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6347
is markedly up-regulated in a subset of adenocarcinoma cells, and
knock down of this receptor subtype inhibits proliferation and
growth of lung adenocarcinoma cells.16
A wealth of S1P-Rs agonists has been described in the literature.
However, the development of subtype selective S1P3-R agonists as
useful pharmacological tools has been limited as a consequence of
targeting the orthosteric binding site for receptor activation. The
orthosteric binding site displays a high level sequence homology
among the S1P-R family subtypes. S1P1-R and S1P3-R are the most
closely related by sequence, particularly at their agonist binding
pockets which consist of a lower hydrophobic and an upper polar
region where Leu-276 in S1P1-R and Phe-263 in S1P3-R are the
main difference. Receptor structure modeling and ligand docking
studies revealed that the S1P3-R binding pocket is contracted be-
tween the lower lipophilic area and the upper polar section by
1.5–1.8 Å compared to the S1P1-R due to the presence of
Phe-263. These differences in steric and space constrains in the
S1P3-R orthosteric binding site may explain the difficulty in
designing S1P3-R agonists devoid of S1P1-R agonist activity.17
Topologically distinct from the conserved orthosteric binding
site, an allosteric site provides a means to overcome important
selectivity issues associated with the orthosteric ligands, in partic-
ular within GPCRs in which the orthosteric site is highly conserved
between subtypes. In addition to offering a potential subtype-
selectivity, allosteric ligands may stabilize different conformations
and functional states, thus activating a distinct repertoire of recep-
tor signaling and regulatory properties that orthosteric ligands are
unable to initiate.18 The S1P binding site is highly conserved
among the S1P-R family thus selective allosteric ligands may be
useful pharmacological tools to decipher individual receptor bio-
logical functions.
Scheme 1. Synthesis of 1a–d. Reagents and conditions: (i) (a) 2 (1 equiv), SOCl2,
benzene, reflux, 3 h; (b) 3a–d (1.5 equiv), DIPEA (1.5 equiv), CH2Cl2, 0 °C–rt, 3 h, 70–
98% (over two steps).
Table 1
S1P3-R agonist activity of compounds 5a–i
a
Compd
Carboxylic acid
R
EC50 (nM)
S1P3-R
S1P1-R
5a
5b
5c
5d
5e
5f
5g
5h
5i
4a
4b
4c
4d
4e
4f
4g
4h
4i
Isobutyl
Isopropyl
Methyl
Cyclohexyl
Cyclopentyl
Cyclopropyl
Phenyl
4-Methoxyphenyl
3,4-Diethoxyphenyl
1375
959
6595
559
339
105
103
323
8070
6420
22,250
>50,000
>50,000
>50,000
33,300
662
859
1104
a
Data are reported as mean of n = 3 determinations.
A high-throughput screening (HTS) of the Molecular Libraries-
Small Molecule Repository (MLSMR) library identified the
N,N-dicyclohexyl-5-propylisoxazole-3-carboxamide 1a (Fig. 1) as
a S1P3-R agonist with acceptable in vitro potency/selectivity pro-
file.17,19 The structural integrity of the hit was corroborated by
the re-synthesis (Scheme 1) of the title compound that showed
confirmed EC50’s of 434 nM at S1P3-R, 7.87
agonist activity at S1P2,4-Rs at concentrations up to 50
l
M at S1P1-R and no
Scheme 2. Synthesis of 5a–i. Reagents and conditions: (i) (a) 4a–h (1 equiv), SOCl2,
benzene, reflux, 3 h; (b) 3a (1.5 equiv), DIPEA (1.5 equiv), CH2Cl2, 0 °C–rt, 3 h, 85–
98% (over two steps).
lM (Fig. 1).
Our SAR studies commenced varying the amide region C. The
synthesis and biological results of 1b–d are outlined in Scheme 1.
The carboxylic acid 2 was transformed into the corresponding acid
chloride and coupled with amines 3b–d to furnish the amide prod-
ucts 1b and 1d. Surprisingly, exchanging a cyclohexyl from 1a for a
cyclopentyl group (1b) led to loss in potency of 18-fold for the
S1P3-R but only of three-fold for the S1P1-R. Furthermore, changing
a cyclohexyl for a phenyl (1d) or hydrogen (1c) led to complete loss
of potency at both receptors. These results underscore the key role
played by the N,N-dicyclohexyl amide for binding to the S1P3-R.
Next we explored region A of the HTS-hit while keeping regions
B and C constant. Compounds 5a–i (Table 1) were synthesized
from a series of isoxazole carboxylic acids 4a–i commercially avail-
able (4a–c and 4f–i) or readily obtained (4d and 4e) according to
literature procedures (Scheme 2).20
Interestingly, the isobutyl 5a was approximately three-fold less
potent than the hit at the S1P3-R and four-fold less selective
against the S1P1-R. The isopropyl derivative 5b was approximately
two-fold less active than 1a for the S1P3-R, but the selectivity
against the S1P1-R remained similar. The methyl derivative 5c
was 15-fold less potent than 1a. Remarkably, the cyclohexyl (5d)
and cyclopentyl (5e) analogs were slightly less and more active
at the S1P3-R and inactive at the S1P1-R. Of note, the cyclopropyl
derivative 5f (CYM5541) was found four-fold more potent than
the hit and nearly 18-fold more selective against the S1P1-R. The
phenyl derivative 5g (CYM5544) was found equipotent to 5f for
the S1P3-R but significantly less selective against the S1P1-R. Inter-
estingly, the 4-methoxyphenyl 5h was slightly more potent than
the hit compound at the S1P3-R, but its selectivity against the
S1P1-R was less than three-fold. Intriguingly, 3,4-diethoxyphenyl
5i was 18-fold less potent than the hit at the S1P3-R but more ac-
tive (ꢀ7-fold) at the S1P1-R. All this information together indicates
that an aromatic ring at position five of the isoxazole hit (portion
A), although favorable for the S1P3-R activity, is also detrimental
for the selectivity against the S1P1-R.
Based on the obtained results, we focused our attention on the
SAR studies of 5f, particularly on the amide region C. The synthesis
of 7a–l is depicted in Scheme 3. The biological results are listed in
Table 2.
Figure 1. HTS S1P3-R agonist hit 1a.
Guerrero, Miguel
