Novel S1P1 receptor agonists - Part 3: From thiophenes to pyridines

Article abstract of DOI:10.1021/jm4014696

In preceding communications we summarized our medicinal chemistry efforts leading to the identification of potent, selective, and orally active S1P ₁ agonists such as the thiophene derivative 1. As a continuation of these efforts, we replaced the thiophene in 1 by a 2-, 3-, or 4-pyridine and obtained less lipophilic, potent, and selective S1P₁ agonists (e.g., 2) efficiently reducing blood lymphocyte count in the rat. Structural features influencing the compounds' receptor affinity profile and pharmacokinetics are discussed. In addition, the ability to penetrate brain tissue has been studied for several compounds. As a typical example for these pyridine based S1P ₁ agonists, compound 53 showed EC₅₀ values of 0.6 and 352 nM for the S1P₁ and S1P₃ receptor, respectively, displayed favorable PK properties, and penetrated well into brain tissue. In the rat, compound 53 maximally reduced the blood lymphocyte count for at least 24 h after oral dosing of 3 mg/kg.

Full text of DOI:10.1021/jm4014696

Article
pubs.acs.org/jmc
Novel S1P1 Receptor Agonists − Part 3: From Thiophenes to
Pyridines
Martin H. Bolli,* Stefan Abele, Magdalena Birker, Roberto Bravo, Daniel Bur, Ruben de Kanter,
Christopher Kohl, Julien Grimont, Patrick Hess, Cyrille Lescop, Boris Mathys, Claus Muller,
̈
Oliver Nayler, Markus Rey, Michael Scherz, Gunther Schmidt, Jurgen Seifert, Beat Steiner, Jorg Velker,
and Thomas Weller
̈
̈
Drug Discovery Chemistry, Actelion Pharmaceuticals Ltd., Gewerbestrasse 16, CH-4123 Allschwil, Switzerland
S
* Supporting Information
ABSTRACT: In preceding communications we summarized
our medicinal chemistry efforts leading to the identification of
potent, selective, and orally active S1P1 agonists such as the
thiophene derivative 1. As a continuation of these efforts, we
replaced the thiophene in 1 by a 2-, 3-, or 4-pyridine and
obtained less lipophilic, potent, and selective S1P1 agonists
(e.g., 2) efficiently reducing blood lymphocyte count in the rat.
Structural features influencing the compounds’ receptor
affinity profile and pharmacokinetics are discussed. In addition,
the ability to penetrate brain tissue has been studied for several
compounds. As a typical example for these pyridine based S1P1
agonists, compound 53 showed EC50 values of 0.6 and 352 nM
for the S1P1 and S1P3 receptor, respectively, displayed
favorable PK properties, and penetrated well into brain tissue. In the rat, compound 53 maximally reduced the blood
lymphocyte count for at least 24 h after oral dosing of 3 mg/kg.
other companies,38−41 but it is not known whether they entered
clinical development yet.
INTRODUCTION
■
Synthetic sphingosine 1-phosphate 1 (S1P1) receptor agonists
hold great promise for treating inflammatory disorders and
autoimmune diseases,1−3 and the number of clinical trials
investigating their safety and efficacy is growing rapidly. While
fingolimod4−7 has approval to treat relapsing multiple sclerosis,
siponimod,8−10 ponesimod,11,12 and ONO-4641 (structure
undisclosed)13,14 successfully completed phase II and RPC-
1063 (structure undisclosed)15 and MT-1303 (structure
undisclosed)16 recently entered phase II clinical trials for this
debilitating disease. The last two compounds are also evaluated
in patients suffering from ulcerative colitis17 and inflammatory
bowel disease,18 respectively. Results of an open-label phase I
study in MS patients treated with CS-0777 have been published
recently.19,20 In addition, siponimod is currently studied in
patients suffering from secondary progressive multiple scle-
rosis21 as well as in patients with polymyositis and
dermatomyositis.22 Ponesimod, on the other hand, successfully
completed a phase II trial for chronic plaque psoriasis.23,24
Furthermore, fingolimod’s ability to reduce graft rejection has
been studied in renal transplant patients,25 and KRP-20326 was
investigated in patients with subacute cutaneous lupus
erythematosus27 and refractory ulcerative colitis.28 APD334
(structure undisclosed)29,30 recently entered phase I clinial
trials. Additional molecules emerged from discovery programs
The lysophospholipid sphingosine 1-phosphate (S1P) is the
natural ligand of five G-protein-coupled receptors named
S1P1−S1P5.42 S1P−S1P receptor signaling is involved in
many cellular responses including survival, proliferation,
differentiation, adhesion, migration, and chemotaxis43−45 and
leads to angiogenesis, endothelial barrier enhancement, airway
and blood vessel constriction, alveolar epithelial barrier
disruption, heart rate modulation, neurite extension, and bone
homeostasis.46−49 The physiological role of S1P receptor
signaling has been studied in several organs and tissues such
as the nervous system,50−52 the lung,46,53 the cardiovascular
system,54−58 the cells of the immune system,59−61 and bone
tissue,62 and S1P receptor activation has been implicated in
many pathological situations such as autoimmunity, inflamma-
tion, cardiovascular disorders, and cancer.3,60,61,63−66
S1P plays an important role in the motility of various cell
types,62,67,68 in particular of the immune system.69−72
Lymphocytes continuously circulate through the body, thereby
monitoring the body for microbial intruders or aberrant cells
and home to lymph nodes and Peyer’s patches.73 In order to
leave these secondary lymphoid organs and return to
Received: September 23, 2013
Published: December 24, 2013
37
at Almirall,31,32 Glaxo,33−35 Bristol-Myers Squibb,36
and
e.g.
© 2013 American Chemical Society
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circulation, lymphocytes follow the gradient of S1P that exists
between lymph and blood. This chemotactic event is driven by
the S1P/S1P1 receptor signaling axis.69,74−76 Synthetic S1P1
agonists have been shown to trigger internalization of the S1P1
receptor, thereby abolishing the lymphocyte’s ability to sense
the S1P gradient.76,77 As a consequence, lymphocytes are
sequestered to lymph nodes and no longer migrate to other
organs. Interfering with lymphocyte trafficking by S1P1 receptor
modulation represents an attractive approach to treat a variety
of lymphocyte dependent autoimmune diseases, as demon-
strated in several clinical trials. In contrast, activation of the
S1P3 receptor is deemed undesirable, as it has been associated
with effects on heart rate,78−84 vaso- and bronchoconstric-
tion,82,85 hypertension,84 and enhancement of fibrosis86−88 in
animal studies.
In addition to the peripheral immunomodulatory action of
S1P1 receptor agonists, more recent studies identified
modulation of the S1P1, S1P3, or S1P5 receptors on neural
cells like astrocytes and oligodendrocytes as a nonimmunologic
central nervous system (CNS) effect possibly contributing to
the efficacy of synthetic S1P receptor agonists in multiple
sclerosis.6,89−95 Following these arguments, brain penetration
of a synthetic S1P1 receptor agonist may be beneficial for a
compound’s efficacy in CNS related diseases such as multiple
sclerosis. On the other hand, for the treatment of peripheral
diseases such as psoriasis or Crohn’s disease, CNS penetration
of a S1P receptor agonist is not needed or may even be
undesired. Hence, the utility of a compound for different
diseases may depend on its ability to penetrate brain tissue, and
we therefore characterized some of our most advanced
compounds with respect to their potential to penetrate the
brain.
making the pyridine an attractive alternative to the thiophene in
1. In the following sections we describe our detailed studies
aiming at the discovery of pyridine containing S1P1 receptor
agonists meeting the following requirements: the compound
has a lower clogP than thiophene 1 (ideally <3), is highly
potent on S1P1 (EC50 < 5 nM) with an affinity of >250 nM for
S1P3 and a more than 100-fold selectivity against this receptor,
and shows maximal lymphocyte count reduction for at least 24
h when administered at a dose of 10 mg/kg to Wistar rats. For
compounds fulfilling these criteria the ability to penetrate the
brain would then be measured to assess their utility in different
diseases.
RESULTS AND DISCUSSION
■
Synthesis. The target compounds 2−54 for this study were
usually prepared by coupling each of the pyridine carboxylic
acids 59−79 with N-hydroxybenzamidine 80 followed by
cyclizing the hydroxyamidine ester intermediate to the desired
oxadiazole (Scheme 1). N-Hydroxybenzamidine 80 was
a
Scheme 1. Assembling the Central Oxadiazole Ring
Previously we described the discovery of a novel series of
selective S1P1 receptor agonists incorporating a 5-isobutyl
substituted thiophene.96 A prototypical example, compound 1
(Figure 1), had EC50 values of 0.7 and 320 nM in a GTPγS
a
Reagents and conditions: (a) HOBt, EDC HCl, THF, rt, 18 h; or
TBTU, Hunig’s base, DMF, rt, 1 h; (b) dioxane, 80 °C, 18 h, 6−76%
(two steps).
̈
prepared by formylating phenol 81 in a Duff reaction to give
benzaldehyde 82 (Scheme 2). Reacting 82 with hydroxylamine
hydrochloride in NMP under microwave irradiation furnished
benzonitrile 83 which was then alkylated with (R)-glycidol
under Mitsunobu conditions. Epoxide 84 was reacted with
ammonia in methanol to give amino alcohol 85. Coupling of 85
with glycolic acid to give 86 established the side chain attached
to the benzene ring. Finally, nitrile 86 was reacted with
hydroxylamine hydrochloride to afford the desired building
block 80. In some cases the polar side chain attached to the
para position of the phenyl moiety was introduced after the
oxadiazole ring had been established. In this case, the
appropriate pyridine carboxylic acid was reacted with 3-ethyl-
N,4-dihydroxy-5-methylbenzamidine and the side chain was
established in analogy to the steps outlined in Scheme 2 (for
details see Supporting Information). For details on the
synthesis of compounds 55−58 (Table 7) see Supporting
Information.
Figure 1. Structures of thiophene based S1P1 receptor agonist 1 and a
first 3-pyridine analogue 2.
assay for S1P1 and S1P3, respectively. With a clogP value of 3.97
this thiophene derivative is rather lipophilic. As high
lipophilicity has been shown to be associated with compound
liabilities such as target promiscuity, hERG inhibition, poor
aqueous solubility, metabolic instability, or high protein
binding,97−101 we embarked on a program in which the
thiophene head of our S1P1 receptor agonists was replaced by a
pyridine. A first such analogue, pyridine derivative 2, has a
clogP of 2.74 and showed an EC50 value of 5.7 nM for S1P1,
Prototypical syntheses of the various pyridinecarboxylic acids
are exemplified in Schemes 3, 4, 5, and 6. Alkyl groups could be
introduced to the pyridines by making use of transition metal
catalyzed cross-coupling reactions developed by Suzuki,102−104
Negishi,104 and Furstner.105,106 The choice of the reaction type
̈
was generally driven by the availability of the corresponding
alkyl electrophile. Alkylamines were introduced either by simple
thermal displacement or by a Pd-catalyzed Buchwald−Hartwig
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a
Scheme 2. Preparation of the N-Hydroxybenzamidine
Building Block 80
Scheme 3. Preparation of 6-Alkylnicotinic Acids
a
a
Reagents and conditions: (a) hexamethylenetetraamine, HOAc, H2O,
120 °C, Dean−Stark 2−3 h, 53−97%; (b) HONH2 HCl, NMP,
microwave 80−100 °C, 0.5−3 h; 86−95%; (c) (R)-glycidol, PPh3,
DEAD, THF, 0−20 °C, 18 h, 72−86%; (d) 7 N NH3 in MeOH, 65
°C, 18 h (sealed vessel), quantitative (crude); (e) glycolic acid, EDC
HCl, HOBt, rt, 18 h, 51−90%; (f) HONH2 HCl, NaHCO3 or Et3N,
MeOH or EtOH 60−80 °C, 18 h, 60−87%.
reaction.107 For more details see Supporting Information.
Scheme 3 illustrates the synthesis of some nicotinic acids. 6-
Alkylnicotinic acid esters 88a−e were obtained by reacting
ethyl 6-chloronicotinate 87 either with the appropriate alkenyl
boronic ester under Suzuki conditions followed by catalytic
hydrogenation or with the appropriate Grignard reagent under
Furstner conditions. Ester cleavage under acidic conditions
̈
afforded the 6-alkylnicotinic acid building blocks 59a−e. 6-
Alkyl-5-methylnicotinic acids (e.g., 60) were prepared in a
similar fashion starting from ethyl 5-methyl-6-chloronicotinate
90 employing 2,4,6-tri-(2-alkenyl)cyclotriboroxanepyridine
complexes or alkenylboronic acid pinacol esters under Suzuki
conditions.108 Catalytic hydrogenation of the ethyl 6-
alkenylnicotinate intermediate furnished the corresponding
ethyl 6-alkyl-5-methylnicotinate (e.g., 91) which upon acidic
ester cleavage afforded the desired nicotinic acid building block
(e.g., 60). Ethyl nicotinate 90 was prepared by oxidizing
aldehyde 89109 with hydrogen peroxide and subsequent
esterification of the resulting nicotinic acid carboxylic acid. In
an alternative approach, isopropyl 5,6-dichloronicotinate 92
was first reacted with cyclopentylmagnesium bromide at room
temperature in the presence of Fe(acac)3 to give isopropyl 5-
chloro-6-cyclopentylnicotinate 93 which was then converted to
nicotinate 94 using dimethylzinc at 75−85 °C. Saponification
yielded nicotinic acid 61. Reacting ethyl 5,6-dichloronicotinate
95 with 2,4,6-tri(2-methylprop-1-en-1-yl)cyclotriboroxane-
pyridine complex under aqueous Suzuki conditions furnished
5-chloronicotinate 96 which was then subjected to anhydrous
Suzuki conditions in the presence of 2,4,6-trivinylcyclo-
triboroxanepyridine complex to afford 5,6-dialkenylnicotinate
97. Hydrogenation and ester cleavage furnished 5-ethyl-6-
isobutylnicotinic acid 62.
a
Reagents and conditions: (a) 2-alkenyl-4,4,5,5-tetramethyl-1,3,2-
dioxaborolane, Pd(PPh3)4, PPh3, 2 M aq K2CO3, 80 °C, 18 h, 66%,
then 5 bar of H2, Pd/C, methanol, 18 h, 46%; (b) alkylmagnesium
bromide, Fe(acac)3, THF, NMP, −75 °C, 1−4 h, 47−93% (c) 4−7 N
aq HCl, 65−80 °C, 18−24 h, 91−97%; (d) 50% H2O2, HCOOH, 0
°C, 15 h, 47−87%; (e) EtOH, H2SO4, or TMSCl, reflux 18−42 h, 65−
92%; (f) 2,4,6-tri(2-methylpropenyl)cyclotriboroxanepyridine complex
or alkenylboronic acid pinacol ester, Ph3P, Pd(PPh3)4, DME or
dioxane, 2 M aq K2CO3, 90 °C, 20 h, 71−73%; (g) Pd/C, H2, MeOH,
THF, rt, 15 h, 61−99%; (h) cyclopentylmagnesium bromide,
Fe(acac)3, THF, NMP, 0 °C, 1 h, rt, 18 h, 78%; (i) Me2Zn,
Pd(dppf)Cl2, dioxane, 75−85 °C, 3−18 h, 36−58%; (j) 2 M aq LiOH,
MeOH or EtOH, rt, 1−2 h, 83−88%; (k) 2,4,6-tri(2-methylpropenyl)-
cyclotriboroxanepyridine complex, Ph3P, Pd(PPh3)4, DME, 2 M aq
K2CO3, 100 °C, 4.5 h, 92%; (l) 2,4,6-trivinylcyclotriboroxanepyridine
complex, Cs2CO3, tri-tert-butylphosphine, Pd2(dba)3, or PPh3, Pd-
(PPh3)4, dioxane, 100 °C, 16−40 h, 59−67%; (m) n-BuLi, 3-
pentanone, THF, −70 °C, 2 h, 37%; (n) KMnO4, acetone, rt, 24 h,
quantitative crude; (o) Burgess reagent, dioxane, 80 °C, 1 h
microwave, 76%; (p) 1 bar of H2, Pd/C, THF/methanol 1:1, rt, 4
h, 80%.
with 3-pentanone to form 5-bromopyridine 99, which was
converted to vinylpyridine 100 using trivinylcyclotriboroxane-
pyridine complex under Suzuki conditions. The olefin in 100
was cleaved by KMnO4, and the resulting acid was esterified to
give nicotinate 101. Treating 101 with Burgess reagent under
microwave irradiation effected dehydration, and the corre-
sponding unsaturated product was hydrogenated using H2 on
In the case of the 3-pentyl derivative 63 the above
approaches were unsuccessful. We therefore followed an
alternative strategy. Thus, 2,5-dibromo-3-methylpyridine 98
was lithiated using 1.1 equiv of n-butyllithium and then reacted
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a
The isomeric picolinic acid 72 was prepared following the same
strategy starting from 2,4-dibromopicoline 119. Thus, a vinyl
group was introduced to position 6 of 119 via Suzuki reaction
to give 120 which was then oxidized and esterified to form
picolinate 121. Suzuki reaction using 2,4,6-tri(2-
methylpropenyl)cyclotriboroxanepyridine complex, catalytic
hydrogenation, and saponification furnished the desired
compound 72. Ethyl 4-bromo-6-methylpicolinate 121 could
also be prepared via an alternative route making use of the
Minisci reaction.110−112 To this end, 4-bromopicoline 123 was
treated with ammonium peroxydisulfate in methanol containing
a small amount of sulfuric acid to give hydroxymethylpyridine
124 in moderate yield. Oxidation of the primary alcohol with
KMnO4 and esterification of the resulting acid gave picolinate
121. The 6-diethylaminopicolinic acid 73 was obtained by
Buchwald reaction of 6-chloropicolinate 125 and subsequent
saponification. The isomeric acid 74 was obtained by thermal
displacement of the 4-bromo substituent of acid 126. To
enhance purification, the acid was temporarily transformed to
its ethyl ester.
Scheme 4. Preparation of 6-Aminonicotinic Acids
a
Reagents and conditions: (a) R1R2NH, 80−100 °C, 1 day to 3 weeks,
85−99%; (b) 4−7 N aq HCl, 65−80 °C, 18−24 h, quantitative; (c)
Et2Zn, Pd(dppf)Cl2, dioxane, 75 °C, 18−24 h, 84% to quantitative;
(d) 3 N aq NaOH, MeOH, 75 °C, 18 h, 79%.
Finally, Scheme 6 illustrates the synthesis of 2-substituted
and 2,6-disubstituted isonicotinic acids. The preparation of the
2-substituted representatives 75a−h started from 2-haloisoni-
Pd/C. Saponification of this material finally furnished the
desired 6-(3-pentyl)nicotinic acid derivative 63.
cotinic ester 127 and involved a Negishi or a Furstner type
̈
cross-coupling reaction with the appropriate alkylzinc or
alkylmagnesium halide, respectively, and subsequent ester
cleavage. The 6-methylisonicotinic acids 76a−g were prepared
in a similar fashion via Negishi or Suzuki reaction of the 2-halo-
6-methylisonicotinate 129. 2-Alkyl-6-ethylisonicotinic acids
77a−d were prepared starting from ethyl 2,6-dichloroisonico-
tinate 131 by two consecutive Negishi couplings. Although the
bulkier alkyl group was introduced first, the monochloro-
isonicotinates 132a−d were usually contaminated with 10−
25% of the corresponding double alkylation product. In the
case of Negishi reactions employing 3-pentylzinc bromide,
formation of a significant amount of the isomeric 2-(pentan-2-
yl)isonicotinates clearly hampered the purification of the
reaction products. Hence, Suzuki reaction and subsequent
catalytic hydrogenation as employed to prepare 6-methyl
isonicotinic esters 130a−e often gave better results when
6-Aminonicotinic acids 64a,b were obtained from their esters
102a,b which were prepared by heating 6-chloronicotinate 90
in a sealed vessel with a large excess of the corresponding amine
(Scheme 4). Alternatively, 5,6-dichloronicotinate 95 was
reacted with diethylamine to form ethyl 5-chloro-6-amino-
nicotinate 103a. When pyrrolidine was used as the amine
component, the ester in 95 was transformed to the
corresponding pyrrolidine amide 103b. Pd-catalyzed Negishi
coupling of 103a,b with diethylzinc and subsequent ester
hydrolysis afforded the 6-amino-5-ethylnicotinic acids 65a,b.
The synthesis of prototypical 4,5-, 5,6-, and 4,6-disubstituted
picolinic acids is outlined in Scheme 5. Reacting 2,5-
dibromopyridine 104 or 108 with trivinylcyclotriboroxane-
pyridine complex selectively furnished the corresponding 2-
vinylpyridines 105 and 109 in good yields. Potassium
permanganate mediated oxidation established the carboxylic
acids which were esterified to deliver 106 and 110. A second
Suzuki reaction using 2,4,6-tri(2-methylpropenyl)-
cyclotriboroxanepyridine complex produced the 5-alkenylpico-
linic acid derivatives 107 and 111. Catalytic hydrogenation and
subsequent ester cleavage completed the synthesis of the two
picolinic acids 66 and 67. Negishi coupling of 5-bromo-4-
methylpicolinate 106 with cyclopentylzinc bromide furnished
ester 112 which was cleaved under basic conditions to give
cyclopentyl-4-methylpicolinic acid 68. 5-Diethylaminopicolinic
acids 69 and 70 were prepared by reacting 5-bromopicolinates
113 and 114, respectively, with diethylamine under Buchwald−
Hartwig conditions and subsequent ester hydrolysis. Although
the yields of these cross-coupling reactions were rather low,
sufficient material could be isolated for the purpose of our
studies. The preparation of 4,6-disubstituted picolinic acids
followed a strategy similar to the one pursued for the 3,4- and
2,3-disubstituted picolinic acids 66 and 67. Hence, 2,6-dichloro-
4-methylpyridine 115 was first converted to 2-chloro-4-methyl-
6-vinylpyridine 116 and then oxidized and esterified to give 2-
chloropicolinate 117. Suzuki cross-coupling of this material
with 2,4,6-tri(2-methylpropenyl)cyclotriboroxanepyridine com-
plex furnished the 6-isobutenylpicolinate 118 which, after
hydrogenation and ester hydrolysis, produced picolinic acid 71.
compared to a Negishi or Furstner cross-coupling approach.
̈
2-Amino-6-methylisonicotinic acids 78a,b and 79 were
prepared starting from 2-chloro-6-methyl- or 2,6-dichloroiso-
nicotinic acid ester 134 or 136, respectively. A large excess of
the amine was usually employed when 2-chloro-6-methyl
isonicotinate 134 was reacted under thermal, noncatalyzed
conditions. However, the use of a large excess of pyrrolidine led
to the formation of the corresponding amide (e.g., 135b) which
then had to be cleaved under basic conditions to give the
desired acid (78b). A much smaller excess of the amine could
be used if the reaction was carried out under Pd-catalyzed
Buchwald conditions (e.g., 135a). In addition, 1 equiv of
diethylamine was sufficient to efficiently convert tert-butyl 2,6-
dichloroisonicotinate 136 to the corresponding monochloride
137. 6-Chloropicolinate 137 was then treated with dimethyl-
zinc to afford the 6-methyl substituted isonicotinate 138 which
upon ester cleavage delivered the isonicotinic acid 79.
In Vitro SAR Discussion. As mentioned in the
introduction, we wanted to reduce the overall lipophilicity
and amphiphilic nature of our S1P1 agonists such as 1 (Figure
1). In our previous studies,113,96 we learned that a large variety
of polar side chains are tolerated in the 4-position of the phenyl
ring of 1. This offered a starting point to improve the
113
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a
Scheme 5. Preparation of Picolinic Acids
a
Reagents and conditions: (a) 2,4,6-trivinylcyclotriboroxanepyridine complex, 2 M aq K2CO3, Pd(PPh3)4, DME, 80 °C, 15 h, 49−99%; (b) KMnO4,
acetone, water, rt, 3 days, quantitative crude; (c) EtOH, H2SO4 or TMSCl, 70 °C, 18 h, 40−95%; (d) 2,4,6-tri(2-methylpropenyl)cycloboroxane-
pyridine complex, Pd(PPh3)4, 2 M aq K2CO3, DME, 80 °C, 6 h, 37−68%; (e) H2, Pd/C, THF, EtOH, rt, 15 h, 95%; (f) 6−7 N aq HCl, 65 °C, 48 h,
64−94%; (g) cyclopentylzinc bromide, Pd(dppf)Cl2, dioxane, 65 °C, 18 h, 28−70% (h) 2 M aq LiOH, dioxane, 75 °C, 5 h, 15−88%; (i) Et2NH,
Pd(OAc)2, xanthphos, Cs2CO3, dioxane, 90−110 °C, 18−24 h, 1−9%; (j) MeOH, H2SO4, ammonium peroxydisulfate, 75 °C, 2 h, 19−28%; (k)
Et2NH, BuOH, reflux 16 h; EtOH, H2SO4, 70 °C, 18 h; 6 N aq HCl, 65 °C, 18 h, 40% (three steps).
compounds’ physicochemical properties. In a next step, we
explored whether polar groups were also tolerated in the
headgroup. In this account, we focus our discussion on the
replacement of the thiophene by a pyridine. For the following
SAR discussion the (S)-3-(3-ethyl-4-(2-hydroxy-3-(2-
hydroxyacetamido)propoxy)-5-methylphenyl)-1,2,4-oxadiazole
part was kept constant, as this moiety reliably brought about
high affinity and selectivity for S1P1 and high in vivo efficacy.
A set of 3-pyridines representing close analogues of
compound 1 is compiled in Table 1. As evident from the
clogP values listed in Table 1, even pyridines bearing large and
lipophilic substituents such as the 3-pentyl derivative 11 or the
cyclopentylpyridines 12 and 14 are less liphophilic than
thiophene 1. The closest analogue of thiophene 1, the
isobutylpyridine 2, had a lower clogP (2.75 vs 3.97) and
showed an EC50 of 5.7 nM for S1P1, demonstrating that
replacing the thiophene in 1 by a pyridine reduces the
compound’s lipophilicity while retaining its affinity for the S1P1
receptor. The S1P1/3 selectivity ratio is comparable for the two
compounds 1 and 2. The potency on S1P1 and S1P3 remained
unaffected when the isobutyl chain in 2 was replaced by an n-
propyl (4) or isopropyl (5) group. However, when the alkyl
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a
Scheme 6. Preparation of Pyridine-4-carboxylic Acids
Table 1. SAR of 3-Pyridines (Nicotinic Acid Derivatives)
a
EC50 [nM]
compd
R1
R2
clogP
S1P1
S1P3
320
1
2
b
3.97
2.75
2.75
2.41
2.37
1.94
1.59
3.06
3.06
2.68
3.61
3.20
3.42
3.56
2.83
3.18
2.24
2.59
0.7
5.7
89
isobutyl
H
1710
5740
1770
2900
>10000
>10000
318
c
3
isobutyl
H
4
5
6
7
8
n-propyl
H
4.6
3.9
23
isopropyl
ethyl
H
H
methyl
H
248
1.0
1.9
0.1
0.9
0.1
0.1
0.2
2.1
0.3
23
isobutyl
Me
Me
Me
Me
Me
Et
c
9
isobutyl
519
10
11
12
13
14
15
16
17
18
isopropyl
pent-3-yl
cyclopentyl
isobutyl
429
2630
25
29
cyclopentyl
diethylamino
diethylamino
N-pyrrolidine
N-pyrrolidine
Et
24
Me
Et
616
30
Me
Et
1230
176
2.8
a
EC50 values as determined in a GTPγS assay using membranes of
CHO cells expressing either S1P1 or S1P3.11 EC50 values represent the
geometric mean of at least three independent measurements. For
structure, see Figure 1. (R)-Enantiomer.
b
c
a
Reagents and conditions: (a) alkylzinc bromide or chloride, or
dialkylzinc, Pd(dppf)Cl2, dioxane, 75−80 °C, 2−24 h, 21−84%; (b)
alkylmagnesium bromide or chloride, Fe(acac)3, THF, NMP, −75 °C,
1 h, rt, 1 h, 26−40%; (c) 6−7 M aq HCl, 60−95 °C, 3−20 h, 37% to
quantitative; (d) 2,4,6-trialkenylcycloboroxanepyridine complex, PPh3,
Pd(PPh3)4, 2 M aq K2CO3, DME, 75−90 °C, 6−20 h, 50−95%; (e)
H2, Pd/C, THF, MeOH, rt, 5−16 h, 98%; (f) Et2Zn, Pd(dppf)Cl2,
dioxane, 75−80 °C, 1−2 h, 79−82%; (g) diethylamine, 70−100 °C,
2−72 h, 95% to quantitative; (h) ethylamine, Cs2CO3, Pd(OAc)2,
Xanthphos, dioxane, 90 °C, 15 h, 44−47%; (i) 3 N aq NaOH, EtOH,
60 °C, 72 h; 87%.
receptors. As observed with the alkyl substituted pyridines, the
5-ethyl analogues 16 and 18 were clearly more potent on S1P1
and S1P3 when compared to their 5-methyl analogues 15 and
17, respectively.
In a next step we turned our attention to replacing the
thiophene in 1 by a number of substituted 2-pyridines bearing
two substituents in either an ortho or a meta relationship. A few
examples illustrating their SAR are listed in Tables 2 and 3. As
judged by their clogP values, the most lipophilic 2-pyridines in
Table 2. SAR of 4,5- and 5,6-Disubstituted Pyridin-2-yl
Derivatives (Picolinic Acid Derivatives)
group was further shortened to an ethyl (6) or a methyl (7)
group, a significant potency loss was observed. As shown with
compounds 8 and 10, introducing a methyl group to position 5
of the pyridine clearly enhanced the compound’s affinity for
S1P1 and S1P3. An ethyl group in position 5 brought a further
potency gain on both receptors, and several compounds (e.g.,
13, 14, 16) reached EC50 values close to 0.1 nM for S1P1 and
about 25 nM for S1P3. While the (R)-enantiomer of 2,
compound 3, was significantly less potent in particular on S1P1,
the two enantiomers 8 and 9 showed an almost identical affinity
profile. As shown with compound 11, replacing the isobutyl
chain in 8 by a 3-pentyl group had no significant effect on S1P1
but significantly increased the compound’s selectivity against
S1P3. Interestingly, the cyclopentyl derivative 12 was clearly
less selective against S1P3 when compared to its open chain
analogue 11. Replacing the 3-pentyl group in 11 by
diethylamine to give aminopyridine 15 was well tolerated by
S1P1 and slightly improved the compound’s affinity for S1P3.
Conversely, exchanging the cyclopentyl group in 12 by a
pyrrolidine to give 17 resulted in a marked affinity loss on both
a
EC50 [nM]
compd
R1
X
Y
clogP
S1P1
S1P3
19
20
21
22
23
24
isobutyl
N
CH
N
3.02
3.12
3.15
3.26
2.41
2.52
0.2
4.4
0.2
0.8
0.2
9.5
154
isobutyl
CH
N
3080
106
cyclopentyl
cyclopentyl
diethylamino
diethylamino
CH
N
CH
N
860
CH
N
617
CH
5960
a
EC50 values as determined in a GTPγS assay using membranes of
CHO cells expressing either S1P1 or S1P3.11 EC50 values represent the
geometric mean of at least three independent measurements.
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Table 3. SAR of 4,6-Disubstituted Pyridin-2-yl Derivatives
(Picolinic Acid Derivatives)
Table 4. SAR of 2- and 2,6-(Di)Substituted Pyridin-4-yl
Derivatives (Isonicotinic Acid Derivatives)
a
a
EC50 [nM]
EC50 [nM]
compd
R1
R2
methyl
clogP
S1P1
S1P3
compd
R1
R2
clogP
S1P1
S1P3
25
26
27
28
29
30
isobutyl
3.12
3.12
3.26
3.26
2.89
2.52
0.5
1.3
0.2
0.9
1.3
54
125
139
866
258
429
5240
31
32
33
34
35
36
37
H
H
1.17
1.59
1.94
2.41
2.37
2.87
2.75
3.21
3.30
2.88
2.36
2.83
3.17
3.71
2.98
3.30
3.62
4.07
3.34
3.65
3.98
2.07
2.93
2.34
1110
86
>10000
>10000
9950
1120
3940
700
757
3730
1340
736
1532
310
33
methyl
isobutyl
methyl
H
cyclopentyl
methyl
methyl
ethyl
H
2.1
0.7
0.3
1.8
0.2
8.4
0.1
0.1
0.3
0.4
0.1
0.2
0.1
0.2
1.0
0.2
0.1
0.3
2.0
0.4
0.6
1.9
cyclopentyl
methyl
n-propyl
H
diethylamino
methyl
isopropyl
n-butyl
H
diethylamino
H
a
isobutyl
H
EC50 values as determined in a GTPγS assay using membranes of
CHO cells expressing either S1P1 or S1P3.11 EC50 values represent the
geometric mean of at least three independent measurements.
38
isopentyl
pent-3-yl
cyclopentyl
ethyl
H
b
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
H
H
Me
Me
Me
Me
Me
Me
Me
Et
n-propyl
Tables 2 and 3 (e.g., 22 and 28) are less lipophilic than
thiophene 1. The 4,5-disubstituted 2-pyridines 19, 21, and 23
are all highly potent S1P1 agonists with affinities and selectivity
profiles very close to those of their 3-pyridine analogues 8, 12,
and 15, respectively (Table 1). Compound 21 appears to be
somewhat more selective against S1P3 when compared to its
analogue 12. The 5,6-disubstituted 2-pyridines 20, 22, and in
particular 24 were less potent on both S1P receptors when
compared to the corresponding 3-pyridine analogues 8, 12, and
15, respectively. In the series of the 4,6-disubstituted 2-
pyridines, compounds 25−28 were highly potent S1P1 agonists,
and no relevant difference in the affinity profile was seen
between isomers (i.e., 25 vs 26, 27 vs 28). This is in contrast to
diethylaminopyridines 29 and 30 where the 6-methyl
substitued isomer 30 was significantly less potent than 4-
methyl isomer 29. A similar trend was observed between
compounds 23 and 24.
isobutyl
pent-3-yl
cyclobutyl
cyclopentyl
cyclohexyl
pent-3-yl
cyclobutyl
cyclopentyl
cyclohexyl
ethylamino
diethylamino
N-pyrrolidinyl
46
24
47
149
3.6
Et
5.0
Et
2.4
Et
25
Me
Me
Me
868
352
307
a
EC50 values as determined in a GTPγS assay using membranes of
CHO cells expressing either S1P1 or S1P3.11 EC50 values represent the
geometric mean of at least three independent measurements.
b
Racemate.
The observation that incorporation of 4,6-disubstituted 2-
pyridines led to potent and selective S1P1 agonists prompted us
to extend our study to 4-pyridine derivatives. Table 4 compiles
some prototypical examples illustrating their SAR. As observed
with the 2- and the 3-pyridine derivatives, the 4-pyridines were
generally less lipophilic when compared to thiophene 1 and
only examples incorporating rather large lipophilic substituents
(e.g., as in 48, 51) showed a clogP as high as the one of
thiophene 1. The least lipophilic compound of this series, the
unsubstituted 4-pyridine 31, gave only weak activity on the
S1P1 receptor. However, the compound’s affinity for S1P1 very
rapidly improved with increasing length of a 2-alkyl substituent,
and the 2-ethylpyridine 33 already represented a highly potent
and selective S1P1 agonist. A further increase in the length of
the alkyl group brought a less profound gain in S1P1 affinity.
The n-butyl (36) and in particular the isopentyl (38) derivative
were in fact less potent when compared to their isomers 37 and
39, indicating that the ideal alkyl substituent may be branched
but should not exceed more than three consecutive carbons in
the chain. In general, there is a trend for compounds 32−40 to
become more potent on S1P3 with increasing size of the alkyl
substituent. As for S1P1, the isopentylpyridine 38 appears to
mark the turn of this trend.
Compounds 41−47 bearing an additional 6-methyl sub-
stituent were all highly potent S1P1 agonists, indicating that the
additional methyl group was well tolerated by this receptor.
The 2-ethyl-6-methyl derivative 41 appears to be slightly more
potent when compared to the corresponding des-methyl
analogue 33. In the case of the n-propylpyridine 42, the
affinity gain on S1P1 was not significant. The additional 6-
methyl group markedly increased the activity of compounds
41−47 on S1P3 (compare 44 with 39, 46 with 40). A further
gain in potency on S1P3 was observed when the 6-methyl group
was replaced by an ethyl group. As a consequence, compounds
48−51 were almost equipotent on both S1P receptors.
The activity of the alkylaminopyridines 52 and 53 on S1P1
was nearly identical to the one of the corresponding
alkylpyridine analogues 42 and 44, while the pyrrolidine
derivative 54 was less potent than its cyclopentane analogue 46.
On the other hand, the aminopyridines had lower clogP values
and showed a clearly reduced affinity for S1P3 when compared
to the corresponding alkylpyridines (compare 53 with 44 or 54
with 46), making them a particularly attractive subclass of
compounds.
In brief, all substituted pyridine isomers constituted potent
S1P1 agonists. More specifically, we have seen that in the 3-
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pyridine series potent S1P1 agonists were obtained if the
substituent at position 6 of the pyridine consisted of at least
three carbon atoms. An additional substituent in position 5
improved the affinity for both S1P1 and S1P3 at least 5-fold. In
the series of the 2-pyridine derivatives, compounds incorporat-
ing a 4,5-, a 5,6-, or a 4,6-substitution pattern have been
studied. While the 4,5- and the 4,6-disubstituted 2-pyridine
compounds led to highly potent S1P1 agonists, compounds
with a 5,6-disubstitution pattern were slightly less active. In the
4-pyridines, an ethyl substituent in position 2 was sufficient to
obtain highly potent S1P1 agonists. A larger substituent in
postion 2 and/or an additional substituent in position 6 mainly
improved the compound’s affinity for S1P3. The amino-4-
pyridine derivatives had lower clogP values and were usually
more selective against S1P3 when compared to the correspond-
ing alkylpyridine analogues.
Pharmacokinetics and Pharmacodynamics. In the
body, S1P1 agonists lead to sequestration of circulating
lymphocytes to lymph nodes and lymphoid tissue. Blood
lymphocyte numbers can be measured easily by means of
hemocytometry and therefore represent an attractive biomarker
to assess a compound’s in vivo activity. Hence, the ability to
reduce the blood lymphocyte count (LC) was studied with
several of the highly potent S1P1 agonists discussed above.
While a high potency on S1P1 was required for a compound to
be included in the pharmacodynamic experiment, we did not
restrict these studies to highly selective compounds only in
order to obtain a representative data set for each pyridine
isomer subseries. The compounds were administered orally at a
dose of 10 mg/kg to Wistar rats and the blood 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 observable under the experimental
conditions. A sustained 24 h plasma exposure resulting in
sustained maximal LC reduction appeared desirable from a
compound efficacy and safety perspective. In humans, a
transient heart rate reduction after oral dosing of (selective)
S1P1 receptor agonists has been observed.8,114−116 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.117 Table 5
summarizes our results.
Table 5. Effect on Blood LC after Oral Administration of 10
mg/kg to Wistar Rats and in Vitro Intrinsic Clearance
Measured in Rat Liver Microsomes
% LC after dose
a
CLint in RLM
compd
type
3-pyridine −68 −61 −69
3-pyridine nd nd nd
3 h
6 h 24 h
[(μL/min)/mg protein]
b
8
nd
253
8
11
12
3-pyridine −75 −81 −85
3-pyridine −55 −61 −66
3-pyridine −59 −61 −67
2-pyridine −70 −74 −65
2-pyridine −68 −68 −71
2-pyridine −69 −74 −61
2-pyridine −64 −67 −20
2-pyridine −67 −69 −50
b
13
nd
56
29
29
17
48
21
85
112
40
2.0
5.7
27
15
19
21
25
26
27
28
39
40
41
42
43
44
46
47
50
51
53
54
2-pyridine −67 −63
4-pyridine −71 −71
9
7
4-pyridine −70 −73 −71
4-pyridine −67 −74 −81
4-pyridine −61 −67 −73
4-pyridine −62 −65 −71
4-pyridine −59 −63 −53
4-pyridine −67 −73 −76
4-pyridine −57 −59 −67
4-pyridine −62 −67 −68
4-pyridine −66 −65 −70
4-pyridine −64 −64 −70
4-pyridine −73 −76 −82
c
65
d
17
nd
nd
nd
e
1.0
nd
a
Intrinsic clearance in the presence of rat liver microsomes (RLM) at
b
1 μM compound concentration. Compound tested as racemate.
c
Intrinsic clearance at 0.5 and 0.1 μM 44 was 107 and 233 μL/(min
d
mg), respectively. Intrinsic clearance at 0.5 and 0.1 μM 46 was 20 and
12 μL/(min mg), respectively. Intrinsic clearance at 0.5 and 0.1 μM
e
53 was 5 and 13 μL/(min mg), respectively.
Table 6. Effect on Lymphocyte Count (LC) and Plasma
Concentration after Oral Administration of 10 mg/kg
Compounds 21 and 28 to Wistar Rats (n = 6)
3 h
6 h
plasma
concn
[nM]
24 h
plasma
concn
[nM]
a
plasma
concn
compd [nM]
CLint in RLM
LC
[%]
LC
[%]
LC [(μL/min)/mg
[%]
protein]
At 3 h all compounds tested displayed maximal LC
reduction, indicating a rapid onset of action irrespective of
the pyridine isomer series the compound belongs to. In general,
maximal LC reduction was maintained for 24 h. Notable
exceptions were the two closely related 2-pyridines 26 and 28
and the 4-pyridine 39. Although these three compounds were
highly potent on S1P1, they did not significantly lower LC at 24
h.
21
28
4910
1890
−68
−67
3690
1070
−68
−63
800
9
−71
9
29
85
a
Intrinsic clearance determined in rat liver microsomes at 1 μM
compound concentration. Experiments at 0.5 and 0.1 μM gave
comparable results indicating that metabolic processes are not
saturated under the assay conditions.
In the LC experiments with the cyclopentyl substituted 2-
pyridines 21 and 28, compound concentrations were
determined in plasma (Table 6). The data show a good PK−
PD correlation. In line with its long duration of action,
compound 21 still showed nearly micromolar plasma
concentrations at 24 h. On the other hand, compound 28
reached micromolar concentrations 3 and 6 h after admin-
istration but was more rapidly cleared and almost completely
removed from circulation at 24 h. Metabolic stability testing
using rat liver microsomes (RLM) revealed that compound 28
is less stable when compared to the corresponding isomer 21
(Tables 5 and 6), explaining its shorter duration of action in
vivo. Similar differences observed between the microsomal
stability of the two isobutyl-2-pyridines 19 and 26 may also
explain the corresponding differences in the compounds’ PD
profiles. Rat microsomal stability data revealed that the pent-3-
ylpyridine 39 is less stable than its cyclopentyl analogue 40
(Table 5), offering an explanation of why compound 39
showed a shorter duration of action relative to analogue 40. A
similar difference in microsomal stability between pent-3-
ylpyridines 11 and 44 and their cyclopentyl analogues 12 and
46, respectively, indicated that the pent-3-yl residue in general
leads to metabolically less stable compounds than the
cyclopentyl analogues (Table 5).
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Table 7. Pharmacokinetic Data of Compounds 44, 46 and 53
a
pharmacokinetic parameters
oral dosing
intravenous dosing
AUC0−inf Cl
dose
Cmax [ng/mL]
(tmax, [h])
AUC0−24h
dose
[mg/kg]
0.5
Vss
[(mL/min)/kg] [L/kg]
t1/2
b
compd
species
rat
[mg/kg]
[ng h/mL]
F [%]
[ng h/mL]
[h]
c
44
10
1
804 (0.5)
BLQ
1760
436
126
69.9
120
3.4
0.6
46
53
rat
1
49 (3.0)
1090 (2.0)
546 (1.0)
97 (2.0)
39
52
32
16
0.5
1
566
16
7.3
3.5
1.2
1.1
5.8
8.3
3.4
4.8
d
rat
10
3
16500
3200
3350
5450
5.4
5.0
3.1
dog
3250
866
1
cynomolgus
1
1
a
F = bioavailability; Cmax = maximal concentration reached in plasma; tmax = time at which Cmax was reached, t1/2 = half-life determined with iv
b
experiment, Cl = clearance, Vss = volume of distribution. Oral dosing in three Wistar rats, two Beagle dogs, or three cynomolgus monkeys; iv dosing
in two Wistar rats, two Beagle dogs, or three cynomolgus monkeys; for experimental details, see Supporting Information. Bioavailability exceeds
100% because of saturation of the metabolism of compound 44 in the 10 mg/kg oral dose experiment. AUC0−32h
c
d
.
□
●
Figure 2. LC (A) and plasma concentrations (B) of a dose response experiment with amino pyridine 53 in Wistar rats: , vehicle (n = 5); , 0.1
◆
■
▲
▼
mg/kg (n = 5); , 0.3 mg/kg (n = 5); , 1 mg/kg (n = 5); , 3 mg/kg (n = 5); , 10 mg/kg (n = 5). (A) LC fully recovered 96 h after
administration of all doses tested (data not shown). (B) 24 h after administration of 0.1 mg/kg and 96 h after administration of 0.3, 1, 3, and 10 mg/
kg plasma concentrations were below the limit of quantification (BLQ) of 1.5 ng/mL.
Interestingly, the enhanced in vitro clearance of 65 (μL/
min)/mg of the pent-3-yl derivative 44 was not clearly reflected
in a significantly shorter duration of action in vivo.
Pharmacokinetic assessment of 44 revealed that this compound
was indeed cleared very rapidly from the body with a clearance
value in the range of liver blood flow (Table 7). When the
compound was administered orally at a dose of 1 mg/kg, no
significant exposure in the plasma was detected. At a dose of 10
mg/kg, however, plasma concentrations reached 804 ng/mL
and therefore clearly exceeded dose proportionality, suggesting
possible saturation of compound metabolism. A closer
inspection of the in vitro clearance revealed that the intrinsic
clearance of compound 44 was already saturated under the
assay conditions chosen (1 μM compound concentration).
Repeating the microsomal stability assay at lower concen-
trations of 44 showed that the unsaturated in vitro clearance is
>250 (μL/min)/mg (see Table 5). From substrate depletion
studies using RLM the Michaelis constant (KM) was estimated
to be <0.5 μM (see Supporting Information). Pent-3-ylpyridine
44 therefore is best characterized as a high clearance compound
for which the metabolic process is saturated at relatively low
concentrations. The pharmacokinetic behavior of the pent-3-yl
derivative 44 is in clear contrast to the one of its close
analogues 46 and 53 (Table 7). Cyclopentyl analogue 46 not
only had a lower in vitro and in vivo clearance than 44 but also
showed no sign of blocking its own metabolism in vitro at
concentrations up to 1 μM (Tables 5 and 7). Interestingly,
replacing the pent-3-yl group in 44 by a diethylamino moiety to
give 53 completely abolished the metabolic instability of 44.
RLM data of 53 indicated very low intrinsic clearance (KM
0.06 μM), although saturation was occurring at 1 μM (Table
5). A PK experiment in the rat revealed that compound 53 was
rapidly and well absorbed, slowly cleared, and easily distributed
into tissue (Table 7).
=
In brief, several compounds listed in Table 4 meet the
requirements we set forth for a compound to qualify for more
detailed studies. For instance, compounds 15, 40, 41, 42, 53
were all highly potent on S1P1, displayed an EC50 of >250 nM
on S1P3, and were >100-fold selective against this receptor. In
particular, the last three compounds were further characterized
by a very low in vitro clearance and produced a maximal LC
reduction in the rat lasting for at least 24 h. In the following
paragraphs we chose compound 53 to illustrate the additional
experiments that were carried out to assess the compounds’
viability as candidates for preclinical development.
First, a clear dose response of compound 53 on LC could be
established in the rat (Figure 2A). At a dose of 0.1 mg/kg,
aminopyridine 53 showed a substantial reduction of LC at 3
and 6 h. Maximal LC count reduction was achieved for 6 h with
a dose of 0.3 mg, and a dose of 3 mg/kg maximally reduced LC
for at least 24 h. At all doses studied, LC completely recovered
within 96 h after compound administration. As shown in Figure
2B, plasma concentrations were dose proportional over the
dose range of 0.1−10 mg/kg at each time point measured. The
exposure data further suggest that a plasma concentration of
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Figure 3. Plasma concentrations (A) and blood LC (B) at days 1 and 7 after repeated daily dosing of 3 (mg/kg)/day compound 53 to Wistar rats.
a
Table 8. Plasma and Brain Concentrations of Compounds 53, 55, 56, 57, and 58 after Oral Administration to Wistar Rats
a
10 mg/kg administered orally as a suspension in 7.5% aqueous gelatin.
about 12 ng/mL (25 nM) pyridine 53 is sufficient to induce
maximal reduction of the number of circulating lymphocytes.
A dose of 3 mg/kg was then selected for a multiple dose
experiment with aminopyridine 53. The compound was
administered once daily (t = 0 h) on 7 consecutive days to
Wistar rats, and plasma concentrations of 53 and LC were
measured (Figure 3). At corresponding time points, plasma
concentrations of 53 were nearly identical on days 1 and 7 and
were in good agreement with the concentrations found in the
single dose experiment, indicating that the compound did not
accumulate upon repeated daily dosing of 3 mg/kg. Sustained
maximal LC reduction was observed between 6 h after the first
dose (day 1) and 24 h after the last dose (day 7) with no sign of
tachyphylaxis.
In a next step, pharmacokinetic studies with compound 53
were extended to the Beagle dog and the cynomolgus monkey
(Table 7). Peak plasma concentrations (Cmax) and total plasma
exposure (AUC) were comparable for all three species when
corrected for the dose. As in the rat, low clearance was observed
in the dog and the monkey. However, the half-life of 53 in the
dog and the monkey was shorter than in the rat because of a
smaller volume of distribution, and bioavailability indicated less
efficient absorption in these two species.
particular in animal models of multiple sclerosis.6,89−95 For the
treatment of CNS related autoimmune diseases, it thus appears
advantageous for a synthetic S1P receptor agonists to be able to
penetrate the brain. On the other hand, CNS penetration of a
S1P receptor agonist is not needed or may even be undesired
when treating peripheral diseases such as psoriasis or Crohn’s
disease. To support the decision on the utility of our most
interesting compounds in different diseases, their ability to
penetrate the brain was studied. For the following discussion,
we selected the aminopyridine 53 and a few close analogues to
illustrate how compound permeability and plasma exposure
influence brain exposure. Similar observations were made with
compound 41 and the corresponding analogues (data not
shown). A number of physicochemical parameters, biochemical
assays, and computational models have been proposed to
predict a compound’s ability to pass the blood−brain barrier
(BBB).118−124 For example, Wager et al. recently proposed the
central nervous system multiparameter optimization (CNS
MPO) tool.125 The clogP, clogD, PSA, Mw, the number of
hydrogen bond donors, and the pKa values of known CNS
drugs were analyzed to design a scoring algorithm to classify the
compounds according to their potential to cross the BBB.
Wager et al. reported that 90% of CNS drugs show a PSA of
<86 Å2 and a Mw of <427 and incorporate one hydrogen bond
donor only.126 Other groups proposed similar values to be
Brain Penetration. Several research groups reported
beneficial effects of S1P1 receptor agonism in brain tissue, in
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Journal of Medicinal Chemistry
Article
optimal for brain penetration.120,122 In view of these
observations, aminopyridine 53 clearly classified as a compound
with a low potential to cross the BBB, as it has a PSA of 134 Å2,
a Mw of 497, and three hydrogen bond donors and scored only
2.9 out of 6 points using the CNS MPO tool. On the other
hand, the smallest cross section of the aminopyridine 53 as
calculated according to Seelig et al.127 is 56 Å2 and thus is
clearly below the proposed maximal projection area of 80 Å2
still allowing good permeability.118 Plasma and brain
concentrations of 53 were then measured after oral
administration of 10 mg/kg to Wistar rats (Table 8). In line
with the previous PK experiment, plasma concentration of 53
reached a Cmax of 2170 ng/mL very rapidly and was still about
400 ng/mL at 24 h. Interestingly, high concentrations of 53
were also found in brain tissue. At first sight, this result
appeared to contradict the above predictions. In the brain,
however, Cmax was reached much later (tmax ≈ 6 h) than in
plasma (tmax = 2 h). At 24 h brain concentrations were even
slightly higher than in plasma, indicating that for compound 53
crossing the BBB is a rather slow process in both directions.
Hence, the predictions were indeed correct in the sense that 53
crosses the BBB only slowly. The high and sustained plasma
exposure of 53 compensated for the low permeability, i.e., the
slow BBB passage of the compound. A similar result was
obtained with the [1,2,4]oxadiazole isomer 55. As with 53,
sustained high plasma concentrations allowed reaching
significant brain concentrations despite slow permeation across
the BBB. In contrast, the [1,3,4]oxadiazole 56 showed a more
rapid decline of the plasma concentrations which clearly
hampered building up of significant brain concentrations. (For
a detailed discussion of property differencies between
pyridine, the compound’s potency on S1P1 and S1P3 was
further enhanced. This time, the affinity gain was more
pronounced on S1P3. Representatives from all three pyridine
isomer series maximally reduced blood LC for at least 24 h
when administered at a dose of 10 mg/kg to Wistar rats.
Enhanced in vitro clearance in rat liver microsomes may explain
the shorter duration of action observed for compounds 26, 28,
and 39. Compound 44 showed a high clearance in vitro and in
vivo. However, the metabolism was saturated at relatively low
compound concentrations, explaining why at a dose of 10 mg/
kg this compound was still able to maximally reduce LC for
almost 24 h. From the above studies several compounds
appeared interesting for further characterization, as they were
less lipophilic than thiophene 1, displayed high affinity and
selectivity for S1P1, and sustained maximal reduction of blood
LC in the rat. As an example, aminopyridine 53 has a clogP
which is 1 log unit lower than the one of thiophene 1, showed
an EC50 of 0.6 nM on S1P1, and was more than 500-fold
selective against S1P3. In addition, this compound was highly
efficacious in reducing blood LC in Wistar rats. In the
following, the aminopyridine 53 was chosen to illustrate the
more detailed charaterization process. A dose response
experiment revealed that for 53 the ED50 for maximal LC
reduction at 24 h after administration is about 0.5 mg/kg in the
rat. Furthermore, compound 53 showed favorable PK proper-
ties in the Wistar rat and Beagle dog, showed acceptable PK
behavior in the cynomolgus monkey, and is considered suitable
for once daily dosing in humans. High concentrations of 53
were found in rat brain tissue after oral administration, making
this compound an interesting candidate for further studies in
particular in CNS related diseases or disorders.
oxadiazole isomers see Bostrom et al.128) Compared to 53,
̈
the two glycerol derivatives 57 and 58 are characterized by a
reduced molecular weight (440) and PSA (105 Å2) and a
smaller number of hydrogen bond donors. With a CNS MPO
score of 3.2 and 3.6 the glycerol derivatives 57 and 58
respectively show a more desirable property profile for brain
penetration. Indeed, brain concentrations of the two glycerol
derivatives 57 and 58 followed plasma concentrations very
closely confirming a more rapid crossing of the BBB of these
two compounds. As a consequence of the enhanced
permeability of 57 and 58, significant brain concentrations
were observed at 2 and 6 h after compound administration but
no sustained 24 h brain exposure was achieved. The different
behavior of compounds 53 and 56 with respect to brain
penetration makes them interesting tools to assess the
contribution of S1P1 agonism to the treatment efficiency in
diseases affecting the brain. In particular, the above studies
suggest that compound 53 might be well suited for the
treatment of diseases for which brain penetration is important,
while compound 56 may be useful for diseases for which brain
penetration is not necessary or even undesirable.
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), gradient 5−95% acetonitrile in water
containing 0.04% of trifluoroacetic acid within 1 min, flow of 4.5 mL/
min. tR is given in min. The asterisk (∗) denotes basic conditions:
gradient 5−95% acetonitrile in water containing 0.04% of ammonium
hydroxide within 1 min, flow of 4.5 mL/min; 40 °C. tR is given in min.
UV detection was at at 230, 254, and 280 nm.
Purity of all final compounds was checked by an additional LC−MS
analysis on a Waters Acquity UPLC system equipped with an ACQ-
PDA detector, an ACQ-ESL detector, and an ACQ-SQ detector:
column ACQUITY UPLC BEH C18 1.7 μm, 2.1 mm × 50 mm;
gradient 2−98% acetonitrile containing 0.045% formic acid in water
containing 0.05% formic acid over 1.8 min; flow of 1.2 mL/min; 60
°C. According to these LC−MS analyses, final compounds showed a
purity of >95% (UV at 230 and at 214 nm).
CONCLUSIONS
■
Chiral integrity was proven by HPLC (chiral stationary phase).
Hardware was from UltiMate instrument series (Dionex): 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) are 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.
Aiming at reducing the lipophilicity of thiophene based S1P1
agonists such as compound 1, we replaced the thiophene in 1
by a 2-, 3-, or 4-pyridine. This led to the discovery of a new
series of less lipophilic S1P1 receptor agonists with all three
pyridine isomers furnishing potent compounds. Increasing the
size of a first alkyl or alkylamino substituent in the para- or
meta-position to the oxadiazole improved the compound’s
affinity rapidly for the S1P1 and to a lesser extent for the S1P3
receptor. When a second alkyl group was attached to the
LC−HRMS parameters were the following: analytical pump Waters
Acquity binary, Solvent Manager, MS, SYNAPT G2 MS, source
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temperature of 150 °C, desolvatation temperature of 400 °C,
desolvatation gas flow of 400 L/h; cone gas flow of 10 L/h, extraction
cone of 4 RF; lens 0.1 V; sampling cone 30; capillary 1.5 kV; high
resolution mode; gain of 1.0, MS function of 0.2 s per scan, 120−1000
amu in full scan, centroid mode. Lock spray: keucine enkephalin, 2 ng/
mL (556.2771 Da), scan time of 0.2 s with interval of 10 s and average
of 5 scans; DAD: Acquity UPLC PDA detector. Column was an
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 at UV 214 nm. For MS, tR is given in min.
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; (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).
For NMR spectroscopy, instruments used were the following:
BSA, using a cell harvester. The filter plates were then washed with ice-
cold 10 mM Na2HPO4/NaH2PO4 (70%/30%, w/w) containing 0.1%
fatty acid free BSA. Then the plates were dried at 50 °C and sealed, 25
μL of MicroScint20 was added, and 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.
Intrinsic metabolic clearance (CLint) was determined by substrate
depletion experiments, with a default starting concentration of 1 μM in
the presence of 0.5 mg/mL rat liver microsomes (RLM) in 100 mM
sodium phosphate buffer at pH 7.4. Incubations were initated by the
addition of an NADPH regenerating system containing D-glucose 6-
phosphate, NADPH, and glucose 6-phosphate dehydrogenase. All
incubations were conducted by shaking reaction mixtures under air at
37 °C. Aliquots (0.1 mL) were removed at 0, 2.5, 5, 10, and 15 min
and terminated by addition to 0.1 mL of methanol on ice. After
protein precipitation by centrifugation, the remaining concentrations
were analyzed by liquid chromatography coupled to mass spectrom-
etry (LC−MS/MS). CLint was calculated from the concentration
remaining versus time, fitted to a first order decay constant versus
time. KM values from substrate consumption experiments were
determined by plotting the first order decay constants versus the
substrate concentration on a linear−log plot and fitting the following
equation:131
⎧
⎨
⎩
⎫
⎬
⎭
[S]
[S] + K
decay constant = CLint[S]→0
1
Varian Oxford, H (300 MHz) or 13C (75 MHz); Bruker Avance II,
M
1
400 MHz UltraShield, H (400 MHz), 13C (100 MHz). Chemical
shifts are reported in parts per million (ppm) relative to
tetramethylsilane (TMS), and multiplicities are given as s (singlet),
d (doublet), t (triplet), q (quartet), quint (quintuplet), h (hextet),
hept (heptuplet), or m (multiplet). br = broad, and coupling constants
are given in Hz. Several compounds have been prepared in a
combinatorial library format on a 15−50 mmol scale. For those
Male Wistar rats (RccHan:WIST) were obtained from Harlan
(Venray, The Netherlands) and used for pharmacokinetic experiments
after an acclimatization period of at least 7 days. 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 of the gas anesthetic
isoflurane (4−5% for induction and 1.5−3% for maintenance) in 100%
O2. Buprenorphine was dosed as analgesic at 0.03 mg/kg sc half an
hour before the operation. Catheters were implanted into jugular vein
and carotid artery under aseptic conditions to allow for multiple serial
blood sampling. Animals foreseen for oral dosing did not undergo
surgery, but blood samples were taken sublingually under light
anesthesia with isoflurane. Compounds were administered intra-
venously via the tail vein at doses of 1 mg/kg body weight formulated
as solutions in an aqueous mixed micellar vehicle based on
phospholipids and bile acids (mixed micelles). Oral administration at
doses of 10 mg/kg was performed by gavage. Oral formulations were
dispersions prepared by addition of a DMSO stock solution of the
compounds to succinylated gelatin (7.5% w/v) in water.
1
compounds H NMR spectra were acquired using nondeuterated 10
mM DMSO stock solutions submitted for biological testing.129 The
solvent and water signals were suppressed by irradiation at 2.54 and
3.54 ppm, respectively. As a consequence, signal integrals close to
those frequencies are not always accurate. The numbers of protons
given in the description represent observed values.
Compounds were purified by flash column chromatography (CC)
on silica gel 60 (Fluka Sigma-Aldrich, Switzerland), by preparative
TLC glass plates coated with silica gel 60 F254 (0.5 mm), 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
10% methanol in water to 100% methanol).
For pharmacokinetics in the dog, the pharmacokinetic profile of
compound 53 was determined in the fasted male Beagle dog (n = 2
dogs). The compound was administered iv at a dose of 1 mg/kg as a
solution in mixed micelles and orally at a dose of 3 mg/kg as a
dispersion in succinylated gelatin (7.5% w/v) in water.
clogP values were calculated using an algorithm developed in-house
and published online at openmolecules.org.130
In Vitro Potency Assessment. Data (EC50) are given as
geometric mean values (Xgeo) with geometric standard deviation
(σg). The upper and lower 95% confidence limits are calculated as
For pharmacokinetics in the monkey, the pharmacokinetic profile of
compound 53 was determined in the male cynomolgus monkey (n =
3). The compound was administered iv as a short infusion at a dose of
1 mg/kg as a solution in mixed micelles and orally at a dose of 1 mg/
kg as a dispersion in 0.25% methyl cellulose in water with 0.05%
Tween 80.
For bioanalysis and pharmacokinetics, serial blood samples of 0.25
mL each were taken from each individual animal to obtain a complete
concentration vs time profile per animal. Blood samples 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 dosing. Plasma was
prepared from blood samples by centrifugation and stored at −20 °C
and analyzed for drug concentrations using LC−MS/MS after protein
precipitation. Pharmacokinetic parameters were estimated with the
WinNonlin software (Pharsight Corporation, Mountain View, CA,
USA) using noncompartmental analysis.
X
geoσg2 and Xgeo/σg2, respectively (results not shown).
GTPγS binding assays were performed in 96-well polypropylene
microtiter plates in a final volume of 200 μL. Membrane preparations
of CHO cells expressing recombinant human S1P1 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
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For brain penetration in the rat, at 2, 6, and 24 h after dosing, male
Wistar rats (n = 2) were anaesthetized with 5% isoflurane and
sacrificed by opening the diaphragm. A blood sample was taken.
Plasma was prepared, and the brain was slowly perfused with 10 mL of
0.9% NaCl through the carotid. The whole brain was then removed
and homogenized in an equal volume of ice-cold 0.1 M Na phosphate
buffer, pH 7.4, using a IKA-WERKE Ultra-Turrax T25 tissue
homogenizer for 10 s, and the brain homogenate was snap frozen in
liquid nitrogen. Drug concentrations were then determined as
described for plasma, using a calibration curve from blanco brain
homogenate.
The in vivo efficacy of the target compounds was assessed by
measuring the circulating lymphocytes after oral administration of 3−
100 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
1.43 (t, J = 7.0 Hz, 3 H), 2.46 (s, 3 H), 4.43 (q, J = 7.3 Hz, 2 H), 8.16
(m, 1 H), 8.84 (d, J = 2.0 Hz, 1 H).
(d) To a solution of 90 (4.98 g, 24.9 mmol), 2,4,6-tri(2-
methylpropenyl)cyclotriboroxanepyridine complex (5.74 g, 17.7
mmol, prepared in analogy to a procedure given by F. Kerins and
D. F. O’Shea108), and PPh3 (1.15 g, 4.4 mmol) in DME (60 mL), a
solution of 2 M aqueous K2CO3 (20 mL) was added. The mixture was
degassed and flushed with N2 before Pd(PPh3)4 (460 mg, 0.4 mmol)
was added. The mixture was stirred at 90 °C for 20 h before it was
cooled to room temperature, diluted with EA (150 mL), and washed
with saturated aqueous NaHCO3 (2 × 50 mL). The organic extract
was dried over MgSO4, filtered, and evaporated. The crude product
was purified by FC (SiO2, heptane−EA) to give 5-methyl-6-(2-
methylpropenyl)nicotinic acid ethyl ester (3.98 g, 73%) as an orange
oil. LC−MS: tR = 0.72 min, [M + 1]+ = 220.15. 1H NMR (CDCl3): δ
9.04 (d, J = 1.9 Hz, 1 H), 8.04 (d, J = 1.6 Hz, 1 H), 6.37 (s, 1 H), 4.41
(q, J = 7.1 Hz, 2 H), 2.34 (s, 3 H), 2.008 (s, 3 H), 2.006 (s, 3 H), 1.42
(t, J = 7.1 Hz, 3 H).
(e) 5-Methyl-6-(2-methylpropenyl)nicotinic acid ethyl ester (3.98 g,
18.2 mmol) was dissolved in THF (100 mL) and MeOH (100 mL).
Pd/C (500 mg, 10% Pd) was added as a slurry in THF (5 mL), and
the mixture was stirred under 1 atm of H2 at room temperature for 15
h. The catalyst was filtered off and the filtrate was evaporated to give 6-
isobutyl-5-methylnicotinic acid ethyl ester 91 (3.76 g, 93%) as a
mean
SEM. Statistical analyses were performed by analysis of
variance (ANOVA) using Statistica (StatSoft) and the Student−
Newman−Keuls procedure for multiple comparisons. The null
hypothesis was rejected when p < 0.05. Because of interindividual
variability and the circadian rhythm of the number of circulating
lymphocytes, a compound showing relative changes in the range of
−20% to +40% is considered inactive. A lymphocyte count (LC)
reduction in the range of −60% to −75% represents the maximal effect
to be observed under the conditions of the experiment. For
formulation, the compounds were dissolved in DMSO. This solution
was added to a stirred solution of succinylated gelatin (7.5% w/v) in
water. The resulting milky suspension containing a final concentration
of 5% of DMSO was administred to the animals by gavage. A mixture
of 95% of succinylated gelatin (7.5% w/v) in water and 5% of DMSO
served as vehicle.
6-Isobutyl-5-methylnicotinic Acid (60). (a) Phosphoroxychlor-
ide (183 mL, 2 mol) was heated at 90 °C, and a mixture of
commercially available 2-methyl-2-butenenitrile (73 g, 0.9 mol) and
DMF (154 mL, 2 mol) was added slowly while keeping the
temperature at 100−110 °C. The mixture was stirred at 110 °C for
15 h, cooled to room temperature, and diluted with DCM (500 mL).
The mixture was cooled at 0 °C and carefully quenched with water
(500 mL). The phases were separated, and the aqueous phase was
extracted with DCM (total of 800 mL). The combined organic extracts
were dried (Na2SO4), filtered, and evaporated. The residue was
crystallized from cyclohexane to provide 6-chloro-3-formyl-5-methyl-
pyridine 89109 (28.3 g, 20%) as slightly yellow crystals. LC−MS: tR =
0.76 min, [M + 1]+ = 156.14. 1H NMR (DMSO-d6): δ 10.09 (s, 1 H),
8.78 (s, 1 H), 8.24 (s, 1 H), 2.43 (s, 3 H).
(b) A solution of 89 (10 g, 64 mmol) in formic acid (200 mL) was
cooled at 0 °C, and an aqueous 50 wt % solution of H2O2 in water (9.6
mL, 360 mmol) was added at this temperature. The mixture was
stirred at 0 °C for 15 h, carefully diluted with water (200 mL), and
extracted with DCM (8 × 100 mL). The combined organic extracts
were washed with 1 M aqueous HCl (100 mL) (checked for remaining
peroxide), dried (MgSO4), filtered, and evaporated. The residue was
dried to give 6-chloro-5-methylnicotinic acid (9.56 g, 87%). LC−MS:
tR = 0.72 min, [M + 1]+ = 172.0. 1H NMR (CDCl3): δ 8.94 (d, J = 2.0
Hz, 1 H), 8.23 (d, J = 2.3 Hz, 1 H), 2.49 (s, 3 H).
(c) A solution of 6-chloro-5-methylnicotinic acid (13.85 g, 80.75
mmol) in dry EtOH (200 mL) containing some drops of concentrated
H2SO4 was stirred at reflux for 2 days. The solution was cooled to
room temperature, the solvent evaporated, the residue dissolved in EA
(200 mL) and washed with a solution of saturated aqueous Na2CO3 (2
× 80 mL), 1 M aqueous KHSO4 (2 × 80 mL), and brine (50 mL).
The organic phase was dried over MgSO4, filtered, and evaporated to
give 6-chloro-5-methylnicotinic acid ethyl ester 90 (12.65 g, 79%) as a
solid. LC−MS: tR = 0.92 min, [M + 1]+ = 200.10. 1H NMR (CDCl3) δ
1
colorless oil. LC−MS: tR = 0.75 min, [M + 1]+ = 222.15. H NMR
(CDCl3) δ 0.97 (d, J = 6.8 Hz, 6 H), 1.42 (t, J = 7.3 Hz, 3 H), 2.20
(hept, J = 6.8 Hz, 1H), 2.38 (s, 3 H), 2.75 (d, J = 7.0 Hz, 2 H), 4.41 (q,
J = 7.3 Hz, 2 H), 8.03 (d, J = 1.8 Hz, 1 H), 9.00 (d, J = 2.0 Hz, 1 H).
(f) A solution of 91 (3.75 g, 16.95 mmol) in 12.5% aqueous HCl
(50 mL) was stirred at 65 °C for 24 h before the solvent was
evaporated. The residue was dried under high vacuum to give 6-
isobutyl-5-methylnicotinic acid hydrochloride 60 (3.55 g, 91%) as a
1
white powder. LC−MS: tR = 0.57 min, [M + 1]+ = 194.25. H NMR
(CDCl3): δ 9.23 (s, 1 H), 8.77 (s, 1 H), 3.17 (d, J = 7.5 Hz, 2 H), 2.62
(s, 3 H), 2.35 (hept, J = 6.3 Hz, 1 H), 1.09 (d, J = 6.6 Hz, 6 H).
6-Cyclopentyl-5-methylnicotinic Acid (61). (a) A solution of
5,6-dichloronicotinic acid (50 g, 260 mmol) and TMSCl (8.49 g, 781
mmol) in isopropanol (500 mL) was stirred at 60 °C for 18 h. The
mixture was concentrated, and the residue was partitioned between EA
and saturated aqueous NaHCO3 solution. The organic extract was
separated, dried over MgSO4, filtered, and concentrated. The crude
product was purified on silica gel, eluting with heptane/EA 9:1 to give
5,6-dichloronicotinic acid isopropyl ester 92 (56.2 g, 92%) as a white
solid. LC−MS: tR = 1.01 min, [M + 1]+ = 233.89. 1H NMR (DMSO-
d6): δ 8.85 (d, J = 2.0 Hz, 1 H), 8.49 (d, J = 2.0 Hz, 1 H), 5.18 (hept, J
= 6.3 Hz, 1 H), 1.35 (d, J = 6.3 Hz, 6 H).
(b) To a solution of 92 (2.15 g, 9.19 mmol) in THF (30 mL) were
added NMP (3 mL) and Fe(acac)3 (162 mg, 0.459 mmol). The
mixture was cooled to 0 °C before cyclopentylmagnesium bromide
(9.2 mL of 2 M solution in diethyl ether) was added. The dark red to
black reaction mixture was stirred at 0 °C for 1 h, then at room
temperature for 18 h. The reaction was quenched carefully by adding
water (30 mL), then extracted twice with EA (2 × 60 mL). The
combined organic extracts were dried over MgSO4, filtered, and
concentrated. The crude product was purified by CC on silica gel,
eluting with heptane/EA 9:1 to give isopropyl 5-chloro-6-cyclopentyl-
nicotinate 93 (1.93 g, 78%) which was still contaminated with 5,6-
dichloronicotinic acid isopropyl ester as a pale yellow oil. LC−MS: tR
= 0.87 min, [M + 1]+ = 268.2. 1H NMR (DMSO-d6): δ 8.95 (d, J = 1.8
Hz, 1 H), 8.21 (d, J = 1.9 Hz, 1 H), 5.16 (hept, J = 6.3 Hz, 1 H), 3.66
(quint, J = 7.8 Hz, 1 H), 1.95−2.05 (m, 2 H), 1.74−1.88 (m, 4 H),
1.59−1.73 (m, 2 H), 1.34 (d, J = 6.2 Hz, 6 H).
(c) To a solution of the above 93 (1.93 g, 7.21 mmol) and
Pd(dppf)Cl2 (60 mg, 74 μmol) in dioxane (30 mL) was added
dimethylzinc (13.3 mL of a 1.2 M solution in toluene). The mixture
was stirred at 75 °C for 18 h before the reaction was quenched at room
temperature by carefully adding water (50 mL). The mixture was
extracted twice with EA (2 × 100 mL). The combined organic extracts
were dried over MgSO4, filtered, and concentrated. The crude product
was purified by CC on silica gel, eluting with heptane/EA 9:1 to give
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isopropyl 6-cyclopentyl-5-methylnicotinate 94 (600 mg, 36%,
eluting with heptane/EA 9:1 to 4:1 to give isopropyl 2-(1-
ethylpropyl)-6-methylisonicotinate 130d (1.6 g, 69%) as a yellow oil
containing a few percent of isopropyl 2-methyl-6-(pentan-2-yl)-
containing the corresponding methyl ester as impurity) as a pale
1
yellow oil. LC−MS: tR = 0.61 min, [M + 1]+ = 248.31. H NMR
1
(CDCl3): δ 9.01 (s, 1 H), 7.99 (s, 1 H), 5.27 (hept, J = 6.5 Hz, 1 H),
3.40 (quint, J = 7.5 Hz, 1 H), 2.41 (s, 3 H), 1.95−2.07 (m, 2 H),
1.83−1.95 (m, 4 H), 1.65−1.78 (m, 2 H), 1.38 (d, J = 6.2 Hz, 6 H).
(d) A solution of the above 94 (600 mg, 2.43 mmol) in MeOH (8
mL) and 2 M aqueous LiOH (2 mL) was stirred at room temperature
for 2 h. The mixture was concentrated, diluted with water (15 mL)
and EA (40 mL), and then neutralized by adding 2 N aqueous HCl
(approximately 2 mL). The organic phase was separated, and the
aqueous phase was extracted three more times with EA (3 × 40 mL).
The organic extracts were combined, dried over MgSO4, filtered,
concentrated, and dried to give the title compound (412 mg, 83%) as a
isonicotinate. LC−MS: tR = 0.79 min, [M + 1]+ = 250.14. H NMR
(DMSO-d6): δ 0.70 (t, J = 7.3 Hz, 6 H), 1.33 (d, J = 6.3 Hz, 6 H),
1.58−1.70 (m, 4 H), 2.51 (s, 3 H), 2.55−2.63 (m, 1 H), 5.15 (hept, J =
5.8 Hz), 7.39 (s, 1 H), 7.49 (s, 1 H).
(c) A solution of 130d (1.54 g, 6.18 mmol) in 25% aqueous HCl
(60 mL) was stirred at 65 °C for 16 h. The solvent was removed in
vacuo and the residue was dissolved in dioxane and concentrated again
to give 2-(1-ethyl-propyl)-6-methylisonicotinic acid 76d as hydro-
chloride salt (1.70 g) in the form of a brownish solid. LC−MS: tR =
1
0.62 min, [M + 1]+ = 208.52. H NMR (CD3OD): δ 8.23 (s, 1 H),
8.19 (s, 1 H), 3.00 (d, J = 7.4 Hz, 2 H), 2.88 (s, 3 H), 2.10−2.25 (m, 1
H), 1.05 (d, J = 6.6 Hz, 6 H).
1
pale yellow oil. LC−MS: tR = 0.35 min, [M + 1]+ = 206.39. H NMR
(CDCl3): δ 9.14 (s, 1 H), 8.13 (s, 1 H), 3.45 (quint, J = 7.3 Hz, 1 H),
2.46 (s, 3 H), 2.01−2.11 (m, 2 H), 1.87−2.01 (m, 4 H), 1.64−1.84
(m, 2 H).
2-Cyclopentyl-6-methylisonicotinic Acid (76f). (a) Under
argon, Pd(dppf)Cl2 (200 mg, 0.245 mmol) was added to a solution
of 2-chloro-6-methylisonicotinic acid ethyl ester 129 (4.80 g, 24.0
mmol) in dioxane (60 mL). A solution of cyclopentylzinc chloride (50
mL, 24.0 mmol, ∼2 M solution in THF) was added dropwise. The
mixture was stirred at 75 °C for 2 h before it was cooled to room
temperature, carefully diluted with water, and extracted twice with EA.
The combined organic extracts were dried over MgSO4, filtered, and
concentrated. The crude product was purified by CC on silica gel,
eluting with heptane/EA 9:1 to give 2-cyclopentyl-6-methylisonico-
tinic acid ethyl ester 130f (3.96 g, 71%) as an oil. LC−MS: tR = 0.72
min, [M + 1]+ = 234.11. 1H NMR (CDCl3): δ 7.56 (s, 1 H), 7.52 (s, 1
H), 4.42 (q, J = 7.1 Hz, 2 H), 3.18−3.29 (m, 1 H), 2.61 (s, 3 H),
2.06−2.18 (m, 2 H), 1.66−1.93 (m, 6 H), 1.43 (t, J = 7.1 Hz, 3 H).
(b) A solution of 130f (3.96 g, 17.0 mmol) in 25% aqueous HCl
(50 mL) was stirred at 75 °C for 16 h. The solvent was removed in
vacuo and the remaining residue was dried under high vacuum to give
76f as a hydrochloride salt (4.12 g, quantitative) in the form of a white
solid. LC−MS: tR = 0.54 min, [M + 1]+ = 206.08. 1H NMR (DMSO-
d6): δ 7.90 (s, 1 H), 7.85 (s, 1 H), 3.79 (s br, 2 H), 3.41−3.53 (m, 1
H), 2.73 (s, 3 H), 2.07−2.17 (m, 2 H), 1.63−1.89 (m, 6 H).
2-Cyclopentyl-6-ethylisonicotinic Acid (77c). The title com-
pound (7.19 g) was prepared in analogy to 77a (see Supporting
5-Cyclopentyl-4-methylpicolinic Acid (68). (a) Under argon,
Pd(dppf)Cl2 (161 mg, 0.198 mmol) and cyclopentylzinc bromide (39
mL of a 0.5 M solution in THF) were added to a solution of ethyl 5-
bromo-4-methylpicolinate 107 (4.73 g, 19.4 mmol) in dioxane (250
mL). The mixture was stirred at 65 °C for 2 h before another portion
of Pd(dppf)Cl2 (161 mg, 0.198 mmol) and cyclopentylzinc bromide
(19.5 mL) was added. Stirring was continued at 65 °C for 18 h. The
mixture was cooled to room temperature before the reaction was
quenched by carefully adding water (200 mL) and saturated aqueous
NaHCO3 solution (150 mL). The mixture was extracted six times with
EA (6 × 100 mL) and DCM (100 mL). The combined organic
extracts were dried over MgSO4, filtered, and concentrated to give
crude ethyl 5-cyclopentyl-4-methylpicolinate 113 (3.96 g) as an
orange oil containing about 10% of the corresponding cyclopentyl
ester. LC−MS: tR = 0.85 min, [M + 1]+ = 234.05. Cyclopentyl ester,
LC−MS: tR = 0.95 min, [M + 1]+ = 274.06.
(b) A solution of 113 (3.96 g, 17.0 mmol) in dioxane (2 mL) and 2
M aqueous LiOH (50 mL) was stirred at 75 °C for 5 h. The mixture
was cooled to room temperature before it was extracted with EA. The
aqueous phase was acidified by adding 1 N HCl and then extracted
with EA. The second organic extract was dried over MgSO4, filtered,
and concentrated. The crude product was purified by MPLC (ODS-
AQ), eluting with a gradient of methanol in water to give 68 (532 mg,
15%) as a beige solid. LC−MS: tR = 0.37 min, [M + 1]+ = 206.31. 1H
NMR (DMSO-d6): δ 8.52 (s, 1 H), 7.92 (s, 1 H), 3.21−3.31 (m, 1 H),
2.44 (s, 3 H), 1.99−2.11 (m, 2 H), 1.75−1.88 (m, 2 H), 1.56−1.75
(m, 4 H).
2-Methyl-6-(pentan-3-yl)isonicotinic Acid (76d). (a) To a
suspension of 2-chloro-6-methylisonicotinic acid (20.0 g, 117 mmol)
in isopropanol (80 mL) was added H2SO4 (5 mL) dropwise. The
mixture became warm (40 °C). The mixture was stirred for 24 h at
room temperature, then at 90 °C for 28 h before the solvent was
removed in vacuo. The residue was dissolved in diethyl ether (200
mL), washed with saturated aqueous NaHCO3 solution (3 × 50 mL)
followed by brine (3 × 50 mL), dried over Na2SO4, filtered, and
concentrated to give 2-chloro-6-methylisonicotinic acid isopropyl ester
(21.0 g) as a colorless oil which slowly crystallizes. LC−MS: tR = 0.97
min, [M + 1]+ = 214.05. 1H NMR (CD3OD). δ 7.64 (s, 1 H), 7.61 (s,
1 H), 5.21 (hept, J = 6.2 Hz, 1 H), 2.54 (s, 3 H), 1.37 (d, J = 6.3 Hz, 6
H).
(b) A solution of 2-chloro-6-methylisonicotinic acid isopropyl ester
(2.0 g, 9.36 mmol) in dioxane (75 mL) was degassed and put under
argon before Pd(dppf)Cl2 (229 mg, 0.281 mmol) was added. At room
temperature, a 0.5 M solution of 1-ethylpropylzinc bromide in THF
(46.8 mL, 23.4 mmol) was added dropwise to the mixture. The
mixture was stirred at 80 °C for 16 h before the reaction was quenched
by adding ice-cold water (200 mL). A precipitate forms, and the
mixture was diluted with EA (200 mL) and filtered through Celite.
The filtrate was transferred into a separatory funnel. The organic phase
was collected, and the aqueous phase was extracted with EA (120 mL).
The combined organic extracts were dried over MgSO4, filtered, and
concentrated. The crude product was purified by CC on silica gel,
1
Information). LC−MS: tR = 0.59 min, [M + 1]+ = 220.00. H NMR
(DMSO-d6): δ 7.85 (s, 1 H), 7.83 (s, 1 H), 3.47−3.57 (m, 1 H), 3.02
(q, J = 7.5 Hz, 2 H), 2.06−2.16 (m, 2 H), 1.71−1.88 (m, 4 H), 1.64−
1.71 (m, 2 H), 1.29 (t, J = 7.5 Hz, 3 H).
2-(Diethylamino)-6-methylisonicotinic Acid (79). (a) A
mixture of tert-butyl 2,6-dichloroisonicotinate 136 (11.3 g, 45.4
mmol) and diethylamine (3.32 g, 45.4 mmol) was stirred at 100 °C for
72 h in a sealed vessel. The mixture was cooled to room temperature,
diluted with water (50 mL), and extracted three times with EA (3 ×
100 mL). The combined organic extracts were dried over MgSO4,
filtered, and concentrated. The crude product was purified by CC on
silica gel, eluting with heptane/EA 9:1 to give tert-butyl 2-chloro-6-
(diethylamino)isonicotinate 137 (13.3 g, quantitative) as a pale yellow
oil. LC−MS: tR = 1.15 min, [M + 1]+ = 285.05. 1H NMR (DMSO-d6):
δ 6.88 (d, J = 0.9 Hz, 1 H), 6.80 (d, J = 0.8 Hz, 1 H), 3.50 (q, J = 7.0
Hz, 4 H), 1.54 (s, 9 H), 1.11 (t, J = 7.0 Hz, 6 H).
(b) To solution of 137 (6.70 g, 23.5 mmol) and Pd(dppf)Cl2 (196
mg, 240 μmol) in dioxane (50 mL) under argon was added
dimethylzinc (70.6 mL, 1 M solution in THF). The mixture was
stirred at 75 °C for 16 h before it was cooled to room temperature.
The reaction was quenched by carefully adding water. The mixture was
extracted twice with EA. The combined organic extracts were dried
over MgSO4, filtered, and concentrated. The crude product was
purified by CC on silica gel, eluting with heptane/EA to give tert-butyl
2-(diethylamino)-6-methylisonicotinate 138 (2.54 g, 41%) as a yellow
oil. LC−MS: tR = 0.81 min, [M + 1]+ = 265.09.
(c) A solution of 138 (2.53 g, 9.57 mmol) in 25% aqueous HCl (50
mL) was stirred at 80 °C for 24 h. The mixture was concentrated and
dried under high vacuum to give the hydrochloride salt of 79 (2.45 g,
quantitative) as a yellow oil. LC−MS: tR = 0.48 min, [M + 1]+
=
1
209.42. H NMR (DMSO-d6): δ 7.17 (s, 1 H), 6.97 (s, 1 H), 3.70 (q
br, J = 7.0 Hz, 2 H), 2.56 (s, 3 H), 1.17 (t, J = 6.8 Hz, 6 H).
123
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Journal of Medicinal Chemistry
Article
N-((S)-3-[2-Ethyl-4-(N-hydroxycarbamimidoyl)-6-methylphe-
noxy]-2-hydroxypropyl)-2-hydroxyacetamide (80). (a) To an
ice-cold solution of H2SO4 (150 mL) in water (250 mL) was added 2-
ethyl-6-methylaniline (15.0 g, 111 mmol). The solution was treated
with ice (150 g) before a solution of NaNO2 (10.7 g, 155 mmol) in
water (150 mL) and ice (50 g) was added dropwise. The mixture was
stirred at 0 °C for 1 h. Then 50% aqueous H2SO4 (200 mL) was added
and stirring was continued at room temperature for 18 h. The mixture
was extracted with DCM. The organic extracts were dried over MgSO4
and evaporated. The crude product was purified by CC on silica gel,
eluting with heptane/EA 9:1 to give 2-ethyl-6-methylphenol 81 (8.6 g,
3.90 (m, 3 H), 4.19 (s, br, 3 H), 7.06 (m, 1 H), 7.36 (s, 1 H), 7.38 (s, 1
H).
(g) To a solution of 86 (19.6 g, 67 mmol) in methanol (500 mL)
were added hydroxylamine hydrochloride (9.32 g, 134 mmol) and
NaHCO3 (11.3 g, 134 mmol).133 The resulting suspension was stirred
at 65 °C for 18 h. The mixture was filtered, and the filtrate was
concentrated. The residue was dissolved in water (20 mL) and EA
(300 mL). The aqueous phase was separated and extracted three times
with EA. The combined organic extracts were dried over MgSO4,
filtered, concentrated, and dried to give the title compound 80 as a
white solid (18.9 g, 87%). LC−MS: tR = 0.51 min, [M + 1]+ = 326.13.
1H NMR (DMSO-d6): δ 1.17 (t, J = 7.4 Hz, 3 H), 2.24 (s, 3H), 2.62
(q, J = 7.4 Hz, 2 H), 3.23 (m, 1 H), 3.43 (m, 1 H), 3.67 (m, 2 H), 3.83
(s, 2 H), 3.93 (m, 1 H), 5.27 (s br, 1 H), 5.58 (s br, 1 H), 5.70 (s, 2
H), 7.34 (s, 1 H), 7.36 (s, 1 H), 7.67 (m, 1 H), 9.46 (s br, 1H).
Method A: (S)-N-(3-(4-(5-(2-Cyclopentyl-6-ethylpyridin-4-yl)-
1,2,4-oxadiazol-3-yl)-2-ethyl-6-methylphenoxy)-2-hydroxy-
propyl)-2-hydroxyacetamide (50). To a solution of 2-cyclopentyl-
6-ethylisonicotinic acid hydrochloride 77c (187 mg, 0.73 mmol) in
1
57%) as a crimson oil. LC−MS: tR = 0.89 min. H NMR (CDCl3): δ
7.03−6.95 (m, 2 H), 6.80 (t, J = 7.6 Hz, 1 H), 4.60 (s, 1 H), 2.64 (q, J
= 7.6 Hz, 2 H), 2.25 (s, 3 H), 1.24 (t, J = 7.6 Hz, 3 H).
(b) A solution of 2-ethyl-6-methylphenol 81 (200 g, 1.47 mol) and
hexamethylenetetraamine (206 g, 1.47 mol) in acetic acid (1600 mL)
and water (264 mL) was heated to reflux. The condensate was
removed using a Dean−Stark apparatus until about 1200 mL of
condensate was collected. The reaction mixture was cooled to room
temperature, and water (1000 mL) was added. The thick suspension
was filtered and the collected solid was dried at 60 °C under vacuum
(10 mbar) to give 3-ethyl-4-hydroxy-5-methylbenzaldehyde 82 (191 g,
79%) as an orange solid. LC−MS: tR = 0.86 min, [M + 1 + CH3CN]+
= 206.27. 1H NMR (CDCl3): δ 9.84 (s, 1 H), 7.59 (s, 1 H), 7.57 (s, 1
H), 4.64 (s br, 1 H), 2.71 (q, J = 7.5 Hz, 2 H), 2.34 (s, 3 H), 1.30 (t, J
= 7.5 Hz, 3 H).
DMF (20 mL) was added Hunig’s base (284 mg, 2.19 mmol) followed
̈
by TBTU (210 mg, 0.65 mmol). The mixture was stirred at room
temperature for 10 min before N-((S)-3-[2-ethyl-4-(N-hydroxycarba-
mimidoyl)-6-methylphenoxy]-2-hydroxypropyl)-2-hydroxyacetamide
80 (251 mg, 0.77 mmol) was added. Stirring was continued at room
temperature for 2 h. The mixture was diluted with EA (100 mL) and
washed three times with saturated NaHCO3 solution (2 × 30 mL).
The organic extract was concentrated, and the residue was dissolved in
dioxane (20 mL). The mixture was stirred at 100 °C for 16 h before it
was concentrated. The crude product was purified on preparative
HPLC (Waters XBridge, gradient of MeCN in water containing 0.5%
NH3) to give 53 (281 mg, 76%) as beige wax. LC−MS: tR = 0.66 min,
[M + 1]+ = 509.28. 1H NMR (CDCl3): δ 7.87 (s, 1 H), 7.86 (s, 1 H),
7.76 (s, 1 H), 7.73 (s, 1 H), 7.21 (t, J = 5.9 Hz, 1 H), 4.18−4.25 (m, 3
H), 3.82−3.93 (m, 2 H), 3.74−3.82 (m, 2 H), 3.60 (s br, 1 H), 3.49−
3.58 (m, 1 H), 3.27−3.39 (m, 1 H), 2.95 (q, J = 7.5 Hz, 2 H), 2.75 (q,
J = 7.5 Hz, 2 H), 2.39 (s, 3 H), 2.13−2.21 (m, 2 H), 1.73−1.95 (m, 6
H), 1.39 (t, J = 7.6 Hz, 3 H), 1.32 (t, J = 7.5 Hz, 3 H). 13C NMR
(CDCl3): δ 174.5, 174.0, 168.8, 166.9, 164.3, 157.3, 137.6, 131.60,
131.55, 128.3, 126.6, 122.5, 116.8, 116.2, 74.2, 69.9, 62.1, 48.1, 42.3,
33.6, 31.4, 25.8, 22.8, 16.4, 14.8, 13.8. LC−HRMS: tR = 2.21 min, [M
+ H]/z = 509.2764, found = 509.2762.
(c) A solution of 82 (5.32 g, 32.4 mmol) and hydroxylamine
hydrochloride (3.38 g, 48.6 mmol) in NMP (35 mL) was stirred for 3
h at 80 °C under microwave irradiation (300 W, continuous
cooling).132 The mixture was diluted with water and extracted twice
with diethyl ether. The organic extracts were washed with 2 N aqueous
HCl, saturated aqueous NaHCO3 solution, and brine. The organic
extracts were combined, dried over Na2SO4, filtered, and concentrated.
The crude product was purified by CC on silica gel, eluting with
heptane/EA 3:2 to give 3-ethyl-4-hydroxy-5-methylbenzonitrile 83 as a
1
pale yellow solid (4.80 g, 92%). LC−MS: tR = 0.90 min. H NMR
(CDCl3): δ 1.24 (t, J = 7.6 Hz, 3 H), 2.26 (s, 3 H), 2.63 (q, J = 7.6 Hz,
2 H), 5.19 (s, 1 H), 7.30 (s, 2 H).
(d) To a solution of 83 (5.06 g, 31.4 mmol) in THF (80 mL) were
added PPh3 (9.06 g, 34.5 mmol) and (R)-glycidol (2.29 mL, 34.5
mmol). The mixture was cooled to 0 °C before DEAD in toluene
(15.8 mL, 34.5 mmol) was added. The mixture was stirred for 18 h
while warming up to room temperature. The solvent was evaporated
and the crude product was purified by CC on silica gel, eluting with
heptane/EA 7:3 to give (S)-3-ethyl-5-methyl-4-(oxiran-2-ylmethoxy)-
benzonitrile 84 (5.85 g, 86%) as a yellow oil. LC−MS: tR = 0.96 min,
[M + 42]+ = 259.08. 1H NMR (CDCl3): δ 7.38 (s, 1 H), 7.35 (s, 1 H),
4.12−4.19 (m, 1 H), 3.73−3.80 (m, 1 H), 3.36−3.42 (m, 1 H), 2.90−
2.96 (m, 1 H), 2.68−2.77 (m, 3 H), 2.34 (s, 3 H), 1.26 (t, J = 7.6 Hz, 3
H).
Method B: N-((S)-3-{4-[5-(2-Diethylamino-6-methylpyridin-
4-yl)[1,2,4]oxadiazol-3-yl]-2-ethyl-6-methylphenoxy}-2-hy-
droxypropyl)-2-hydroxyacetamide (53). For an alternative, large
scale synthesis, see Schmidt et al.134 (a) A solution of 2-
(diethylamino)-6-methylisonicotinic acid hydrochloride 79 (6.96 g,
28.4 mmol), Hunig’s base (11.0 g, 85.3 mmol), and TBTU (9.13 g,
̈
28.4 mmol) in DCM (100 mL) was stirred at room temperature for 10
min before 3-ethyl-N,4-dihydroxy-5-methylbenzimidamide (5.52 g,
28.4 mmol) was added. Stirring was continued at room temperature
for 1 h. The mixture was diluted with DCM, washed with saturated
aqueous NaHCO3 solution, dried over MgSO4, filtered, and
concentrated. The residue was dissolved in dioxane (100 mL), and
the mixture was stirred at 100 °C for 18 h. The solvent was evaporated
and the crude product was purified by CC on silica gel, eluting with
heptane/EA 5:1 to give 4-(5-(2-(diethylamino)-6-methylpyridin-4-yl)-
1,2,4-oxadiazol-3-yl)-2-ethyl-6-methylphenol (6.40 g, 61%) as a yellow
solid. LC−MS: tR = 0.90 min, [M + 1]+ = 367.16. 1H NMR (CDCl3):
δ: 7.85 (s, 2 H), 7.10 (s, 1 H), 7.01 (s, 1 H), 4.98 (s, 1 H), 3.63 (q, J =
7.0 Hz, 4 H), 2.73 (q, J = 7.4 Hz, 2 H), 2.49 (s, 3 H), 2.36 (s, 3 H),
1.33 (t, J = 7.5 Hz, 3 H), 1.25 (t, J = 6.9 Hz, 6 H).
(e) The above epoxide 84 (5.85 g, 26.9 mmol) was dissolved in 7 N
NH3 in methanol (250 mL), and the solution was stirred at 65 °C for
18 h. The solvent was evaporated to give crude (S)-4-(3-amino-2-
hydroxypropoxy)-3-ethyl-5-methylbenzonitrile 85 (6.23 g, quantita-
1
tive) as a yellow oil. LC−MS: tR = 0.66 min, [M + 1]+ = 235.11. H
NMR (DMSO-d6): δ 7.54 (s, 2 H), 4.96 (s br, 1 H), 3.77−3.83 (m, 1
H), 3.68−3.77 (m, 2 H), 2.59−2.75 (m, 4 H), 2.28 (s, 3 H), 1.58 (s br,
2 H), 1.17 (t, J = 7.5 Hz, 3 H).
(f) To a solution 85 (6.23 g, 26.6 mmol) were added glycolic acid
(2.43 g, 31.9 mmol), HOBt (4.31 g, 31.9 mmol), and EDC
hydrochloride (6.12 g, 31.9 mmol). The mixture was stirred at room
temperature for 18 h before it was diluted with saturated aqueous
NaHCO3 and extracted twice with EA. The combined organic extracts
were dried over MgSO4, filtered, and concentrated. The crude product
was purified by CC with DCM containing 8% of methanol to give (S)-
N-[3-(4-cyano-2-ethyl-6-methylphenoxy)-2-hydroxypropyl]-2-hydrox-
yacetamide 86 (7.03 g, 90%) as a yellow oil. LC−MS: tR = 0.74 min,
(b) To a solution of 4-(5-(2-(diethylamino)-6-methylpyridin-4-yl)-
1,2,4-oxadiazol-3-yl)-2-ethyl-6-methylphenol (580 mg, 1.58 mmol) in
isopropanol (15 mL) and 3 M aqueous NaOH (3 mL) was added (R)-
epichlorohydrine (439 mg, 4.75 mmol), and the mixture was stirred at
room temperature for 24 h before another portion of (R)-
epichlorohydrine (439 mg, 4.75 mmol) was added. Stirring was
continued for 24 h. The mixture was diluted with EA (100 mL) and
washed with saturated aqueous NaHCO3 solution. The organic extract
was dried over MgSO4, filtered, and concentrated. The crude product
1
[M + 1]+ = 293.10. H NMR (CDCl3): δ 1.25 (t, J = 7.5 Hz, 3 H),
2.32 (s, 3 H), 2.69 (q, J = 7.5 Hz, 2 H), 3.48−3.56 (m, 3 H), 3.70−
124
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Journal of Medicinal Chemistry
Article
was purified by CC on silica gel using heptane/EA 4:1 to give (S)-
N,N-diethyl-4-(3-(3-ethyl-5-methyl-4-(oxiran-2-ylmethoxy)phenyl)-
1,2,4-oxadiazol-5-yl)-6-methylpyridin-2-amine (450 mg, 67%) as a
2-hydroxyacetamide (21). 21 was obtained with method A, using
picolinic acid 68. Beige resin (79 mg, 22%). LC−MS: tR = 0.75 min,
[M + 1]+ = 495.34. 1H NMR (CD3OD): δ 8.62 (s, 1 H), 8.18 (s, 1 H),
8.00 (t, J = 6.0 Hz, 1 H), 7.90 (s, 1 H), 7.87 (s, 1 H), 4.11−4.19 (m, 1
H), 4.04 (s, 2 H), 3.84−3.92 (m, 2 H), 3.63−3.71 (m, 1 H), 3.37−3.52
(m, 2 H), 2.81 (q, J = 7.5 Hz, 2 H), 2.55 (s, 3 H), 2.41 (s, 3 H), 2.13−
2.24 (m, 2 H), 1.68−1.99 (m, 6 H), 1.32 (t, J = 7.5 Hz, 3 H). LC−
HRMS: tR = 1.95 min, [M + H]/z = 495.2607, found = 495.2609.
N-[(S)-3-(2-Ethyl-4-{5-[2-(1-ethylpropyl)-6-methylpyridin-4-
yl][1,2,4]oxadiazol-3-yl}-6-methylphenoxy)-2-hydroxypropyl]-
2-hydroxyacetamide (44). 44 was obtained with method A, using
isonicotinic acid 76d. Pale yellow solid (836 mg, 34%). LC−MS: tR =
0.94 min, [M + 1]+ = 497.19. HPLC with chiral stationary phase
(Chiralpak AD-H 250 mm × 4.6 mm i.d., 5 μm; 80% heptane
containing 0.05% DEA, 20% ethanol containing 0.05% DEA): tR = 9.5
min, 100% ((R)-enantiomer, tR = 7.5 min). 1H NMR (CDCl3): δ 7.90
(s, 1 H), 7.89 (s, 1 H), 7.75 (s, 1 H), 7.67 (s, 1 H), 6.99 (t, J = 5.5 Hz,
1 H), 4.19−4.27 (m, 3 H), 3.92 (dd, J1 = 9.6 Hz, J2 = 4.7 Hz, 1 H),
3.85 (dd, J1 = 9.5 Hz, J2 = 6.3 Hz, 1 H), 3.80 (ddd, J1 = 13.8 Hz, J2 =
6.0 Hz, J3 = 2.5 Hz, 1 H), 3.54 (ddd, J1 = 14.1 Hz, J2 = 7.3 Hz, J3 = 5.5
Hz, 1 H), 3.32 (d, J = 4.5 Hz, 1 H), 2.76 (q, J = 7.5 Hz, 2 H), 2.70 (s, 3
H), 2.55 (s br, 1 H), 2.41 (s, 3 H), 1.80 (quint, J = 7.4 Hz, 4 H), 1.33
(t, J = 7.6 Hz, 3 H), 0.85 (t, J = 7.4 Hz, 6 H). 13C NMR (CDCl3): δ
174.4, 173.8, 168.9, 166.7, 159.3, 157.3, 137.7, 131.6, 131.5, 128.4,
126.6, 122.5, 118.3, 116.9, 74.2, 70.0, 62.1, 51.5, 42.3, 28.1, 24.6, 22.9,
16.5, 14.8, 12.1. LC−HRMS: tR = 2.01 min, [M + H]/z = 497.2764,
found = 497.2767.
N-((S)-3-{4-[5-(2-Cyclopentyl-6-methylpyridin-4-yl)[1,2,4]-
oxadiazol-3-yl]-2-ethyl-6-methylphenoxy}-2-hydroxypropyl)-
2-hydroxyacetamide (46). 46 was obtained with method A, using
isonicotinic acid 76f. Colorless oil (40 mg, 47%). LC−MS: tR = 0.83
min, [M + 1]+ = 495.30. HPLC with chiral stationary phase (Chiralpak
AD-H 250 mm × 4.6 mm i.d., 5 μm; 80% heptane containing 0.05%
DEA, 20% ethanol containing 0.05% DEA): tR = 13.6 min, 100% ((R)-
enantiomer, tR = 10.5 min). 1H NMR (CDCl3): δ: 7.89 (s, 1 H), 7.88
(s, 1 H), 7.76 (s, 1 H), 7.73 (s, 1 H), 7.03 (t, J = 5.5 Hz, 1 H), 4.18−
4.27 (m, 3 H), 3.91 (dd, J1 = 9.6 Hz, J2 = 4.7 Hz, 1 H), 3.85 (dd, J1 =
9.5 Hz, J2 = 6.3 Hz, 1 H), 3.80 (ddd, J1 = 14.2 Hz, J2 = 6.7 Hz, J3 = 3.3
Hz, 1 H), 3.49−3.58 (m, 1 H), 3.38 (s br, 1 H), 3.25−3.35 (m, 1 H),
2.76 (q, J = 7.6 Hz, 2 H), 2.68 (s, 3 H), 2.40 (s, 3 H), 2.12−2.22 (m, 2
H), 1.73−1.95 (m, 6 H), 1.33 (t, J = 7.5 Hz, 3 H). 13C NMR (CDCl3):
δ 174.4, 173.3, 168.9, 167.0, 159.2, 157.3, 137.7, 131.7, 131.6, 128.4,
126.7, 122.5, 118.2, 116.0, 74.1, 70.2, 62.2, 48.2, 42.3, 33.7, 25.8, 24.6,
22.9, 16.5, 14.8. LC−HRMS: tR = 1.98 min, [M + H]/z = 495.2607,
found = 495.2600.
1
yellow oil. LC−MS: tR = 0.94 min, [M + 1]+ = 423.19. H NMR
(CDCl3): δ 7.88 (s, 1 H), 7.87 (s, 1 H), 7.10 (s, 1 H), 7.01 (s, 1 H),
4.18−4.27 (m, 1 H), 3.92 (d, J = 5.2 Hz, 2 H), 3.58−3.70 (m, 6 H),
2.77 (q, J = 7.6 Hz, 2 H), 2.50 (s, 3 H), 2.41 (s, 3 H), 1.33 (t, J = 7.5
Hz, 3 H), 1.25 (t, J = 7.0 Hz, 6 H).
(c) A solution of (S)-N,N-diethyl-4-(3-(3-ethyl-5-methyl-4-(oxiran-
2-ylmethoxy)phenyl)-1,2,4-oxadiazol-5-yl)-6-methylpyridin-2-amine
(5.70 g, 13.5 mmol) in 7 M NH3 in MeOH (100 mL) was stirred in a
sealed vessel at 60 °C for 24 h. The mixture was concentrated and the
crude product was purified by CC on silica gel, eluting with DCM/
MeOH 10:1 containing a small amount of 7 M NH3 in MeOH to give
(S)-1-amino-3-(4-(5-(2-(diethylamino)-6-methylpyridin-4-yl)-1,2,4-
oxadiazol-3-yl)-2-ethyl-6-methylphenoxy)propan-2-ol (2.33 g, 39%) as
1
a yellow oil. LC−MS: tR = 0.70 min, [M + 1]+ = 440.25. H NMR δ:
7.88 (s, 1 H), 7.87 (s, 1 H), 7.10 (s, 1 H), 7.00 (s, 1 H), 3.97−4.05 (m,
1 H), 3.85−3.93 (m, 2 H), 3.62 (q, J = 7.0 Hz, 4 H), 3.23 (s, 1 H),
3.03 (dd, J1 = 12.8 Hz, J2 = 3.8 Hz, 1 H), 2.93 (dd, J1 = 12.8 Hz, J2 =
7.0 Hz, 1 H), 2.77 (q, J = 7.5 Hz, 2 H), 2.49 (s, 3 H), 2.40 (s, 3 H),
1.32 (t, J = 7.5 Hz, 3 H), 1.24 (t, J = 7.0 Hz, 6 H).
(d) To a solution of (S)-1-amino-3-(4-(5-(2-(diethylamino)-6-
methylpyridin-4-yl)-1,2,4-oxadiazol-3-yl)-2-ethyl-6-methylphenoxy)-
propan-2-ol (2.33 g, 5.30 mmol) in DCM (25 mL) were added
glycolic acid (443 mg, 5.83 mmol) and HOBt (788 mg, 5.83 mmol).
The mixture was stirred for 10 min before EDC·HCl (1.12 g, 5.83
mmol) was added. The mixture was stirred at room temperature for 1
h before it was diluted with water and brine. The mixture was extracted
twice with EA. The combined organic extracts were dried over MgSO4,
filtered, and concentrated. The crude product was purified by CC on
silica gel, eluting with DCM/MeOH 10:1 to give 56 (2.20 g, 83%) as a
yellow foam. LC−MS: tR = 0.78 min, [M + 1]+ = 498.22. HPLC with
chiral stationary phase (Chiralpak AD-H 250 mm × 4.6 mm i.d., 5 μm;
85% hexane, 15% ethanol containing 0.1% DEA): tR = 12.3 min, 100%
1
((R)-enantiomer, tR = 10.2 min). H NMR (DMSO-d6): δ 7.79 (s, 2
H), 7.70 (t, J = 5.5 Hz, 1 H), 7.07 (s, 1 H), 6.99 (s, 1 H), 5.56 (t, J =
5.5 Hz, 1 H), 5.31 (d, J = 5.1 Hz, 1 H), 3.93−4.00 (m, 1 H), 3.84 (d, J
= 5.2 Hz, 2 H), 3.70−3.81 (m, 2 H), 3.58 (q, J = 6.5 Hz, 4 H), 3.39−
3.48 (m, 1 H), 3.20−3.29 (m, 1 H), 2.73 (q, J = 7.3 Hz, 2 H), 2.43 (s,
3 H), 2.35 (s, 3 H), 1.22 (t, J = 7.5 Hz, 3 H), 1.16 (t, J = 6.9 Hz, 6 H).
LC-HRMS: tR = 1.86 min, [M + H]/z = 498.2716, found = 498.2717.
N-((S)-3-{2-Ethyl-4-[5-(6-isobutyl-5-methylpyridin-3-yl)-
[1,2,4]oxadiazol-3-yl]-6-methylphenoxy}-2-hydroxypropyl)-2-
hydroxyacetamide (8). 8 was obtained with method A, using
nicotinic acid 60. Colorless resin (17 mg, 20%). LC−MS: tR = 0.83
min, [M + 1]+ = 483.23. HPLC with chiral stationary phase (Chiralpak
AD-H 250 × 4.6 mm i.d., 5 μm; 80% heptane containing 0.05% DEA,
20% ethanol containing 0.05% DEA): tR = 12.9 min, 98% ((R)-
enantiomer, tR = 11.3 min, 2%). 1H NMR (DMSO-d6): δ 9.08 (d, J =
2.0 Hz, 1 H), 8.30 (d, J = 1.7 Hz, 1 H), 7.79 (s, 2 H), 7.72 (t, J = 5.8
Hz, 1 H), 5.58 (t, J = 5.7 Hz, 1 H), 5.33 (d, J = 5.2 Hz, 1 H), 3.93−
4.01 (m, 1 H), 3.84 (d, J = 5.7 Hz, 2 H), 3.70−3.79 (m, 2 H), 3.40−
3.48 (m, 1 H), 3.20−3.29 (m, 1 H), 2.69−2.78 (m, 4 H), 2.42 (s, 3 H),
2.34 (s, 3 H), 2.19 (hept, J = 6.8 Hz, 1 H), 1.22 (t, J = 7.5 Hz, 3 H),
0.94 (d, J = 6.6 Hz, 6 H). LC−HRMS: tR = 1.93 min, [M + H]/z =
483.2607, found = 483.2612.
ASSOCIATED CONTENT
* Supporting Information
■
S
Additional experimental details on the synthesis and character-
ization of all target compounds and all intermediates not given
above as well as data and a brief comment on mean arterial
blood pressure and heart rate effects of compound 53 in
spontaneously hypertensive rats are given. 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.
■
N-((S)-3-{4-[5-(6-Cyclopentyl-5-methylpyridin-3-yl)[1,2,4]-
oxadiazol-3-yl]-2-ethyl-6-methylphenoxy}-2-hydroxypropyl)-
2-hydroxyacetamide (12). 12 was obtained with method A, using
nicotinic acid 61. Pale yellow oil (105 mg, 73%). LC−MS: tR = 0.75
1
min, [M + 1]+ = 495.31. H NMR (CDCl3): δ 9.20 (d, J = 2.0 Hz, 1
Notes
H), 8.20 (d, J = 1.5 Hz, 1 H), 7.87 (s, 1 H), 7.85 (s, 1 H), 7.17 (t, J =
5.8 Hz, 1 H), 4.18−4.25 (m, 3 H), 3.76−3.93 (m, 3 H), 3.40−3.57 (m,
2 H), 2.74 (q, J = 7.5 Hz, 2 H), 2.49 (s, 3 H), 2.38 (s, 3 H), 2.00−2.11
(m, 2 H), 1.88−2.00 (m, 4 H), 1.70−1.81 (m, 2 H), 1.31 (t, J = 7.5
Hz, 3 H). LC−HRMS: tR = 2.11 min, [M + H]/z = 495.2607, found =
495.2609.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
The authors gratefully acknowledge their co-workers Maxime
■
Boucher, Cel
́
ine Bortolamiol, Step
́
hane Delahaye, Patrick
Dorrwachter, Alexandre Flock, Hakim Hadana, Julie Hoerner,
̈
̈
N-((S)-3-{4-[5-(5-Cyclopentyl-4-methylpyridin-2-yl)[1,2,4]-
oxadiazol-3-yl]-2-ethyl-6-methylphenoxy}-2-hydroxypropyl)-
Benedikt Hofstetter, Franco̧ is Le Goff, Daniel Leuenberger,
125
dx.doi.org/10.1021/jm4014696 | J. Med. Chem. 2014, 57, 110−130

Journal of Medicinal Chemistry
Claire Maciejasz, Celine Mangold, Katalin Menyhart, Matthias
Merrettig, Christine Metzger, Markus Rey, Virginie Sippel,
Mireille Tena Stern, Marco Tschanz, Gaby von Aesch, Daniel
Wanner, Aude Weigel, and Rolf Wuest for the excellent work
done and Martine Clozel for support.
Article
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C. Dose-Dependent Effect of Ponesimod, an Oral, Selective
Sphingosine 1-Phosphate Receptor-1 Modulator, on Magnetic
Resonance Imaging Outcomes in Patients with Relapsing−Remitting
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ABBREVIATIONS USED
■
AUC, area under the curve; BuLi, butyllithium; CC, column
chromatography; DCM, dichloromethane; DEAD, diethyl
azodicarbocylate; DMF, dimethylformamide; EA, ethyl acetate;
EDC, 1-ethyl-3-(3-dimethyllaminopropyl)carbodiimide; HOBt,
N-hydroxybenzotriazole; LC, lymphocyte count; LC−MS,
(high pressure) liquid chromatography combined with mass
spectrometry; PK, pharmacokinetics; PD, pharmacodynamics;
RLM, rat liver microsome; SAR, structure−activity relationship;
TBTU, O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium
tetrafluoroborate; THF, tetrahydrofuran; S1P, sphingosine 1-
phosphate
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