Novel S1P1 receptor agonists - Part 2: From bicyclo[3.1.0] hexane-fused thiophenes to isobutyl substituted thiophenes

Article abstract of DOI:10.1021/jm401456d

Previously, we reported on the discovery of a novel series of bicyclo[3.1.0]hexane fused thiophene derivatives that serve as potent and selective S1P₁ receptor agonists. Here, we discuss our efforts to simplify the bicyclohexane fused thiophene

Full text of DOI:10.1021/jm401456d

Article
pubs.acs.org/jmc
Novel S1P1 Receptor Agonists - Part 2: From Bicyclo[3.1.0]hexane-
Fused Thiophenes to Isobutyl Substituted Thiophenes
Martin H. Bolli,* Jorg Velker, Claus Muller, Boris Mathys, Magdalena Birker, Roberto Bravo, Daniel Bur,
̈
̈
Ruben de Kanter, Patrick Hess, Christopher Kohl, David Lehmann, Solange Meyer, Oliver Nayler,
Markus Rey, Michael Scherz, and Beat Steiner
Drug Discovery Chemistry, Actelion Pharmaceuticals Ltd., Gewerbestrasse 16, CH-4123 Allschwil, Switzerland
S
* Supporting Information
ABSTRACT: Previously, we reported on the discovery of a
novel series of bicyclo[3.1.0]hexane fused thiophene deriva-
tives that serve as potent and selective S1P1 receptor agonists.
Here, we discuss our efforts to simplify the bicyclohexane
fused thiophene head. In a first step the bicyclohexane moiety
could be replaced by a simpler, less rigid cyclohexane ring
without compromising the S1P receptor affinity profile of
these novel compounds. In a second step, the thiophene head
was simplified even further by replacing the cyclohexane ring
with an isobutyl group attached either to position 4 or position
5 of the thiophene. These structurally much simpler headgroups again furnished potent and selective S1P1 agonists (e.g., 87),
which efficiently and dose dependently reduced the number of circulating lymphocytes upon oral administration to male Wistar
rats. For several compounds discussed in this report lymphatic transport is an important route of absorption that may offer
opportunities for a tissue targeted approach with minimal plasma exposure.
be useful in a large number of pathological situations including
autoimmune diseases, graft versus host disease, asthma,
cardiovascular diseases, and cancer.30−32
INTRODUCTION
Sphingosine 1-phosphate (S1P, Figure 1) exerts a wealth of
cellular effects such as cell survival, proliferation, or migration
■
Of particular interest is that lymphocytes exposed to a
(synthetic) S1P1 receptor agonist are sequestered to lymphoid
organs and removed from circulation. This has first been
described for fingolimod (Figure 1), which is phosphorylated in
vivo to become a potent nonselective S1P1−5 receptor
agonist.33−40 The S1P concentration gradient41 that exists
between blood plasma and lymph is seen as the driving force
for T-lymphocytes to exit the lymph nodes and re-enter
systemic circulation.11,42,43 Activation of the S1P1 receptor is
thought to lead to receptor internalization and thus
desensitization of the cell toward the external S1P gradient.
The desensitized lymphocytes can no longer leave the lymph
node, and according to this model, the S1P1 agonist acts as a
“functional antagonist”. Indeed, recent studies demonstrated
that S1P1 antagonists are able to sequester lymphocytes from
circulation.44,45 By use of S1P1 receptor knockout mice46,47 and
S1P1 selective agonists,48−50 it has been demonstrated that
targeting S1P1 is sufficient to cause lymphocyte sequestration to
lymphoid organs. On the other hand, activation of the S1P3
pathway has been reported to cause heart rate reduction51−53
and vaso- and bronchoconstriction in rodents.17,54,55 It is
noteworthy, however, that in the rat selectivty against the S1P3
receptor is not sufficient for a compound to be devoid of effects
Figure 1. Structures of sphingosine 1-phosphate and fingolimod
(FTY720).
by acting intracellularly as well as extracellularly.1−8 The
extracellular signaling of S1P through five S1P receptors,
9
numbered S1P1 through S1P5, has been characterized in great
detail, and the large body of data has been summarized in
several review articles.7,10−13 The pharmacological consequen-
ces of the different S1P−S1P receptor signaling cascades have
been studied in many organs and tissues such as the nervous
system,14,15 the lung,16,17 the cardiovascular system,18−26 the
cells of the immune system,11,27,28 and bone tissue,29 and as a
consequence, S1P receptor modulators have been proposed to
Received: September 20, 2013
Published: December 17, 2013
© 2013 American Chemical Society
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Figure 2. Structures of the original HTS hit 1 and compounds 2, 3, and 4 as examples of novel lead series derived thereof.
on heart rate and that the experimental design (e.g., single dose
vs multiple dose regimen, conscious vs anesthetized animals)
impacts the study outcome.56,57 In fact, the recent study by
Fryer et al.57 suggests that in the rat, the observed heart rate
reduction is caused by S1P1 receptor agonism while the arterial
blood pressure increase is triggered by S1P3 activation. In
addition, there is growing evidence that the transient heart rate
reduction observed in humans is triggered by S1P1 receptor
activation.58−61
Scheme 1. Preparation of the Cyclohexane Fused Thiophen-
2-ylcarboxylic Acids 8−12 and Methyl Ketones 13−17
a
Today fingolimod is approved to treat patients suffering from
multiple sclerosis (MS),62−64 and several S1P1 agonists such as
ponesimod,65−68 siponimod (BAF312),69−71 ONO-4641
(structure undisclosed),72 CS-0777,73,74 KRP-203,75−77 RPC-
1063 (structure undisclosed),78 and MT-1303 (structure
undisclosed)79 entered clinical trials for MS and other diseases.
In addition, an overwhelming number of patents claiming novel
selective S1P1 receptor agonists have been published in the past
few years.80−83
In our previous account84 we described the discovery of a
novel series of selective S1P1 receptor agonists. Efforts aimed at
replacing the chemically unstable acylpyrazole moiety present
in the original HTS hit structure 1 (Figure 2) led to the
discovery of a series of bicyclo[3.1.0]hexane fused thiophene
derivatives (e.g., 2, Figure 2) with high affinity and selectivity
for the S1P1 receptor. In addition, several representatives of this
class of 3-carene derivatives not only exhibited good PK
properties but also efficiently reduced the number of circulating
lymphocytes in the rat. In this report we discuss our attempts to
simplify the structure of these S1P1 receptor agonists without
compromising their favorable pharmacological profile. First, we
considered opening of the 3-carene derived bicyclo[3.1.0]-
hexane moiety in 2 to a cyclohexane ring to give analogue 3
(Figure 2). Indeed, the cyclohexane fused thiophene 3 was as
potent on the S1P1 receptor (EC50 = 0.5 nM) as the
bicyclo[3.1.0]hexane derivative 2 (EC50 1.2 nM). In a second
step, the cyclohexane ring was opened to give the 4-
isopropylthiophene 4. This further simplified compound
showed an EC50 value of 2.7 nM for S1P1. On the basis of
these observations, we decided to explore the scope of the
cyclohexane fused and the isobutyl substituted thiophene
derivatives in detail.
a
Reagents and conditions: (a) ethyl formate, KOtBu, THF, rt, 1−24 h,
73−89% (distillation); (b) oxalyl chloride, DCM or CHCl3, 0−30 °C,
1−18 h, 81−89% crude; (c) NaOEt, HSCH2COOEt, EtOH, THF, rt,
1 h; (d) NaOEt, EtOH, 50−75 °C, 1−18 h; (e) 2 N aqueous LiOH or
NaOH, H2O, 70 °C, 2−3 h, 57−64% (over four steps); (f) tert- or sec-
BuLi, THF, iodoalkane, −78 to −70 °C, 2−18 h, 53−86%; (g) NIS,
CH3Cl/HOAc 1:1, rt, 24 h, 58%; or 8 as ethyl ester, Br2, AcOH, 50
°C, 4 h, 81%; (h) Cu(I) triflate, Cs2CO3, MeOH, 95 °C, 24 h (sealed
vessel), 16−58%; (i) MeLi, diethyl ether, 20−30 °C, 1−2 h, 17−80%.
tautomer) in good yield. Chlorination to the vinyl chloride 7
was effected by treating 6 with oxalyl chloride in chloroform.
The chloride 7 was then reacted using ethyl 2-mercaptoace-
tate,85 and subsequent cyclization and ester hydrolysis
produced the thiophenecarboxylic acid derivative 8 in good
yield. It is important to note that we occasionally observed
rapid, spontaneous decomposition of concentrated crude 7
when it was allowed to stand at room temperature. We
therefore did not purify crude 7 any further but immediately
subjected it to the substitution−cyclization sequence using
ethyl mercaptoacetate. Deprotonating 8 using an excess (2.3−
2.7 equiv) of tert- or sec-butyllithium at −70 to −78 °C gave the
corresponding dianion which was then reacted with the
appriopriate iodoalkane to yield the 5-alkylated thiophenecar-
boxylic acids 9−11. On the other hand, exposing 8 to N-
iodosuccinimide led to the corresponding 5-iodothiophene-2-
carboxylic acid derivative which could be transformed to the 5-
methoxythiophene 12 in an Ullmann-type reaction.86 Treating
the carboxylic acids 8−12 with methyllithium furnished the
RESULTS AND DISCUSSION
■
Synthesis. The synthesis of the 4,5,6,7-tetrahydrobenzo-
thiophene-1-carboxylic acid scaffold is analogous to the one of
the bicyclo[3.1.0]hexane fused thiophene we discussed in our
previous report84 (Scheme 1). Thus, 4,4-dimethyl-
cyclohexanone 5 is formylated with ethyl formate in THF in
the presence of potassium tert-butylate to give aldehyde 6 (or a
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a
Scheme 2. Preparation of 5-Trifluoromethylthiophen-2-yl Methyl Ketone Derivative 20
a
Reagents and conditions: (a) N,O-dimethylhydroxylamine HCl, TBTU, Hunig’s base, DMSO, DMF, rt, 2 h, 95%; (b) LDA, I , THF, −78 °C, 30
̈
2
min, 52%; (c) ClF2COOMe, CuI, KF, DMF, 135 °C, 4 h, 92%; (d) MeLi, diethyl ether, 20−30 °C, 1−2 h, 75%.
corresponding methyl ketones 13−17. The 5-trifluorome-
thylthiophene derivative 20 was prepared by first transforming
the carboxylic acid 8 to the corresponding Weinreb amide87
(Scheme 2). Subsequent electrophilic substitution of the
thiophene carbanion with iodine gave 5-iodothiophene 18
which was reacted with methyl chlorodifluoroacetate88 to
furnish 5-trifluoromethylthiophene 19 in good yield. Finally,
transforming the Weinreb amide to the corresponding methyl
ketone using methyl lithium established compound 20.
Scheme 4. Preparation of Target Compounds Incorporating
a [1,2,4]Oxadiazole Linker
a
The preparation of target compounds 21−33 is shown in
Scheme 3. First, the methyl ketone 14 was reacted with 4-
a
Reagents and conditions: (a) 3-ethyl-4,N-dihydroxy-5-methylbenza-
a
Scheme 3
midine, TBTU, DMF, rt, 18 h, then dioxane, 100 °C, 20 h, 59−71%;
(b) (R)- or rac-epichlorohydrin, isopropanol, 3 N aqueous NaOH, rt,
20 h, 53−84%; (c) 7 N NH3 in methanol, 65 °C, 18 h, 86%
quantitative (crude); (d) glycolic acid, TBTU, Hunig’s base, DCM, rt,
̈
2−4 h, 36−70%; (e) (for 44) (S)-N-(3-(2-ethyl-4-(N-hydroxycarba-
mimidoyl)-6-methylphenoxy)-2-hydroxypropyl)-2-hydroxyacetamide,
EDC, HOBt, DMF, rt, 24 h, then dioxane, 100 °C, 24 h, 6%.
(step a) and subsequent elaboration of the polar side chain
(steps b−d) produced the compounds 40−43. Alternatively, as
illustrated with the methoxythiophene derivative 44, the
thiophene-2-carboxylic acid can also be coupled and cyclized
with an N-hydroxybenzamidine building block already incor-
porating the glycolamide side chain.
a
Reagents and conditions: (a) 4-hydroxy-3,5-dimethylbenzaldehyde, 1
N HCl in 2-propanol, EtOH, rt, 1−2 h, 65−87%; (b) 1 bar H2, Pd/C,
EtOH, rt, 1−5 h, 78−96% (21); or 3-(4-formyl-2,6-dimethylphenyl)-
propenoic acid, NaOH, MeOH, rt, 3−18 h, 60−75%; (b) 10 bar H2,
Pd/C, EtOH, Hunig’s base, 50−65 °C, 2−4 d; 45−78% (33); (c) 2-
̈
bromoethanol, 3-bromopropanol or (R)- or (S)-3-chloropropane-1,2-
diol, 2 N aqueous NaOH, 2-propanol, 70 °C, 4−8 h, 57−74% (22−
The thiophenecarboxylic acids 59, 62, 66, 69, and 72 and the
thienyl methyl ketones 60, 63, 67, 70, and 73 required to
prepare target compounds 54−57 and 45−50 (Table 3),
respectively, were obtained following the pathway outlined in
Scheme 5. The precursor of 59, 4,5,6,7-tetrahydrobenzo[c]-
thiophene-1-carboxylic acid 58 (for target compounds 45 and
54), is commercially available. Preparation of 62 (for 47 and
55) relied on 4,4-diethylcyclohexanone 61.90,91 Spiro[2.5]-
octan-6-one 65 needed for the preparation of 66 (for 48 and
56) was prepared starting from acetal 64 following literature
procedures.92−94 The starting material for thiophenecarboxylic
acid 69 (for 49 and 57), spiro[4.5]decan-8-one 68, was also
prepared following literature procedures.91,95 4-Methylcyclo-
hexanone 71 needed for the synthesis of thiophene 72 (for 50)
is commercially available. The carboxylic acids 59, 62, 66, 69,
and 72 were then transformed to the corresponding ketones
60, 63, 67, 70, and 73, respectively, by treating them with
methyllithium. Scheme 6 outlines the synthetic access to 5-
hydroxytetrahydrobenzo[c]thiophenes 77 and 78 which served
as starting materials for the preparation of 51 and 52,
respectively. In brief, thiophene-2-carboxylic acid 74 was
obtained from cyclohexanone 64 following our standard
formylation−chlorination−substitution−cyclization−alkylation
sequence. Weinreb amide87 formation followed by acetal
25); or 22 or 23, methanesulfonyl chloride, Hunig’s base, DCM, rt, 1
̈
h, quantitative crude, then appropriate amine, Hunig’s base, DMF, 75
̈
°C, 7 h, 58−67% (26, 27, 28); or epichlorohydrin, 3 N aqueous
NaOH, 2-propanol, rt, 16−21 h, 57−91%, then 7 N NH3 in MeOH,
65 °C, 18 h (63−91%, 29), then glycolic acid, Hunig’s base, TBTU,
̈
DCM, rt, 1−2 h, 45−67% (3); or epichlorohydrin as for 3, then 2-
aminoethanol, β-alanine or azetidine-3-carboxylic acid, EtOH, H2O,
Hunig’s base, 85 °C, 5 h, 58−67% (30, 31, 32).
̈
hydroxy-3,5-dimethylbenzaldehyde or 3-(4-formyl-2,6-
dimethylphenyl)propenoic acid,89 and the corresponding
condensation product was hydrogenated to give phenol 21 or
carboxylic acid 33, respectively. Further elaboration of side
chains attached to phenol 21 as summarized in step c furnished
the target compounds 22−32. Starting from the appropriate
thiophene derivatives 13−17, the preparation of the corre-
sponding glycolamide derivatives 34−39 was carried out in
analogy to the pathway outlined in Scheme 3. The
corresponding oxadiazole analogues 40−44 were prepared as
shown in Scheme 4. Thus, coupling of the thiophenecarboxylic
acids 8−11 with 3-ethyl-4,N-dihydroxy-5-methylbenzamidine
followed by thermal dehydration to form the oxadiazole ring
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Scheme 5. Preparation of 5-Mono- or Disubstituted 3-Ethyl-4,5,6,7-tetrahydrobenzo[c]thiophene-1-carboxylic Acids and 1-(3-
a
Ethyl-4,5,6,7-tetrahydrobenzo[c]thiophen-1-yl)ethanones
a
Reagents and conditions: (a) tBuLi, THF, EtI, −100 to −70 °C, 2−18 h, 31−85%; (b) MeLi, diethyl ether, 20−30 °C, 1−2 h, 33−66%; (c) H2SO4,
rt, 24 h, 70%, then 1 bar of H2, Pd/C, EA, rt, 24 h, quantitative;90,91 (d) ethyl formate, KOtBu, THF, rt, 24 h; (e) oxalyl chloride, DCM or CHCl3,
0−30 °C, 1−18 h; (f) (1) NaOEt, HSCH2COOEt, EtOH, rt, 1 h; (2) NaOEt, EtOH, 65−75 °C, 18 h; (3) 2 N aqueous LiOH or NaOH, H2O, 70
°C, 2−3 h; 23−47% (over steps d, e, f, b); (g) (1) Ph3PMeBr, NaH, toluene, DMSO, rt, 1 h, quantitative (crude);92,94 (2) Et2Zn, CH2I2, toluene,
−40 °C to rt, 18 h, then TFA, H2O, rt, 30 min, 90% (crude);93,94 (h) (1) piperidine, ethanol, 85 °C, 24 h, then NaOAc, HOAc, 85 °C, 24 h, 63%;
(2) 1 atm of H2, Pd/C, EA, rt, 90 min, 97%.91,95
.
cleavage delivered ketone 75 which was then reduced to the
corresponding alcohol 76. Exposing Weinreb amides 76 and 75
to the methyl Grignard reagent produced methyl ketones 77
and 78, respectively. On the other hand, reductive amination of
ketone 75 with dimethylamine followed by Grignard reaction
furnished the racemic ketone 79 in good yield (steps k and l).
Starting from the appropriate thiophenyl methyl ketones and
thiophene-2-carboxylic acids shown in Schemes 5 and 6,
compounds 45−57 (Table 3) were prepared in analogy to the
reactions outlined in Schemes 3 and 4.
obtained. Treating this material with ethyl mercaptoacetate
delivered 4-isobutyl-5-methylthiophene-2-carboxylic acid 96
which could be further alkylated using tert-butyllithium
followed by methyl iodide to give the 3,5-dimethyl substituted
thiophene derivative 97. Finally, alkylation of thiophene-2-
carboxylic acid 98 using LDA or tert-butyllithium and an alkyl
halide to give the 5-alkylated thiophene-2-carboxylic acids 99−
102 was performed following literature procedures.96 The
target compounds 81−90 were then prepared in analogy to the
pathway shown in Scheme 4.
Synthesis of alkylated thiophene-2-carboxylic acids 94, 96,
97, and 99−102, which were used to prepare the target
compounds 81−90 (Table 5), is shown in Scheme 7.
Formylation of isopentyl methyl ketone 91 using ethyl formate
under basic conditions furnished enol 92 (or tautomer) which
was treated with oxalyl chloride at room temperature to give
vinyl chloride 93. Reacting 93 with ethyl mercaptoacetate in the
presence of a base furnished 4-isobutyl-3-methylthiophene-2-
carboxylic acid 94. Conversely, if ketone 91 was formylated
under Vilsmeyer conditions, the isomeric vinyl chloride 95 was
The target compounds 4 and 80 were prepared starting from
thiophenecarboxylic acids 97 and 102, respectively, in analogy
to the pathways outlined in Schemes 5 and 3.
In Vitro SAR Discussion. For the following discussion, the
compounds’ potencies were assessed using a GTPγS assay with
membranes from CHO cells expressing either the recombinant
S1P1 or S1P3 receptor. Table 1 compiles a first set of
compounds wherein the polar side chain attached to the phenyl
ring has been varied. As already observed in the 3-carene
derived series,84 a large variety of polar side chains was
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a
Scheme 6
a
Reagents and conditions: (a) ethyl formate, KOtBu, THF, rt, 24 h, 60−65%; (b) oxalyl chloride, CHCl3, rt, 30 min, 88% crude; (c) NaOEt,
HSCH2COOEt, EtOH, 30 °C, 2 h, 46%; (d) 1 N aqueous NaOH, EtOH, 70 °C, 1 h, 98%; (e) tBuLi, EtI, THF, −100 to −80 °C, 8 h, 50%; (f) N,O-
dimethylhydroxylamine HCl, TBTU, Hunig’s base, DMF, rt, 3 h, 92%; (g) 2 N aqueous HCl, THF, 40 °C, 2 h, 96%; (h) NaBH , MeOH, rt, 1 h,
quantitative crude; (i) MeMgBr, THF, rt, 18 h, 82%; (j) MeMgBr, THF, rt, 3 h, 40−45%; (k) HNMe2, NaBH(OAc)3, EtOH, rt, 18 h, 50−66%; (l)
MeMgBr, THF, rt, 3 h, 80% to quantitative (crude).
̈
4
a
Scheme 7. Preparation of Thiophene-2-carboxylic Acids
a
Reagents and conditions: (a) ethyl formate, KOtBu, THF, 0 °C to rt, 15 h, 66%; (b) oxalyl chloride, CHCl3, rt, 2 h, quantitative crude; (c) (1)
NaOEt, HSCH2COOEt, EtOH, rt, 15 h; (2) NaOEt, EtOH, 85 °C, 1 h; (3) 2 N aqueous LiOH, EtOH, rt, 48 h, 28−41%; (d) POCl3, DMF, 0 °C,
30 min, rt, 3 h, quantitative crude; (e) tBuLi, THF, MeI, −78 °C, 2 h, 65%; (f) LDA or tBuLi, THF, alkyl iodide or bromide, −78 °C, 2 h, 15−68%.
tolerated at the phenyl ring and the relative potency order was
preserved between the two series. In general, the cyclohexane
fused thiophene derivatives were slightly less potent on both
S1P1 and S1P3.
compound 30 by forming glycolamide 3 improved the
compound’s affinity for S1P1 and S1P3 significantly. Interest-
ingly, compounds 31 and 32 combining a basic nitrogen with a
carboxylic acid in their side chain again showed high affinity for
the S1P1 receptor and maintained good selectivity against S1P3.
The much shorter propanoic acid side chain in 33 led to a
reduced potency on both receptors.
In contrast to the carene derivatives, the 4,5,6,7-tetrahydro-
benzo[c]thiophene series allowed for easy synthetic access to a
variety of scaffolds with different 3-substituents (benzo[c]-
thiophene numbering). Therefore, the influence of the 3-
substituent could be studied in more detail. The SAR is
illustrated with a set of compounds incorporating a
“glycolamide” side chain and includes propanone (34−39) or
oxadiazole (40−44) linked derivatives (Table 2). In general,
Thus, while both glycerol derivatives 24 and 25 reached
single digit nanomolar activity with several-hundred-fold
selectivity against S1P3, the phenol 21 and the ethylene and
propylene glycol derivatives 22 and 23 showed a slightly
reduced affinity for S1P1. The amine 26 was more potent on
S1P1 when compared to its alcohol analogue 22. The opposite
trend was observed when the (racemic) amine 29 was
compared to the corresponding glycerol analogues 24 and
25. Adding a hydroxyethyl chain to amines 26 and 29
(compounds 27, 28, 30) reduced the compound’s affinity for
S1P1. On the other hand, masking the basic nitrogen in
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Table 1. SAR of Polar Side Chains in the 4-Position of the
Phenyl Ring
Table 2. SAR of the 3-Substituent at the 4,5,6,7-
Tetrahydrobenzo[c]thiophene
a
a
EC50
S1P1
EC50
S1P3
[nM]
EC50
S1P1
EC50
S1P3
[nM]
R
propanone [nM]
oxadiazole [nM]
H
34
35
36
37
38
39
4.8
1.2
0.6
1.3
1.3
1.3
5270
4418
141
40
41
42
43
b
1.7
1.5
4.6
20
314
122
8.9
11
methyl
ethyl
c
n-propyl
CF3
25
242
OCH3
3910
44
1.8
83
a
EC50 values as determined in a GTPγS assay using membranes of
CHO cells expressing either S1P1 or S1P3. EC50 values represent
geometric mean values of at least three independent measurements in
duplicate. All compounds with EC50 ≤ 1 μM behaved as full agonists
on S1P1 and S1P3 (Emax > 85%). For compounds with EC50 > 1 μM it
was not possible to determine whether or not they are full agonists
because no activity plateau was reached at the highest compound
concentration tested (10 μM). For details see Experimental Section.
b
c
Compound not prepared. Pure (S)-enantiomer only. Studies on
earlier series and close analogues of compounds discussed in this
account (cf. compounds 23 and 24) showed only little differences
between the two enantiomers with respect to S1P receptor affinity.
Several 4-substituted cyclohexanones are either commercially
available or easily accessible by short syntheses. Using these
cyclohexanones as starting materials in our standard thiophene
synthesis allowed for a rapid assembly of a collection of 4,5,6,7-
tetrahydrobenzo[c]thiophene scaffolds with a variety of
substituents in position 5. Compounds with either a propanone
or an oxadiazole linker were prepared. For the following SAR
discussion propapone derivatives with a glycerol side chain and
oxadiazoles incorporating a glycolamide side chain have been
compiled in Table 3. As already seen with the compounds in
Table 2, the propanone linked derivatives were clearly more
selective against S1P3. In the propanone series, removal of the
two 5-methyl groups (compound 45) led to a clear loss in
affinity for S1P1, while the same modification in the oxadiazole
linked series (compound 54) slightly improved the compound’s
potency on S1P1. The various 5-substituents showed little
difference on the S1P1 receptor affinity of propanones 46−50
and oxadiazoles 42 and 55−57. On the other hand, the affinity
for the S1P3 receptor increased with increasing size of the
substituents in position 5. As a consequence, the two diethyl
substituted compounds 47 and 55 are the least selective
representatives of the propanone and the oxadiazole series,
respectively. Interestingly, forming a ring between the two alkyl
groups in position 5 clearly improved the selectivity against
S1P3 (compare 48 with 46, 56 with 42, 49 with 47, and 57 with
55), and the cyclopropyl derivative 48 was the most selective
example of the propanone series. In the oxadiazole series, the
cyclopropyl derivative 56 was clearly more selective than its
dimethyl analogue 42 but did not reach the selectivity of the
unsubstituted compound 54. An alcohol function (compounds
a
EC50 values as determined in a GTPγS assay using membranes of
CHO cells expressing either S1P1 or S1P3. EC50 values represent
geometric mean values of at least three independent measurements in
duplicate. All compounds with EC50 ≤ 1 μM behaved as full agonists
on S1P1 and S1P3 (Emax > 85%). For compounds with EC50 > 1 μM it
was not possible to determine whether or not they are full agonists
because no activity plateau was reached at the highest compound
concentration tested (10 μM). For details see Experimental Section.
b
For comparison, for structure see Figure 2.
both linkers led to potent S1P1 agonists. The propanone linked
compounds were consistently more selective against S1P3. In
both linker series, the 3-unsubstituted compounds (34 and 40)
showed high affinity and more than 100-fold selectivity for the
S1P1 receptor. In the propanone linked series, increasing the
size of the 3-substituent from hydrogen to an n-propyl group
had little effect on the S1P1 receptor affinity while the potency
on S1P3 increased steadily. The affinity profile of the
trifluoromethyl derivative 38 resembled the one of the ethyl
analogue 36, while the methoxythiophene 39 showed almost
identical potencies as the methyl analogue 35. The oxadiazole
derivatives, too, gained potency on S1P3 with increasing chain
length of the 3-substituent. But they lost potency on S1P1 in
going from 40 to 43 making the n-propyl derivative 43 the least
selective compound of this set. As before, the affinity profile of
the methoxythiophene 44 was very similar to the one of the
methyl analogue 41.
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a
Table 3. SAR of the Substitutent at the Cyclohexane Ring
a
EC50 values as determined in a GTPγS assay using membranes of CHO cells expressing either S1P1 or S1P3. EC50 values represent geometric mean
values of at least three independent measurements in duplicate. All compounds with EC50 ≤ 1 μM behaved as full agonists on S1P1 and S1P3 (Emax
>
85%). For compounds with EC50 > 1 μM it was not possible to determine whether or not they are full agonists because no activity plateau was
b
c
reached at the highest compound concentration tested (10 μM). For details see Experimental Section. Compound not prepared. As an
approximately 1:1 mixture of epimers.
51 and 52) or a dimethylamino group (compound 53) in
position 5 of the tetrahydrobenzo[c]thiophene scaffold was
obviously not tolerated.
Knowing that opening of the bicyclo[3.1.0]hexane system (as
in 2) to a cyclohexane yielded highly potent and selective S1P1
agonists, we then studied further simplifications of the
cyclohexane fused thiophene moiety. We therefore prepared a
series of compounds wherein the cyclohexane ring was opened
(Figure 3). The compounds compiled in Table 4 and Table 5
substituent to position 5 and omitting additional methyl groups
at the thiophene head to give compound 80 reduced the
compound’s affinity for S1P1 only slightly and increased the
potency on S1P3. In the oxadiazole linked series (Table 5), the
bicyclo[3.1.0]hexane 81 and the cyclohexane 82 were highly
potent on S1P1. The exact ring-opened analogue of cyclohexane
82, compound 83, was slightly less active on both receptors
than compound 82. Replacing the ethyl group in R3 by a
methyl group (84) or a hydrogen (86) restored the
compound’s affinity for S1P1 and reduced the potency on
S1P3. On the other hand, omitting the 5-methyl group (R1) in
84 to give compound 85 not only enhanced the potency on
S1P1 but also significantly improved the selectivity against S1P3,
a trend already observed in the cyclohexane fused thiophene
series (see Table 2). On the other hand, removing the 3-methyl
group in 84 to give 86 had a less pronounced effect on the
compound’s affinity profile. In contrast to the observations with
the two propanone derivatives 80 and 4, the 5-isobutyl-
thiophene 87 retained the potency on S1P1 and lost affinity for
S1P3 when compared to the 4-isobutylthiophene 86. While the
5-n-butyl and the 5-ethyl analogues 88 and 90, respectively,
were both less potent on S1P1 and S1P3, the 5-n-propyl
substituted thiophene 89 is comparable to 87 on S1P1 but more
selective against S1P3. In brief, the data on the oxadiazoles in
Table 5 illustrate that while the S1P1 receptor tolerates a large
Figure 3. Simplifying the bicyclo[3.1.0]hexane fused thiophene head.
illustrate our findings. In the propanone linked series (Table 4),
the carene derivative 2 and the cyclohexane fused thiophene 3
showed a nearly identical affinity profile for S1P1 and S1P3.
Also the 4-isobutyl-3,5-dimethylthiophene 4 was almost
identical in potency on S1P1. This compound, however, was
clearly more selective against S1P3 when compared to its
(bi)cyclic analogues 2 and 3. Interestingly, moving the isobutyl
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oxadiazole analogue 42 to illustrate the typical PK and PD
behavior of the two subseries. In vitro and in vivo PK data of
the two compounds are listed in Table 6. In rat liver
microsomes and hepatocytes propanone 36 showed a much
higher in vitro clearance than oxadiazole 42. Similarly, a marked
difference in in vivo clearance was observed between the two
compounds upon iv administration of 1 mg/kg to Wistar rats.
As both compounds had a comparable volume of distribution
(Vss), plasma concentrations of compound 36 dropped with a
half-life (t1/2) of 1.7 h while compound 42 showed a half-life of
21 h. The high clearance observed with propanone 36 results in
low exposure (Cmax, AUC0−24h) after oral dosing (Table 6).
At this point, we decided to compare the pharmacodynamic
behavior of compounds 36 and 42 with their respective PK
profile. Hence, the compounds were administered at a dose of
10 mg/kg to male Wistar rats and the blood lymphocyte count
(LC) was measured shortly before and 3, 6, and 24 h after
compound administration. A LC reduction of ≥60% was
considered to be the maximal effect (plateau) to be observed
under the conditions of the experiment.65 The different
pharmacokinetic properties of the two compounds clearly
reflected their pharmacodynamic behavior (Figure 5). While
the highly potent propanone 36 maximally reduced LC at 3 and
6 h (plasma concentrations of 32 and 10 ng/mL, respectively)
and showed complete recovery of the LC at 24 h (plasma
concentratoin of <1.5 ng/mL) after compound administration,
the less potent oxadiazole 42 showed maximal LC reduction for
at least 24 h (plasma concentration at 24 h of 530 ng/mL) and
a still significant effect on LC after 3 and 6 days (plasma
concentrations of 236 and 66 ng/mL, respectively) because of
significantly higher plasma concentrations.
In two recent studies, compounds that did not produce a
sustained 24 h maximal LC reduction after single dose
administration have been shown to be efficacious in the
mouse EAE model of multiple sclerosis, suggesting that
sustained maximal LC reduction is not mandatory to obtain
in vivo efficacy in this model.97,98 On the other hand, in clinical
studies transient heart rate reduction after oral dosing of
(selective) S1P1 receptor agonists has been observed.58−61
Model studies in guinea pigs suggested that the S1P1 receptor is
rapidly desensitized upon activation by a S1P1 receptor agonist
and sustained plasma concentrations of the agonist protect the
animal from second dose effects on heart rate.99 A sustained 24
h plasma exposure, and as a consequence a sustained LC count
reduction in the rat, therefore appeared desirable from a
compound safety and efficacy perspective. Bearing this in mind,
we studied the in vivo efficacy of the oxadiazole derivatives 81−
90 (Table 5) at an oral dose of 10 mg/kg. All compounds
showed a rapid onset of action, and with the exception of
compound 83, all compounds reached maximal LC reduction 3
h after compound administration. The duration of action,
however, varied significantly between the various thiophene
derivatives. While compound 81 maximally reduced LC for at
least 24 h and complete recovery of LC was observed at 96 h,
cyclohexane derivative 82 fully sequestered circulating
lymphocytes for at least 72 h and LC recovery was seen after
168 h only. The 4-isobutyl substituted thiophenes 84, 85, and
86 maximally reduced LC for at least 24 h. Rapid reversibility of
the LC reduction could be observed when compound 85 was
administered at a lower dose of 3 mg/kg. At this dose, LC was
maximally reduced at 6 h but was already close to baseline
values 24 h after compound administration.
Table 4. SAR of Substitution Pattern around the Thiophene
Ring in the Propanone Series
a
a
EC50 values as determined in a GTPγS assay using membranes of
CHO cells expressing either S1P1 or S1P3. EC50 values represent
geometric mean values of at least three independent measurements in
duplicate. All compounds with EC50 ≤ 1 μM behaved as full agonists
on S1P1 and S1P3 (Emax > 85%). For compounds with EC50 > 1 μM it
was not possible to determine whether or not they are full agonists
because no activity plateau was reached at the highest compound
concentration tested (10 μM). For details see Experimental Section.
b
Compound represents a 1:1 mixture of epimers with respect to polar
c
side chain. Compound represents racemate.
variety of alkyl substituted thiophene heads, the S1P3 receptor
is clearly more susceptible to changes in this part of the
molecule. This is most impressively demonstrated by
comparing the cyclohexane fused thiophene derivative 82
with the 5-propyl substituted analogue 89. The large structural
difference between these two compounds has no effect on S1P1
affinity but evokes an 80-fold potency shift on S1P3. The EC50
values for the propanone derivatives compiled in Table 4
confirm earlier observations that in this series the affinity
toward the S1P3 receptor is consistently lower than in the
oxadiazole series.
The similarity of the various thiophene headgroups becomes
apparent when the X-ray crystal structures of the corresponding
thiophene-2-carboxylic acids 103, 9, and 97 are superimposed
(Figure 4). The substituents decorating the three thiophene-2-
carboxylic acids assume conformations that overlap consid-
erably suggesting overall very similar steric demands and space
filling properties of these thiophene derivatives. While 103
comprises literally no degree of rotational freedom, cyclohexane
9 is more flexible because the ring pucker in 9 could switch,
leading to a minor change in space filling. In 97 two preferred
orientations of the isobutyl side chain (above or below the
thiophene plane) are conceivable; however, the two neighbor-
ing methyl groups constrain its free rotation.
In Vivo Studies. In the course of establishing the SAR on
the S1P1 and S1P3 receptor, we also studied the DMPK
properties of selected compounds. In vitro metabolic stability
data showed a clear difference between the propanone and the
oxadiazole linked compounds, and we anticipated that the
propanone linked derivatives would be clearly inferior with
respect to pharmacokinetics (PK) and in particular pharmaco-
dynamics (PD). We therefore selected compound 36 as the
most potent representative of the propanone series and its
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a
Table 5. SAR of Substitution Pattern around the Thiophene Ring in the Oxadiazole Series
a
EC50 values as determined in a GTPγS assay using membranes of CHO cells expressing either S1P1 or S1P3. EC50 values represent geometric mean
values of at least three independent measurements in duplicate. All compounds with EC50 ≤ 1 μM behaved as full agonists on S1P1 and S1P3 (Emax
>
85%). For compounds with EC50 > 1 μM it was not possible to determine whether or not they are full agonists because no activity plateau was
b
reached at the highest compound concentration tested (10 μM). For details see Experimental Section. Compound represents 1:1 mixture of
c
d
e
f
epimers at polar side chain. Racemic compound. Lymphocyte counts in Wistar rats receiving 10 mg/kg compound po. +30% at 96 h. −73% at 72
g
h
i
h, −7% at 144 h. After administration of 3 mg/kg. +14% at 72 h. In vivo data of pure (S)-enantiomer.
a
Table 6. Rat PK Data of Propanone 36 and Oxadiazole 42
parameter
Experiment: In Vitro
36
42
intrinsic clearance, rat microsomes [(μL/min)/mg]
rat hepatocytes [(μL/min)/106 cells]
Experiment: In Vivo, 1 mg/kg iv
clearance in vivo [(mL/min)/kg]
Vss [L/kg]
>1250
107
0
3.1
46
2.0
3.5
21
2.8
1.7
t1/2 in vivo [h]
Experiment: In Vivo, 10 mg/kg po
AUC0−24h [ng·h/mL]
Cmax [ng/mL]
276
141
0.5
8
28100
1450
6.0
Tmax [h]
b
bioavailability F [%]
>33
a
PK parameters obtained in Wistar rats after oral administration of 10
Figure 4. Structures (a) and superposition of the X-ray structures (b,
c) of the three thiophene-2-carboxylic acids 103 (red), 9 (yellow), and
97 (blue) using the thiophene ring as the anchor unit.
mg/kg and iv administration of 1 mg/kg of either 36 or 42; for details
see Experimental Section. Bioavailability cannot be determined
accurately, as sampling time was up to 24 h only.
b
In the series of the 5-alkylthiophenes 87−90, only the
isobutyl derivative 87 was able to maximally sequester blood
lymphocytes for 24 h. The n-butyl (88), the n-propyl (89), and
the ethyl (90) thiophenes all showed complete recovery of LC
24 h after compound administration. This observation could be
rationalized on the basis of the compounds’ plasma exposures.
For instance, while the isopropylthiophene 87 showed plasma
concentrations of 1010, 680, and 23 ng/mL at 3, 6, and 24 h
after oral administration, respectively, the concentrations of the
ethyl analogue 90 were 490, 190, and <2 ng/mL at these time
points.
Lymphocytes leave lymph nodes by migrating along a
gradient of S1P that exists between plasma and lymph.
Synthetic S1P1 receptor agonists lead to S1P1 receptor
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Figure 5. Lymphocyte counts measured after administration of 10 mg/kg of either ketone 36 (A) or oxadiazole 42 (B) to male Wistar rats. Note the
different range of the time axis for the two graphs.
internalization and thus abolish the lymphocyte’s ability to
sense the S1P gradient and to leave the lymph node. The
concentration of the synthetic S1P1 agonist in lymph can be
expected to influence the agonist’s pharmacodynamic behav-
ior.11,42,43 We thus extended the above rat PK studies and
measured the concentration of compound 42 in mesenteric
lymph fluid that was collected over periods of 20 min at several
time points after oral administration of 10 mg/kg compound
42. In the middle of each lymph collection period, a plasma
sample was drawn as well. The compound concentrations
summarized in Table 7 demonstrate that for the first few hours
lipophilicity is a clear driver of lymphatic transport.100,101 The
high lipophilicity (log D = 4.3) of compound 42 and its lipid-
like structure may thus explain its propensity for high lymphatic
transport. Prompted by this interesting observation, the
lymphatic route of absorption was studied for a few closely
related analogues of amide 42 (Table 8, EC50 values on S1P1
and S1P3 are given for completeness). Not surprisingly, the
more lipophilic diol 104 (log D = 5.4) showed a very similar
behavior. The compound was rapidly absorbed into mesenteric
lymph, reaching a Cmax that is remarkably higher than in plasma.
Mesenteric lymph exposure was again significantly higher than
plasma exposure. Despite their ionic/zwitter ionic nature, the
acid 105 and the amino acid 106 appear rather lipophilic (log D
of 3.6 and 4.9, respectively) and showed significant lymphatic
transport as well.
Table 7. Concentration of Compound 42 in Plasma and
Mesenteric Lymph after Oral Administration of 10 mg/kg to
Wistar Rats
42 concentration [ng/mL]
CONCLUSIONS
a
■
sample time [h]
plasma
mesenteric lymph
By replacing the bicyclo[3.1.0]hexane by a cyclohexane or an
isobutyl group, we were able to reduce the structural
complexity and rigidity of our S1P1 agonists such as 2 without
compromising their in vitro potency and in vivo efficacy. For
these novel thiophene derivatives the SARs of the polar side
chain and the linker between the thiophene and the phenyl ring
bearing the polar side chain were similar to those previously
reported for the bicyclo[3.1.0]hexane fused thiophene series.84
The easier and much more versatile access to the current
thiophene headgroups, however, allowed detailed SAR studies
that were not available for the (+)-3-carene derived
bicyclo[3.1.0]hexane fused thiophenes. For instance, the
influence of the substituent in position 5 of the thiophene as
well as of the two methyl groups attached to the cyclohexane
ring could be studied easily. In the cyclohexane fused thiophene
series, the 5-substituent strongly influenced the compound’s
affinity for the S1P3 receptor. With increasing length of this
alkyl chain the compounds became less selective for the S1P1
receptor. For instance, attaching an n-propyl group to position
5 of the thiophene head furnished dual S1P1/S1P3 receptor
agonists (e.g., 43). The affinity for the S1P3 receptor also
increased with increasing size of the substituents attached to the
cyclohexane ring, and the two diethyl substituted compounds
47 and 55 were the least selective representatives in the
propanone and the oxadiazole series, respectively. These
observations illustrate that the S1P1 receptor is rather tolerant,
while the S1P3 receptor is much more sensitive toward changes
in size and shape of the thiophene headgroup. As observed
earlier,84 the linker between the thiophene head and the phenyl
1.0
1.7
2.3
3.0
6.0
24.8
44.4
58.1
123
132
911
680
5790
4340
4600
8090
4590
684
a
Mean of collection interval given for mesenteric lymph.
the concentration of compound 42 in mesenteric lymph fluid
exceeded the one in plasma by far, indicating that there is
significant lymphatic transport of the compound after oral
dosing. The compound was quickly absorbed into mesenteric
lymph, reaching a concentration of nearly 6000 ng/mL at 1 h
after administration. Plasma concentrations increased much
more slowly and reached a significantly lower Cmax. Compound
concentrations in plasma and mesenteric lymph were equal at
24 h after compound administration, suggesting equilibration of
the compound between the two compartments. As a
consequence of the rapid and pronounced lymphatic transport
of compound 42, the exposure (AUC0−t) in mesenteric lymph
was significantly higher than in plasma. Although our
experimental setup does not allow calculation of the fraction
of the dose that undergoes lymphatic transport, lymphatic
exposure and thus lymphatic transport are significant. The high
lymph exposure of compound 42 may contribute to the
compound’s in vivo efficacy.
Compound concentrations in lymph fluids are not
determined regularly. However, it is generally accepted that
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Table 8. Mesenteric Lymph and Plasma Exposure after Oral Administration of 10 mg/kg of Compounds 42, 104, 105, and 106
to Male Wistar Rats
a
EC50 values as determined in a GTPγS assay using membranes of CHO cells expressing either S1P1 or S1P3. EC50 values represent geometric mean
values of at least three independent measurements in duplicate. All compounds behaved as full agonists on S1P1 and S1P3 (Emax > 85%). For details
b
c
see Experimental Section. tmax given in parentheses. 0−t = from 0 h to last quantifiable measurement (t = 24 h except for 104, t = 6.4 h).
ring bearing the polar side chain further influenced the
compound’s selectivity against the S1P3 receptor. In general,
the oxadiazole linked compounds were more potent on S1P3
and thus less selective for S1P1 than their propanone linked
analogues. On the other hand, the oxadiazole derivatives usually
showed lower in vitro and in vivo clearance and as a
consequence a higher efficacy in sequestering lymphocytes
from circulation. Several oxadiazole derivatives (e.g., 42, 81, 82,
and 84−87) maximally reduced LC for at least 24 h after oral
dosing of 10 mg/kg to Wistar rats. Among these, compounds
85 and 87 appear particularly interesting, as they incorporate a
simple alkyl substituted thiophene head and combine high
affinity and selectivity for S1P1 with sustained maximal LC
reduction.
Finally, we discovered that lymphatic transport is a significant
route of absorption for several of the compounds discussed in
this study. Compounds that undergo lymphatic transport may
offer several advantages. First, although these compounds may
ultimately end up in the systemic circulation, they avoid hepatic
first pass metabolism. Lymphatic transport may therefore
positively influence the bioavailability of a compound.100,101
Second, lymphatic transport may also offer opportunities for a
tissue targeted approach wherein plasma exposure is minimal.
The PKC inhibitor sotrastaurin, for instance, showed superior
immunosuppressor efficacy in vivo when compared to a
structurally close analogue reaching similar plasma exposure
but lacking significant lymphatic absorption.102 Mesenteric
lymph concentrations of compounds 42 and 104−106
exceeded those in plasma significantly. These compounds
may therefore be particularly useful in diseases where
mesenteric lymph represents the target compartment. This
may indeed be the case for autoimmune diseases affecting the
gastrointestinal tract such as ulcerative colitis or Crohn’s
disease. More detailed studies to understand the correlation
between tissue specific pharmacokinetic behavior and efficacy
in relevant disease models are certainly warranted for the
compounds described above.
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 LC−MS. LC−
MS parameters were the following: 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) or Zorbax extended C18, 1.8 μm, 4.6 mm × 20
mm (Agilent); gradient, 5−95% acetonitrile in water containing 0.04%
of trifluoroacetic acid, within 1 min; flow, 4.5 mL/min; 40 °C; tR is
given in min; UV detection at 230, 254, and 280 nm. LC−MS analysis
was done 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.
HPLC Using Chiral Stationary Phase. Hardware from UltiMate
instrument series (Dionex) and parameters were the following: HPG-
3200SD binary pump, WPS-3000 autosampler, TCC-3200 thermo-
stated column compartment, DAD-3000 detector, SRD-3400 degasser,
ValveMate 2 (Gilson) solvent valves; column, solvent, and retention
time (tR) as indicated, DEA = diethylamine, TFA = trifluoroacetic acid,
at 25 °C, flow 1 mL/min. No racemization was observed during the
synthesis of the target compounds. LC−HRMS parameters were the
following: analytical pump, Waters Acquity Binary Solvent Manager;
MS, SYNAPT G2MS, source temperature 150 °C; desolvatation
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 five scans; DAD, Acquity UPLC PDA detector;
column: Acquity UPLC BEH C18 1.7 μm, 2.1 mm × 50 mm from
Waters, thermostated in the Acquity UPLC column manager at 60 °C.
Eluents were the following: water + 0.05% formic acid; B, acetonitrile
+ 0.05% formic acid. Gradient was 2−98% B over 3.0 min. Flow was
0.6 mL/min. Detection was with UV 214 nm and MS. tR is given in
min.
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1
NMR Spectroscopy. Equipment used was Varian Oxford for H
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. An amount of 25 μL of MicroScint20 was
added, and 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.
(300 MHz) or 13C (75 MHz) or Bruker Avance II, 400 MHz
1
UltraShield, for H (400 MHz) and 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),
m (multiplet), or br (broad). Coupling constants are given in Hz.
Several compounds have been prepared in a combinatorial library
format on a 15−50 mmol scale. For those compounds 1H NMR
spectra were acquired using nondeuterated 10 mM DMSO stock
solutions submitted for biological testing.103 The solvent and water
signal were suppressed by irradiation at 2.54 and 3.54 ppm,
respectively. As a consequence, signal integrals close to those
frequencies are not always accurate. The numbers of protons given
in the description represent observed values.
Compound Purification. Compounds were purified by 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 of 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 of 10% methanol in water to 100% methanol).
X-ray Diffraction. To determine the molecular structure of
thiophenecarboxylic acids 9, 97, and 103, 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. Refinement was performed with CRYSTALS. Full matrix least-
squares refinement was performed with anisotropic temperature
factors for all atoms except hydrogen which were included at
calculated positions with isotropic temperature factors. Coordinates,
anisotropic temperature factors, bond lengths, and bond angles have
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 numbers: 972174 (9),
972173 (97), 972175 (103).
Purity. The 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 of 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 of
1.2 mL/min. In addition, important compounds were analyzed by
LC−HRMS as described above. Purity and identity of the target
compounds were further corroborated by NMR spectroscopy, and
chiral integrity was proven by HPLC using chiral stationary phases. No
racemization/epimerization was observed during the synthesis of the
target compounds. According to these LC−MS analyses, final
compounds showed a purity of ≥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 Xgeoσg2 and
Xgeo/σg2, respectively (results not shown).
GTPγS Binding Assays. 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 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,
1 or 3 μM GDP (for S1P1 or S1P3, respectively), 2.5% DMSO, and 50
pM 35S-GTPγS. Test compounds were dissolved and 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 filter plates
Metabolic Stability in Liver Microsomes and Hepatocytes.
Glucose 6-phosphate (disodium salt) and NADP were purchased from
Sigma (Buchs, Switzerland). Glucose 6-phosphate dehydrogenase was
supplied by Boehringer Mannheim (Rotkreuz, Switzerland). All other
chemicals and solvents used throughout this study were of the highest
commercially available quality. Liver microsomal preparations from
humans (pool of 48 donors), male Wistar rat (pool of 4 animals), and
Beagle dog (pool of several animals) were purchased from Becton
Dickinson (Basel, Switzerland). They were employed at a microsomal
protein concentration of 0.5 mg/mL. The NADPH-regenerating
system used for the microsomal incubations was prepared as a 10-fold
concentrated stock solution and kept at −20 °C. It consisted of 11
mM NADP, 100 mM glucose 6-phosphate, and 50 mM MgCl2 in 0.1
M phosphate buffer (pH 7.4). An amount of 20 UI/mL glucose 6-
phosphate dehydrogenase was added before use. Incubations of
compounds were performed at a single concentration of 1 μM in a
total reaction volume of 1 mL at 37 °C in an Eppendorf thermomixer
at 450 rpm. The reaction was initiated by addition of the NADPH-
regenerating system (100 μL) containing the glucose 6-phosphate
dehydrogenase and terminated after appropriate time periods up to 15
min by addition of ice-cold methanol (100 μL) to aliquots (100 μL) of
the reaction mixture. The samples were centrifuged at 3220g for 20
min at 4 °C, and an aliquot (10 μL) of the supernatant was submitted
to LC−MS−MS analysis. The total concentration of DMSO in the
assay did not exceed 0.1%. Male rat hepatocytes were prepared
following the standard two-step collagenase perfusion method.104
Freshly prepared hepatocytes were cultured in phenol red-free
William’s medium E supplemented with 10% fetal calf serum, 0.7
μM insulin, 10.000 UI/mL penicillin, and 10 mg/mL streptomycin
(preincubation medium). Freshly isolated rat hepatocytes were
centrifuged at 50g for 4 min at 4 °C and resuspended in preincubation
medium, and the cell number and initial viability were determined
using the erythrosine exclusion test. Cell viabilities below 85% were
rejected for use. After a second centrifugation step, cells were
resuspended in preincubation medium at a nominal density of 5.0 ×
105 viable cells/mL. Aliquots (400 μL) of this suspension were
dispensed into collagen-coated 24-well plates and incubated at 37 °C
for a period of about 3 h in a humidified atmosphere containing 5%
CO2. At the end of the preincubation period, the medium was
removed from each well and replaced with 200 μL of prewarmed (37
°C) incubation medium containing compounds at a final concen-
tration of 1 μM. Duplicate wells were sampled at 0, 0.5, 1, 2, 4, and 24
h by addition of 200 μL of methanol containing 0.3% SDS, and the
entire well content was transferred into cryovials. Samples were stored
frozen at −20 °C pending analysis. Compound disappearance was
monitored by LC−MS−MS (MRM). Intrinsic clearances were
determined by initial first order disappearance rates according to
Obach et al.105
Pharmacokinetics in the Rat. Male Wistar rats (RCC Ltd.,
Biotechnology and Animal Breeding Division, Fullinsdorf, Switzer-
̈
land) were used for pharmacokinetic experiments after an acclimatiza-
tion period of at least 7 days after delivery. The body weight of the rats
was about 250 g at the day of the experiment. Two days prior to
dosing, rats were anesthetized via inhalation by the gas anesthetic
isoflurane. Buprenorphine was dosed as analgesic at 0.03 mg/kg sc half
an hour before the operation. Catheters were implanted into the
jugular vein and carotid artery under aseptic conditions to allow for
multiple serial blood sampling. Animals foreseen for oral dosing did
not undergo surgery, and blood samples were taken sublingually under
light anesthesia with isoflurane. Compounds were administered
intravenously as a 5 min infusion via the jugular vein at a dose of 1
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mg/kg body weight formulated as solutions in an aqueous mixed
micellar vehicle based on phospholipids and bile acids. Oral
administration at a dose of 10 mg/kg was performed by gavage. For
that, the compounds were dissolved in DMSO. This solution was
added to a stirred solution of succinylated gelatin (7.5%) in water. The
resulting milky suspension contained 5% of DMSO. Serial blood
samples of 0.25 mL each were taken predose and at 30 min and 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 the end of infusion. Plasma was separated by centrifugation
and stored at −20 °C. By use of 1.25 μL of plasma on column, lower
and upper limits of quantification were 1.52 and 1.52 ng/mL and 3300
and 10 000 ng/mL for compounds 36 and 42, respectively.
Pharmacokinetic parameters were estimated with the WinNonlin
software (Pharsight Corporation, Mountain View, CA, USA) using
noncompartmental analysis.
For lymph sampling, rats were put under anesthesia by 150 mg/kg
thiobutobarbital sodium salt, ip, at selected times after oral dosing and
a catheter was inserted into the thoracic lymph duct after a
laparotomy. The mesenteric lymph was collected for at least 10 min.
Also a blood sample was taken at the mean time of the lymph
collection period. At the end of the sampling period rats were
euthanized with pentobarbital (iv or ip). All animals had free access to
food and water during the entire duration of the experiments. Plasma
and lymph samples from the rat were analyzed after protein
precipitation with methanol and centrifugation at 3220g for 20 min
at 4 °C using liquid chromatography coupled to mass spectrometry
(LC−MS−MS) using appropriate calibration curves, internal stand-
ards (close analogues), and bioanalytical quality controls.
min. The orange suspension is poured onto 10% aqueous citric acid
solution (200 mL) and brine (200 mL) and extracted with EA (2 ×
200 mL). The organic extracts are washed with 0.2 N aqueous NaOH
and brine, dried over Na2SO4, and evaporated to dryness to give 5,5-
dimethyl-2-oxocyclohexanecarbaldehyde 6 (52 g, 85%) as a yellow oil.
1
LC−MS: tR = 0.89 min, [M + 1 + CH3CN]+ = 196.15. H NMR
(CDCl3): δ 14.42 (d, J = 2.1 Hz, 1 H), 8.59 (d, J = 1.8 Hz, 1 H), 2.40
(t, J = 6.8 Hz, 2 H), 2.14 (s, 2 H), 1.50 (t, J = 6.8 Hz, 2 H), 1.01 (s, 6
H).
(c) To a solution of 6 (51 g, 331 mmol) in chloroform (250 mL),
oxalyl chloride (40 mL, 465 mmol) is rapidly added. Gas evolution is
observed, and the mixture becomes warm (35 °C). The mixture is
cooled with a water bath to 20−25 °C. After the mixture was stirred
for 1 h, ice followed by 3 N aqueous NaOH (100 mL) is added
carefully. Again, gas formation is observed. The mixture is stirred for
45 min until gas evolution has ceased. The organic phase is separated,
and the aqueous phase is extracted once more with chloroform. The
combined organic extracts are washed with water and dried over
Na2SO4. The solvent is removed in vacuo to give crude 2-
chloromethylene-4,4-dimethylcyclohexanone 7 (50 g, 87%) as a
1
brown oil. LC−MS: tR = 0.96 min. H NMR (CDCl3): δ 7.15 (t, J
= 2.1 Hz, 1 H), 2.47 (t, J = 7.0 Hz, 2 H), 2.43 (s, 2 H), 1.71 (t, J = 7.0
Hz, 2 H), 1.06 (s, 6 H).
(d) To a part (300 mL) of a freshly prepared solution of sodium
(21 g, 875 mmol) in ethanol (500 mL), mercaptoacetic acid ethyl ester
(50 mL) is added. The resulting solution is added over a period of 10
min to a solution of 7 (50 g, 290 mmol) in THF (170 mL). The
mixture becomes warm (50 °C). Upon complete addition, the
remaining part of the freshly prepared solution of sodium in ethanol
(200 mL) is added to the reaction mixture. The mixture is stirred at
room temperature for 15 min before 1 N aqueous LiOH solution (300
mL) is added. The solution is refluxed for 3 h, then stirred at room
temperature for 16 h. The THF and ethanol are removed under
reduced pressure, and the remaining dark solution is extracted with
heptane/EA 3:1 (2 × 200 mL). The aqueous phase is acidified by
adding citric acid (30 g) and 2 N aqueous HCl (200 mL) and then
extracted three times with EA. The combined organic extracts are
washed three times with saturated aqueous NaHCO3 solution, dried
over Na2SO4, and evaporated. The resulting dark brown oil is
dissolved in acetonitrile at 60 °C and crystallized at 5 °C. The crystals
are collected, washed with acetonitrile, and dried to give 8 (31 g, 51%)
as a slightly gray powder. LC−MS: tR = 0.95 min, [M + 1 + CH3CN]+
= 252.18. 1H NMR (CDCl3): δ 7.15 (s, 1 H), 3.05 (t, J = 7.0 Hz, 2 H),
2.47 (s, 2 H), 1.58 (t, J = 7.0 Hz, 2 H), 0.97 (s, 6 H).
In vivo efficacy of the target compounds was assessed by measuring
the circulating lymphocytes after oral administration of 1−10 mg/kg of
a 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 LC at a given time
point and the predose value (=100%). All data are presented as the
mean
SEM. Statistical analyses were performed by analysis of
variance (ANOVA) using Statistica (StatSoft) and the Student−
Newman−Keuls procedure for multiple comparisons. 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 lymphocyte count
reduction in the range of −60% to −75% represents the maximal effect
to be observed under the conditions of the experiment. The null
hypothesis was rejected when p < 0.05. For formulation, the
compounds were dissolved in DMSO. This solution was added to a
stirred solution of succinylated gelatin (7.5%) in water. The resulting
milky suspension containing a final concentration of 5% of DMSO was
administred to the animals by gavage. A mixture of 95% of
succinylated gelatin (7.5%) in water and 5% of DMSO served as
vehicle.
5,5-Dimethyl-4,5,6,7-tetrahydrobenzo[c]thiophene-1-car-
boxylic Acid (8). (a) To a solution of 4,4-dimethylcyclohex-2-enone
(50 g, 403 mmol) in EA (230 mL) a suspension of Pd/C (2.5 g, 10%
Pd) in EA is added. The suspension is stirred at room temperature for
2 h under 1 bar of H2. The catalyst is filtered off and the solvent of the
filtrate is carefully evaporated to give 4,4-dimethyl-cyclohexanone 5
(50 g, 98%) as a colorless oil which slowly crystallizes. 1H NMR
(CDCl3): δ 2.34 (t, J = 6.4 Hz, 4 H), 1.66 (t, J = 6.4 Hz, 4 H), 1.09 (s,
6 H).
3,5,5-Trimethyl-4,5,6,7-tetrahydrobenzo[c]thiophene-1-car-
boxylic Acid (9). At −78 °C a solution of 8 (5 g, 23.8 mmol) in THF
is treated with tert-butyllithium (41 mL, 1.5 M in pentane). The
mixture is stirred at −78 °C for 15 min before methyl iodide (17.1 g,
120 mmol) is added dropwise. Stirring is continued at −78 °C for 30
min. The mixture is warmed to room temperature, diluted with water
(400 mL), acidified with 10% aqueous citric acid solution, and
extracted three times with EA. The combined organic extracts are
dried over MgSO4 and evaporated. The remaining solid is suspended
in heptane/diethyl ether, filtered, and dried under HV to give 9 (4.01
g, 75%) as a beige powder. LC−MS: tR = 0.97 min, [M + 1] = 225.13.
1H NMR (DMSO-d6): δ 12.49 (s br, 1 H), 2.87 (t, J = 6.7 Hz, 2 H),
2.26 (s, 5 H), 1.45 (t, J = 6.7 Hz, 2 H), 0.91 (s, 6 H). 13C NMR
(DMSO-d6): δ 164.0, 145.4, 139.1, 136.4, 122.7, 38.3, 35.3, 29.6, 28.1,
24.2, 13.4. Crystals suitable for single crystal X-ray structure analysis
were obtained by crystallizing 9 from acetonitrile. Crystals of 9
(C12H16O2S, formula weight 224.09) formed in the triclinic space
group P1. A total of 12 337 reflections was measured at 293 K. Results
̅
were the following: molecules/unit cell Z = 2, cell dimensions a =
6.6024(6) Å, b = 8.6950(8) Å, c = 9.8154(10) Å, α = 96.056(7)°, β =
96.461(7)°, γ = 94.944(6)°; calculated density of 1.34 g cm−3. The
final R-factor of 0.031 was obtained for 2416 observed reflections (I >
3σ(I)). CCDC code is 972174.
(b) To an ice-cold solution of potassium tert-butylate (24.5 g, 109
mmol, 50% solution in tert-butanol) in THF (700 mL), ethyl formate
(120 mL, 123 mmol) is slowly added. The mixture is stirred at room
temperature for 30 min before a solution of 5 (50 g, 396 mmol) in
ethyl formate (50 mL) and THF (70 mL) is added over a period of 20
min. Upon complete addition, stirring is continued at 15−20 °C for 30
3-Ethyl-5,5-dimethyl-4,5,6,7-tetrahydrobenzo[c]thiophene-
1-carboxylic Acid (10). To a cooled solution (−78 °C) of 8 (960
mg, 4.57 mmol) in THF (19 mL), tert-butyllithium (8 mL, 1.5 M
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solution in pentane) is added. The mixture is stirred at −78 °C for 10
min before ethyl iodide (3.80 g, 24.37 mmol) is added. The reaction
mixture is stirred at −78 °C for 3 h. Water/methanol 1:1 (8 mL)
followed by 10% aqueous citric acid solution is added, and the mixture
is extracted with EA. The combined organic extracts are washed with
brine, dried over Na2SO4, and evaporated. The remaining solid is
suspended in acetonitrile (6 mL), heated to 60 °C, cooled to room
temperature, filtered, and dried to give 10 (640 mg, 50%) as a slightly
at room temperature for 48 h before it is extracted with diethyl ether
(1 × 400 mL, 2 × 150 mL). The organic extracts are washed with 1 N
aqueous NaOH (3 × 100 mL). The aqueous extracts are carefully
acidified with 25% aqueous HCl and then extracted with DCM (3 ×
150 mL). The combined DCM extracts are dried over MgSO4, filtered,
and evaporated. The crude product is purified by crystallization from
acetonitrile (150 mL) at 4 °C to give 94 (16.0 g, 41%) as a beige-
brown crystalline powder. LC−MS: tR = 0.95 min. 1H NMR
(CD3OD): δ 7.21 (s, 1 H), 2.43 (s, 3 H), 2.42 (d, J = 7.0 Hz, 2
H), 1.83 (nonet, J = 7.0 Hz, 1 H), 0.92 (d, J = 7.0 Hz, 6 H).
4-Isobutyl-5-methylthiophene-2-carboxylic Acid (96). (a)
Phosphorus oxychloride (53.7 g, 350 mmol) is slowly added to
DMF (60 mL) stirred at 5 °C. Upon complete addition, the clear
solution is stirred for further 30 min at 5 °C before 5-methyl-2-
hexanone 91 (20 g, 175 mmol) is added dropwise. The yellow solution
is stirred for 30 min at 0 °C, then for 90 min at room temperature. The
mixture becomes warm (40 °C), and a thick suspension forms. The
mixture is cooled to 25 °C, and stirring is continued for 1 h before it is
poured into an aqueous solution of NaOAc (80 g)/ice mixture. The
mixture is extracted twice with diethyl ether. The organic extracts are
washed with water, combined, dried over MgSO4, filtered, and
evaporated to give crude 3-chloro-2-isobutylbut-2-enal 95 (35.4 g,
quantitative) as a 2:3 mixture of E/Z isomers as a yellow oil. LC−MS:
tR = 0.97 min. 1H NMR (CDCl3): δ major isomer 10.02 (s, 1 H), 2.60
(s, 3 H), 2.33 (d, J = 7.3 Hz, 2 H), 1.82 (hept, J = 7.0 Hz, 1 H), 0.87
(d, J = 6.7 Hz, 6 H); δ minor isomer 10.22 (s, 1 H), 2.38 (s, 3 H), 2.20
(d, J = 7.3 Hz, 2 H), 1.69 (hept, J = 6.4 Hz, 1 H), 0.86 (d, J = 6.7 Hz, 6
H).
(b) Sodium (10.7 g, 467 mmol) is dissolved in ethanol (500 mL),
and the resulting solution is diluted with THF (100 mL) before
mercaptoacetic acid ethyl ester (33.7 g, 280 mmol) dissolved in THF
(70 mL) is slowly added at 5 °C. The mixture is stirred at room
temperature for 1 h before a solution of 95 (30 g, 187 mmol) in THF
(100 mL) is slowly added at 8 °C. The resulting yellow suspension is
stirred at room temperature for 16 h. The reaction mixture is diluted
with diethyl ether (500 mL) and is washed with dilute aqueous NaOCl
solution followed by aqueous 1 N HCl and water. The organic extract
is dried over MgSO4, filtered, and evaporated. The remaining orange
oil is dissolved in ethanol (150 mL), and 2 N aqueous LiOH (50 mL)
is added. The mixture is stirred for 16 h at 50 °C, acidified with 2 N
aqueous HCl, and extracted with EA. The organic extract is dried over
MgSO4, filtered, and evaporated. The crude product is recrystallized
from EA/heptane to give 96 (10.5 g, 28%) as colorless crystals. LC−
MS: tR = 0.92 min, [M + 1 + CH3CN] = 240.16. 1H NMR (CDCl3): δ
7.59 (s, 1 H), 2.40−2.37 (m, 5 H), 1.84 (hept, J = 7.0 Hz, 1 H), 0.90
(d, J = 7.0 Hz, 6 H). 13C NMR (CDCl3): δ 167.8, 144.0, 139.3, 137.4,
127.4, 37.3, 29.6, 22.4, 14.0.
1
beige solid. LC−MS: tR = 1.01 min, [M + 1 + CH3CN] = 280.10. H
NMR (DMSO-d6): δ 12.48 (s br, 1 H), 2.87 (t, J = 6.8 Hz, 2 H), 2.66
(q, J = 7.5 Hz, 2 H), 2.27 (s, 2 H), 1.46 (t, J = 6.7 Hz, 2 H), 1.15 (t, J =
7.5 Hz, 3 H), 0.91 (s, 6 H). 13C NMR (CDCl3): δ 168.6, 149.23,
149.21, 134.6, 121.4, 40.8, 35.3, 29.5, 28.0, 21.9, 21.5, 14.7.
1-(3,5,5-Trimethyl-4,5,6,7-tetrahydrobenzo[c]thiophen-1-
yl)ethanone (14). To a suspension of 9 (4.10 g, 18.28 mmol) in
diethyl ether (300 mL), methyllithium (23 mL, 1.6 M solution in
diethyl ether) is slowly added at room temperature. The reaction
mixture becomes clear, yellow, and slightly warm (26 °C) and is stirred
for 15 min before it is quenched with water. The organic layer is
separated, washed once more with water, dried over MgSO4, and
evaporated. The crude product is purified by CC on silica gel, eluting
with heptane/EA 4:1 to give 14 (2.80 g, 69%) as a pale yellow
1
crystalline solid. LC−MS: tR = 0.97 min, [M + 1] = 223.26. H NMR
(CDCl3): δ 3.00 (t, J = 7.0 Hz, 2 H), 2.43 (s, 3 H), 2.31 (s, 3 H), 2.26
(s, 2 H), 1.51 (t, J = 7.0 Hz, 2 H), 0.95 (s, 6 H). 13C NMR (CDCl3): δ
190.6, 145.6, 140.0, 137.3, 131.7, 38.6, 35.4, 29.4, 27.9, 25.2, 13.5.
4-Isobutyl-3-methylthiophene-2-carboxylic Acid (94). (a) To
a solution of KOtBu (50 g, 446 mmol) in THF (400 mL) is added
during 30 min ethyl formate (92 g, 1.25 mol). Strong gas evolution
occurs. The mixture is cooled during the addition with a water bath at
10 °C. After complete addition, the mixture is stirred until the gas
evolution ceases (15 min). The mixture is cooled with ice at 0 °C, and
a mixture of 5-methyl-2-hexanone 91 (34.25 g, 300 mmol) and ethyl
formate (41 g, 0.55 mol) is added slowly during 30 min. The mixture
is stirred for 15 h, diluted with EA (500 mL), and washed with 1 N
aqueous HCl (100 mL), 1 M aqueous NaH2PO4 solution (100 mL),
and brine (100 mL). The organic extract is dried (MgSO4), filtered,
and evaporated to give crude 4-hydroxy-3-isobutylbut-3-en-2-one 92
(28 g, 66%) which is used without further purification. LC−MS: tR =
0.80 min, [M + 1] = 143.39. 1H NMR (CDCl3): δ 7.99 (d, J = 6.4 Hz,
1 H), 2.11 (s, 3 H), 2.00 (d, J = 7.2 Hz, 2 H), 1.60 (hept, J = 7.0 Hz, 1
H), 0.89 (d, J = 6.6 Hz, 6 H).
(b) To a solution of 92 (28 g, 197 mmol) in chloroform (350 mL)
a solution of oxalyl chloride (44.3 g, 349 mmol) in chloroform (50
mL) is added slowly during 5 min. The resulting dark brown mixture is
stirred at room temperature for 2 h before it is cooled to 0 °C and
treated with ice (100 g) followed by 1 N aqueous NaOH (100 mL).
When the quite violent gas evolution ceases, the phases are separated
(the still acidic aqueous phase is discarded). The organic phase is
washed with 1 N aqueous NaOH (3 × 75 mL) and 1 N aqueous
NaH2PO4 (75 mL), dried (MgSO4), filtered, and evaporated to give
crude 4-chloro-3-isobutylbut-3-en-2-one 93 (31.6 g, quantitative) as a
dark brown oil. LC−MS: tR = 0.97 min. 1H NMR (CDCl3): δ 7.29 (s,
1 H), 2.36 (d, J = 7.3 Hz, 2 H), 2.31 (s, 3 H), 1.81 (hept, J = 6.8 Hz, 1
H), 0.87 (d, J = 6.7 Hz, 6 H).
4-Isobutyl-3,5-dimethylthiophene-2-carboxylic Acid (97).
Under argon, a solution of 96 (2.00 g, 10.1 mmol) in THF (150
mL) is allowed to stand over 3 Å molecular sieves for 30 min before it
is transferred to the reaction vessel. The solution is cooled to −78 °C,
t
and BuLi (20.2 mL of 1.5 M solution in pentane) is added. Upon
complete addition, stirring is continued at −78 °C for 45 min before
MeI (7.16 g, 50.4 mmol) is added. Stirring is continued at −70 °C for
30 min. The mixture is allowed to warm to room temperature. The
reaction is quenched by adding 10% aqueous citric acid solution and
water. The mixture is extracted three times with EtOAc (3 × 200 mL).
The organic extracts are combined, dried over MgSO4, filtered, and
concentrated. The crude product is purified by column chromatog-
raphy on silica gel, eluting with heptane/EtOAc 4:1 to give 97 (1.40 g,
65%) as a white solid. LC−MS: tR = 0.97 min, [M + 1 + CH3CN] =
254.26. 1H NMR (CDCl3): δ 2.46 (s, 3 H), 2.39 (s, 3 H), 2.36 (d, J =
7.0 Hz, 2 H), 1.78 (hept, J = 7.0 Hz, 1 H), 0.91 (d, J = 7.0 Hz, 6 H).
13C NMR (CDCl3): δ 168.7, 148.0, 142.3, 139.9, 122.2, 36.0, 29.2,
22.5, 14.9, 14.5. Crystals suitable for single crystal X-ray structure
analysis were obtained by crystallizing 97 by the isothermal distillation
method106,107 using EA as solvent and hexane as the precipitating
agent. Crystals of 97 (C11H16O2S, formula weight 212.09) formed in
the monoclinic space group C2/c, and 13 680 reflections were
measured at 293 K. Results were the following: molecules/unit cell
(c) KOtBu (44.2 g, 394 mmol) is added portionwise to ethanol
(200 mL). The mixture is stirred for 30 min at 20 °C to dissolve all
KOtBu. Mercaptoacetic acid ethyl ester (47.3 g, 394 mmol) is added,
and the temperature is maintained at 20 °C. This solution is slowly
added at 20 °C to a solution of the crude 93 (31.6 g, 197 mmol) in
THF (350 mL). The mixture is stirred at room temperature for 15 h
before sodium ethylate (13.4 g, 197 mmol) is added, and stirring is
continued at reflux for 1 h. The mixture is cooled to room
temperature, and the solvents are evaporated on a rotavap. The
residue is diluted with diethyl ether (500 mL), washed with 1 M
aqueous NaH2PO4 (200 mL), 1 N aqueous NaOH (2 × 100 mL),
saturated aqueous NaHCO3 (35 mL) containing 10% aqueous NaOCl
(15 mL), and brine (100 mL), dried over Na2SO4, filtered, and
evaporated. The resulting residue (36.3 g) is dissolved in EtOH (250
mL). 2 N aqueous LiOH (100 mL) is added, and the mixture is stirred
91
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Z = 8, cell dimensions a = 15.852(3) Å, b = 7.0898(10) Å, c =
20.177(3) Å, α = 90°, β = 93.945(16)°, γ = 90°; calculated density of
1.24 g cm−3. The final R-factor of 0.035 was obtained for 2450
observed reflections (I > 3σ(I)). CCDC code is 972173.
192.5, 150.5, 145.9, 139.7, 137.3, 132.9, 131.1, 128.5, 123.0, 43.9, 38.6,
35.5, 30.0, 29.5, 27.9, 25.3, 16.0, 13.5.
rac-3-[4-(3-Amino-2-hydroxypropoxy)-3,5-dimethylphenyl]-
1-(3,5,5-trimethyl-4,5,6,7-tetrahydrobenzo[c]thiophen-1-yl)-
propan-1-one (29). (a) To a solution of 21 (420 mg, 1.18 mmol) in
isopropanol (15 mL) and 3 N aqueous NaOH (6 mL) rac-
epichlorohydrin (545 mg, 5.89 mmol) is added. The mixture is stirred
at room temperature for 20 h before it is diluted with diethyl ether
(150 mL) and washed with saturated NaHCO3 solution (50 mL). The
washing is extracted back with diethyl ether (150 mL). The combined
organic extracts are dried over MgSO4, filtered, and concentrated. The
crude product is purified by column chromatography on silica gel,
eluting with heptane/EtOAc 4:1 to give rac-3-(3,5-dimethyl-4-(oxiran-
2-ylmethoxy)phenyl)-1-(3,5,5-trimethyl-4,5,6,7-tetrahydrobenzo[c]-
thiophen-1-yl)propan-1-one (443 mg, 91%) as an almost colorless oil.
LC−MS: tR = 1.19 min, [M + 1]+ = 413.36. 1H NMR (CDCl3): δ 6.87
(s, 2 H), 4.00 (dd, J1 = 11.1 Hz, J2 = 3.3 Hz, 1 H), 3.73 (dd, J1 = 11.1
Hz, J2 = 5.9 Hz, 1 H), 3.31−3.39 (m, 1 H), 2.98−3.08 (m, 4 H), 2.85−
2.96 (m, 3 H), 2.70 (dd, J1 = 4.9 Hz, J2 = 2.6 Hz, 1 H), 2.33 (s, 3 H),
2.28 (s, 2 H), 2.26 (s, 6 H), 1.53 (t, J = 6.7 Hz, 2 H), 0.97 (s, 6 H).
(b) A solution of the above epoxide (443 mg, 1.07 mmol) in 7 N
NH3 in methanol (15 mL) is stirred at 65 °C for 18 h in a sealed
vessel. The mixture is concentrated and dried to give 29 (443 mg,
5-Isobutylthiophene-2-carboxylic Acid (102). To a solution of
thiophene-2-carboxylic acid 98 (4.16 g, 32.1 mmol) in THF (200 mL)
tert-butyllithium (49 mL, 1.7 M solution in pentane, 83.6 mmol) is
slowly added at −78 °C. The mixture is stirred at −78 °C for 30 min
before isobutyl bromide (22.7 g, 160.7 mmol) is carefully added. The
mixture is stirred at −78 °C for 5 h, then at room temperature for 16
h. The reaction is quenched by the addition of water (400 mL). The
mixture is acidified and extracted with EA. The organic extract is dried
over MgSO4, filtered, and evaporated. The crude product is purified by
MPLC on reverse phase silica gel to give 102 (1.67 g, 28%) as a
brownish oil. LC−MS: tR = 0.91 min. 1 H NMR (CDCl3): δ 7.73 (d, J
= 3.8 Hz, 1 H), 6.80 (d, J = 3.8 Hz, 1 H), 2.72 (d, J = 7.0 Hz, 2 H),
1.94 (hept, J = 6.7 Hz, 1H), 0.96 (d, J = 6.7 Hz, 6 H). 13C NMR
(CDCl3): δ 168.0, 154.5, 135.2, 130.3, 126.4, 39.8, 30.7, 22.2.
rac-N-(3-(2,6-Dimethyl-4-(3-oxo-3-(3,5,5-trimethyl-4,5,6,7-
tetrahydrobenzo[c]thiophen-1-yl)propyl)phenoxy)-2-hydroxy-
propyl)-2-hydroxyacetamide (3). A solution of glycolic acid (61
mg, 0.806 mmol), TBTU (224 mg, 0.698 mmol), and Hunig’s base
̈
(278 mg, 2.15 mmol) in DCM (5 mL) is stirred at room temperature
for 10 min before a solution of 29 (231 mg, 0.537 mmol) in DCM (5
mL) is added. The mixture is stirred at room temperature for 1 h.
Another portion of glycolic acid (61 mg, 0.806 mmol) and TBTU
(100 mg, 0.311 mmol) is added, and stirring is continued for 2 h. The
mixture is diluted with DCM and washed with saturated aqueous
NaHCO3 solution. The washing is extracted back two times with
DCM. The organic extracts are combined, dried over MgSO4, filtered,
and concentrated. The crude product is purified on preparative TLC
plates using DCM containing 5% of methanol to give 3 (143 mg, 55%)
as a colorless foam. LC−MS: tR = 1.00 min, [M + 1]+ = 488.17. HPLC
with chiral stationary phase (Chiralpak IA 250 mm × 4.6 mm i.d., 5
μm; 90% heptane containing 0.05% DEA, 10% ethanol containing
96%) as a pale yellow foam. LC−MS: tR = 0.89 min, [M + 1]+
=
1
430.33. H NMR (DMSO-d6, solvent suppression): δ 6.87 (s, 2 H),
3.90−3.97 (m, 1 H), 3.63−3.65 (m, 1 H), 2.87−2.93 (m, 2 H), 2.73−
2.82 (m, 2 H), 2.30 (s, 3 H), 2.27 (s, 2 H), 2.17 (s, 6 H-), 1.43−1.50
(m, 2 H), 0.92 (s, 6 H).
rac-N-(3-{2-Ethyl-4-[3-(3-ethyl-5,5-dimethyl-4,5,6,7-
tetrahydrobenzo[c]thiophen-1-yl)-3-oxopropyl]-6-methylphe-
noxy}-2-hydroxypropyl)-2-hydroxyacetamide (36). 36 was
prepared in analogy to 3 using 15, giving a beige foam. LC−MS: tR
= 1.05 min, [M + 1] = 516.42. HPLC with chiral stationary phase
(Chiralpak IE 250 mm × 4.6 mm i.d., 5 μm; 70% heptane containing
0.05% DEA, 30% ethanol containing 0.05% DEA): tR = 11.0 min, 50%,
tR = 11.8 min, 50%. 1H NMR (CDCl3): δ 6.86−6.95 (m, 3 H), 4.16 (s,
2 H), 4.09−4.15 (m, 1 H), 3.69−3.84 (m, 3 H), 3.40−3.52 (m, 1 H),
3.20 (s br, 1 H), 3.00−3.10 (m, 4 H), 2.90−2.98 (m, 2 H), 2.72 (q, J =
7.6 Hz, 2 H), 2.61 (q, J = 7.5 Hz, 2 H), 2.30 (s, 2 H), 2.25 (s, 3 H),
1.54 (t, J = 7.0 Hz, 2 H), 1.26 (t, J = 7.6 Hz, 3 H), 1.21 (t, J = 7.8 Hz, 3
H), 0.97 (s, 6 H). 13C NMR (CDCl3): δ 192.4, 172.8, 152.7, 147.6,
146.1, 137.4, 136.6, 136.4, 130.9, 130.5, 129.0, 127.2, 74.0, 70.2, 62.2,
43.5, 42.2, 38.6, 35.6, 30.2, 29.5, 27.9, 25.3, 22.9, 21.7, 16.4, 15.0, 14.9.
LC−HRMS: tR = 2.25 min, [M + H]/z = 516.2784, found = 516.2787.
N-((S)-3-{2-Ethyl-4-[5-(3-ethyl-5,5-dimethyl-4,5,6,7-
tetrahydrobenzo[c]thiophen-1-yl)[1,2,4]oxadiazol-3-yl]-6-
methylphenoxy}-2-hydroxypropyl)-2-hydroxyacetamide (42).
(a) To a solution of 10 (2.00 g, 8.39 mmol) in DMF (25 mL)
1
0.05% DEA): tR = 19.7 min, 48%, tR = 22.0 min, 52%. H NMR
(CDCl3): δ 6.97 (t br, J = 5.0 Hz, 1 H), 6.87 (s, 2 H), 4.08−4.15 (m, 1
H), 4.15 (s, 2 H), 3.69−3.84 (m, 3 H), 3.40−3.52 (m, 1 H), 2.97−3.08
(m, 4 H), 2.86−2.96 (m, 2 H), 2.33 (s, 3 H), 2.28 (s, 2 H), 2.24 (s, 6
H), 1.53 (t, J = 6.7 Hz, 2 H), 0.97 (s, 6 H). 13C NMR (CDCl3): δ
192.3, 172.9, 153.2, 146.0, 139.9, 137.3, 137.1, 131.0, 130.5, 129.0,
73.4, 70.2, 62.2, 43.5, 42.3, 38.6, 35.4, 30.0, 29.5, 27.9, 25.3, 16.3, 13.5.
LC−HRMS: tR = 2.08 min, [M + H]/z = 488.2471, found = 488.2470.
3-(4-Hydroxy-3,5-dimethyl-phenyl)-1-(3,5,5-trimethyl-
4,5,6,7-tetrahydro-benzo[c]thiophen-1-yl)-propan-1-one (21).
(a) A solution of 1-(3,5,5-trimethyl-4,5,6,7-tetrahydrobenzo[c]-
thiophen-1-yl)ethanone 14 (1.35 g, 6.07 mmol) and 4-hydroxy-3,5-
dimethylbenzaldehyde (1.09 g, 7.29 mmol) in ethanol (20 mL) and 5
N HCl in isopropanol (10 mL) is stirred at room temperature for 100
min. The dark solution is diluted with diethyl ether (300 mL), washed
with water followed by a 1:1 mixture of 1 N aqueous NaOH and
saturated aqueous NaHCO3, dried over MgSO4, and evaporated. The
crude product is purified by CC on silica gel, eluting with heptane/EA
7:3 to give 3-(4-hydroxy-3,5-dimethylphenyl)-1-(3,5,5-trimethyl-
4,5,6,7-tetrahydrobenzo[c]thiophen-1-yl)propenone (1.86 g, 87%) as
Hunig’s base (2.17 g, 16.8 mmol) followed by TBTU (2.96 g, 9.23
̈
mmol) is added. The mixture is stirred at room temperature for 10
min before 3-ethyl-4,N-dihydroxy-5-methylbenzamidine (1.63 g, 8.39
mmol, Supporting Information) is added. Stirring is continued at room
temperature for 18 h. The mixture is diluted with EA and washed with
water. The organic extract is dried over MgSO4, filtered, and
concentrated. The residue is dissolved in dioxane (20 mL) and stirred
under reflux for 20 h. The mixture is cooled to room temperature and
concentrated, and the crude product is purified by CC on silica gel,
eluting with heptane/EA 9:1 to give 2-ethyl-4-(5-(3-ethyl-5,5-
dimethyl-4,5,6,7-tetrahydrobenzo[c]thiophen-1-yl)-1,2,4-oxadiazol-3-
yl)-6-methylphenol (2.36 g, 71%) as a yellow solid. LC−MS: tR = 1.16
1
a yellow-orange solid. LC−MS: tR = 1.15 min, [M + 1] = 355.26. H
NMR (CDCl3): δ 7.65 (d, J = 15.2 Hz, 1 H), 7.26 (s, 2 H), 7.13 (d, J =
15.2 Hz, 1 H), 5.04 (s, 1 H), 3.15 (t, J = 6.4 Hz, 2 H), 2.37 (s, 3 H),
2.31 (s, 2 H), 2.28 (s, 6 H), 1.56 (t, J = 6.4 Hz, 2 H), 0.99 (s, 6 H).
(b) A solution of 3-(4-hydroxy-3,5-dimethylphenyl)-1-(3,5,5-
trimethyl-4,5,6,7-tetrahydrobenzo[c]thiophen-1-yl)propenone (1.86
g, 5.25 mmol) in THF (50 mL) and ethanol (50 mL) is treated
with Pd/C (400 mg, 10% Pd), and the resulting slurry is stirred at
room temperature for 5 h under 1.5 bar of H2. The catalyst is filtered
off, and the solvent of the filtrate is evaporated. The crude product is
purified by CC on silica gel, eluting with heptane/EA 1:1 to give 21
(1.80 g, 96%) as a pale red foam. LC−MS: tR = 1.15 min, [M + 1] =
1
min, [M + 1] = 396.16. H NMR (CDCl3: δ 7.82 (s, 2 H), 7.28 (s, 1
H), 4.99 (s, 1 H), 3.17 (t, J = 6.8 Hz, 2 H), 2.80 (q, J = 7.5 Hz, 2 H),
2.72 (q, J = 7.6 Hz, 2 H), 2.38 (s, 2 H), 2.35 (s, 3 H), 1.66 (t, J = 6.7
Hz, 2 H), 1.33 (t, J = 7.5 Hz, 3 H), 1.32 (t, J = 7.3 Hz, 3 H).
(b) The title compound is then prepared from the above phenol
following the procedures described for compounds 3 and 29, giving a
1
white solid. LC−MS: tR = 1.05 min, [M + 1] = 528.23. H NMR
1
357.27. H NMR (CDCl3): δ 6.85 (s, 2 H), 4.53 (s, 1 H), 3.07−2.98
(CDCl3): δ 7.85 (s, 1 H), 7.84 (s, 1 H), 7.08 (t br, J = 5.8 Hz, 1 H),
4.18−4.24 (m, 3 H), 3.90 (dd, J1 = 9.6 Hz, J2 = 4.6 Hz, 1 H), 3.76−
3.86 (m, 2 H), 3.48−3.56 (m, 1 H), 3.16 (t, J = 6.7 Hz, 2 H), 2.80 (q, J
(m, 4 H), 2.94−2.86 (m, 2 H), 2.33 (s, 3 H), 2.28 (s, 2 H), 2.22 (s, 6
H), 1.53 (t, J = 7.0 Hz, 2 H), 0.97 (s, 6 H). 13C NMR (CDCl3): δ
92
dx.doi.org/10.1021/jm401456d | J. Med. Chem. 2014, 57, 78−97

Journal of Medicinal Chemistry
Article
= 7.5 Hz, 3 H), 2.74 (q, J = 7.5 Hz, 2 H), 2.38 (s, 5 H), 1.66 (t, J = 6.7
Hz, 2 H), 1.33 (t, J = 7.5 Hz, 3 H), 1.31 (t, J = 7.5 Hz, 3 H), 1.04 (s, 6
H). 13C NMR (CDCl3): δ 173.3, 172.2, 168.0, 157.0, 147.7, 144.7,
137.4, 135.7, 131.4, 128.4, 126.7, 123.1, 115.2, 74.1, 70.1, 62.2, 42.3,
38.5, 35.4, 29.6, 27.9, 24.3, 22.9, 21.6, 16.5, 14.91, 14.87. LC−HRMS:
tR = 2.47 min, [M + H]/z = 528.2532, found = 528.2523.
34.0, 29.7, 28.0, 25.7, 24.6, 24.3, 21.4, 19.9, 16.1, 15.2. LC−HRMS: tR
= 1.85 min, [M + H]/z = 453.2212, found = 453.2209.
3-((2-(2-Ethyl-4-(5-(3-ethyl-5,5-dimethyl-4,5,6,7-
tetrahydrobenzo[c]thiophen-1-yl)-1,2,4-oxadiazol-3-yl)-6-
methylphenoxy)ethyl)amino)propanoic Acid (106). (a) To a
solution of 2-ethyl-4-(5-(3-ethyl-5,5-dimethyl-4,5,6,7-tetrahydrobenzo-
[c]thiophen-1-yl)-1,2,4-oxadiazol-3-yl)-6-methylphenol (1.60 g, 4.03
mmol) in isopropanol (100 mL) and 3 M aqueous NaOH (40 mL) 2-
bromoethanol (2.02 g, 16.1 mmol) is added, and the mixture is stirred
at 65 °C for 18 h. Another portion of 2-bromoethanol (2.02 g, 16.1
mmol) is added, and stirring is continued at 65 °C for 48 h. The
mixture is diluted with EA (250 mL) and washed with water (100
mL). The organic extract is dried over MgSO4, filtered, and
concentrated. The crude product is purified by CC on silica gel,
eluting with heptane/EA to give 2-(2-ethyl-4-(5-(3-ethyl-5,5-dimethyl-
4,5,6,7-tetrahydrobenzo[c]thiophen-1-yl)-1,2,4-oxadiazol-3-yl)-6-
rac-2-Hydroxy-N-(2-hydroxy-3-{4-[5-(5-isobutylthiophen-2-
yl)[1,2,4]oxadiazol-3-yl]-2,6-dimethylphenoxy}propyl)-
acetamide (87). 87 was prepared in analogy to 42 using 102, giving a
1
white solid. LC−MS: tR = 1.01 min, [M + 1] = 460.18. H NMR δ:
7.83 (s, 2 H), 7.80 (d, J = 3.7 Hz, 1 H), 7.00 (t br, J = 6.5 Hz, 1 H),
6.90 (d, J = 3.7 Hz, 1 H), 4.17−4.25 (m, 3 H), 3.90 (dd, J1 = 9.5 Hz, J2
= 4.5 Hz, 1 H), 3.83 (dd, J1 = 9.5 Hz, J2 = 6.3 Hz, 1 H), 3.76−3.83 (m,
1 H), 3.48−3.57 (m, 1 H), 2.79 (d, J = 7.1 Hz, 2 H), 2.37 (s, 6 H),
1.93−2.05 (m, 1 H), 1.01 (d, J = 6.6 Hz, 6 H). LC−HRMS: tR = 1.45
min, [M + H]/z = 460.1906, found = 460.1909.
methylphenoxy)ethanol (1.15 g, 65%) as a beige oil. LC−MS: tR
1.15 min, [M + 1] = 440.94.
=
(S)-3-(2-Ethyl-4-(5-(3-ethyl-5,5-dimethyl-4,5,6,7-tetrahydro-
benzo[ c]thiophen-1-yl)-1, 2, 4-oxadiazol-3-yl)-6-
methylphenoxy)propane-1,2-diol (104). To a solution of 3-ethyl-
5,5-dimethyl-4,5,6,7-tetrahydrobenzo[c]thiophene-1-carboxylic acid 10
(200 mg, 0.839 mmol) in DMF (5 mL) DIPEA (217 mg, 1.68 mmol)
followed by TBTU (296 mg, 0.923 mmol) is added. The mixture is
stirred at room temperature for 10 min before (R)-4-(2,2-dimethyl-
[1,3]dioxolan-4-ylmethoxy)-3-ethyl-N-hydroxy-5-methylbenzamidine
(259 mg, 0.839 mmol) is added. Stirring is continued at room
temperature for 1 h. The mixture is diluted with EA (100 mL), washed
with water (50 mL), dried over MgSO4, filtered, and concentrated.
The remaining residue is dissolved in dioxane (10 mL), and the
mixture is stirred at 80 °C for 18 h, then at 100 °C for 48 h. The
mixture is concentrated, and the crude product is purified on
preparative TLC plates using heptane/EA 9:1 to give (R)-3-(4-((2,2-
dimethyl-1,3-dioxolan-4-yl)methoxy)-3-ethyl-5-methylphenyl)-5-(3-
ethyl-5,5-dimethyl-4,5,6,7-tetrahydrobenzo[c]thiophen-1-yl)-1,2,4-oxa-
diazole (217 mg, 51%) as a pale beige resin. LC−MS: tR = 1.25 min,
[M + 1] = 511.27. This material is dissolved in 4 M HCl in dioxane
(10 mL), and the mixture is stirred at room temperature for 18 h. The
solvent is removed in vacuo and the crude product is purified by
preparative HPLC to give the title compound 104 (160 mg, 80%).
LC−MS: tR = 1.10 min, [M + 1] = 471.28. 1H NMR (CDCl3): δ 7.86
(s, 1 H), 7.85 (s, 1 H), 4.13−4.20 (m, 1 H), 3.82−3.99 (m, 4 H), 3.17
(t, J = 6.7 Hz, 2 H), 2.80 (q, J = 7.5 Hz, 2 H), 2.76 (q, J = 7.8 Hz, 2
H), 2.40 (s, 3 H), 2.38 (s, 2 H), 2.10 (s br, 1 H), 1.67 (t, J = 6.7 Hz, 2
H), 1.34 (t, J = 7.8 Hz, 3 H), 1.32 (t, J = 7.0 Hz, 3 H), 1.05 (s, 6 H).
13C NMR (CDCl3): δ 172.2, 168.1, 157.0, 147.7, 144.6, 137.5, 135.7,
(b) To a solution of 2-(2-ethyl-4-(5-(3-ethyl-5,5-dimethyl-4,5,6,7-
tetrahydrobenzo[c]thiophen-1-yl)-1,2,4-oxadiazol-3-yl)-6-
methylphenoxy)ethanol (1.10 g, 2.50 mmol) in DCM triethylamine
(354 mg, 3.50 mmol) is added. The mixture is cooled to 0 °C before
methanesulfonyl chloride (343 mg, 3.00 mmol) is added. The mixture
is stirred at 0 °C for 1 h, then at room temperature for 2 h before it is
diluted with DCM (50 mL), washed with H2O (50 mL), dried over
MgSO4, filtered, and concentrated to give a crude mesylate
intermediate (1.46 g, quantitative). LC−MS: tR = 1.14 min, [M + 1]
= 511.99. This material is added to a solution of β-alanine methyl ester
obtained by filtering a solution of the corresponding hydrochloride salt
(471 mg, 3.37 mmol) in acetonitrile over Dowex Marathon ion-
exchange resin (4 g, OH form). Triethylamine (49 mg, 0.482 mmol) is
added, and the mixture is stirred at 80 °C for 96 h in a sealed vessel.
The mixture is concentrated and the crude product is purified by
preparative HPLC to give methyl 3-((2-(2-ethyl-4-(5-(3-ethyl-5,5-
dimethyl-4,5,6,7-tetrahydrobenzo[c]thiophen-1-yl)-1,2,4-oxadiazol-3-
yl)-6-methylphenoxy)ethyl)amino)propanoate which is dissolved in
THF (5 mL), methanol (4 mL), and 3 M aqueous NaOH (1 mL).
The mixture is stirred at room temperature for 8 h. The mixture is
diluted with water acidified with aqueous HCl and extracted twice with
DCM (2 × 50 mL). The organic extracts are washed with brine, dried
over MgSO4, filtered, concentrated, and dried to give the title
compound 106 (115 mg, 47%) as a colorless resin. LC−MS: tR = 0.92
min, [M + 1] = 512.05. 1H NMR (CDCl3): δ 9.41 (s br, 2 H), 7.78 (s,
1 H), 7.74 (s, 1 H), 4.26 (t, J = 4.8 Hz, 2 H), 3.44−3.61 (m, 4 H),
3.08−3.20 (m, 4 H), 2.79 (q, J = 7.5 Hz, 2 H), 2.71 (q, J = 7.4 Hz, 2
H), 2.37−2.44 (m, 2 H), 2.36 (s, 2 H), 2.35 (s, 3 H), 1.65 (t, J = 6.8
Hz, 2 H), 1.32 (t, J = 7.5 Hz, 3 H), 1.29 (t, J = 8.0 Hz, 3 H), 1.04 (s, 6
131.5, 128.4, 126.7, 123.2, 115.2, 73.9, 71.0, 63.8, 38.5, 35.5, 29.6, 27.9,
24.3, 22.9, 21.6, 16.4, 14.9. LC−HRMS: tR = 1.80 min, [M + H]/z =
471.2318, found = 471.2315.
1
H). H NMR (DMSO-d6): δ 7.76 (s, 2 H), 4.09 (t, J = 4.5 Hz, 2 H),
3-(2-Ethyl-4-(5-(3-ethyl-5,5-dimethyl-4,5,6,7-tetrahydro-
benzo[c]thiophen-1-yl)-1,2,4-oxadiazol-3-yl)-6-methylphenyl)-
propanoic Acid (105). To a solution of 3-ethyl-5,5-dimethyl-4,5,6,7-
tetrahydrobenzo[c]thiophene-1-carboxylic acid 10 (2.00 g, 8.39 mmol)
in DMF (20 mL) DIPEA (3.25 g, 25.2 mmol) followed by TBTU
(2.83 g, 8.81 mmol) is added. The mixture is stirred at room
temperature for 5 min before 3-[2-ethyl-4-(N-hydroxycarbamimidoyl)-
6-methylphenyl]propanoic acid (2.10 g, 8.39 mmol, Supporting
Information) is added. Stirring is continued at room temperature for
2 h. Water (50 mL) and saturated aqueous NaHCO3 solution (50 mL)
are added, and the mixture is extracted twice with EA (2 × 100 mL).
The organic extracts are combined, dried over MgSO4, filtered, and
concentrated. The residue is dissolved in dioxane (50 mL), and the
mixture is stirred at 80 °C for 18 h. The solvent is evaporated and the
crude product is purified by preparative HPLC to give the title
compound 105 (2.85 g, 75%) as a white solid. LC−MS: tR = 1.14 min,
[M + 1] = 453.13. 1H NMR (DMSO-d6): δ 12.28 (s br, 1 H), 7.69 (s,
1 H), 7.69 (s, 1 H), 3.07 (t, J = 6.6 Hz, 2 H), 2.90−2.96 (m, 2 H), 2.80
(q, J = 7.4 Hz, 2 H), 2.72 (q, J = 7.5 Hz, 2 H), 2.36−2.42 (m, 7 H),
1.62 (t, J = 6.6 Hz, 2 H), 1.25 (t, J = 7.5 Hz, 3 H), 1.22 (t, J = 7.5 Hz, 3
H), 0.99 (s, 6 H). 13C NMR (DMSO-d6): δ 174.2, 171.9, 168.1, 148.2,
144.9, 143.3, 141.2, 137.7, 136.3, 127.0, 125.3, 124.4, 114.6, 38.2, 35.1,
3.27 (t, J = 7.0 Hz, 2 H), 3.06 (t, J = 6.5 Hz, 2 H), 2.70−2.84 (m, 6 H),
2.38 (s, 5 H), 1.61 (t, J = 6.5 Hz, 2 H), 1.25 (t, J = 7.5 Hz, 3 H), 1.24
(t, J = 7.5 Hz, 3 H), 0.99 (s, 6 H). 13C NMR (DMSO-d6): δ 172.4,
172.0, 167.8, 157.3, 148.3, 145.1, 138.1, 136.4, 132.4, 128.3, 126.4,
122.9, 114.5, 68.3, 47.4, 43.6, 38.2, 35.1, 30.9, 29.7, 28.0, 24.3, 22.6,
21.4, 16.7, 15.3, 15.2. LC−HRMS: tR = 1.35 min, [M + H]/z =
512.2583, found = 512.2584.
ASSOCIATED CONTENT
■
S
* Supporting Information
Experimental details on the synthesis and characterization of all
target compounds and intermediates not described in the main
text. This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: +41 61 565 65 65. Fax: +41 61 565 65 00. E-mail:
martin.bolli@actelion.com.
93
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors gratefully acknowledge their colleagues Stefan
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ABBREVIATIONS USED
■
AcOH, acetic acid; BuLi, butyllithium; CC, column chromatog-
raphy; DCM, dichloromethane; DEAD, diethyl azodicarbocy-
late; DMF, dimethylformamide; EA, ethyl acetate; EDC, 1-
ethyl-3-(3-dimethylaminopropyl)carbodiimide; HOBt, N-hy-
droxybenzotriazole; HTS, high-throughput screening; HV,
high vacuum; LC, lymphocyte count; LDA, lithium diisopro-
pylamide; PK, pharmacokinetics; PD, pharmacodynamics; SAR,
structure−activity relationship; TBTU, O-(benzotriazol-1-yl)-
N,N,N′,N′-tetramethyluronium tetrafluoroborate; THF, tetra-
hydrofuran; S1P, sphingosine 1-phosphate
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