Discovery, design and synthesis of the first reported potent and selective sphingosine-1-phosphate 4 (S1P4) receptor antagonists

Article abstract of DOI:10.1016/j.bmcl.2011.04.097

Selective S1P₄ receptor antagonists could be novel therapeutic agents for the treatment of influenza infection in addition to serving as a useful tool for understanding S1P₄ receptor biological functions. 5-(2,5-Dichlorophenyl)-N-(2,6-dimethylphenyl)furan-2-carboxamide was identified from screening the Molecular Libraries-Small Molecule Repository (MLSMR) collection and selected as a promising S1P₄ antagonist hit with moderate in vitro potency and high selectivity against the other family receptor subtypes (S1P_1-3,5). Rational chemical modifications of the hit allowed the disclosure of the first reported highly selective S1P₄ antagonists with low nanomolar activity and adequate physicochemical properties suitable for further lead-optimization studies.

Full text of DOI:10.1016/j.bmcl.2011.04.097

Bioorganic & Medicinal Chemistry Letters 21 (2011) 3632–3636
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery, design and synthesis of the first reported potent and selective
sphingosine-1-phosphate 4 (S1P4) receptor antagonists
Miguel Guerrero a, Mariangela Urbano a, Subash Velaparthi a, Jian Zhao a, Marie-Therese Schaeffer b,c
,
Steven Brown b,c, Hugh Rosen b,c, Edward Roberts a,c,
⇑
a Department of Chemistry, The Scripps Research Institute, 10550 N. Torrey Pines Rd, La Jolla, CA 92037, United States
b Department of Immunology, The Scripps Research Institute, 10550 N. Torrey Pines Rd, La Jolla, CA 92037, United States
c The Scripps Research Institute Molecular Screening Center, 10550 N. Torrey Pines Rd, La Jolla, CA 92037, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Selective S1P4 receptor antagonists could be novel therapeutic agents for the treatment of influenza
infection in addition to serving as a useful tool for understanding S1P4 receptor biological functions.
5-(2,5-Dichlorophenyl)-N-(2,6-dimethylphenyl)furan-2-carboxamide was identified from screening the
Molecular Libraries-Small Molecule Repository (MLSMR) collection and selected as a promising S1P4
antagonist hit with moderate in vitro potency and high selectivity against the other family receptor sub-
types (S1P1–3,5). Rational chemical modifications of the hit allowed the disclosure of the first reported
highly selective S1P4 antagonists with low nanomolar activity and adequate physicochemical properties
suitable for further lead-optimization studies.
Received 15 March 2011
Revised 18 April 2011
Accepted 21 April 2011
Available online 28 April 2011
Keywords:
S1P4 receptor antagonists
S1P1–3,5 receptor family
5-Aryl furan-2-arylcarboxamide
Ó 2011 Elsevier Ltd. All rights reserved.
Sphingosine-1-phosphate (S1P) is a pleiotropic lysophospho-
lipid that is formed in various cells including platelets, mast cells
and macrophages in response to various stimuli such as grow fac-
tors, cytokines and antigens. It serves as an important biological
mediator that influences endothelial and lung epithelial integrity1,2
as well as lymphocyte recirculation.3–6 S1P exerts its biological ef-
fects via extracellular signaling through five G-protein coupled
receptor subtypes, namely S1P1 through S1P5.7 While S1P1–3 recep-
tors are expressed ubiquitously, S1P4 expression is highly re-
stricted to hematopoietic and lymphatic tissue and S1P5 is
expressed in the central nervous system.8 S1P4 is coupled to Gai
agonists nor antagonists, with high selectivity against the
S1P1–3,5 subtypes. Herein we report on the discovery, synthesis
and structure–activity relationships (SAR) of novel S1P4 selective
antagonists as a valuable tool to elucidate the biological and phar-
macological profile of the target receptor.
In house high-throughput screening (HTS) of the Molecular Li-
braries-Small Molecule Repository (MLSMR) collection identified
a 5-aryl furan-2-arylcarboxamide derivative 1 (Fig. 1) as a S1P4
antagonist hit with acceptable in vitro potency/selectivity profile.
With the aim to validate the identified hit, 1 was re-synthesized
(Scheme 1) confirming IC50’s of 78 nM at S1P4, 14
lM at S1P5
and G
a
o proteins and activates the ERK, MAPK, PLC cascades.9 Local
and no activity at concentrations up to 25 M towards S1P1–3 sub-
l
modulation of S1P receptors in the lungs has been shown to alter
dendritic cell activation and accumulation in the mediastinal
lymph nodes, resulting in blunted T cell responses and control of
immunopathological features of influenza virus infection, without
involvement of the S1P1 immunosuppressive activities.10 Reports
showing that in dendritic cells S1P5 expression is very low whereas
S1P4 is highly expressed,11 suggest that chemical activation of the
S1P4 subtype in the airways may be effective at controlling the
immunopathological response to viral infections. It has been re-
ported that S1P4 is upregulated during megakaryocytes differenti-
ation suggesting that S1P4 antagonists are a suitable target for
reactive thrombocytosis.12 However, aspects of the biological role
of S1P4 remain unclear partly due to the lack of ligands, neither
types. However, inadequate values of logarithm of partition coeffi-
cient and total polar surface area (c Log P = 4.8, tPSA = 38.5) were
calculated for 1. We sought to increase potency and reduce lipo-
philicity (c Log P and tPSA optimal values ranges of 2.5–3.5 and
60–90, respectively),13 through rational chemical modifications of
⇑
Corresponding author. Tel.: +1 858 784 7770; fax: +1 858 784 7745.
E-mail address: eroberts@scripps.edu (E. Roberts).
Figure 1.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.04.097

M. Guerrero et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3632–3636
3633
Table 1 (continued)
Compd
R
Ar
c Log Pb tPSAb IC50
4k
H
4.5
6.2
38.3
38.3
6400
Scheme 1. Synthesis of 5-(2,5-dichlorophenyl)-2-arylcarboxamides 1, 4a–z.
Reagents and conditions: (i) 2 (1 equiv), 3 (2 equiv), DIPEA (2 equiv), CH2Cl2, rt,
2–4 h, 60–95%.
4l
H
253
the hit structure, thereby altering the predicted physicochemical
properties and S1P4 binding affinity. 1 was formally fragmented
into two regions to investigate its SAR (Fig. 1): (A) aryl ring
C-linked to furan, (B) arylamide.
The preparation of various analogs with modified substituents
on the phenyl ring of region B was easily achieved as presented
in Scheme 1. Coupling of commercially available acylchloride 2
with a variety of anilines provided, in good yields, a structurally
and electronically diverse set of analogs 4a–4z. The obtained com-
pounds were submitted to S1P4 functional assay (Table 1).14
Potency and lipophilicity were not significantly affected by
attaching small alkylic groups on positions 2 and 6 (4b, 4c, 4f)
4m
4n
H
H
5.0
4.0
47.6
47.6
931
589
4o
4p
4q
4r
H
4.2
4.9
5.4
5.5
58.6
47.6
38.3
29.5
58
H
80
Table 1
S1P4 antagonists (IC50, nM)a
Compd
R
Ar
c Log Pb tPSAb IC50
H
63
4a
H
5.5
5.2
38.3
38.3
165
41
Me
8100
4b
4c
H
H
4s
4t
H
H
3.4
3.8
90.7
58.6
52
21
5.5
38.3
89
4d
4e
H
H
4.0
3.3
56.8
58.6
417
573
4u
4v
4w
4x
H
H
H
H
4.7
3.8
3.7
4.7
64.6
64.3
67.4
41.6
34
25
4f
H
6.2
38.3
105
94
348
4g
4h
H
H
3.8
5.1
72.5
38.3
302
169
4y
4z
H
H
6.7
4.0
38.3
67.8
115
44
4i
4j
H
4.5
5.6
47.6
29.5
163
NA
a
Data are reported as mean for n = 3 determinations. NA = no active at concen-
trations up to 25 M.
l
b
c Log P and tPSA values are obtained from ChemDraw 12.0 V.
–CH2–CH2–
compared to the hit. When polar substituents, hydrogen donors
or acceptors, were attached in the same positions the potency
decreased more substantially (4d, 4e, 4h, 4i). Similar activity to

3634
M. Guerrero et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3632–3636
Scheme 2. Synthesis of 5-aryl-N-(2,6-dialkylphenyl)furan-2-carboxamides 9a–9q. Reagents and conditions: (i) 5 (1 equiv), 6 (1.2 equiv), EDCl (1.2 equiv), HOBt (1.2 equiv),
DMF, rt, overnight, 90%; (ii) 7 (1 equiv), 8 (1.5 equiv), Pd(PPh3)4 (0.1 equiv), 2 M aq NaCO3 (2 equiv), 1,4-dioxane, 80 °C, 4–6 h, 20–85%.
Table 2 (continued)
the hit was observed for the 2,4,6-trimethyl derivative 4q. Incorpo-
Compd
R
Ar
c Log Pb
tPSAb
38.3
IC50
575
rating the amidic nitrogen into a five-membered ring suppressed
completely the activity (4j). Two major reasons were formulated
to explain this phenomenon: (a) the constriction of the C–N bond
rotation, (b) the loss of hydrogen bond donor capability. To evalu-
ate the contribution of the N–H hydrogen bond donor, a N-methyl-
ated derivative 4r was synthesized. 4r was approximately 128-fold
less potent than the homolog 4q; therefore the hydrogen-bond do-
nor capability of the amide group was hypothesized to be essential
for the potency. Noteworthy, position 4 of the phenyl ring tolerated
lipophilic substituents (4q, 4y) as well as polar and ionizable
groups as observed for 4n, 4p, 4w, 4x, 4y, and 4o, 4s–4v, 4z, the lat-
ter showing enhancement of both potency and physicochemical
properties. Particularly, the amine 4v(CYM50358) [c Log P = 3.8,
tPSA = 64.4] and alcohols 4t [c Log P = 3.8, tPSA = 58.6], 4z
[c Log P = 4.0, tPSA = 67.8] were more potent than the hit com-
pound 1 with more desirable physicochemical properties.
9d
H
3.6
9e
9f
H
4.0
5.5
2.4
3.4
38.3
38.3
78.8
56.8
308
NA
H
9g
9h
Me
Me
NA
Next, SAR studies of the aryl ring A were performed while main-
taining 2,6-dialkylanilines in the pendant moiety B. The general
synthetic route to afford 9a–9q is outlined in Scheme 2. Amide
coupling of 2-furoic acid 5 with appropriate aniline 6 followed by
Suzuki cross coupling under standard conditions15 with a wide
range of aryl boronic acids afforded 9a–9q.
513
S1P4 activity of 9a–9q is reported in Table 2. The 2,5-dimethyl
analogue 9i showed similar potency to the hit compound. Deletion
of 2-chlorine (9b) had a higher impact on the loss of activity than
the removal of 5-chlorine (9c) (6- vs 3-fold). Notably, the 2,4-
dichlorophenyl regioisomer 9a was 25- and 7-fold less potent than
the hit and the mono-chlorinated 9c, respectively, thus indicating
that substitution at position 4 was detrimental for the potency.
2,6-Dimethylated derivative 9j was found to be inactive probably
due to the anti-coplanar orientation of the phenyl ring. In an at-
tempt to reduce lipophilicity, polar substitutions at positions 2
and 5 were installed, but were found detrimental for the activity
9i
9j
H
H
4.3
4.0
38.3
38.3
64
NA
9k
9l
H
H
3.3
3.5
38.3
38.3
448
286
Table 2
S1P4 antagonists (IC50, nM)a
Compd
R
Ar
c Log Pb
tPSAb
38.3
IC50
9m
9n
H
H
3.5
4.3
38.3
38.3
118
515
9a
H
4.9
2000
9o
9p
H
3.7
3.3
38.3
47.6
93
9b
9c
H
4.4
4.4
38.3
38.3
497
269
Me
130
Me
a
Data are reported as mean for n = 3 determinations. NA = no active at concen-
trations up to 25 M.
c Log P and tPSA values are obtained from ChemDraw 12.0 V.
l
b

M. Guerrero et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3632–3636
3635
Scheme 3. Synthesis of molecules 15, 16. Reagents and conditions: (i) 10 (1 equiv), 11 (1.5 equiv), Pd(PPh3)4 (0.1 equiv), 2 M aq Na2CO3 (2 equiv), 1,4-dioxane, 80 °C,
overnight; (ii) LiOH (1.6 equiv), THF/MeOH/H2O (2:2:1), rt, 3 h, 65% (over two steps); (iii) 12 (1 equiv), 13 or 14 (1.5 equiv), EDCl (1.5 equiv), HOBt (1.5 equiv), DMF, overnight,
60–70%.
(9e, 9f, 9g, 9h), suggesting that region A binds to a lipophilic pock-
et. Successively, a new set of analogs was prepared by replacing the
phenyl ring. Interestingly, thiophene and furan rings were found to
be good bioisosteres. The 3-thienyl 9k and 2-thienyl 9m analogs
were slightly more potent than the phenyl derivative 9d. As the
presence of either chlorine or methyl groups in positions 2 and 5
of the phenyl ring were found to be essential for the activity, meth-
ylated and chlorinated thienyl derivatives (9l, 9n and 9o) were
synthesized. Interestingly, 4-methyl-3-thienyl 9l was 1.5-fold
more potent than 9k (similar trend was observed in the phenyl ser-
ies, 9c vs 9d) whereas 3-methyl-2-thienyl 9o was equipotent to
9m. Moreover, equipotency was found for 3-chlorophenyl 9b and
2-furanyl 9p compared to 5-chloro-2-thienyl 9n and thienyl 9m,
respectively. Interestingly, thienyl and furanyl derivatives showed
more suitable physicochemical properties (3.3 6 c Log P 6 4.3)
within the hit class.
To merge SAR studies of region A and B, hybrid molecules 15
and 16 (CYM50374) were synthesized (Scheme 3). 5-Bromofuran
10 underwent Suzuki cross coupling with thiophene boronic acid
11 followed by ester hydrolysis to afford carboxylic acid 12 in good
yields. Amide coupling of 12 with the opportune anilines 13 and 14
yielded the final compounds in moderate yields.
Indeed, 15 (c Log P = 3.0, tPSA = 58.6) and 16 (c Log P = 2.7,
tPSA = 58.6) were potent S1P4 antagonists (IC50 = 46 and 34 nM,
respectively), with lower lipophilicity compared to the hit
compound.
erties were selected as lead compounds to initiate a lead-optimiza-
tion program.
In summary, we conducted a systematic SAR analysis of novel
selective S1P4 antagonists based on 5-aryl furan-2-arylcarboxa-
mide 1 scaffold, identified by our HTS efforts. Physicochemical
properties (c Log P and tPSA) were calculated for a preliminary
evaluation of drug-like properties. Notably, introduction of differ-
ent ionizable and polar groups at position 4 of region B led to the
identification of molecules of particular interest (4v and 16) with
attractive in vitro biological profile and adequate physicochemical
properties. As the first disclosure of S1P4 antagonists with low
nanomolar potency and high selectivity against S1P1–3,5 receptor
subtypes, the class of molecules herein reported represents a sig-
nificant milestone that may allow experiments aimed to gain more
insights into the biological functions of S1P4 in fundamental
immunological processes. Details of more in-depth ongoing SAR
for lead-optimization of the identified lead molecules will be com-
municated in due course.
Acknowledgments
This work was supported by the National Institute of Health
Molecular Library Screen Center Network grant U54 MH084512A.
We thank Mark Southern for data management with Pub Chem.
References and notes
A set of the most active compounds was selected for functional
assays at S1P1–3,5 subtypes (Table 3). Notably, all the selected com-
pounds displayed an exquisite selectivity for the S1P4 receptor ver-
sus the other receptor subtypes; among them 4v (CYM50358) and
16 (CYM50374) showing the most suitable physicochemical prop-
1. Marsolais, D.; Hahm, B.; Walsh, K. B.; Edelmann, K. H.; McGavern, D.; Hatta, Y.;
Kawaoka, Y.; Rosen, H.; Oldstone, M. B. A. Proc. Natl. Acad. Sci. U.S.A. 2009, 106,
1560.
2. Marsolais, D.; Rosen, H. Nat. Rev. Drug Discov. 2009, 8, 297–307.
3. Alfonso, C.; McHeyzer-Williams, M. G.; Rosen, H. Eur. J. Immunol. 2006, 36, 149.
4. Jo, E.; Sanna, M. G.; Gonzalez-Cabrera, P. J.; Thangada, S.; Tigyi, G.; Osborne, D.
A.; Hla, T.; Parrill, A. L.; Rosen, H. Chem. Biol. 2005, 12, 703.
5. Sanna, G.; Liao, J.; Jo, E.; Alfonso, C.; Ahn, M.; Peterson, M. S.; Webb, B.;
Lefebvre, S.; Chun, J.; Gray, N.; Rosen, H. J. Biol. Chem. 2004, 279, 13839.
6. Wei, S. H.; Rosen, H.; Matheu, M. P.; Sanna, M. G.; Wang, S.; Jo, E.; Wong, C.;
Parker, I.; Cahalan, M. Nat. Immunol. 2005, 6, 1228.
Table 3
S1P selectivity counter screen (IC50, nM, percentage of inhibition)a,b,c
Compd
S1P4 IC50
S1P1 IC50
S1P2 IC50
S1P3 IC50
S1P5 IC50
7. Rosen, H.; Goetzl, E. J. Nat. Rev. Immunol. 2005, 5, 560.
4b
4c
4o
4p
4t
4v
4z
9o
15
16
41
89
58
80
36
25
44
93
46
34
NA
NA
50%c
NA
55%c
NA
50%c
NA
3000
40%c
40%
8. Gräler, M. H.; Bernhardt, G.; Lipp, M. Genomics 1998, 53, 164; (b) Van Brocklyn,
J. R.; Gräler, M. H.; Bernhardt, G.; Hobson, J. P.; Lipp, M.; Spiegel, S. Blood 2000,
53, 164; (c) Im, D. S.; Heise, C. E.; Ancellin, N.; O’Dowd, B. F.; Shei, G. J.; Heavens,
R. P.; Rigby, M. R.; Hla, T.; Mandala, S.; McAllister, G.; George, S. R.; Lynch, K. R.
J. Biol. Chem. 2000, 275, 14281.
5300
20%c
45%c
6400
35%c
95%c
75%c
80%c
2800 (90%)b
2800 (70%)b
3000 (85%)b
3900 (90%)b
2400 (90%)b
2600
5400
30%c
60%
95%
70%c
20%c
10%c
NA
9. Toman, R. E.; Spiegel, S. Neurochem. Res. 2002, 27, 619.
5500 (90%)b
60%c
40%c
NA
10. Marsolais, D.; Hahm, B.; Edelmann, K. H.; Walsh, K. B.; Guerrero, M.; Hatta, Y.;
Kawaoka, Y.; Roberts, E.; Oldstone, M. B.; Rosen, H. Mol. Pharmacol. 2008, 74,
896.
11. Maeda, Y.; Matsuyuki, H.; Shimano, K.; Kataoka, H.; Sugahara, K.; Kenji Chiba,
K. J. Immunol. 2007, 178, 3437.
80%c
90%c
NA
12. Golfier, S.; Kondo, S.; Schulze, T.; Takeuchi, T.; Vassileva, G.; Achtman, A. H.;
Gräler, M. H.; Abbondanzo, S. J.; Wiekowski, M.; Kremmer, E.; Endo, Y.; Lira, S.
A.; Bacon, K. B.; Lipp, M. FASEB J. 2010, 24, 4701.
13. (a) Hughes, J. D.; Blagg, J.; Price, D. A.; Bailey, S.; DeCrescenzo, G. A.; Devraj, R.
V.; Ellsworth, E.; Fobian, Y. M.; Gibbs, M. E.; Gilles, R. W.; Greene, N.; Huang, E.;
a
b
c
Data are reported as mean for n = 3 determinations.
Percentage of inhibition.
Percentage of inhibition at 25
lM.
lM. NA = no active at concentrations up to
25

3636
M. Guerrero et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3632–3636
Krieger-Burke, T.; Loesel, J.; Wager, T.; Whiteley, L.; Zhang, Y. Bioorg. Med.
Chem. Lett. 2008, 18, 4872; (b) Muchmore, S. W.; Edmunds, J. J.; Stewart, K. D.;
Hajduk, P. J. J. Med. Chem. 2010, 53, 4830.
TEV protease fusion protein and a b-lactamase (BLA) reporter gene under the
control of a UAS response element. BLA expression is monitored by measuring
fluorescence resonance energy transfer (FRET) of a cleavable, fluorogenic, cell-
permeable BLA substrate.
14. The activity was measured by using Tango™ EDG6-bla U2OS cells which
contain the human Endothelial Differentiation Gene 6 linked to a GAL4-VP16
transcription factor via a TEV protease site. The cells also express a b-arrestin/
15. McAllister, L. A.; Hixon, M. S.; Kennedy, K. P.; Dickerson, T. J.; Janda, K. D. J. Am.
Chem. Soc. 2006, 128, 4176.