epichlorohydrin, 3 N aq NaOH, 2-propanol, rt, 18 h, 63−77%, then
ethanolamine, glycine or azetidine-3-carboxylic acid, EtOH, H2O,
Hunig’s base, 65 °C, 3−5 h, 34−67% (57, 61, and 62); 54, glycolic
̈
acid, Hunig’s base, TBTU, DCM, rt, 2 h, 35% (63); or 31 and
̈
epichlorohydrin as for 61, then 7 N NH3 in MeOH, 60−65 °C, 2−4 h,
64−85%, then glycolic acid, Hunig’s base, TBTU, DCM, rt, 1 h, 57%
̈
(64); or bromoacetic acid, 3 N NaOH, 2-propanol, 70 °C, 24 h; 15−
24% (65); or 65, appropriate amine, TBTU, Hunig’s base, DMF, rt, 30
̈
min, 53−75% (67−69); or 50, methanesulfonyl chloride, Hunig’s
base, DCM, rt, 0.5−2 h, 34% (70).
̈
to the 1,2,4-oxadiazole 71 was achieved by heating the crude
coupling product in dioxane at 100 °C.72,73 The glycerol side
chain in 78 was introduced by alkylating the phenol 71 with
(R)-glycerol acetonide under Mitsunobu conditions followed
by acid-mediated cleavage of the acetal protective group or by
reacting 71 with (S)-3-chloropropane-1,2-diol. Coupling
thiophene-2-carboxylic acid 20 with 4-(allyloxy)-3,5-dimethyl-
benzoic acid hydrazide followed by cyclization using Burgess’
reagent furnished the 1,3,4-oxadiazole 72. On the other hand,
using Lawesson’s reagent in the cyclization step gave access to
the corresponding 1,3,4-thiadiazole 73.73−75 Osmium tetroxide
mediated dihydroxylation of the allylic side chain delivered
compounds 80 and 82 as 1:1 mixtures of the two epimers at the
glycerol side chain. Coupling of thiophene-2-carboxylic acid 20
with 2-amino-4′-allyloxy-3′,5′-dimethyl-acetophenone followed
by cyclization effected by either Burgess’ or Lawesson’s reagent
gave access to the oxazole 76 or the thiazole 77,
respectively.75−77 As before, dihydroxylation of the allylic
ether furnished the desired diols 81 and 83 as a 1:1 mixture of
epimers. The 3-thiophen-2-yl-1,2,4-oxadiazole derivative 79
(Scheme 5) was prepared in analogy to 78. Thus, reaction of
chlorovinyl ketone 14 with thioacetic acid cyanomethyl ester
under basic conditions furnished the 2-cyano-thiophene
derivative that, after reaction with hydroxylamine hydro-
chloride, gave N-hydroxyamidine 74.73,78 Coupling of 74 with
3,5-dimethyl-4-hydroxy-benzoic acid followed by cyclization
under Dean−Stark conditions produced the 1,2,4-oxadiazole 75
C
dx.doi.org/10.1021/jm4014373 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Scheme 4. Preparation of Thiophene Derivatives with Five-
Membered Heteroaromatic Linkers
Scheme 5. Preparation of Thiophene Derivatives with Five-
Membered Heteroaromatic Linkers
a
a
a
Reagents and conditions: (a) thioacetic acid S-cyanomethylester, 2 N
aq NaOH, THF, rt, 3 h; (b) 2 N aq NaOH, THF, 90 °C, 4 h, 59%
(over 2 steps); (c) KOtBu; HONH2−HCl, MeOH, rt, 5 h, 85%; (d)
3,5-dimethyl-4-hydroxy benzoic acid, TBTU, Hunig’s base, DMF, rt,
̈
2−3 h, 46%; (e) toluene, 110 °C, 35 h, 86%; or dioxane, 60 °C, 24 h,
41%; (f) (S)-3-chloro-propane-1,2-diol, 2 N aq NaOH, 2-propanol, 65
°C, 10 h, 18−38%.
but did not significantly ameliorate the instability problem.
Similarly, replacing the pyrazole moiety by a pyrrole ring (e.g.,
pyrrole 6, Figure 3) delivered potent S1P1 agonists for which in
vivo efficacy on blood lymphocyte count reduction could be
measured after oral dosing, but the compounds were still prone
to hydrolyze in aqueous media. Analogues wherein the acyl
group was attached to a carbon rather than a nitrogen atom of
the five-membered heterocycle, as in compounds 7 and 8, for
instance, lost affinity for the S1P1 receptor (Figure 3).
However, replacing the pyrrole in compound 8 by a
thiophene furnished stable compounds with improved affinity
for the S1P1 receptor (e.g., compound 9, Figure 3). We
therefore set out to explore the structure−activity relationship
(SAR) of these novel thiophene derivatives in more detail.
In Vitro SAR of Bicyclo[3.1.0]hexane-fused Thiophene
Derivatives. Easy access to the thiophene-2-carboxamides,
such as 9, facilitated exploration of the SAR around the phenyl
ring. First, several examples incorporating either a chlorine or a
fluorine atom, a methyl or a methoxy group were prepared.
Among these monosubstituted derivatives, the 2-methoxy
compound 9 was clearly the most potent representative (data
not shown). In a second step, we introduced another methoxy
group to the phenyl ring (Table 1). As it turned out, this
additional group was only tolerated in the 4-position
(compound 22). In order to reduce the lipophilicity of the
compound, we attached polar chains to the phenyl moiety. As
illustrated by compound 25, a glycolic acid moiety was not
tolerated in position 4. Similarly, an ethylene glycol attached to
position 2 or 3 led to inactive compounds (26, 27). However,
attaching the ethylene glycol to position 4 of the phenyl ring
was tolerated (compound 28) and in combination with a 2-
methoxy substituent gave rise to compound 28 that showed a
similar S1P1 receptor affinity but a clearly reduced lipophilicity
(clogP = 4.4) when compared to analogues 9 and 22 (both
clogP = 5.2).
a
Reagents and conditions: (a) N,4-Dihydroxy-3,5-dimethylbenzami-
dine, TBTU, Hunig’s base, DMF, rt, 1−6 h; (b) dioxane or toluene,
̈
100 °C, 18−24 h, 39−71% (over 2 steps); (c) (1) (R)-(2,2-dimethyl-
1,3-dioxolan-4-yl)methanol, Ph3P, DEAD, THF, rt, 24 h, 66% (2) 6 N
HCl in 2-propanol, H2O, THF, rt, 30 min, 59%; or 3-chloropropane-
1,2-diol, 2 N NaOH, isopropanol, 65 °C, 10 h, 50%; (d) 4-(allyloxy)-
3,5-dimethylbenzoic acid hydrazide, HOBt, EDC, Et3N, DCM, 0 °C,
16 h, 82%; (e) Burgess reagent, THF, microwave irradiation 110 °C, 5
min, 58-70% (for 72 and 76) or Lawesson reagent, THF, microwave
irradiation 110 °C, 5 min, 30−41% (for 73 and 77); (f) OsO4, NMO,
acetone, H2O, rt, 16−18 h, 15−48%; (g) 2-amino-4′-allyloxy-3′,5′-
dimethyl-acetophenone,77 HOBt, EDC HCl, Et3N, DCM, rt, 5 h, 51%.
in good yield. Alkylation of the phenol moiety with (S)-3-
chloro-propane-1,2-diol concluded the synthesis of 79.
Early in Vitro Structure−Activity Relationship Studies
Addressing the Instability of Pyrazole 2. From a high-
throughput screening (HTS) campaign based on FLIPR
technology using CHO cells stably expressing a chimeric
S1P1-Gαq5 construct, compound 2 emerged as a hit structure. In
a GTPγS assay using membranes of CHO cells overexpressing
the human S1P1 receptor, this compound showed an EC50
value of 16 nM, and small changes in its structure led to
analogues, such as compounds 3 or 4, with single digit
nanomolar activities on S1P1 (Figure 2). Compounds 3 and 4
were more than 100-fold selective against S1P3. However, it
soon became evident that the stability of the acyl pyrazole
moiety, in particular in aqueous media, is insufficient for these
molecules to serve as drugs.79 Urea analogues such as
compound 5 retained at least some of the affinity for S1P1
Meanwhile, we established the chemistry to get access to
thiophene derivatives incorporating a ketone function in the
linker (Scheme 3), and we prepared a set of compounds
making use of the SAR knowledge acquired with the thiophene-
2-carboxamides (Table 2). We therefore prepared compound
32 incorporating an ethylene glycol side chain in position 4 of
the phenyl ring. As already observed with the two pyrazoles 4
and 5 (Figure 2), compound 32 lacking a nitrogen in the linker
D
dx.doi.org/10.1021/jm4014373 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Figure 2. Structure and in vitro activity of the HTS hit 2 and close analogues thereof.
Figure 3. Some examples of close analogues of the HTS hit 1 prepared to overcome the instability of 2 in aqueous media.
Table 1. SAR of the Substituents around the Phenyl Ring in
the Thiophene-2-carboxamide Series
Table 2. SAR of the Ethylene Glycol Substituted Phenyl Ring
in the 1-Thien-2-yl propan-1-one Series
a
a
a
a
EC50 S1P1
[nM]
EC50 S1P3
[nM]
compound
R
EC50 S1P1 [nM] EC50 S1P3 [nM]
compound
R
32
33
34
35
36
37
38
39
40
41
42
43
44
45
none
15.0
4.6
1610
9
2-methoxy
520
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
2-methoxy
2780
21
22
23
24
25
2,3-dimethoxy
2,4-dimethoxy
2,5-dimethoxy
2,6-dimethoxy
2-methoxy-4-
(carboxymethoxy)
2-(2-hydroxyethoxy)
3-(2-hydroxyethoxy)
4-(2-hydroxyethoxy)
7490
2-chloro
6.5
5200
450
2,6-dimethoxy
2,6-dimethyl
3-methoxy
3110
902
28.6
5.8
>10 000
>10 000
>10 000
2590
7280
>10 000
>10 000
3-methyl
3-chloro
9.8
4630
26
27
28
29
>10 000
>10 000
1800
>10 000
>10 000
>10 000
>10 000
3,5-dimethoxy
3,5-dimethyl
3,5-dichloro
3-chloro-5-methyl
3-ethyl-5-methyl
2,3,5-trimethyl
232
2.6
>10 000
879
1.1
4250
2-methoxy-4-(2-
hydroxyethoxy)
282
1.8
1350
4.4
>10 000
>10 000
a
EC50 values as determined in a GTPγS assay using membranes of
CHO cells expressing either S1P1 or S1P3;80 EC50 values represent
geometric mean values of at least three independent measurements in
duplicate. For details see the Experimental Section.
23.7
a
EC50 values as determined in a GTPγS assay using membranes of
CHO cells expressing either S1P1 or S1P3;80 EC50 values represent
geometric mean values of at least three independent measurements in
duplicate. For details see the Experimental Section.
turned out to be considerably more potent on S1P1 when
compared to its amide linked analogue 28. While keeping the
ethylene glycol moiety in position 4 constant, we therefore
evaluated the effect of an additional substituent at the phenyl
ring in more detail. As illustrated with compounds 33 and 34, a
substituent in position 2 further improved the potency of the
compound on S1P1 and slightly decreased the activity on S1P3.
In contrast, as shown with compounds 35 and 36, a 2,6-
disubstitution seriously hampered the affinity of the compound
for S1P1. A substituent in the 3-position had a moderate effect
on the S1P1 potency (compounds 37−39) only. Interestingly,
while a 3,5-dimethoxy (40) substitution pattern resulted in a
marked loss in affinity for S1P1, 3,5-dichloro (42) and in
particular 3,5-dimethyl (41) and 3-chloro-5-methyl (43)
substitutions furnished highly potent S1P1 receptor agonists
with good selectivity against S1P3. Among the various 3,5-
disubstitutions evaluated, the 3,5-dichloro (42) and the 3-ethyl-
5-methyl (44) patterns gave the best selectivity against S1P3.
Compound 45 illustrates that combining the 3,5-disubstitution
E
dx.doi.org/10.1021/jm4014373 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Table 3. SAR of Polar Side Chains in the 4-Position of the Phenyl Ring
a
EC50 values as determined in a GTPγS assay using membranes of CHO cells expressing either S1P1 or S1P3;80 EC50 values represent geometric
b
mean values of at least three independent measurements in duplicate. For details see the Experimental Section. Compound represents a 1:1 mixture
of epimers with respect to the polar side chain.
with a 2-substituent reduces the activity of the compound on
S1P1, indicating that, at the phenyl ring, either a 2- or a 3,5-
substitution is optimal for S1P1 receptor affinity.
Keeping the 3,5-dimethyl substitution pattern at the phenyl
ring constant, we then evaluated the effect of a large variety of
polar side chains in position 4 (Table 3). As compared to the
ethylene glycol 41, the propylene glycol analogue 46 was
slightly less potent on both S1P1 and S1P3. Introducing an
additional hydroxy group to this side chain gave the 2,3-
dihydroxy-propoxy derivatives 47 and 48. As illustrated by
these two compounds, the chirality in the side chain had a
minimal effect on the potency of the compound. As observed in
other series (data not shown), the (S)-configured side chain
was slightly more potent on S1P1 while both epimers showed
comparable affinity for S1P3. The achiral diol 49 was less potent
than diols 47 and 48. Replacing the alcohol function in 46 by a
primary (compound 50) or secondary amine (compounds 51
and 52) again furnished highly potent S1P1 agonists with good
selectivity against S1P3. The tertiary amine 53 represented a
slightly less potent S1P1 receptor agonist. Compounds 54, 55,
F
dx.doi.org/10.1021/jm4014373 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
and 56 incorporating an amino-propoxy chain were less potent
on S1P1 but also more selective against S1P3 when compared to
their corresponding amino-ethoxy analogues 50, 51, and 52,
respectively. As seen with compounds 46 and 47, introducing
an additional hydroxy group to the side chain to give
compound 57 improved the affinity for S1P1. The three
examples 58−60 illustrate that incorporating an amino acid
into the side chain was tolerated and furnished highly potent
S1P1 receptor agonists. Even the two carboxylic acid groups in
glutamate derivative 60 were well tolerated by the receptor. In
this series, however, an additional hydroxy group attached to
the amino-propoxy unit had only minimal effect on the potency
of the compound and selectivity (compare 61 and 62 with 58
and 59, respectively). In view of the above data, it is not
surprising that also amides 63 and 64 were potent and selective
S1P1 receptor agonists. Interestingly, there is a marked
difference in potency between the two carboxylic acids 65
and 66. While the glycolic acid derivative 65 showed only
moderate potency on the S1P1 receptor (in line with
observations made with compound 25), the propanoic acid
derivative 66 showed significant affinity for S1P1. The glycolic
acid amide derivatives 68, 69, and in particular, 67 represent
highly potent and selective S1P1 receptor agonists. Finally, as
illustrated by compound 70, a sulfonamide moiety was also
tolerated by S1P1. In brief, the compounds compiled in Table 3
illustrate that a large variety of polar side chains attached to the
4-position of the phenyl ring are tolerated by the S1P1 receptor.
The wide range of functional groups tolerated in this part of the
molecule clearly allows for fine-tuning of the physicochemical
properties of the compound.
Table 4. SAR of the Linker between the Thiophene and the
Phenyl Ring
With a last set of compounds compiled in Table 4, we
explored the SAR of the linker between the thiophene and the
phenyl ring. Inspired by the structure of S1P1 selective agonist
SEW287111, the propanone moiety was replaced by an
oxadiazole and other five-membered heteroaromates. Interest-
ingly, as illustrated by compounds 78−80, replacing the
propanone linker by an oxadiazole had little impact on the
affinity of the compound for S1P1. However, all three
oxadiazole derivatives were markedly more potent on S1P3
and were therefore clearly less selective when compared to 47.
The oxazole 81 had a similar receptor affinity profile as its
structurally closest oxadiazole analogues 78 and 80. In contrast,
the thiadiazole 82 and the thiazole 83 were both significantly
less potent S1P1 agonists.
a
EC50 values as determined in a GTPγS assay using membranes of
CHO cells expressing either S1P1 or S1P3;80 EC50 values represent
geometric mean values of at least three independent measurements in
duplicate. For details see the Experimental Section. 1:1 mixture of
b
In Vivo Efficacy Assessment. Several compounds
incorporating a carene-derived thiophene scaffold were assessed
for their ability to reduce the peripheral blood LC in the rat,
and a few examples are listed in Table 5. The compounds were
administered orally at a dose of 10 mg/kg to male Wistar rats,
and the blood LC was measured shortly before and 3, 6, and 24
h after compound administration. A LC reduction of greater
than or equal to 60% was considered to be the maximal effect
that is observed under the described experimental conditions.80
As illustrated by the compounds in Table 5, a large variety of
side chains attached to position 4 of the phenyl ring led to in
vivo active compounds. Clear differences in the pharmacody-
namic profiles could be observed. For instance, while the
propanone-linked compounds incorporating a mono- (46) or
dialcohol (47), an amino acid (61), or a glycolamide (64) in
the side chain reached maximal efficacy at 3 h and already
showed partial recovery of LC at 6 h, compounds incorporating
an amine or an amino alcohol side chain (52 and 55−57)
reached maximal efficacy at 6 h. At 24 h, only amino alcohols
epimers at 2,3-dihydroxy-propoxy side chain.
56 and 57 showed significant LC reductions. On the other
hand, the four oxadiazole-linked compounds 78−80 and 84
already reached maximal efficacy at 3 h post-administration, and
the LC remained maximally reduced at 6 h. Similar to the
propanone-linked analogue 47, the LC recovered 24 h after
administration of the three compounds incorporating a glycerol
side chain (78−80). On the other hand, the glycolamide 84
showed maximal LC reductions for 24 h and therefore
displayed a much longer duration of action when compared
to the propanone-linked analogue 64. Studies with additional
analogues revealed that compounds 47 and 64 and 78 and 84
are prototypical with respect to the in vivo behavior of
propanone- and oxadiazole-linked thiophene derivatives,
respectively. In part two of this communication series, we
further illustrate that oxadiazole-linked compounds consistently
show a higher LC reduction efficacy due to a more sustained
PK when compared to their propanone analogues.81
G
dx.doi.org/10.1021/jm4014373 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Table 5. Effect on Blood LC after Oral Administration of 10 mg/kg Compound to Male Wistar Rats
a
b
Data of pure (S)-enantiomer. This compound showed EC50 values of 1.3 and 7.0 nM for S1P1 and S1P3, respectively.
The potency on S1P1 and selectivity against S1P3 was then
of 1, 3, and 10 mg/kg had a clear, dose-dependent effect on the
number of circulating lymphocytes. Rapid reversibility of the
LC was observed at 1 and 3 mg/kg. At 10 mg/kg, the LC was
maximally reduced for 24 h. For the three active doses of 85,
the reduction of the LC measured at 3 h demonstrates rapid
onset of action. A PK experiment with oxadiazole 85 in the rat
revealed that the compound was absorbed almost completely
and reached a Cmax of 4000 ng/mL at 2 h after administration
(Table 7). A rather low clearance and a volume of distribution
further optimized in the oxadiazole series by combining a 3-
ethyl-5-methyl substitution pattern with a propanoic acid chain
in position 4 at the phenyl ring. This led to compound 85
showing EC50 values of 7 and 2880 nM for S1P1 and S1P3,
respectively (Figure 4 and Table 6). A dose response of LC
change in rats was established with oxadiazole 85 (Figure 4).
Administration of 0.3 mg/kg 85 to Wistar rats displayed
minimal if any blood lymphocyte sequestration. Administration
H
dx.doi.org/10.1021/jm4014373 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
24 h, respectively, after oral administration of 10 mg/kg to
Wistar rats.
Cardiovascular Effects in Spontaneously Hyperten-
sive Rats. In a next step, compound 85 was administered to
conscious spontaneously hypertensive rats (SHR) equipped
with a telemetry system allowing noninvasive monitoring of
mean arterial blood pressure (MAP) and heart rate (HR).82,83
The compound was administered at doses of 3, 10, 30, and 100
mg/kg in the morning, that is, at the end of the wake phase of
the animals. None of the doses induced a MAP increase that
was observed with 1 using the same experimental conditions
(Figures B and C, Supporting Information). However, the HR
recordings 24 h before (control) and after administration of
compound 85 shown in Figure 5 clearly document that oral
administration of compound 85 to SHR affects HR. As
illustrated by the data summarized in Figure 6, the amplitude of
the HR reduction as well as the overall extent of the effect as
expressed by the area between the curves (ABC) followed a
clear dose response. A dose of 3 mg/kg had no significant effect
on HR. With the doses of 10, 30, and 100 mg/kg, the HR
remained unchanged for the first 8 h after oral administration
(sleep phase of the rats). However, about 8 h after dosing, a
dose-dependent difference (ABC) between the HR of control
and treated rats started to manifest, and the effect persisted for
8−10 h (i.e., for the entire wake phase of the animals). In fact,
the HR increase normally observed during the wake phase of
nontreated rats was completely abolished in animals treated
with 30 and 100 mg/kg of compound 85. At a dose of 10 mg/
kg, the HR was maximally 23 bpm lower than that of the
control at about 9 h after compound administration. On the
basis of the PK experiment and the plasma exposure data
obtained from the LC experiment, plasma concentrations of 85
were 4−6 μM at this time point and therefore at best reached
the EC50 value for the rat S1P3 receptor. At 12 h, plasma
concentrations were clearly below the EC50 value on S1P3. At
30 and 100 mg/kg, the HRs were 32 and 37 bpm lower than
those in the control rats, respectively. The ABC reached 250
bpm × h at 10 mg/kg and appeared to reach a plateau with 350
and 380 bpm × h at 30 and 100 mg/kg, respectively.
Figure 4. Change of blood lymphocyte count after oral administration
□
●
▲
1
of 85 to male Wistar rats. vehicle (n = 5); 0.3 mg/kg (n = 5);
■
○
mg/kg (n = 5);
3 mg/kg (n = 5);
10 mg/kg (n = 5). For
experimental details see the Supporting Information.
Table 6. In Vitro Potency Profile (GTPγS EC50 Values) of
Compound 85 on Human (h) and Rat (r) S1P Receptors
hS1P1 hS1P2
hS1P3
hS1P4 hS1P5 rS1P1 rS1P3
a
EC50
[nM]
7.0
>10 000 2880 >10 000 275
4.4
6110
b
σg
1.8
11
−
1.8
12
−
2.6
10
2.3
3
1.7
3
c
n
7
7
a
EC50 values as determined in a GTPγS assay using membranes of
CHO cells expressing either S1P1 or S1P3;80 EC50 values represent
geometric mean values of at least three independent measurements in
duplicate. For details see the Experimental Section. Geometric
We then speculated that the late onset of the effect on HR
could be due to the slow formation of an active metabolite.
However, the observation that compounds 56 and 57 (both
tested at 100 mg/kg, for 57 see Figure D Supporting
Information), but also 1 (Figure 6, Figure E Supporting
Information) displayed a very similar behavior in SH rats
precluded this interpretation. It is important to note that
maximal HR changes were clearly more pronounced with 1
than with compound 85 (Figure 6) and that with 1 first signs of
HR changes manifested earlier, that is, 3 h after dosing already.
As p-1 is a potent agonist of the S1P3 receptor4,5,45 while
compound 85 is highly selective for S1P1 (Table 6), part of the
HR effect observed with 1 can be attributed to S1P3 receptor
b
c
standard deviation. Number of independent measurements.
clearly exceeding total body water resulted in a half-life of 4.9 h.
The PK profile therefore showed a good correlation with the
LC reductions in the rat.
The PK profile in the Beagle dog was comparable (Table 7).
The compound was rapidly absorbed reaching high plasma
concentrations. Clearance in the dog was lower than in the rat
leading to an even longer half-life despite a smaller volume of
distribution. Compound 85 distributed well into brain tissue
reaching concentrations of 220, 2880, and 410 ng/g at 2, 6, and
Table 7. Pharmacokinetic Data of Compound 85
a
pharmacokinetic parameters
AUC0−24h [(ng·h)/mL]
species
F [%]
Cmax (tmax) [ng/mL]([h])
t1/2 [h]
clearance [mL/(min·kg)]
Vss [L/kg]
rat
97
62
4000 (2)
3160 (2)
40 800
39 300
4.9
6.9
4.0
0.8
1.6
dog
0.45
a
F = bioavailability; Cmax = maximal plasma concentration; tmax = time at which Cmax was reached; t1/2 = half-life determined with iv experiment; Vss =
volume of distribution; oral dosing: 10 mg/kg in 3 Wistar rats, 3 mg/kg in 2 Beagle dogs; iv dosing: 1 mg/kg in 2 rats, 2 dogs; for experimental
details see the Supporting Information.
I
dx.doi.org/10.1021/jm4014373 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Figure 5. HR in beats per minute (bpm) after oral administration of (A) vehicle, (B) 3, (C) 10, (D) 30, and (E) 100 mg/kg 85 to male SHR. The
HR increase observed during the first 2 h is an artifact of the oral gavage. For experimental details see the Supporting Information and Hess et al.84
agonism. Fryer et al.45 observed a clear but only short-lasting
HR reduction in anesthetized normotensive Sprague−Dawley
rats after infusion of nonselective 1 and S1P1/5 selective
BAF312. However, these authors report that 1 and BAF312 had
no significant effect on HR after oral dosing to conscious
Sprague−Dawley rats. In contrast, we observed a significant,
dose-dependent effect on HR during the wake phase of male
SH rats. Our experiments therefore corroborate the observation
by Hamada et al.47 that, in the rat, selectivity against S1P3 alone
is not sufficient for a compound to be devoid of effects on HR.
At this point, it remains unclear whether the HR effects
observed in the SH rat with compound 85 are indeed
associated with S1P1 receptor agonism. Further experiments
will be needed to address this question and may shed light on
why effects on HR manifest during the wake phase of the rats
only and hence significantly differ from the rapid but only
transient HR reduction observed in humans.49−54
studies of these novel S1P1 agonists revealed that a ketone
linker yields significantly more potent compounds as compared
to analogues incorporating an amide between the thiophene
and the phenyl ring. Replacing the linear propanone linker by
an oxadiazole maintained the affinity for S1P1 but also increased
the potency on S1P3. A large variety of side chains in position 4
of the phenyl ring are tolerated, and either an additional 2- or a
3,5-substitution is optimal for S1P1 agonistic activity. Within a
given series of analogues, the 3-ethyl-5-methyl substitution
provided compounds with the most pronounced selectivity
against S1P3. The tolerance of a diverse set of polar side chains
in position 4 as well as various linkers between the thiophene
and the phenyl ring offered a handle to fine-tuning the
physicochemical and pharmacodynamic properties of the
compound. From the above SAR studies, the acid 85 emerged
as a potent S1P1 receptor agonist with high selectivity against
S1P3. In Wistar rats, this compound showed an ED50 of about 5
mg/kg for the sequestration of circulating lymphocytes at 24 h
after oral compound administration. The compound was well
absorbed in the rat and the dog, rapidly reached high plasma
concentrations, and was cleared slowly. Despite its high
selectivity against S1P3, compound 85 showed a marked
CONCLUSIONS
■
Starting from the hydrolytically susceptible HTS hit 2, we
developed stable, potent, and selective S1P1 receptor agonists
wherein the pyrazole in 2 was replaced by a thiophene. SAR
J
dx.doi.org/10.1021/jm4014373 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Figure 6. Results of dose response experiments of compounds (A, B) 85 and (C, D) 1 in male SHR. Maximal HR changes (ΔHR in bpm, A, C) and
area between curves (ABC, in bpm h; B, D).
3400 degasser. ValveMate 2 (Gilson) solvent valves; column, solvent
and retention time (tR) as indicated, DEA = diethyl amine, TFA =
trifluoroacetic acid, at 25 °C, flow 1 mL/min. No racemisation/
epimerization was observed during the synthesis of the target
compounds. LC-HRMS: Analytical pump: Waters Acquity Binary,
Solvent Manager, MS: SYNAPT G2MS, source temperature: 150 °C,
desolvation temperature: 400 °C; desolvatation gas flow: 400 L/h;
cone gas flow: 10 L/h; extraction cone: 4 RF; lens: 0.1 V; sampling
cone: 30; capillary: 1.5 kV; high-resolution mode; gain: 1.0; MS
function: 0.2 s per scan, 120−1000 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 μm, 2.1 mm ×
50 mm from Waters, thermostatted in the Acquity UPLC Column
Manager at 60 °C. Eluents: water and 0.05% formic acid; B:
acetonitrile and 0.05% formic acid. Gradient: 2−98% B over 3.0 min.
Flow: 0.6 mL/min. Detection: UV 214 nm and MS, tR is given in
minutes. Compound purity and identity was further confirmed by
NMR spectroscopy (Varian Oxford; 1H (300 MHz) or 13C (75 MHz)
inhibition of the HR increase that is usually observed during the
wake phase of nontreated male SH rats. Further studies to
better understand the factors influencing the HR and efforts to
identify compounds devoid of this activity are certainly
warranted.
EXPERIMENTAL SECTION
■
Chemistry. All reagents and solvents were used as purchased from
commercial sources (Sigma−Aldrich, Switzerland, Lancaster Synthesis
GmbH, Germany, Acros Organics, USA). Moisture sensitive reactions
were carried out under an argon atmosphere. Progress of the reactions
was followed either by thin-layer chromatography (TLC) analysis
(Merck, 0.2 mm silica gel 60 F254 on glass plates) or by liquid
chromatography−mass spectrometry (LC-MS). LC-MS: Finnigan
MSQ plus or MSQ surveyor (Dionex, Switzerland), with HP 1100
Binary Pump and DAD (Agilent, Switzerland); column: Zorbax SB-
AQ, 3.5 μm, 120 Å, 4.6 mm × 50 mm (Agilent); gradient: 5−95%
acetonitrile in water containing 0.04% of trifluoroacetic acid (TFA),
within 1 min; flow: 4.5 mL/min; 40 °C; tR is given in min. Purity of all
final compounds was checked by an additional LC-MS analysis on a
Waters Acquity UPLC system equipped with an ACQ-PDA detector,
an ACQ-ESL detector, and an ACQ-SQ detector; column: ACQUITY
UPLC BEH C18 1.7 μm, 2.1 mm × 50 mm; gradient: 2−98%
acetonitrile containing 0.045% formic acid in water containing 0.05%
formic acid over 1.8 min; flow: 1.2 mL/min; 60 °C. According to these
LC-MS analyses, final compounds showed a purity of greater than 95%
(UV at 230 and at 214 nm). Chiral integrity was proven by HPLC
(chiral stationary phase): Hardware from UltiMate instrument series
(Dionex): HPG-3200SD binary pump, WPS-3000 autosampler, TCC-
3200 thermostatted column compartment, DAD-3000 detector, SRD-
1
or Bruker Avance II, 400 MHz UltraShield, H (400 MHz), 13C (100
MHz); chemical shifts are reported in parts per million (ppm) relative
to tetramethylsilane (TMS), and multiplicities are given as s (singlet),
d (doublet), t (triplet), q (quartet), quint (quintuplet), h (hextet),
hept (heptuplet), or m (multiplet), br = broad, coupling constants are
given in Hz). Several compounds have been prepared in a
combinatorial library format on a 15−50 μmol scale. For those
1
compounds, H NMR spectra were acquired using nondeuterated 10
mM DMSO stock solutions submitted for biological testing.85 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 two frequencies are not always accurate. The numbers of
K
dx.doi.org/10.1021/jm4014373 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
protons given in the description represent observed values. In some
cases, signals close to either of the two solvent signals were not visible
as they were suppressed by irradiation. Compound purification:
compounds were purified by either flash column chromatography
(CC) on silica gel 60 (Fluka Sigma−Aldrich, Switzerland), or by
preparative HPLC (Waters XBridge Prep C18, 5 μm, OBD, 19 mm ×
50 mm, or Waters X-terra RP18, 19 mm × 50 mm, 5 μm, 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 mm × 18 mm, Labogel-RP-18−5s-100,
gradient: 10% MeOH in water to 100% MeOH). X-ray diffraction:
To determine the molecular structure of the 2-methyl isomer of
compound 17, a crystal of the compound was mounted on a Bruker
Nonius diffractometer equipped with a CCD detector, and reflections
were measured using monochromatic Mo Kα radiation. The structure
was solved by direct methods using SIR92, and refinement was
performed with CRYSTALS. Full matrix least-squares refinement was
performed with anisotropic temperature factors for all atoms except
hydrogen that were included at calculated positions with isotropic
temperature factors. Coordinates, anisotropic temperature factors, and
bond lengths and angles were deposited with the Cambridge
Crystrallographic Data Centre; CCDC code: 969024. Purity of all
target compounds was assessed using the two independent LC-MS
methods described above: (1) a Zorbax SB-AQ, 5 μm, 120 Å, 4.6 mm
× 50 mm (Agilent) column eluting with a gradient of 5−95%
acetonitrile in water containing 0.04% of trifluoroacetic acid, within 1
min, flow: 4.5 mL/min; and (2) an ACQUITY UPLC BEH C18 1.7
μm, 2.1 mm × 50 mm column eluting with a gradient of 2−98%
acetonitrile containing 0.045% formic acid in water containing 0.05%
formic acid over 1.8 min; flow 1.2 mL/min. In addition, important
compounds were analyzed by LC-HRMS as described above. Purity
and identity of the target compounds was further corroborated by
NMR spectroscopy, and chiral integrity was proven by HPLC using
chiral stationary phases. No racemisation/epimerization was observed
during the synthesis of the target compounds. According to these LC-
MS analyses, final compounds showed a purity of greater than or equal
to 95% (UV at 230 and at 214 nm).
In Vitro Potency Assessment. Data (EC50) are given as
geometric means (Xgeo) with geometric standard deviation (σg). The
upper and lower 95% confidence limits are calculated as Xgeo*σg2 and
Xgeo/σg2, respectively (results not shown). GTPγS binding assays were
performed in 96-well polypropylene microtiter plates in a final volume
of 200 μL. Membrane preparations of CHO cells expressing
recombinant human S1P1, S1P2, S1P3, S1P4, or S1P5 or rat S1P1 or
S1P3 receptors were used. Assay conditions were 20 mM Hepes, pH
7.4, 100 mM NaCl, 5 mM MgCl2, 0.1% fatty acid free BSA, 0.5 μM
(for S1P2, S1P5), 1 μM (for S1P1, S1P4) or 3 μM GDP (for S1P3),
2.5% DMSO, and 50 pM 35S-GTPγS. Test compounds were dissolved,
diluted, and preincubated with the membranes, in the absence of 35S-
GTPγS, in 150 μL of assay buffer at room temperature for 30 min.
After addition of 50 μL of 35S-GTPγS in assay buffer, the reaction
mixture was incubated for 1 h at room temperature. The assay was
terminated by filtration of the reaction mixture through a Multiscreen
GF/C plate, prewetted 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 BSA.
Then, the plates were dried at 50 °C and sealed, 25 μL of
MicroScint20 was added, and the membrane-bound 35S-GTPγS was
determined on the TopCount. Specific 35S-GTPγS binding was
determined by subtracting nonspecific binding (the signal obtained in
the absence of agonist) from maximal binding (the signal obtained
with 10 μM S1P). The EC50 of a test compound is the concentration
of a compound inducing 50% of specific binding. PK in the rat. Wistar
rats (RCC Ltd., Biotechnology and Animal Breeding Division,
anesthesia with isoflurane. Compounds were administered intra-
venously via the tail vein at doses of 1 mg/kg body weight formulated
as solutions in an aqueous mixed micellar vehicle based on
phospholipids and bile acids. Oral administration at doses of 10 mg/
kg was performed by gavage. Oral formulations were dispersions
prepared by addition of a DMSO stock solution of the compounds to
succinylated gelatin (7.5% w/v) in water. Serial blood samples of 0.25
mL each were taken predose and at 30 min, 1, 2, 3, 4, 6, 8, and 24 h
postdose into vials containing EDTA as anticoagulant. For the iv
applications, additional samples were obtained 2, 10, and 20 min after
dosing. Plasma was separated by centrifugation and stored at −20 °C.
All animals had free access to food during the entire duration of the
experiments. Plasma samples from the rat were analyzed using liquid
chromatography coupled to mass spectrometry (LC-MS-MS) after
protein precipitation with MeOH and centrifugation at 3220g for 20
min at 4 °C. Using 1.25 μL of plasma on the column, lower and upper
limits of quantification were 41 and 10 000 ng/mL for compound 85,
respectively. PK parameters were estimated with the WinNonlin
software (Pharsight Corporation, Mountain View, CA, USA) using
noncompartmental analysis. Brain penetration in the rat. At 2, 6, and
24 h after dosing, male Wistar rats (n = 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 of 0.9% NaCl trough the carotid. The whole brain was
then removed and homogenized in an equal volume of ice-cold 0.1 M
sodium phosphate buffer, pH 7.4, using a IKA-WERKE ultraturrax
T25 tissue homogenizer 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 from blanco brain
homogenate. PK in the dog. The PK profile of compound 85 was
determined in the male Beagle dog. The compound was administered
iv at a dose of 1 mg/kg as a solution in miced micelles and orally at a
dose of 3 mg/kg as a dispersion in succinylated gelatin (7.5% w/v) in
water. In vivo efficacy of the target compounds was assessed by
measuring the circulating lymphocytes after oral administration of 3−
100 mg/kg target compound to normotensive male Wistar rats. 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 before and 3, 6, and 24 h after drug
administration. 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 the LC at a given
time point and the predose value (= 100%). All data are presented as
mean
SEM. Statistical analyses were performed by analysis of
variance (ANOVA) using Statistica (StatSoft) and the Student−
Newman−Keuls procedure for multiple comparisons. The null
hypothesis was rejected when p < 0.05. Because of interindividual
variability and the circadian rhythm of the number of circulating
lymphocytes, a compound showing relative changes in the range of
−20% to +40% is considered inactive. A LC reduction in the range of
−60 to −75% represents the maximal effect to be observed under the
conditions of the experiment. For formulation, the compounds were
dissolved in DMSO. This solution was added to a stirred solution of
succinylated gelatin (7.5% w/v) in water. The resulting milky
suspension containing a final concentration of 5% DMSO was
administred to the animals by gavage. A mixture of 95% succinylated
gelatin (7.5% w/v) in water and 5% DMSO served as the vehicle.
Telemetric in vivo studies.82−84 The effect of compound 85 on MAP
and HR was assessed by oral administration of 85 as a suspension in
succinylated gelatin (7.5% w/v) in water to male SHR equipped with a
telemetric system recording arterial blood pressure and HR.
Compound administation took place in the morning, that is, during
the sleep phase of the animals. For both of these parameters, each
animal served as its own control by using the data of the last 24 h
before treatment. An area between curve (ABC) was calculated
between the control and the treatment period to assess the efficacy of
the compound. Maximal mean arterial blood pressure change was
extracted from the moving average over 6 h of the blood pressure
recordings.
Fullinsdorf, Switzerland) were used for PK experiments after an
̈
acclimatization period of at least 7 days after delivery. Animals foreseen
for iv dosing underwent surgery to receive a catheter implanted into
the jugular vein. Animals foreseen for oral dosing did not undergo
surgery, and blood samples were taken sublingually under light
L
dx.doi.org/10.1021/jm4014373 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(3bS,4aR)-3,4,4-Trimethyl-3b,4,4a,5-tetrahydrocyclopropa-
[3,4]cyclopenta[1,2-c]thiophene-1-carboxylic Acid (20). To a
solution of sodium (2.80 g, 122 mmol) in ethanol (400 mL), a
solution of mercapto-acetic acid ethyl ester (14.64 g, 122 mmol) in
ethanol (40 mL) was added. The solution was stirred for 5 min before
(1S,5R)-2-(1-chloro-(E)-ethylidene)-6,6-dimethyl-bicyclo[3.1.0]-
hexan-3-one (15.0 g, 81.2 mmol) in ethanol (40 mL) was added
dropwise. The solution became slightly warm (approximately 30 °C)
and turned orange to brown. A fine precipitate formed. Stirring was
continued at rt for 1 h. Then, a solution of sodium (2.24 g, 97.5 mmol)
in ethanol (75 mL) was added rapidly, and the mixture was heated to
75 °C for 1 h. A 2 N aq solution of LiOH (75 mL) was added and
stirring was continued at 75 °C for 2 h and then at rt for 16 h. About
two-thirds of the solvent was removed in vacuo, and the remaining
mixture was diluted with water (250 mL) and extracted with DCM
(200 mL). The organic extract was washed twice with 1 N aq (100
mL). The combined aqueous layers are acidified by adding 2N aq HCl
and extracted three times with diethyl ether (3 × 300 mL). The
organic extracts are dried over MgSO4 and evaporated. The remaining
residue was suspended in acetonitrile, filtered, washed with additional
acetonitrile, and dried under high vacuum to give thiophene carboxylic
acid 20 (12.02 g, 67%) as a pale yellow to beige crystalline powder.
XTerra Prep MS C18 19 mm × 50 mm 5 μm, gradient of acetonitrile
in water containing 0.85% of Et2NH) to give 27 (1.83 mg, 21%) as a
colorless resin. LC-MS: tR = 0.97 min, [M + 1]+ = 372.15 (calcd
372.16); 1H NMR (H6-DMSO, solvent suppression): δ 7.96 (t, J = 5.9
Hz, 1 H), 7.21 (t, J = 8.1 Hz, 1 H), 6.78−6.87 (m, 2 H), 6.78 (d, J =
7.8 Hz, 1 H), 4.90 (t, J = 4.9 Hz, 1 H), 4.32 (d, J = 5.1 Hz, 2 H), 3.91−
3.97 (m, 2 H), 3.65−3.73 (m, 2 H), 2.92 (dd, J1 = 18.6 Hz, J2 = 6.1 Hz,
1 H), 2.75 (d, J = 18.6 Hz, 1 H), 2.31 (s, 3 H), 1.86−1.96 (m, 2 H),
1.07 (s, 3 H), 0.66 (s, 3 H).
1-((3bS,4aR)-3,4,4-Trimethyl-3b,4,4a,5-tetrahydrocyclo-
propa[3,4]cyclo-penta[1,2-c]thiophen-1-yl)ethanone (30). To a
solution of 20 (12.0 g, 54 mmol) in diethyl ether (600 mL), MeLi (1.6
M, 72 mL, 115 mmol, 1.6 M in diethyl ether) was slowly added.
Progress of the reaction was carefully monitored by LC-MS. Upon
completion of the addition, stirring was continued at rt for 10 min.
The reaction was quenched by carefully adding the mixture to ice/
water (400 mL). The organic layer was separated, dried over MgSO4,
and filtered, and the solvent was evaporated. The crude product was
purified by CC on silica gel eluting with heptane/EA 4:1 to give the
title compound (8.81 g, 74%) as a pale yellow oil. LC-MS: tR = 1.03
1
min, [M + 1]+ = 221.20 (calcd 221.10). H NMR (CDCl3): δ 3.00
(ddd, J = 1.8, 4.7, 18.8 Hz, 1 H), 2.80 (d, J = 18.8 Hz, 1 H), 2.38 (s, 6
H), 1.93−1.90 (m, 2 H), 1.14 (s, 3 H), 0.74 (s, 3 H).
1
LC-MS: tR = 0.95 min, [M + 1]+ = 223.00 (calcd 223.08). H NMR
(CDCl3): δ 3.04−2.92 (m, 1 H), 2.83 (d, J = 19.3 Hz, 1 H), 2.39 (s, 3
H), 1.91−1.87 (m, 2 H), 1.13 (s, 3 H), 0.73 (s, 3 H). 13C NMR
(CDCl3): δ 168.4, 160.1, 146.8, 137.2, 117.5, 36.2, 30.0, 29.1, 26.6,
22.8, 14.4, 14.1.
(3bS,4aR)-N-(2-Methoxybenzyl)-3,4,4-trimethyl-3b,4,4a,5-
tetrahydrocyclopropa[3,4]cyclopenta[1,2-c]thiophene-1-car-
boxamide (9). A solution of (3bS,4aR)-3,4,4-trimethyl-3b,4,4a,5-
tetrahydrocyclo-propa[3,4]cyclopenta[1,2-c]thiophene-1-carboxylic
acid 20 (30.0 mg, 135 μmol), TBTU (47.7 mg, 148 μmol), and
3-(4-(2-Hydroxyethoxy)phenyl)-1-((3bS,4aR)-3,4,4-trimeth-
yl-3b,4,4a,5-tetrahydrocyclopropa[3,4]cyclopenta[1,2-c]-
thiophen-1-yl)propan-1-one (32). (a) A solution of 4-hydrox-
ybenzaldehyde (346 mg, 2.84 mmol) and 30 (500 mg, 2.27 mmol) in
ethanol (20 mL) and approximately 6 N HCl in isopropanol (4 mL)
was stirred at rt for 20 h. The dark brown solution was diluted with
diethyl ether and washed with saturated aq NaHCO3 solution and
water. The aqueous phases are extracted with diethyl ether. The
combined organic extracts are dried over MgSO4 and evaporated. The
crude product was purified by crystallization from MeOH to give (E)-
3-(4-hydroxyphenyl)-1-((3bS,4aR)-3,4,4-trimethyl-3b,4,4a,5-tetrahy-
dro-cyclopropa[3,4]cyclopenta[1,2-c]thio-phen-1-yl)prop-2-en-1-one
(694 mg, 75%) as an olive powder. LC-MS: tR = 1.05 min, [M + 1]+ =
Hunig’s base (57.6 mg, 445 μmol) in DMF (1.2 mL) was stirred at rt
̈
for 30 min before a solution of 2-methoxy-benzylamine (3.3 mg, 0.022
mmol) in DMF (0.2 mL) was added. The mixture was allowed to
stand at rt for 1 h before it was subjected to purification by preparative
HPLC (Grom-Sil 120 ODS-4-HE, 30 mm × 75 mm, 10 μm particle
size, gradient of 20−95% acetonitrile in water containing 0.5% formic
acid) to give 9 (45 mg, 98%) as a colorless lyophilizate. LC-MS: tR =
1
325.22 (calcd 325.13). H NMR (CDCl3): δ 7.70 (d, J = 15.8 Hz, 1
H), 7.51 (d, J = 8.8 Hz, 2 H), 7.10 (d, J = 15.2 Hz, 1 H), 6.89 (d, J =
8.2 Hz, 2 H), 5.72 (s, 1 H), 3.13 (dd, J = 5.9, 18.8 Hz, 1 H), 2.94 (d, J
= 18.8 Hz, 1 H), 2.42 (s, 3 H), 1.98−1.89 (m, 2 H), 1.13 (s, 3 H), 0.74
(s, 3 H).
1
1.08 min, [M + 1]+ = 342.18 (calcd 342.15). H NMR (H6-DMSO,
solvent suppression): δ 7.64 (t br, J = 4.8 Hz, 1 H), 7.17 (t, J = 7.7 Hz,
1 H), 7.06 (d, J = 7.3 Hz, 1 H), 6.83 (t, J = 7.3 Hz, 1 H), 4.32 (d, J =
5.2 Hz, 2 H), 3.79 (s, 3 H), 2.93 (dd, J = 6.3, 18.3 Hz, 1 H), 2.74 (d, J
= 18.5 Hz, 1 H), 2.31 (s, 3 H), 1.86−1.99 (m, 2 H), 1.08 (s, 3 H), 0.68
(s, 3 H). LC-HRMS: tR = 2.14 min, [M + H]/z = 342.1528, found
342.1530.
(3bS,4aR)-N-(3-(2-Hydroxyethoxy)benzyl)-3,4,4-trimethyl-
3b,4,4a,5-tetrahydrocyclopropa[3,4]cyclopenta[1,2-c]-
thiophene-1-carboxamide (27). (a) To a solution of thiophene
carboxylic acid 20 (222 mg, 1.00 mmol) in DMF (15 mL), TBTU
(b) A mixture of (E)-3-(4-hydroxyphenyl)-1-((3bS,4aR)-3,4,4-
trimethyl-3b,4,4a,5-tetrahydro-cyclopropa[3,4]cyclopenta[1,2-c]-
thiophen-1-yl)prop-2-en-1-one (690 mg, 2.13 mmol) and Pd/C (200
mg, 10% Pd) in ethanol (25 mL) and THF (25 mL) was stirred at rt
for 3 h under H2 (1.5 bar). The mixture was filtered, the filtrate was
evaporated, and the crude product was purified by CC on silica gel
eluting with heptane/EA 7:3 to give 3-(4-hydroxyphenyl)-1-
((3bS,4aR)-3,4,4-trimethyl-3b,4,4a,5-tetrahydrocyclo-propa[3,4]
cyclopenta[1,2-c]thiophen-1-yl)propan-1-one 31 (616 mg, 88%) as a
colorless foam. LC-MS: tR = 1.05 min, [M + 1]+ = 327.24 (calcd
(353 mg, 1.10 mmol) and Hunig’s base (427 mg, 3.30 mmol) was
̈
1
added. The mixture was allowed to stand at rt for 20 min before 3-
327.14). H NMR (CDCl3): δ 7.11−7.05 (m, 2 H), 6.78−6.70 (m, 2
hydroxybenzylamine (160 mg, 1.00 mmol) and Hunig’s base (142 mg,
̈
H), 4.75 (s, 1 H), 3.04−2.90 (m, 5 H), 2.78 (d, J = 18.8 Hz, 1 H), 2.37
(s, 3 H), 1.91−1.85 (m, 2 H), 1.11 (s, 3 H), 0.70 (s, 3 H).
1.10 mmol) were added as a solution in DMF (1.5 mL). The mixture
was stirred at rt for 3 h, treated with formic acid (2 mL), and directly
separated by preparative HPLC (Phenomenex AQUA, 30 mm × 75
mm, gradient of acetonitrile in water containing 0.5% of formic acid)
to give (3bS,4aR)-N-(3-hydroxybenzyl)-3,4,4-trimethyl-3b,4,4a,5-
tetrahydrocyclopropa[3,4]cyclopenta[1,2-c]thiophene-1-carboxamide
(126 mg, 38%) as a pale yellow resin. LC-MS: tR = 0.97 min, [M + 1]+
(c) Alkylation was carried out as described for 33. LC-MS: tR = 1.05
min, [M + 1]+ = 371.19 (calcd 371.17). 1H NMR (H6-DMSO, solvent
suppression): δ 7.08 (d, J = 8.0 Hz, 2 H), 6.77 (d, J = 7.9 Hz, 2 H),
4.88 (t, J = 5.4 Hz, 1 H), 3.91 (t, J = 4.4 Hz, 2 H), 3.64−3.72 (m, 2 H),
2.92−2.96 (m, 1 H), 2.76 (t, J = 7.5 Hz, 2 H), 2.71 (d, J = 18.8 Hz, 1
H), 2.32 (s, 3 H), 1.88−1.92 (m, 2 H), 1.08 (s, 3 H), 0.65 (s, 3 H).
LC-HRMS: tR = 1.44 min, [M + H]/z = 371.1680, found 371.1684.
3-(4-(2-Hydroxyethoxy)-2-methoxyphenyl)-1-((3bS,4aR)-
3,4,4-trimethyl-3b,4,4a,5-tetrahydrocyclopropa[3,4]cyclo-
penta[1,2-c]thiophen-1-yl)propan-1-one (33). (a) A stirred
solution of (1aS,5aR)-1-(1,1,2-trimethyl-1,1a,5,5a-tetrahydro-3-thia-
cyclopropa[a]pen-talen-4-yl)-ethanone (535 mg, 1.70 mmol), 4-
hydroxy-2-methoxybenzaldehyde (335 mg, 2.20 mmol), and NaOH
(2.60 g, 65.0 mmol) in MeOH (25 mL) was heated to 70 °C for 4 h.
The mixture was diluted with water (150 mL) and extracted with
diethyl ether (75 mL). The pH of the aqueous phase was adjusted to
1
= 328.25 (calcd 328.14). H NMR (H6-DMSO, solvent suppression):
δ 9.35 (s, 1 H), 7.94 (t br, J = 5.6 Hz, 1 H), 7.09 (t, J = 7.8 Hz, 1 H),
6.64−6.71 (m, 2 H), 6.60 (d, J = 8.3 Hz, 1 H), 4.28 (d, J = 6.1 Hz, 2
H), 2.92 (dd, J1 = 18.6 Hz, J2 = 6.6 Hz, 1 H), 2.77 (d, J = 18.3 Hz, 1
H), 2.32 (s, 3 H), 1.84−2.02 (m, 2 H), 1.08 (s, 3 H), 0.68 (s, 3 H).
(b) To a solution of the above phenol (7.5 mg, 23 μmol) in MeOH
(1 mL) and 2 M aq NaOH (0.1 mL), a catalytic amount of NaI and 2-
bromoethanol (11.5 mg, 92 μmol) were added. The mixture was
stirred at 85 °C for 24 h before another portion of 2 M aq NaOH was
added, and the mixture was separated by preparative HPLC (Waters
M
dx.doi.org/10.1021/jm4014373 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
pH = 10 by adding saturated aq NH4Cl solution, and the solution was
extracted twice with DCM (2 × 70 mL). The combined organic
extracts were dried over MgSO4, filtered, and concentrated. The crude
product was separated by preparative HPLC to afford 3-(4-hydroxy-2-
methoxy-phenyl)-1-((1aS,5aR)-1,1,2-trimethyl-1,1a,5,5a-tetrahydro-3-
thia-cyclo-propa[a]pentalen-4-yl)-propenone (450 mg, 75%) as a
yellow solid. LC-MS: tR = 1.06 min, [M + 1]+ = 355.20 (calcd
355.14). 1H NMR (D6-DMSO): δ 10.15 (s, 1 H), 7.73 (d, J = 15.2 Hz,
1 H), 7.55 (d, J = 8.8 Hz, 1 H), 7.23 (d, J = 15.8 Hz, 1 H), 6.47−6.40
(m, 2 H), 3.84 (s, 3 H), 3.12 (dd, J = 6.4, 18.8 Hz, 1 H), 2.88 (d, J =
18.8 Hz, 1 H), 2.07 (s, 3 H), 2.02−1.93 (m, 2 H), 1.11 (s, 3 H), 0.70
(s, 3 H).
3.05−2.76 (m, 5 H), 2.39 (s, 3 H), 2.23 (s, 6 H), 1.94−1.87 (m, 2 H),
1.13 (s, 3 H), 0.72 (s, 3 H).
(c) A solution of 31 (10.6 g, 29.9 mmol) in isopropanol (100 mL)
and 3 N aq. NaOH (50 mL) was treated with epichlorohydrin (8.31 g,
89.8 mmol). The dark red reaction mixture was stirred at rt for 24 h.
The mixture was diluted with diethyl ether (300 mL) and washed with
saturated aq. NaHCO3 followed by water. The organic layer was dried
over MgSO4 and evaporated. The crude product was purified by CC
on silica gel eluting with heptane/EA 4:1 to give 3-(3,5-dimethyl-4-
(oxiran-2-ylmethoxy)phenyl)-1-((3bS,4aR)-3,4,4-trimethyl-3b,4,4a,5-
tetrahydrocyclopropa[3,4]cyclopenta[1,2-c]thiophen-1-yl)propan-1-
one (9.57 g, 78%, mixture of diastereoisomers) as an almost colorless
1
oil. LC-MS: tR = 1.16 min, [M + 1]+ = 411.17 (calcd 411.20). H
(b) To a solution of 3-(4-hydroxy-2-methoxy-phenyl)-1-
((1aS,5aR)-1,1,2-trimethyl-1,1a,5,5a-tetrahydro-3-thia-cyclo-propa[a]-
pentalen-4-yl)-propenone (443 mg, 1.25 mmol) in ethanol (30 mL)
was added Pd/C (450 mg, 10% Pd). The resulting suspension was
stirred at rt for 2 h under 1 atm H2. The mixture was filtered over
Celite, and the filtrate was evaporated. The residue was purified by
preparative HPLC to give 3-(4-hydroxy-2-methoxy-phenyl)-1-
((1aS,5aR)-1,1,2-trimethyl-1,1a,5,5a-tetrahydro-3-thia-cyclopropa[a]-
pent-alen-4-yl)-propan-1-one (162 mg, 36%) as a slightly yellow resin.
NMR (CDCl3): δ 6.85 (s, 2 H), 4.04−3.97 (m, 1 H), 3.77−3.69 (s, 1
H), 3.52−3.44 and 3.38−3.32 (2m, 1 H), 3.05−2.84 (m, 6 H), 2.79 (d,
J = 18.8 Hz, 1 H), 2.72−2.68 (m, 1 H), 2.38 (s, 3 H), 2.26 (s, 6 H),
1.92−1.86 (m, 2 H), 1.11 (s, 3 H), 0.71 (s, 3 H).
(d) A solution of 3-(3,5-dimethyl-4-(oxiran-2-ylmethoxy)phenyl)-1-
((3bS,4aR)-3,4,4-trimethyl-3b,4,4a,5-tetrahydrocyclopropa[3,4]cyclo-
penta[1,2-c]thiophen-1-yl)propan-1-one (4.55 g, 11.1 mmol) in
ethanol (50 mL) was treated with ethanolamine (2.71 g, 44.3
mmol) The reaction mixture was stirred at 65 °C for 3 h before it was
diluted with diethyl ether (400 mL) and washed twice with water (2 ×
200 mL). The aqueous washings were extracted back with diethyl
ether (400 mL). The combined organic extracts were dried over
MgSO4, filtered, and concentrated. The crude product was purified by
MPLC eluting with 90−50% water in MeOH. Product containing
fractions were extracted with diethyl ether. The organic extract was
dried over MgSO4, filtered, and evaporated to give the title compound
57 (3.51 g, 67%) as a pale yellow foam. LC-MS: tR = 0.88 min, [M +
1]+ = 472.27 (calcd 472.25). HPLC with chiral stationary phase
(Chiralpak AD-H 250 mm × 4.6 mm i.d., 5 μm; 50% ethanol, 50%
MeOH containing 0.05% DEA): tR = 6.7 min, 52%, tR = 8.0 min, 48%.
1H NMR (CDCl3): δ 6.85 (s, 2 H), 4.21−4.30 (m, 1 H), 3.81 (t, J =
1
LC-MS: tR = 1.06 min, [M + 1]+ = 357.10 (calcd 357.15). H NMR
(CD3OD): δ 6.88 (d, J = 8.2 Hz, 1 H), 6.37 (d, J = 2.3 Hz, 1 H), 6.26
(dd, J = 2.3, 8.2 Hz, 1 H), 3.76 (s, 3 H), 3.00−2.74 (m, 6 H), 2.04 (s, 3
H), 1.98−1.89 (m, 2 H), 1.12 (s, 3 H), 0.70 (s, 3 H).
(c) To a solution of the above phenol (80 mg, 0.225 mmol) in
isopropanol (7 mL) and 2 N aq NaOH (2.8 mL), NaI (10 mg) and 2-
bromoethanol (156 mg, 1.25 mmol) were added. The mixture was
stirred at 65 °C for 24 h before it was treated with acetic acid (0.3 mL)
and concentrated. The crude product was dissolved in MeOH (8 mL)
and purified by HPLC (Waters Xterra MS 18, 75 mm × 30 mm i.d., 10
μm, gradient of acetonitrile in water containing 0.74% diethylamine)
to give the title compound (81 mg, 90%) as a pale yellow resin. LC-
1
MS: tR = 1.06 min, [M + 1]+ = 401.18 (calcd 401.18). H NMR
5.0 Hz, 2 H), 3.77 (d, J = 5.2 Hz, 2 H), 3.55 (s br, 4 H), 2.85−3.07 (m,
9 H), 2.81 (d, J = 18.8 Hz, 1 H), 2.39 (s, 3 H), 2.24 (s, 6 H), 1.88−
1.93 (m, 2 H), 1.13 (s, 3 H), 0.72 (s, 3 H). 13C NMR (CDCl3): δ
191.9, 156.0, 153.4, 147.2, 136.9, 136.5, 130.6, 129.2, 128.9, 74.1, 68.6,
60.1, 51.7, 51.2, 42.2, 36.1, 29.9, 29.8, 29.7, 25.4, 22.7, 16.3, 14.4, 14.1.
LC-HRMS: tR = 1.52 min, [M + H]/z = 472.2521, found 472.2530.
N-((2R/S)-3-(2,6-Dimethyl-4-(3-oxo-3-((3bS,4aR)-3,4,4-tri-
methyl-3b,4,4a,5-tetrahydro-cyclopropa[3,4]cyclopenta[1,2-
c]thiophen-1-yl)propyl)phenoxy)-2-hydroxy-propyl)-2-hydrox-
yacetamide (64). (a) A solution of 3-(3,5-dimethyl-4-(oxiran-2-
ylmethoxy)phenyl)-1-((3bS,4aR)-3,4,4-trimethyl-3b,4,4a,5-tetra-
hydrocyclopropa[3,4]cyclopenta[1,2-c]thiophen-1-yl)propan-1-one
(800 mg, 1.95 mmol) in 6 N NH3 in MeOH (10 mL) was stirred at 60
°C for 4 h in a sealed vessel. The crude product was purified on
preparative TLC plates using DCM containing 15% of MeOH to give
3-(4-(3-amino-2-hydroxypropoxy)-3,5-dimethylphenyl)-1-((3bS,4aR)-
3,4,4-trimethyl-3b,4,4a,5-tetrahydro-cyclopropa[3,4] cyclopenta[1,2-
c]thiophen-1-yl)propan-1-one (554 mg, 66%) as a colorless foam.
LC-MS: tR = 0.87 min, [M + 1]+ = 428.05 (calcd 428.23).
(CDCl3): δ 7.06 (d, J = 8.2 Hz, 1 H), 6.47 (d, J = 2.3 Hz, 1 H), 6.41
(dd, J1 = 8.2 Hz, J2 = 2.3 Hz, 1 H), 4.05−4.09 (m, 2 H), 3.92−3.98 (m,
2 H), 3.81 (s, 3 H), 2.90−3.01 (m, 5 H), 2.79 (d, J = 18.8 Hz, 1 H),
2.38 (s, 3 H), 1.86−1.90 (m, 2 H), 1.12 (s, 3 H), 0.72 (s, 3 H). LC-
HRMS: tR = 2.11 min, [M + H]/z = 401.1786, found 401.1790.
(2R/S)-3-((2-Hydroxy-3-((2-hydroxyethyl)amino)propoxy)-
3,5-dimethylphenyl)-1-((3bS,4aR)-3,4,4-trimethyl-3b,4,4a,5-
tetrahydrocyclopropa[3,4]cyclopenta[1,2-c]thiophen-1-yl)-
propan-1-one (57). (a) A solution of 3,5-dimethyl-4-hydroxyben-
zaldehyde (2.21 g, 14.7 mmol) and 30 (2.70 g, 12.3 mmol) in ethanol
(50 mL) and approximately 6 N HCl in isopropanol (25 mL) was
stirred at rt for 90 min. The dark brown solution was diluted with
diethyl ether and washed with a 1:1 mixtue of 1 N aq NaOH and
saturated aq NaHCO3 solution and water. The aqueous phases were
extracted with diethyl ether. The combined organic extracts were dried
over MgSO4 and evaporated. The crude product was purified by CC
on silica gel eluting with heptane/EA 7:3 to give (E)-3-(4-hydroxy-3,5-
dimethylphenyl)-1-((3bS,4aR)-3,4,4-trimethyl-3b,4,4a,5-tetrahydrocy-
clo-propa[3,4]cyclopenta[1,2-c]thio-phen-1-yl)prop-2-en-1-one (3.28
(b) To a solution of the above amine (70 mg, 164 μmol) in DCM
g, 76%) as a yellow powder. LC-MS: tR = 1.12 min, [M + 1]+
=
(3 mL) was added Hunig’s base (85 mg, 655 μmol), TBTU (74 mg,
̈
1
353.31 (calcd 353.16). H NMR (CDCl3): δ 7.65 (d, J = 15.8 Hz, 1
H), 7.24 (s, 2 H), 7.06 (d, J = 15.8 Hz, 1 H), 5.08 (s, 1 H), 3.12 (dd, J
= 5.9, 18.8, 1 H), 2.95 (d, J = 18.8 Hz, 1 H), 2.41 (s, 3 H), 2.28 (s, 6
H), 1.96−1.89 (m, 2 H), 1.13 (s, 3 H), 0.75 (s, 3 H).
229 μmol), and glycolic acid (19 mg, 246 μmol), and the reaction
mixture was stirred at rt for 1 h before it was separated on preparative
TLC plates with DCM containing 10% of MeOH to give 64 (45 mg,
57%) as a pale yellow resin. LC-MS: tR = 0.98 min, [M + 1]+ = 486.04
(calcd 486.23). HPLC with chiral stationary phase (Chiralpak AD-H
250 mm × 4.6 mm i.d., 5 μm; 80% heptane containing 0.05% DEA,
20% ethanol containing 0.05% DEA): tR = 12.1 min, 47%, tR = 13.9
min, 53%. 1H NMR (CDCl3): δ 6.91 (t br, J = 5.0 Hz, 1 H), 6.86 (s, 2
H), 4.09−4.20 (m, 3 H), 3.68−3.85 (m, 3 H), 3.41−3.52 (m, 1 H),
3.19 (s br, 1 H), 2.84−3.01 (m, 5 H), 2.79 (d, J = 18.8 Hz, 1 H), 2.38
(s, 3 H), 2.24 (s, 6 H), 1.84−1.94 (m, 2 H), 1.11 (s, 3 H), 0.71 (s, 3
H). LC-HRMS: tR = 1.36 min, [M + H]/z = 486.2314, found
486.2318.
(b) A mixture of (E)-3-(4-hydroxy-3,5-dimethylphenyl)-1-
((3bS,4aR)-3,4,4-trimethyl-3b,4,4a,5-tetrahydrocyclopropa[3,4]cyclo-
penta[1,2-c]thiophen-1-yl)prop-2-en-1-one (3.0 g, 8.51 mmol) and
Pd/C (500 mg, 10% Pd) in ethanol (50 mL) and THF (50 mL) was
stirred at rt for 4 h under H2 (1.5 bar). The mixture was filtered, the
filtrate was evaporated, and the crude product was purified by CC on
silica gel eluting with heptane/EA 1:1 to give 3-(4-hydroxy-3,5-
dimethylphenyl)-1-((3bS,4aR)-3,4,4-trimethyl-3b,4,4a,5-tetrahydro-
cyclopropa[3,4]cyclopenta[1,2-c]thiophen-1-yl)propan-1-one 31 (3.0
g, 99%) as a yellow foam. LC-MS: tR = 1.11 min, [M + 1]+ = 355.33
(S)-3-(2,6-Dimethyl-4-(5-((3bS,4aR)-3,4,4-trimethyl-
3b,4,4a,5-tetrahydro-cyclopropa[3,4]cyclopenta[1,2-c]-
1
(calcd 355.17). H NMR (CDCl3): δ 6.84 (s, 2 H), 4.62 (s, 1 H),
N
dx.doi.org/10.1021/jm4014373 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
thiophen-1-yl)-1,2,4-oxadiazol-3-yl)phenoxy)-propane-1,2-
diol (78). (a) To dry MeOH (190 mL) was carefully added K-tert-
butylate (18.68 g, 166 mmol) followed by hydroxylamine hydro-
chloride (9.92 g, 143 mmol). The suspension was stirred for 30 min
before 3,5-dimethyl-4-hydroxybenzonitrile (7.00 g, 47.6 mmol) was
added. The mixture was refluxed for 32 h; then, the suspension was
diluted by adding 2 N aq HCl. The solution was extracted twice with
DCM (100 mL). The aqueous layer was basified (pH 9) by adding
solid NaHCO3 and extracted five times with DCM followed by four
times with EA. The combined organic layers were dried over Na2SO4
and evaporated to dryness to give 4,N-dihydroxy-3,5-dimethyl-
benzamidine (7.9 g, 92%) as a colorless solid. LC-MS: tR = 0.62
min, [M + 1]+ = 181.14 (calcd 181.10). 1H NMR (D6-DMSO): δ 9.25
(s, 1 H), 8.36 (s, 1 H), 7.21 (s, 2 H), 5.54 (s, 2 H), 2.14 (s, 6 H).
(b) A solution of 20 (3.00 g, 13.5 mmol), TBTU (4.77 g, 14.9
mmol), and DIPEA (7.62 mL, 44.5 mmol) in DMF (30 mL) was
stirred for 10 min at rt before 4,N-dihydroxy-3,5-dimethyl-
benzamidine (2.68 g, 14.9 mmol) was added. The solution was stirred
for further 20 min, formic acid (6 mL) was added, and the solution
was chromatographed by preparative HPLC (Grom-Sil 120 ODS-4-
HE, 30 mm × 75 mm, gradient of acetonitrile in water containing 0.5%
formic acid) to give 4-hydroxy-3,5-dimethyl-N-(((3bS,4aR)-3,4,4-
trimethyl-3b,4,4a,5-tetrahydrocyclopropa[3,4]cyclopenta[1,2-c]-
thiophene-1-carbonyl) oxy)benzimidamide (4.1 g, 79%) as a colorless
(b) A solution of 2-ethyl-6-methyl-phenol (8.40 g, 61.7 mmol) and
hexamethylene tetraamine (12.97 g, 92.5 mmol) in acetic acid (60
mL) and water (14 mL) was heated to 115 °C. The water was distilled
off at 117 °C and collected with a Dean−Stark apparatus. Then, the
water separator was replaced by a reflux condensor, and the mixture
was refluxed for 3 h. The mixture was cooled to rt, diluted with water
(100 mL), and extracted with EA. The organic extract was washed
with saturated aq NaHCO3, dried over MgSO4, and evaporated. The
remaining solid was dissolved in EA and treated with heptane to
initialize crystallization. The solid material was collected and dried to
give 3-ethyl-4-hydroxy-5-methyl-benzaldehyde (3.13 g, 31%) as a
colorless crystalline powder. 1H NMR (CDCl3): δ 9.83 (s, 1 H), 7.58−
7.53 (m, 2 H), 5.30 (s br, 1 H), 2.69 (q, J = 7.6 Hz, 2 H), 2.32 (s, 3
H), 1.28 (t, J = 7.6 Hz, 3 H).
(c) To an ice-cooled solution of 5-ethyl-4-hydroxy-3-methylbenzal-
dehyde (10.0 g, 60.9 mmol) in DCM (50 mL) and pyridine (15 mL),
trifluoromethanesulfonic acid anhydride (18.9 g, 67 mmol) was added
over a period of 20 min. Upon complete addition, the ice bath was
removed, and the reaction was stirred for a further 2 h at rt. The
mixture was diluted with DCM (150 mL), washed three times with
water, dried over MgSO4, filtered, and evaporated. The residue was
purified by flash chromatography on silica gel eluting with heptane/EA
9:1 to give trifluoro-methanesulfonic acid 2-ethyl-4-formyl-6-methyl-
phenyl ester (10.75 g, 60%) as a pale yellow oil. LC-MS: tR = 1.07 min.
1H NMR (CDCl3): δ 9.98 (s, 1 H), 7.70 (s, 1 H), 7.66 (s, 1 H), 2.85
(q, J = 10.1 Hz, 2 H), 2.48 (s, 3 H), 1.30 (t, J = 10.2 Hz, 3 H).
(d) To a stirred solution of the above triflate (10.7 g, 36.1 mmol) in
dry DMF (75 mL) was sequentially added triethylamine (7.3 g, 72.2
mmol), methyl acrylate (31.1 g, 361 mmol), DPPP (819 mg, 1.99
mmol), and Pd(OAc)2 (405 mg, 1.81 mmol) under nitrogen. The
mixture was stirred at 115 °C for 5 h, cooled to rt, diluted with diethyl
ether (350 mL), and washed twice with 1 N aq HCl and once with
saturated aq NaHCO3 solution. The organic extract was dried over
MgSO4, filtered, and evaporated. The residue was purified by flash
chromatography on silica gel eluting with heptane/EA 19:1 to give 3-
(2-ethyl-4-formyl-6-methyl-phenyl)-acrylic acid methyl ester (5.93 g,
71%) as a colorless liquid. LC-MS: tR = 0.99 min. 1H NMR (CDCl3):
δ 9.97 (s, 1 H), 7.83 (d, J = 16.4 Hz, 1 H), 7.60 (s, 1 H), 7.57 (s, 1 H),
6.09 (d, J = 16.4 Hz, 1 H), 3.84 (s, 3 H), 2.72 (q, J = 7.5 Hz, 2 H), 2.39
(s, 3 H), 1.22 (t, J = 7.5 Hz, 3 H).
1
solid. LC-MS: tR = 1.03 min, [M + 1]+ = 385.18 (calcd 385.16). H
NMR (D6-DMSO): δ 8.65 (s, 1 H), 7.30 (s, 2 H), 6.38 (s br, 2 H),
3.04(dd, J = 5.9, 18.8 Hz, 1 H), 2.75 (d, J = 18.8 Hz, 1 H), 2.36 (s, 3
H), 2.18 (s, 6 H), 2.01−1.88 (m, 2 H), 1.10 (s, 3 H), 0.70 (s, 3 H).
(c) A suspension of the above hydroxyamidine ester (4.0 g, 10.4
mmol) in toluene (400 mL) was stirred at 100 °C for 24 h before the
solvent was removed under reduced pressure. The residue was
dissolved in DCM and purified by CC on silica gel eluting with
hexane/EA 5:1 to give 71 (1.5 g, 39%) as a colorless solid. LC-MS: tR
1
= 1.37 min, [M + 1]+ = 367.13 (calcd 367.15). H NMR (CDCl3): δ
7.75 (s, 2 H), 4.90 (s, 1 H), 3.12 (dd, J = 5.9, 18.8 Hz, 1 H), 2.94 (d, J
= 18.8 Hz, 1 H), 2.44 (s, 3 H), 2.32 (s, 6 H), 2.02−1.94 (m, 2 H), 1.16
(s, 3 H), 0.77 (s, 3 H).
(d) To a solution of 71 (10 mg, 27 μmol) in isopropanol (1 mL), 3-
chloropropane-1,2-diol (15 mg, 135 μmol) and 2 N aq NaOH (0.2
mL) were added. The reaction mixture was shaken for 10 h at 65 °C
and then directly separated by preparative HPLC (Waters XTerra Prep
MS C18 19 mm × 50 mm, 5 μm, gradient of acetonitrile in water
containing 0.85% of diethylamine) to give 78 (6 mg, 50%) as a
colorless lyophilizate. LC-MS: tR = 1.02 min, [M + 1]+ = 440.88 (calcd
441.18). HPLC with chiral stationary phase (Chiralpak AD-H 250 mm
× 4.6 mm i.d., 5 μm; 50% ethanol, 50% MeOH containing 0.1% TFA):
tR = 10.3 min, 100%, ((R)-epimer: tR = 12.4 min). 1H NMR (CDCl3):
δ 7.78 (s, 2 H), 4.08−4.18 (m, 1 H), 3.78−3.92 (m, 4 H), 3.11 (dd, J1
= 6.0 Hz, J2 = 18.8 Hz, 1 H), 2.94 (d, J = 18.7 Hz, 1 H), 2.71 (d, J =
4.4 Hz, 1 H), 2.43 (s, 3 H), 2.35 (s, 6 H), 2.06 (s br, 1 H), 1.92−2.01
(m, 2 H), 1.15 (s, 3 H), 0.76 (s, 3 H). 13C NMR (CDCl3): δ 171.4,
168.2, 157.5, 156.7, 147.0, 135.2, 131.4, 128.4, 122.8, 111.0, 73.3, 71.0,
63.8, 36.4, 30.1, 28.8, 26.6, 22.9, 16.3, 14.5, 13.9. LC-HRMS: tR = 2.39
min, [M + H]/z = 441.1848, found 441.1848.
3-{2-Ethyl-6-methyl-4-[5-((1aS,5aR)-1,1,2-trimethyl-
1,1a,5,5a-tetrahydro-3-thia-cyclopropa[a]pentalen-4-yl)-
[1,2,4]oxadiazol-3-yl]-phenyl}-propionic Acid (85). (a) 2-Ethyl-
6-methylaniline (15.0 g, 111 mmol) was added to an ice-cold solution
of H2SO4 (150 mL) in water (250 mL). The solution was treated with
ice (150 g) before a solution of NaNO2 (10.7 g, 155 mmol) in water
(150 mL) and ice (50 g) was added dropwise. The mixture was stirred
at 0 °C for 1 h. 50% aq. H2SO4 (200 mL) was added, and stirring was
continued at rt for 18 h. The mixture was extracted with DCM, and
the organic extracts were dried over MgSO4 and evaporated. The
crude product was purified by CC on silica gel eluting with heptane/
EA 9:1 to give 2-ethyl-6-methyl-phenol (8.6 g, 57%) as a crimson oil.
LC-MS: tR = 0.89 min. 1H NMR (CDCl3): δ 7.03−6.95 (m, 2 H), 6.80
(t, J = 7.6 Hz, 1 H), 4.60 (s, 1 H), 2.64 (q, J = 7.6 Hz, 2 H), 2.25 (s, 3
H), 1.24 (t, J = 7.6 Hz, 3 H).
(e) A suspension of 3-(2-ethyl-4-formyl-6-methyl-phenyl)-acrylic
acid methyl ester (5.93 g, 25.53 mmol) in MeOH (140 mL) and 2 N
aq NaOH (45 mL) was stirred at rt for 1 h. The MeOH was
evaporated, and the aqueous solution was extracted twice with DCM.
The aqueous layer was acidified with 37% aq HCl. The precipitate that
formed was collected, washed with water, and dried. The product was
further purified by recrystallization from EA (100 mL) to give 3-(2-
ethyl-4-formyl-6-methyl-phenyl)-acrylic acid (4.2 g, 75%) as yellow
1
crystals. LC-MS: tR = 0.87 min. H NMR (CDCl3): δ 9.98 (s, 1 H),
7.97 (d, J = 16.4 Hz, 1 H), 7.62 (s, 1 H), 7.59 (s, 1 H), 6.14 (d, J =
16.4 Hz, 1 H), 2.75 (q, J = 7.5 Hz, 2 H), 2.42 (s, 3 H), 1.24 (t, J = 7.5
Hz, 3 H).
(f) To a solution of 3-(2-ethyl-4-formyl-6-methyl-phenyl)-acrylic
acid (2.75 g, 12.6 mmol) and DIPEA (1.8 g, 13.8 mmol) in ethanol
(80 mL), Pd/C (275 mg, 10% Pd, moistened with 50% water) was
added. The mixture was stirred for 16 h at rt under 1 atm of H2. The
catalyst was filtered off, and the filtrate was concentrated. The residue
was dissolved in EA, washed with 2 N aq HCl followed by 1 N aq HCl
and brine. The organic extract was dried over Na2SO4, filtered, and
evaporated to give 3-(2-ethyl-4-hydroxymethyl-6-methyl-phenyl)-
propionic acid (2.8 g, 99%) as a white solid. LC-MS: tR = 0.76 min.
1H NMR (CDCl3): δ 7.06 (s, 1 H), 7.04 (s, 1 H), 4.62 (s, 2 H), 2.97−
3.05 (m, 2 H), 2.67 (q, J = 7.5 Hz, 2 H), 2.47−2.55 (m, 2 H), 2.35 (s,
3 H), 1.24 (t, J = 7.5 Hz, 3 H).
(g) A solution of 3-(2-ethyl-4-hydroxymethyl-6-methyl-phenyl)-
propionic acid (2.8 g, 12.6 mmol) in acetic acid (50 mL) was treated
with MnO2 (3.9 g, 45.4 mmol), and the resulting mixture was stirred at
80 °C for 4 h. The mixture was filtered, and the filtrate was
concentrated. The crude product was purified by CC on silica gel
eluting with DCM to give 3-(2-ethyl-4-formyl-6-methyl-phenyl)-
O
dx.doi.org/10.1021/jm4014373 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
propionic acid (1.76 g, 63%) as a beige solid. LC-MS: tR = 0.86 min.
1H NMR (CDCl3): δ 9.93 (s, 1 H), 7.57 (s, 1 H), 7.53 (s, 1 H), 3.03−
3.12 (m, 2 H), 2.74 (q, J = 7.5 Hz, 2 H), 2.49−2.57 (m, 3 H), 2.43 (s,
3 H), 1.28 (t, J = 7.6 Hz, 3 H).
showing mean arterial blood pressure (MAP) recordings in
telemetrized SHR after administration of compound 85, and
heart rate recordings in SHR after administration of
compounds 1 and 57. This material is available free of charge
via the Internet at http://pubs.acs.org.
(h) A solution of 3-(2-ethyl-4-formyl-6-methyl-phenyl)-propionic
acid (1.67 g, 7.58 mmol) and hydroxylamine hydrochloride (780 mg,
11.36 mmol) in 1-methyl-2-pyrrolidone was heated to 80 °C for 30
min in the microwave (300 W, active cooling during irradiation). The
reaction mixture was diluted with diethyl ether and washed with water
and brine. The organic extract was dried over Na2SO4, filtered, and
evaporated to give 3-(4-cyano-2-ethyl-6-methyl-phenyl)-propionic acid
Accession Codes
The corresponding data set has been deposited at the
Cambridge Crystallographic Data Centre, 12 Union Road,
Cambridge CB2 1EZ, United Kingdom, http://www.ccdc.cam.
ac.uk/, under the following deposition number: 969024
(isomer of compound 17, see the Supporting Information).
1
(1.55 g, 94%) as a beige solid. LC-MS: tR = 0.89 min. H NMR (D6-
DMSO): δ 12.25 (s, 1 H), 7.45 (s, 2 H), 2.91−2.84 (m, 2 H), 2.67−
2.59 (m, 2 H), 2.35−2.30 (m, 5 H), 1.14 (t, J = 7.6 Hz, 3 H).
(i) Potassium tert-butoxide (2.71 g, 24.1 mmol) was carefully
dissolved in MeOH (25 mL). To this solution, hydroxylamine
hydrochloride (1.44 g, 20.7 mmol) followed by 3-(4-cyano-2-ethyl-6-
methyl-phenyl)-propionic acid (1.50 g, 6.90 mmol) dissolved in
MeOH (7.5 mL) was added. The mixture was refluxed for 8 h, and the
solvent was evaporated. The residue was dissolved in 2 N aq HCl and
extracted with EA. The pH of the aqueous phase was adjusted to pH 5
by adding saturated aq NaHCO3, and the mixture was extracted three
times with EA. The combined organic extracts were dried over
Na2SO4, filtered, evaporated, and dried to give 3-[2-ethyl-4-(N-
hydroxycarbamimidoyl)-6-methyl-phenyl]-propionic acid (1.4 g, 81%)
as a white solid. LC-MS: tR = 0.60 min, [M + 1]+ = 251.17 (calcd
251.14). 1H NMR (D6-DMSO): δ 9.48 (s br, 1 H), 7.33 (s, 1 H), 7.31
(s, 1 H), 5.72 (s br, 1 H), 2.83−2.89 (m, 2 H), 2.63 (q, J = 7.4 Hz, 2
H), 2.31−2.37 (m, 2 H), 2.30 (s, 3 H), 1.18 (t, J = 7.4 Hz, 3 H).
(j) To a solution of (1aS, 5aR)-1,1,2-trimethyl-1,1a,5,5a-tetrahydro-
3-thia-cyclopropa[a]pentalene-4-carboxylic acid 20 (270 mg 1.22
mmol) in DMF (3 mL), TBTU (390 mg, 1.22 mmol) and DIPEA
(518 mg, 4.0 mmol) were added. The reaction mixture was stirred at rt
for 5 min before a solution of 3-[2-ethyl-4-(N-hydroxycarbamimi-
doyl)-6-methyl-phenyl]-propionic acid (305 mg, 1.22 mmol) in DMF
(2 mL) was added. Stirring was continued at rt for 1 h. The mixture
was diluted with formic acid (0.5 mL) and acetonitrile (5 mL) and
separated by preparative HPLC (Grom-Sil 120 ODS-4-HE, 30 mm ×
75 mm, 10 μm, 10−95% acetonitrile in water containing 0.5% formic
acid) to afford (1aS,5aR)-1,1,2-trimethyl-1,1a,5,5a-tetrahydro-3-thia-
cyclopropa[a]pentalene-4-carboxylic acid N-(3-ethyl-5-methyl-4-(2-
carboxy-ethyl)-N-hydroxybenzamidine) ester (260 mg, 47%) as a
white solid. LC-MS: tR = 1.05 min, [M + 1]+ = 455.32.
AUTHOR INFORMATION
Corresponding Author
*Phone: +41 61 565 65 65; fax: +41 61 565 65 00; e-mail:
martin.bolli@actelion.com.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
The authors gratefully acknowledge their colleagues Cel
Bortolamiol, Maxime Boucher, Step
■
́
ine
́
hane Delahaye, Julien
Grimont, Hakim Hadana, Julie Hoerner, Benedikt Hofstetter,
Francois LeGoff, Heidemarie Kletzl, Daniel Leuenberger, Claire
Maciejasz, Celine Mangold, Katalin Menyhart, Matthias
̧
́
Merrettig, Christine Metzger, Christine Seeger, Jurgen Seifert,
̈
Virginie Sippel, Mireille Tena Stern, Marco Tschanz, Gaby von
Aesch, Daniel Wanner, and Aude Weigel for the excellent work
done and Martine Clozel and Walter Fischli for support.
ABBREVIATIONS USED
■
AcOH, acetic acid; AUC, area under the curve; BuLi, butyl
lithium; CC, column chromatography; CDI, carbonyldiimida-
zole; DCM, dichloromethane; DEAD, diethyl azodicarboxylate;
D M F , d i m e t h y l f o r m a m i d e ; D P P P , 1 , 3 - b i s -
(diphenylphosphino)propane; EA, ethyl acetate; EDC, 1-
ethyl-3-(3-dimethylamino-propyl)carbodiimide; HOBt, N-hy-
droxybenzotriazole; LC, lymphocyte count; LDA, lithium
diisopropyl amide; MeOH, methanol; NMO, N-methyl-
morpholine-N-oxide; PK, pharmacokinetics; PD, pharmacody-
namics; PPh3, triphenylphosphine; SAR, structure−activity
relationship; SHR, spontaneously hypertensive rats; TBTU,
O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetra-
fluoroborate; THF, tetrahydrofuran; S1P, sphingosine 1-
phosphate
(k) A suspension of (1aS,5aR)-1,1,2-trimethyl-1,1a,5,5a-tetrahydro-
3-thia-cyclopropa[a]pentalene-4-carboxylic acid N-(3-ethyl-5-methyl-
4-(2-(hydroxy-carboxy)-ethyl)-N-hydroxybenzamidine) ester (255 mg,
0.561 mmol) in toluene (10 mL) was heated to 85 °C for 24 h and
then at 105 °C for 3 days. The mixture was cooled to rt, and the
solvent was evaporated. The residue was dissolved in DMF and
separated by preparative HPLC (Grom-Sil 120 ODS-4-HE, 30 mm ×
75 mm, 10 μm, 70−100% acetonitrile in water containing 0.5% formic
acid) to afford title compound 85 (150 mg, 61%) as a white crystalline
1
solid. LC-MS: tR = 1.19 min, [M + 1]+ = 437.28 (calcd 437.19). H
REFERENCES
NMR (D6-DMSO): δ 12.26 (s, 1 H), 7.64 (s, 1 H), 7.63 (s, 1 H), 3.08
(dd, J = 6.4, 19.3 Hz, 1 H), 2.94−2.89 (m, 2 H), 2.84 (d, J = 18.2 Hz, 1
H), 2.69 (q, J = 7.6 Hz, 2 H), 2.41 (s, 3 H), 2.39−2.33 (m, 5 H), 2.05
(d, J = 5.8 Hz, 1 H), 1.99 (t, J = 5.8 Hz, 1 H), 1.19 (t, J = 7.6 Hz, 3 H),
1.11 (s, 3 H), 0.70 (s, 3 H). 13C NMR (D6-DMSO): δ 174.1, 171.1,
168.3, 156.9, 147.2, 143.2, 141.2, 137.6, 135.8, 126.9, 125.3, 124.3,
110.5, 36.2, 34.0, 29.9, 28.7, 26.6, 25.7, 24.6, 22.8, 19.9, 16.0, 14.8,
14.0. LC-HRMS: tR = 2.56 min, [M + H]/z = 437.1899, found
437.1896.
■
(1) Kappos, L.; Antel, J.; Comi, G.; Montalban, X.; O’Connor, P.;
Polman, C. H.; Haas, T.; Korn, A. A.; Karlsson, G.; Radue, E. W. Oral
Fingolimod (FTY720) for Relapsing Multiple Sclerosis. N. Engl. J.
Med. 2006, 355, 1124−1140.
(2) Kappos, L.; Radue, E.-W.; O’Connor, P.; Polman, C.; Hohlfeld,
R.; Calabresi, P.; Selmaj, K.; Agoropoulou, C.; Leyk, M.; Zhang-
Auberson, L.; Burtin, P. A Placebo-Controlled Trial of Oral
Fingolimod in Relapsing Multiple Sclerosis. N. Engl. J. Med. 2010,
362, 387−401.
(3) Cohen, J. A.; Chun, J. Mechanisms of Fingolimod’s Efficacy and
Adverse Effects in Multiple Sclerosis. Ann. Neurol. 2011, 69, 759−777.
(4) Brinkmann, V.; Davis, M. D.; Heise, C. E.; Albert, R.; Cottens, S.;
Hof, R.; Bruns, C.; Prieschl, E.; Baumruker, T.; Hiestand, P.; Foster, C.
A.; Zollinger, M.; Lynch, K. R. The Immune Modulator FTY720
Targets Sphingosine 1-Phosphate Receptors. J. Biol. Chem. 2002, 277,
21453−21457.
ASSOCIATED CONTENT
* Supporting Information
Experimental details on synthesis and characterization of all
target compounds and corresponding building blocks not
described in the main text are given; the structure of compound
17 as determined by single crystal X-ray analysis; figures
■
S
P
dx.doi.org/10.1021/jm4014373 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(5) Mandala, S.; Hajdu, R.; Bergstrom, J.; Quackenbush, E.; Xie, J.;
Milligan, J.; Thornton, R.; Shei, G.-J.; Card, D.; Keohane, C. A.;
Rosenbach, M.; Hale, J.; Lynch, C. L.; Rupprecht, K.; Parsons, W.;
Rosen, H. Alteration of Lymphocyte Trafficking by Sphingosine-1-
Phosphate Receptor Agonists. Science 2002, 296, 346−349.
(22) Berst, F.; Grosche, P.; Janser, P.; Zecri, F.; Bollbuck, B. N-Biaryl
(Hetero) Arylsulphonamide Derivatives Useful in the Treatment of
Diseases Mediated by Lymphocyte Interactions. WO2008028937 A1,
March 13, 2008.
(23) Tarrason
M. T.; Prats, N.; Domínguez, M.; Lop
́
, G.; Aulí, M.; Mustafa, S.; Dolgachev, V.; Domen
̀
ech,
(6) Billich, A.; Bornancin, F.; Dev
́
ay, P.; Mechtcheriakova, D.; Urtz,
́
ez, R.; Auilar, N.; Calbet, M.;
N.; Baumruker, T. Phosphorylation of the Immunomodulatory Drug
FTY720 by Sphingosine Kinases. J. Biol. Chem. 2003, 278, 47408−
47415.
Pont, M.; Milligan, G.; Kunkel, S. L.; Godessart, N. The Sphingosine-
1-phosphate Receptor-1 Antagonist, W146, Causes Early and Short-
Lasting Peripheral Blood Lymphophenia in Mice. Int. Immunopharma-
col. 2011, 11, 1773−1779.
(7) Sobel, K.; Menyhart, K.; Killer, N.; Renault, B.; Bauer, Y.; Studer,
R.; Steiner, B.; Bolli, M. H.; Nayler, O.; Gatfield, J. Sphingosine-1-
Phosphate (S1P) Receptor Agonists Mediate Pro-fibrotic Responses in
Normal Human Lung Fibroblasts via S1P2 and S1P3 Receptors and
Smad-Independent Signaling. J. Biol. Chem. 2013, 288, 14839−14851.
(8) Allende, M. L.; Dreier, J. L.; Mandala, S.; Proia, R. L. Expression
of the Sphingosine 1-Phosphate Receptor, S1P1, on T-cells Controls
Thymic Emigration. J. Biol. Chem. 2004, 279, 15396−15401.
(9) Matloubian, M.; Lo, C. G.; Cinamon, G.; Lesneski, M. J.; Xu, Y.;
Brinkmann, V.; Allende, M. L.; Proia, R. L.; Cyster, J. G. Lymphocyte
Egress from Thymus and Peripheral Lymphoid Organs Is Dependent
on S1P Receptor 1. Nature 2004, 427, 355−360.
(10) Hale, J. J.; Neway, W.; Mills, S. G.; Hajdu, R.; Keohane, C. A.;
Rosenbach, M.; Milligan, J.; Shei, G.-J.; Chrebet, G.; Bergstrom, J.;
Card, D.; Koo, G. C.; Koprak, S. L.; Jackson, J. J.; Rosen, H.; Mandala,
S. Potent S1P Receptor Agonists Replicate the Pharmacologic Actions
of the Novel Immune Modulator FTY720. Bioorg. Med. Chem. Lett.
2004, 14, 3351−3355.
(11) Jo, E.; Sanna, M. G.; Gonzalez-Cabrera, P. J.; Thangada, S.;
Tigyi, G.; Osborne, D. A.; Hla, T.; Parrill, A. L.; Rosen, H. S1P1-
Selective In Vivo-Active Agonists from High-Throughput Screening:
Off-the-Shelf Chemical Probes of Receptor Interactions, Signaling, and
Fate. Chem. Biol. 2005, 12, 703−715.
(12) McVerry, B. J.; Garcia, J. G. N. In vitro and in Vivo Modulation
of Vascular Barrier Integrity by Sphingosine 1-Phosphate: Mechanistic
Insights. Cell. Signalling 2005, 17, 131−139.
(13) Wei, S. H.; Rosen, H.; Matheu, M. P.; Sanna, M. G.; Wang, S.-
K.; Jo, E.; Wong, C.-H.; Parker, I.; Cahalan, M. D. Sphingosine 1-
Phosphate Type 1 Receptor Agonism Inhibits Transendothelial
Migration of Medullary T Cells to Lymphatic Sinuses. Nat. Immunol.
2005, 6, 1228−1235.
(14) Rosen, H.; Alfonso, C.; Surh, C. D.; McHeyzer-Williams, M. G.
Rapid Induction of Medullary Thymocyte Phenotypic Maturation and
Egress Inhibition by Nanomolar Sphingosine 1-Phosphate Receptor
Agonist. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 10907−10912.
(15) Sanna, M. G.; Wang, S.-K.; Gonzalez-Cabrera, P. J.; Don, A.;
Marsolais, D.; Matheu, M. P.; Wei, S. H.; Parker, I.; Jo, E.; Cheng, W.-
C.; Cahalan, M. D.; Wong, C.-H.; Rosen, H. Enhancement of Capillary
Leakage and Restoration of Lymphocyte Egress by a Chiral S1P1
Antagonist in Vivo. Nat. Chem. Biol. 2006, 8, 434−441.
(16) Marsolais, D.; Rosen, H. Chemical Modulators of Sphingosine-
1-phosphate Receptors as Barrier-Oriented Therapeutic Molecules.
Nat. Rev. Drug Discovery 2009, 8, 297−307.
(17) Hla, T.; Venkataraman, K.; Michaud, J. The Vascular S1P
GradientCellular Sources and Biological Significance. Biochim.
Biophys. Acta 2008, 1781, 477−482.
(24) Fujii, Y.; Ohtake, H.; Ono, N.; Hara, T.; Sakurai, T.; Takahashi,
S.; Takayama, T.; Fukasawa, Y.; Shiozawa, F.; Tsukahara, N.;
Hirayama, T.; Igarashi, Y.; Goitsuka, R. Lymphopenia Induced by a
Novel Selective S1P1 Antagonist Structurally Unrelated to S1P.
Biochim. Biophys. Acta 2012, 1821, 600−606.
(25) Fujii, Y.; Hirayama, T.; Ohtake, H.; Ono, N.; Inoue, T.; Sakurai,
T.; Takayama, T.; Matsumoto, K.; Tsukahara, N.; Hidano, S.; Harima,
N.; Nakazawa, K.; Igarashi, Y.; Goitsuka, R. Amelioration of Collagen-
Induced Arthritis by a Novel S1P1 Antagonist with Immunomodula-
tory Activities. J. Immunol. 2012, 188, 206−215.
(26) Foster, C. A.; Howard, L. M.; Schweitzer, A.; Persohn, E.;
Hiestand, P. C.; Balatoni, B.; Reuschel, R.; Beerli, C.; Schwartz, M.;
Billich, A. Brain Penetration of the Oral Immunomodulatory Drug
FTY720 and Its Phosphorylation in the Central Nervous System
during Experimental Autoimmune Encephalomyelitis: Consequences
for Mode of Action in Multiple Sclerosis. J. Pharmcol. Exp. Ther. 2007,
323, 469−476.
(27) Balatoni, B.; Storch, M. K.; Swoboda, E.-M.; Schonborn, V.;
̈
Koziel, A.; Lambrou, G. N.; Hiestand, P. C.; Weissert, R.; Foster, C. A.
FTY720 Sustains and Restores Neuronal Function in the DA Rat
Model of MOG-Induced Experimental Autoimmune Encephalomye-
litis. Brain Res. Bull. 2007, 74, 307−316.
(28) Dev, K. K.; Mullershausen, F.; Mattes, H.; Kuhn, R. R.; Bilbe,
G.; Hoyer, D.; Mir, A. Brain Sphingosine-1-phosphate Receptors:
Implication for FTY720 in the Treatment of Multiple Sclerosis.
Pharmacol. Ther. 2008, 117, 77−93.
(29) Miron, V. E.; Schubart, A.; Antel, J. P. Central Nervous System-
Directed Effects of FTY720 (Fingolimod). J. Neurol. Sci. 2008, 274,
13−17.
(30) Miron, V. E.; Hall, J. A.; Kennedy, T. E.; Soliven, B.; Antel, J. P.
Cyclical and Dose-Dependent Responses of Adult Human Mature
Oligodendrocytes to Fingolimod. Am. J. Pathol. 2008, 173, 1143−
1152.
(31) Chun, J.; Hartung, H.-P. Mechanism of Action of Oral
Fingolimod (FTY720) in Multiple Sclerosis. Clin. Neuropharmacol.
2010, 33, 91−101.
(32) Lee, C. W.; Choi, J. W.; Chun, J. Neurological S1P Signalling as
an Emerging Mechanism of Action of Oral FTY720 (Fingolimod) in
Multiple Sclerosis. Arch. Pharm. Res. 2010, 33, 1567−1574.
(33) Kipp, M.; Amor, S. FTY720 on the Way from the Base Camp to
the Summit of the Mountain: Relevance for Remyelination. Mult. Scler.
J. 2012, 18, 258−263.
(34) Awad, A. S.; Rouse, M. D.; Khutsishvili, K.; Huang, L.; Bolton,
W. K.; Lynch, K. R.; Okusa, M. D. Chronic Sphingosine 1 - Phosphate
1 Receptor Activation Attenuates Early-Stage Diabetic Nephropathy
Independent of Lymphocytes. Kidney Int. 2011, 79, 1090−1098.
(35) Miron, V. E.; Ludwin, S. K.; Darlington, P. J.; Jarjour, A. A.;
Soliven, B.; Kennedy, T. E.; Antel, J. P. Fingolimod (FTY720)
Enhances Remyelination Following Demyelination of Organotypic
Cerebellar Slices. Am. J. Pathol. 2010, 176, 2682−2694.
(36) Mattes, H.; Dev, K. K.; Bouhelal, R.; Barske, C.; Gasparini, F.;
Guerini, D.; Mir, A. K.; Orain, D.; Osinde, M.; Picard, A.; Dubois, C.;
Tasdelen, E.; Haessig, S. Design and Synthesis of Selective and Potent
Orally Active S1P5 Agonists. ChemMedChem 2010, 5, 1693−1696.
(37) Choi, J. W.; Gardell, S. E.; Herr, D. R.; Rivera, R.; Lee, C.-W.;
Noguchi, K.; Teo, S. T.; Yung, Y. C.; Lu, M.; Kennedy, G.; Chun, J.
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. 2011, 108, 751−756.
(18) Dorsam, G.; Graeler, M. H.; Seroogy, C.; Kong, Y.; Voice, J. K.;
Goetzl, E. J. Transduction of Multiple Effects of Sphingosine 1-
Phosphate (S1P) on T Cell Functions by the S1P1 G Protein-Coupled
Receptor. J. Immunol. 2003, 171, 3500−3507.
(19) Schwab, S. R.; Cyster, J. G. Finding a Way Out: Lymphocyte
Egress from Lymphoid Organs. Nat. Immunol. 2007, 8, 1295−1301.
(20) Pappu, R.; Schwab, S. R.; Cornelissen, I.; Pereira, J. P.; Regard, J.
B.; Xu, Y.; Camerer, E.; Zheng, Y.-W.; Huang, Y.; Cyster, J. G.;
Coughlin, S. R. Promotion of Lymphocyte Egress into Blood and
Lymph by Distinct Sources of Sphingosine-1-Phosphate. Science 2007,
316, 295−298.
(21) Sensken, S.-C.; Nagarajan, M.; Bode, C.; Graeler, M. H. Local
Inactivation of Spingosine 1-Phosphate in Lymph Nodes Induces
Lymphopenia. J. Immunol. 2011, 186, 3432−3440.
Q
dx.doi.org/10.1021/jm4014373 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(38) Hale, J. J.; Doherty, G.; Toth, L.; Mills, S. G.; Hajdu, R.;
Keohane, C. A.; Rosenbach, M.; Milligan, J.; Shei, G.-J.; Chrebet, G.;
Bergstrom, J.; Card, D.; Forrest, M.; Sun, S.-Y.; West, S.; Xie, H.;
Nomura, N.; Rosen, H.; Mandala, S. Selecting against S1P3 Enhances
the Acute Cardiovascular Tolerability of 3-(N-Benzyl)-
aminopropylphosphonic Acid S1P Receptor Agonists. Bioorg. Med.
Chem. Lett. 2004, 14, 3501−3505.
(39) Forrest, M.; Sun, S.-Y.; Hajdu, R.; Bergstrom, J.; Card, D.;
Doherty, G.; Hale, J.; Keohane, C.; Meyers, C.; Milligan, J.; Mills, S.;
Nomura, N.; Rosen, H.; Rosenbach, M.; Shei, G.-J.; Singer, I. I.; Tian,
M.; West, S.; White, V.; Xie, J.; Proia, R. L.; Mandala, S. Immune Cell
Regulation and Cardiovascular Effects of Sphingosine 1-Phosphate
Receptor Agonists in Rodents Are Mediated via Distinct Receptor
Subtypes. J. Pharmacol. Exp. Ther. 2004, 309, 758−768.
(40) Sanna, M. G.; Liao, J.; Jo, E.; Alfonso, C.; Ahn, M.-Y.; Peterson,
M. S.; Webb, B.; Lefebvre, S.; Chun, J.; Gray, N.; Rosen, H.
Sphingosine 1-Phosphate (S1P) Receptor Subtypes S1P1 and S1P3,
Respectively, Regulate Lymphocyte Recirculation and Heart Rate. J.
Biol. Chem. 2004, 279, 13839−13848.
(41) Salomone, S.; Potts, E. M.; Tyndall, S.; Ip, P. C.; Chun, J.;
Brinkmann, V.; Waeber, C. Analysis of Sphingosine 1-Phosphate
Receptors Involved in Constriction of Isolated Cerebral Arteries with
Receptor Null Mice and Pharmacological Tools. Br. J. Pharmacol.
2008, 153, 140−147.
Pharmacology & Therapeutics, March 18−21, 2009, Washington
DC, USA; ASCPT:Alexandria, VA.
(51) Gergely, P.; Wallstrom, E.; Nuesslein-Hildesheim, B.; Bruns, C.;
̈
́
Zecri, F. J.; Cooke, N. G.; Traebert, M.; Tuntland, T.; Rosenberg, M.;
Saltzman, M. Phase I Study with the Selective S1P1/S1P5 Receptor
Modulator BAF312 Indicates That S1P1 Rather Than S1P3 Mediates
Transient Heart Rate Reduction in Humans. 25th Congress of the
European Committe for Treatment and Research in Multiple Sclerosis
(ECTRIMS), Sept 9−12, 2009, Dusseldorf, Germany; p P437.
̈
́
(52) Wallstrom, E.; Gergely, P.; Nuesslein-Hildesheim, B.; Zecri, F.
̈
J.; Cooke, N. G.; Bruns, C.; Luttringer, O.; Sing, T.; Groenewegen, A.;
Rosenberg, M.; Saltzman, M. BAF312, a Selective S1P1/S1P5
Receptor Modulator, Effectively Reduces Absolute Lymphocyte
Counts in Human Volunteers and Demonstrates the Relevance of
S1P1 in Mediating a Transient Heart Rate Reduction. In 62nd Annual
Meeting of the American Academy of Neurology, Toronto, Ontario,
Canada, 2010; p PO5.052.
(53) Gergely, P.; Nuesslein-Hildesheim, B.; Guerini, D.; Brinkmann,
V.; Traebert, M.; Bruns, C.; Pan, S.; Gray, N. S.; Hinterding, K.;
Cooke, N. G.; Groenewegen, A.; Vitaliti, A.; Sing, T.; Luttringer, O.;
Yang, J.; Gardin, A.; Wang, N.; Crumb, W. J. J.; Saltzmann, M.;
Rosenberg, M.; Wallstrom, E. The Selective S1P Receptor Modulator
̈
BAF312 Redirects Lymphocyte Distribution and Has Species-Specific
Effects on Heart Rate: Translation from Preclinical to Clinical Studies.
Br. J. Pharmacol. 2012, 167, 1035−1047.
(42) Murakami, A.; Taksugi, H.; Ohnuma, S.; Koide, Y.; Sakurai, A.;
Takeda, S.; Hasegawa, T.; Sasamori, J.; Konno, T.; Hayashi, K.;
Watanabe, Y.; Mori, K.; Sato, Y.; Takahashi, A.; Mochizuki, N.;
Takakura, N. Sphingosine 1-Phosphate (S1P) Regulates Vascular
Contraction via S1P3 Receptor: Investigation Based on a New S1P3
Receptor Antagonist. Mol. Pharmacol. 2010, 77, 704−713.
(54) Brossard, P.; Derendorf, H.; Xu, J.; Maatouk, H.; Halabi, A.;
Dingemanse, J. Pharmacokinetics and Pharmacodynamics of Ponesi-
mod, a Selective S1P Receptor Modulator, in the First-in-Human
Study. Br. J. Clin. Pharmacol. [Online early access]. DOI 10.1111/
bcp.12129. Published Online: April 18, 2013. http://onlinelibrary.
wiley.com/doi/10.1111/bcp.12129/abstract;jsessionid=
BB7009BA3B1862F516180A38B14839B0.f03t03
(43) Saha, A. K.; Yu, X.; Lin, J.; Lobera, M.; Shardendu, A.; Chereku,
S.; Schutz, N.; Segal, D.; Marantz, Y.; McCauley, D.; Middleton, S.;
(55) Popov, S. A.; Denisov, A. Y.; Gatilov, Y. V.; Bagryanskaya, I. Y.;
Tkachev, A. V. Synthesis of New Chiral Heterocycles of the Pyrazole
and 2-Isoxazoline Types Form (+)-3-Carene. Tetrahedron: Asymmetry
1994, 5, 479−489.
(56) Popov, S. A.; Tkachev, A. V. Synthesis of 2-Alkyl and 2-Aryl
Pyrimidines from β-Chlorovinyl Ketones of Cyclopentanone Type.
Synth. Commun. 2001, 31, 233−243.
(57) Gajcy, K.; Librowski, T.; Lochynski, S. Concise Synthesis fo
Non-protein Amino Acid from Natural Product via Schmidt Reaction.
Lett. Org. Chem. 2009, 6, 186−188.
Siu, J.; Burli, R. W.; Buys, J.; Horner, M.; Salyers, K.; Schrag, M.;
̈
Vargas, H. M.; Xu, Y.; McElvain, M.; Xu, H. Benzofuran Derivatives as
Potent, Orally Active S1P1 Receptor Agonists: A Preclinical Lead
Molecule for MS. ACS Med. Chem. Lett. 2011, 2, 97−101.
(44) Lucke, S.; Levkau, B. Endothelial Functions of Sphingosine-1-
phosphate. Cell. Physiol. Biochem. 2010, 26, 87−96.
(45) Fryer, R. M.; Muthukumarana, A.; Harrison, P. C.; Nodop
Mazurek, S.; Chen, R. R.; Harrington, K. E.; Dinallo, R. M.; Horan, J.
C.; Patnaude, L.; Modis, L. K.; Reinhart, G. A. The Clinically-Tested
S1P Receptor Agonists, FTY720 and BAF312, Demonstrate Subtype-
Specific Bradycardia (S1P(1)) and Hypertension (S1P(3)) in Rat.
PLoS One 2012, 7, e52985.
(46) Gon, Y.; Wood, M. R.; Kiosses, W. B.; Jo, E.; Sanna, M. G.;
Chun, J.; Rosen, H. S1P3 Receptor-Induced Reorganization of
Epithelial Tight Junctions Compromises Lung Barrier Integrity and
Is Potentiated by TNF. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 9270−
9275.
(47) Hamada, M.; Nakamura, M.; Kiuchi, M.; Marukawa, K.;
Tomatsu, A.; Shimano, K.; Sato, N.; Sugahara, K.; Asayama, M.;
Takagi, K.; Adachi, K. Removal of Sphingosine 1-Phosphate Receptor-
3 (S1P3) Agonism Is Essential, but Inadequate to Obtain
Immunomodulating 2-Aminopropane-1,3-diol S1P1 Agonists with
Reduced Effect on Heart Rate. J. Med. Chem. 2010, 53, 3154−3168.
(48) Pan, S.; Gray, N.; Gao, W.; Mi, Y.; Fan, Y.; Wang, X.; Tuntland,
T.; Che, J.; Lefebvre, S.; Chen, Y.; Chu, A.; Hinterding, K.; Gardin, A.;
End, P.; Heining, P.; Bruns, C.; Cooke, N. G.; Nuesslein-Hildesheim,
B. Discovery of BAF312 (Siponimod), a Potent and Selective S1P
Receptor Modulator. ACS Med. Chem. Lett. 2013, 4, 333−337.
(49) Brinkmann, V. FTY720 (Fingolimod) in Multiple Sclerosis:
Therapeutic Effects in the Immune and Central Nervous System. Br. J.
Pharmacol. 2009, 158, 1173−1182.
(58) Gajcy, K.; Pekala, J.; Frackowiak-Wojtasek, B.; Librowski, T.;
Lochynski, S. Stereochemistry of Terpene Derivatives. Part 7: Novel
Rigidified Amino Acids from (+)-3-Carene Designed as Chiral GABA
Analogues. Tetrahedron: Asymmetry 2010, 21, 2015−2020.
(59) Appel, R. Tertiary Phosphane/Tetrachloromethane, a Versatile
Reagent for Chlorination, Dehydration, and P-N Linkage. Angew.
Chem., Int. Ed. 1975, 14, 801−811.
(60) Popov, S. A.; Tkachev, A. V. New Chiral Agents for Resolution
of Racemic cis-Permethric and cis-Z-Cyhalothric Acids. Tetrahedron:
Asymmetry 1995, 6, 1013−1018.
(61) Walkowicz, M.; Lochynski, S.; Walkowicz, C. Stereochemistry of
Bicyclo[3.1.0]hexane Derivatives. Part XII. Optically Active 6,6-
Dimethylbicyclo[3.1.0]hexan-2-ols. Pol. J. Chem. 1981, 55, 135−140.
(62) Malkov, A. V.; Pernazza, D.; Bell, M.; Bella, M.; Massa, A.;
Teply, F.; Meghani, P.; Kocovsky, P. Synthesis of New Chiral 2,2′-
Bipyridine Ligands and Their Application in Copper-Catalyzed
Asymmetric Allylic Oxidation and Cyclopropanation. J. Org. Chem.
2003, 68, 4727−4742.
(63) Boatman, D. P.; Schrader, T. O.; Semple, G.; Skinner, P. J.;
Jung, J.-K. Fused Pyrazole Derivatives and Methods of Treatment of
Metabolic-Related Disorders Thereof. WO2006069242, Jun 29, 2006.
(64) Boatman, P. D.; Schrader, T. O.; Kasem, M.; Johnson, B. R.;
Skinner, P. J.; Jung, J.-K.; Xu, J.; Cherrier, M. C.; Webb, P. J.; Semple,
G.; Sage, C. R.; Knudsen, J.; Chen, R.; Taggart, A. K.; Carballo-Jane,
E.; Richman, J. G. Potent Tricyclic Pyrazole Tetrazole Agonists of the
Nicotinic Acid Receptor (GPR109a). Bioorg. Med. Chem. Lett. 2010,
20, 2797−2800.
(50) Brossard, P.; Hofmann, S.; Cavallaro, M.; Halabi, A.;
Dingemanse, J. Entry-into-Humans Study with ACT-128800, a
Selective S1P1 Receptor Agonist: Tolerability, Safety, Pharmacoki-
netics, and Pharmacodynamics. Clin. Pharmcol. Ther. 2009, S63−S64,
PII-87; Annual Meeting of the American Society for Clinical
R
dx.doi.org/10.1021/jm4014373 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(65) Paine, J. B.; Brough, J. R.; Buller, K. K.; Erikson, E. E.
Regioselectivity of Pyrrole Synthesis from Diethyl Aminomalonate and
1,3-Diketones: Further Observations. J. Org. Chem. 1987, 52, 3986−
3993.
(66) Bolli, M.; Bur, D.; Clozel, M.; Fischli, W.; Lehmann, D.; Mathys,
B.; Mueller, C.; Nayler, O.; Scherz, M.; Weller, T. 4-Carbonyl
Substituted 1,1,2-Trimethyl-1A,4,5,5A-Tetrahydro-1H-4-Aza-
Cyclopropa[a]pentalene Derivatives as Agonists for the G-Protein
Coupled Receptor S1P1/EDG1 and Immunsuppressant Agents.
WO2005123677, Dec 29, 2005.
(81) Bolli, M. H.; Velker, J.; Muller, C.; Mathys, B.; Birker, M.; Bravo,
̈
R.; Bur, D.; de Kanter, R.; Hess, P.; Kohl, C.; Lehmann, D.; Meyer, S.;
Nayler, O.; Rey, M.; Scherz, M.; Steiner, B., unpublished work.
(82) Brockway, B. P.; Mills, P. A.; Azar, S. H. A New Method for
Continuous Chronic Measurement and Recording of Blood Pressure,
Heart Rate and Activity in the Rat via Radio-Telemetry. Clin. Exp.
Hypertens., Part A 1991, A13, 885−895.
́
(83) Guiol, C.; Ledoussal, C.; Surge, J.-M. A Radiotelemetry System
for Chronic Measurement of Blood Pressure and Heart Rate in the
Unrestrained Rat Validation of the Method. J. Pharmcol. Toxicol.
Methods 1992, 28, 99−105.
(67) Alberola, A.; Andres, J. M.; Gonzalez, A.; Pedrosa, R.; Vicente,
M. The Reaction of β-Aminoenones with α-Amino Derivatives.
Synthesis of 2-Functionalized Pyrroles. Heterocycles 1990, 31, 1049−
1058.
(84) Hess, P.; Clozel, M.; Clozel, J.-P. Telemetry Monitoring of
Pulmonary Arterial Pressure in Freely Moving Rats. J. Appl. Physiol.
1996, 81, 1027−1032.
(85) Grimont, J.; Le Goff, F.; Bourquin, G.; Silva, J. Quality
Assessment and Concentration Determination of Actelion’s Com-
pound Solutions by Automated LCMS and Quantitative NMR.
MipTec - The Leading European Event for Drug Discovery, Basel,
Switzerland, Sept 20−24, 2010.
(68) Hombrecher, H. K.; Horter, G. Synthesis of Pyrroles via Ethyl
N-(3-Oxo-1-alkenyl)glycinates. Synthesis 1990, 1990, 389−391.
(69) Alberola, A.; Andres, J. M.; Gonzalez, A.; Pedrosa, R.; Pradanos,
P. Regioselective Synthesis of 2-Functionalized Thiophenes by
Condensation of α-Mercapto Compounds with β-Amino Enone
Derivatives. Synth. Commun. 1990, 20, 2537−2547.
(70) Bolli, M.; Bur, D.; Clozel, M.; Fischli, W.; Lehmann, D.; Mathys,
B.; Mueller, C.; Nayler, O.; Scherz, M.; Weller, T. 1, 1A, 5, 5A-
Tetrahydro-3-thia-cycloprop[a] pentalenes: Tricyclic Thiophene De-
rivatives as S1P1/EDG1 Receptor Agonists. WO2006010544, Feb 2,
2006.
(71) Bolli, M.; Lehmann, D.; Mathys, B.; Mueller, C.; Nayler, O.;
Velker, J.; Weller, T. Novel Thiophene Derivatives. WO2006100635,
Sep 28, 2006.
(72) Diana, G. D.; Rudewicz, P.; Pevear, D. C.; Nitz, T. J.; Aldous, S.
C.; Aldous, D. J.; Robinson, D. T.; Draper, R.; Dutko, F. J.; Aldi, C.;
Gendron, G.; Oglesby, R. C.; Volkots, D. L.; Reuman, M.; Bailey, R.
R.; Czerniak, R.; Block, T.; Roland, R.; Oppermann, J. Picornavirus
Inhibitors: Trifluoromethyl Substitution Provides a Global Protective
Effect agains Hepatic Metabolism. J. Med. Chem. 1995, 38, 1355−
1371.
(73) Bolli, M.; Lehmann, D.; Mathys, B.; Mueller, C.; Nayler, O.;
Velker, J.; Weller, T. Novel Thiophene Derivatives as Sphingosine-1-
phosphate Receptor Agonists. WO2006100633, Sep 28, 2006.
(74) Rasmussen, P. B.; Pedersen, U.; Thomsen, I.; Yde, B.; Lawesson,
S.-O. Studies on Organophosphorus Compounds XLIX. An Improved
Method for the Preparation of 2,5-Disubstituted 1,3,4-Thiadiazoles
and 1,3,4-Thiadiazole-2(3H)-Thiones. Bull. Soc. Chim. Fr. 1985, 1985,
II/62−II/65.
(75) Kiryanov, A. A.; Sampson, P.; Seed, A. J. Synthesis of 2-Alkoxy-
Substituted Thiophenes, 1,3-Thiazoles, and Related S-Heterocycles via
Lawesson’s Reagent-Mediated Cyclization under Microwave Irradi-
ation: Applications for Liquid Crystal Synthesis. J. Org. Chem. 2001,
66, 7925−7929.
(76) Brain, C. T.; Paul, J. M. Rapid Synthesis of Oxazoles under
Microwave Conditions. Synlett 1999, 1999, 1642−1644.
(77) Holub, J. M.; O’Toole-Colin, K.; Getzel, A.; Argenti, A.; Evans,
M. A.; Smith, D. C.; Dalglish, G. A.; Rifat, S.; Wilson, D. L.; Taylor, B.
M.; Miott, U.; Glersaye, J.; Lam, K. S.; McCranor, B. J.; Berkowitz, J.
D.; Miller, R. B.; Lukens, J. R.; Krumpe, K.; Gupton, J. T.; Burnham, B.
S. Lipid-Lower Effects of Ethyl 2-Phenacyl-3-aryl-1H-pyrrole-4-
carboxylates in Rodents. Molecules 2004, 2004, 135−157.
(78) Hedegaard, B.; Mortensen, J. Z.; Lawesson, S.-O. Tetrahedron
1971, 27, 3853−3859.
́
(79) Zecri, F. J.; Albert, R.; Landrum, G.; Hinterding, K.; Cooke, N.
G.; Guerini, D.; Streiff, M.; Bruns, C.; Nuesslein-Hildesheim, B.
Pyrazole Derived from (+)-3-Carene; a Novel Potent, Selective
Scaffold for Sphingosine-1-phosphate (S1P1) Receptor Agonists.
Bioorg. Med. Chem. Lett. 2010, 20, 35−37.
(80) Bolli, M. H.; Abele, S.; Binkert, C.; Bravo, R.; Buchmann, S.;
Bur, D.; Gatfield, J.; Hess, P.; Kohl, C.; Mangold, C.; Mathys, B.;
Menyhart, K.; Muller, C.; Nayler, O.; Scherz, M.; Schmidt, G.; Sippel,
̈
V.; Steiner, B.; Strasser, D.; Treiber, A.; Weller, T. 2-Imino-thiazolidin-
4-one Derivatives as Potent, Orally Active S1P1 Receptor Agonists. J.
Med. Chem. 2010, 53, 4198−4211.
S
dx.doi.org/10.1021/jm4014373 | J. Med. Chem. XXXX, XXX, XXX−XXX