SAR analysis of innovative selective small molecule antagonists of sphingosine-1-phosphate 4 (S1P4) receptor

Article abstract of DOI:10.1016/j.bmcl.2011.06.132

Recent evidence suggests an innovative application of chemical modulators targeting the S1P₄ receptor as novel mechanism-based drugs for the treatment of influenza virus infection. Modulation of the S1P₄ receptor may also represent an alternative therapeutic approach for clinical conditions where reactive thrombocytosis is an undesired effect or increased megakaryopoiesis is required. With the exception of our recent research program disclosure, we are not aware of any selective S1P₄ antagonists reported in the literature to date. Herein, we describe complementary structure-activity relationships (SAR) of the high-throughput screening (HTS)-derived hit 5-(2,5-dichlorophenyl)-N-(2,6-dimethylphenyl)furan-2- carboxamide and its 2,5-dimethylphenyl analog. Systematic structural modifications of the furan ring showed that both steric and electronic factors in this region have a significant impact on the potency. The furan moiety was successfully replaced with a thiophene or phenyl ring maintaining potency in the low nanomolar range and high selectivity against the other S1P receptor subtypes. By expanding the molecular diversity within the hit-derived class, our SAR study provides innovative small molecule potent and selective S1P ₄ antagonists suitable for in vivo pharmacological validation of the target receptor.

Full text of DOI:10.1016/j.bmcl.2011.06.132

Bioorganic & Medicinal Chemistry Letters 21 (2011) 5470–5474
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
SAR analysis of innovative selective small molecule antagonists
of sphingosine-1-phosphate 4 (S1P4) receptor
Mariangela Urbano a, Miguel Guerrero a, Jian Zhao a, Subash Velaparthi a, Marie-Therese Schaeffer b,d
,
Steven Brown b,d, Hugh Rosen b,c,d, Edward Roberts a,b,
⇑
a Department of Chemistry, The Scripps Research Institute, 10550 N. Torrey Pines Rd, La Jolla, CA 92037, United States
b Department of Chemical Physiology, The Scripps Research Institute, 10550 N. Torrey Pines Rd, La Jolla, CA 92037, United States
c Department of Immunology, The Scripps Research Institute, 10550 N. Torrey Pines Rd, La Jolla, CA 92037, United States
d 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:
Recent evidence suggests an innovative application of chemical modulators targeting the S1P4 receptor as
novel mechanism-based drugs for the treatment of influenza virus infection. Modulation of the S1P4
receptor may also represent an alternative therapeutic approach for clinical conditions where reactive
thrombocytosis is an undesired effect or increased megakaryopoiesis is required. With the exception of
our recent research program disclosure, we are not aware of any selective S1P4 antagonists reported in
the literature to date. Herein, we describe complementary structure–activity relationships (SAR) of the
high-throughput screening (HTS)-derived hit 5-(2,5-dichlorophenyl)-N-(2,6-dimethylphenyl)furan-2-
carboxamide and its 2,5-dimethylphenyl analog. Systematic structural modifications of the furan ring
showed that both steric and electronic factors in this region have a significant impact on the potency.
The furan moiety was successfully replaced with a thiophene or phenyl ring maintaining potency in
the low nanomolar range and high selectivity against the other S1P receptor subtypes. By expanding
the molecular diversity within the hit-derived class, our SAR study provides innovative small molecule
potent and selective S1P4 antagonists suitable for in vivo pharmacological validation of the target
receptor.
Received 20 May 2011
Revised 24 June 2011
Accepted 27 June 2011
Available online 13 July 2011
Keywords:
S1P4 receptor antagonists
S1P1–3,5 receptor family
Megakaryocyte differentiation
Viral infections
Ó 2011 Elsevier Ltd. All rights reserved.
Sphingosine-1-phosphate (S1P) is a pleiotropic lysophospho-
lipid involved in a wide range of cellular functions including prolif-
eration, apoptosis, differentiation and migration.1–6 High levels of
S1P are present in lymph (30–300 nM) and plasma (100 nM–
closely related lysophosphatidic acid (LPA) Edg2,4,7 receptors fam-
ily.11 The coupling of S1P1–5 to different signaling cascades along
with their differential expression pattern explains the broad
biological effects of S1P. S1P1 is expressed ubiquitously, particu-
larly in brain, lung, spleen, heart, liver as well in a variety of im-
mune system cells lineages.7 High affinity S1P1 agonists have
been widely studied and emerged as major immunomodulatory
1 l
M), whereas those found in tissues are significantly lower.7
Although platelets possess the highest sphingosine kinase activity,
accumulating evidence suggests that the plasma levels of S1P are
mainly the result of its release from erythrocytes due to their great
number, capability to store S1P produced from other tissues, and
lack of S1P-degrading enzymes. Furthermore, mast cells have been
described as additional vascular source of S1P upon IgE dependent
activation, while the vascular endothelium could be the main
source of lymph S1P.7,8
S1P can function intracellularly as a second messenger or extra-
cellularly by binding with nanomolar affinities to five S1P G-pro-
tein coupled receptor subtypes named S1P1–5 (formerly Edg1,5,3,6,8
receptors).9,10 S1P1–5 members share approximately 40% sequence
identity, with a conserved glutamic acid residue at the third trans-
menbrane domain responsible for the ligand specificity versus the
⇑
Corresponding author. Tel.: +1 858 784 2396; fax: +1 858 784 2988.
E-mail address: eroberts@scripps.edu (E. Roberts).
Figure 1. Novel S1P4 antagonists based on
scaffold.
a 5-aryl furan-2-arylcarboxamide
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.06.132

M. Urbano et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5470–5474
5471
Table 1
S1P4 antagonist activity of synthesized molecules
Scheme 1. Synthesis of aniline derivative 5. Reagents and conditions: (i) Pd(PPh3)4
(0.1 equiv), 3 (1.5 equiv), 2 M aq Na2C03 (2 equiv), 1,4-dioxane, 80 °C, 12 h, 65%; (ii)
4 (1.2 equiv), NaBH(OAc)3 (1.3 equiv), AcOH (1.5 equiv), DCE, rt overnight, 75%.
R1
IC50 (nM)
a
Compd
R
d
1a
Cl
Me
78
64
1b
9
Me
Cl
Me
Et
NA
14
19
Me
Me
Me
Me
6300
59
Scheme 2. Synthesis of 1,3,4-oxadiazole derivative 9. Reagents and conditions: (i) 7
(1.0 equiv), Et3N (2 equiv), CH2CI2, 0 °C, 1 h; (ii) Burgess reagent (1.5 equiv), THF,
80 °C, 10 h, 40% (over two steps); (iii) 2 N aq NaOH, MeOH, rt, 2 h; (iv) 4 (1.2 equiv),
EDCI (1.2 equiv), HOBt (1.2 equiv), DMF, rt, overnight, 70% (over two steps).
23
Me
Me
233
28
33
Me
Me
Me
Me
211
173
37
47
48
49
Me
Me
Me
Cl
Me
Me
Me
Me
911
75
Scheme 3. Synthesis of N-methyl pyrrole analog 14. Reagents and conditions: (i)
Pd(PPh3)4 (0.05 equiv), Na2C03 (2 equiv), 1,4-dioxane/H2O (10:1), 100 °C. 4 h, 50%;
(ii) (a) Mel (1.5 equiv), NaH (1.5 equiv), DMF, 0 °C to rt, overnight. 65%, (b) LiOH
(1.3 equiv), THF/MeOH/H2O (2:2:1), rt, 2 h; (iii) (a) (COCI)2 (1.3 equiv), DMF, CH2CI2,
0 °C to rt, 8 h, (b) Et3N (2 equiv), 4 (2 equiv), CH2CI2, 0 °C, 2 h. 50% (over three steps).
2400
NA
a
Data are reported as mean for n = 3 determinations. NA = not active at con-
centrations up to 25 M.
l
Scheme 4. Synthesis of thiophene analog 19. Reagents and conditions: (i) (a) concd
H2SO4, MeOH, reflux, 12 h; (b) Pd(OAc)2 (0.05 equiv), Cy3P (0.1 equiv), 16
(1.4 equiv), K3PO4 (3 equiv), toluene/H2O (8:1), 100 °C, 12 h, 50% (over two steps);
(ii) 1 M aq LiOH (1 equiv), THF, 24 h, 25 °C; (iii) (a) (COCI)2 (1.3 equiv), DMF, CH2CI2,
rt, 8 h, (b) Et3N (2 equiv), 4 (1.2 equiv), CH2CI2, rt, overnight, 75% (over three steps).
molecules with therapeutic applications being tested in multiple
sclerosis and allogenic transplantation.12 S1P3 is also widely dis-
tributed with highest levels in the heart where it is co-expressed
with S1P1 and S1P2. S1P2 has been shown to be expressed in rat
brain during embryogenesis as well as in most other developing
tissues. S1P5 is highly present in adult rat brain, while in human
and mouse high expression of the receptor is also found in the
spleen.13
S1P4 has been shown to bind S1P with lower affinity and have a
narrower tissue distribution than the other family members. First
isolated from human and mouse dendritic cells (DCs), S1P4 is
highly expressed in lymphoid and hematopoietic tissue.13 S1P4
S1P-metabolizing enzymes have been recently proposed as inno-
vative potential therapeutics for viral diseases.1,12a,16 Consistent
with these data, local S1P receptor modulation in the lung has been
demonstrated to control immunopathological features of influenza
virus infections by impairing the accumulation of DCs and cytokine
release in the draining lymph nodes without altering the essential
activity of virus-specific T-cells toward virus-infected cells.12a
Therefore, regulation of pulmonary immune response by S1P
receptor modulators may have therapeutic implications for allevi-
ating excessive immune response responsible for exacerbating
have been reported to couple to Gai,
Ga
o, and G
proteins
a
12/13
leading to the stimulation of MAPK/ERK signaling pathways, as
well as PLC and Rho-Cdc42 activation.14,15 Molecules targeting

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M. Urbano et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5470–5474
Scheme 5. Synthesis of 5-aryl furan-3-carboxamide regioisomer 23. Reagents and conditions: (i) concd H2SO4, MeOH, reflux. 12 h, 60%; (ii) 16 (1.5 equiv), Pd(OAc)2
(0.05 equiv), Cy3P (0.1 equiv), K3PO4 (3 equiv), toluene/H2O (8:1), 100 °C, 12 h overnight, 78%; (iii) LiOH (1.2 equiv) THF/MeOH/H2O (2:2:1), rt. 3 h; (iv) (a) (COCI)2. DMF,
CH2CI2 rt, 8 h, (b) 4 (12 equiv), Et3N (1.5 equiv) rt overnight, 88% (over three steps).
airway diseases. Based on the evidence that modulation of S1P1
alone did not inhibit DC-dependent T cell activation, and that the
sphingosine analog used in the experiments did not bind to S1P2,
it was hypothesized that either the single activation of S1P3,
S1P4, S1P5 or the combined activity on S1P1,3,4,5 is responsible for
the functional impairment of DCs.12a Reports showing that, in con-
trast to S1P5 and S1P2, S1P4 is highly expressed in DCs10 confirm
that the S1P4 chemical activation in the airway may be effective
at controlling the immunopathological response to viral infections,
thus offering novel mechanism-based potential therapeutics for
airway viral diseases.
Both in vitro and in vivo experiments have recently provided
Scheme 6. Synthesis of 4-aryl furan-2-carboxamide regioisomer 28. Reagents and
strong evidence that S1P4 is involved in the late stage of megak-
aryocyte differentiation. In S1P4–deficient mice the bone marrow
is characterized by the presence of morphologically aberrant
megakaryocytes, and platelet repopulation of the peripheral blood
after thrombocytopenia is delayed. Indeed, S1P4 has been proposed
as a suitable target either for increasing thrombocyte production in
clinical conditions requiring increased platelets number, or for
inhibiting a potentially detrimental reactive thrombocytosis.8
Despite the emerging therapeutic potential, aspects of the bio-
logical role of S1P4 remain unclear, partly due to the lack of ligands
with high selectivity against the S1P1–3,5 subtypes. Herein we re-
port on the synthesis, biological evaluation and structure–activity
relationships (SAR) of the first class of selective S1P4 antagonists.
Recently, investigations from our laboratories have led to the
discovery of the first class of potent and selective S1P4 antago-
nists.17 Synthesis and SAR analysis of various derivatives based
on a 5-aryl furan-2-arylcarboxamide scaffold were carried out on
regions A and C of the original hit 1a identified through a high-
throughput screening campaign (Fig. 1, Table 1). Similar biological
properties were found for the 2,5-dimethylphenyl analog 1b (
Fig. 1). It was postulated that disubstitution on positions 2 and 6
of the phenyl ring C with small alkyl groups (e.g., methyl, ethyl)
was essential to increase the potency. Remarkably, steric and elec-
tronic effects at position 4 of the phenyl ring C did not affect the
functional activity to any appreciable extent, thus allowing the
installation of solubility enhancing features such as alcohols and
amines. However, safety concerns might arise from the presence
of the furan ring given the number of furan-containing drug candi-
dates demonstrating hepatotoxic and hepatocarcinogenic effects as
a result of furan cytochrome P450-catalyzed oxidative metabolism
and the covalent binding of the electrophilic metabolites to macro-
molecules.18 Thus, our chemistry efforts were successively focused
on the SAR analysis of the central moiety B with the aim to acquire
more insight into the receptor binding mode and identify new
chemotypes to address potential metabolic and toxicity issues.
For investigational purposes we fragmented the moiety B into aryl
ring d and amide group e (Fig. 1) while conserving regions A and C
as in 1a and 1b.
conditions: (i) aq NH4OH, Zn (1 equiv), rt, 3 h, 80%; (ii) concd H2SO4 MeOH, reflux,
12 h, 90%; (iii) 16 (1.4 equiv), Pd(OAc)2 (0.05 equiv), Cy3P (0.1 equiv) K3PO4
(3 equiv), toluene/H2O (8:1), 100 °C, 12 h, 60%; (iv) LiOH (1.3 equiv) THF/MeOH/
H2O (2:2:1), rt, 3 h; (v) (a) (COCI)2 (1.3 equiv), DMF, CH2CI2, rt overnight; (b) Et3N
(2.0 equiv), 4 (1.4 equiv), CH2CI2, rt, 2 h, 85% (over three steps).
Scheme 7. Synthesis of 2,3,5-trisubstituted furan analog 33. Reagents and condi-
tions: (i) NBS (1.1 equiv), MeOH/THF, 0 °C to rt, 2 h, 60%; (ii) Pd(OAc)2 (0.05 equiv),
Cy3P (0.01 equiv), 16 (1.4 equiv), K3PO4 (3 equiv), toluene/H2O (8:1), 100 °C, 12 h,
50%; (iii) LiOH (1.3 equiv), THF/MeOH/H2O (2:2:1), rt, 3 h; (iv) (a) (COCI)2
(1.3 equiv), DMF, CH2CI2. rt, overnight; (b) Et3N (2 equiv), 4 (1.5 equiv), CH2CI2, rt,
overnight, 80% (over three steps).
Scheme 8. Synthesis of 2,4,5-trisubstituted furan analog 37. Reagents and condi-
tions: (i) Pd(OAc)2 (0.05 equiv), Cy3P (0.1 equiv), 16 (1.4 equiv), K3PO4 (3 equiv),
toluene/H2O (8:1), 100 °C, 12 h, 40%; (ii) Pd[(Po-Tol)3]2CI2 (0.05 equiv), Me4Sn
(2 equiv), DMA, 90 °C, 6 h, 65%; (iii) LiOH (1.2 equiv), THF/MeOH/H2O (2:2:1); (iv)
(a) (COCI)2 (1.3 equiv), DMF, CH2CI2, rt, 2 h; (b) Et3N (2 equiv), 4 (2 equiv), rt,
overnight, 78% (over eight steps).
In our previous studies17 it was hypothesized that the hydro-
gen-bond donor capability of the amide group was important for
the functional activity. Herein, the impact of the carbonyl group
as hydrogen-bond acceptor was explored by replacing the amide
functionality by a methyleneaniline as in 5. Suzuki coupling19 be-
tween bromide 2 and boronic acid 3 followed by reductive amina-
tion with 2,6-dimethylaniline 4 furnished the final compound 5
(Scheme 1).

M. Urbano et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5470–5474
5473
Scheme 9. Synthesis of phenyl and pyridine analogs 47–49. Reagents and conditions: (i) 3 or 16 (1.5 equiv), Pd(OAc)2 (0.05 equiv), Cy3P (0.1 equiv), K3PO4 (3 equiv), toluene/
H2O (8:1), 100 °C, 12 h, 40–75%; (ii) LiOH (1.3 equiv), THF/MeOH/H2O (2:2:1), rt, 3 h;(iii) (a) (COCI)2 (1.3 equiv), DMF, CH2CI2. rt, 6 h; (b) Et3N (2 equiv), 4 (2 equiv), CH2CI2, rt,
overnight, 60–85% (over three steps).
Notably, 5 was found to be inactive in the S1P4 antagonist as-
Table 2
say,20 indicating that the amide function is an essential molecular
S1P selectivity counter screen of compounds 19, 47
feature in the binding mode.
a
Compd
IC50 (nM)
A systematic SAR analysis of the furan ring d was performed by
the synthesis of molecules containing variously substituted aro-
matic rings as outlined in Schemes 2–9. Initially, the furan ring
was replaced by 1,3,4-oxadiazole as a substructure with poten-
tially improved toxicity profile18 (9, Scheme 2). Condensation of
hydrazide 6 with ethyl oxalyl chloride 7 followed by cyclization
using Burgess reagent afforded oxadiazole ethyl ester 8. Subse-
quent ester hydrolysis of 8 followed by amidation with 4 furnished
the final compound 9.
S1P4
S1P1
S1P2
S1P3
S1P5
19 (CYM50333)
47 (CYM50367)
59
75
NA
1100 (40%)b
25%c
35%c
40%c
30%c
40%c
30%c
a
b
c
Data are reported as mean for n = 3 determinations.
Percentage of inhibition.
Percentage of inhibition at 25
lM.
lM. NA = not active at concentrations up to
25
Aware of literature evidence of pyrrole and thiophene toxicity
which mostly arises from hydroxylation at C2 and C5 positions,18
N-methyl pyrrole 14 and thiophene 19 analogs were prepared
(Schemes 3 and 4) primarily to elucidate the electronic effects
of the oxygen atom on the functional activity. Suzuki coupling
of phenyl iodide 10 with N-Boc-pyrrole boronic acid 11 fur-
nished the N-deprotected pyrrole 12 as the major product.19
Alkylation of 12 with methyl iodide under strong basic condi-
tions followed by hydrolysis of the methyl ester afforded carbox-
ylic acid 13. Coupling the acyl chloride of 13 with 4 furnished
compound 14.
The synthesis of intermediate 17 commenced with the esterifi-
cation of carboxylic acid 15 followed by Suzuki coupling with 2,5-
dimethylphenyl boronic acid 16. Ester hydrolysis of 17, formation
of the acyl chloride and coupling with 4 furnished thiophene deriv-
ative 19 (CYM50333) (Scheme 4).
preparation and amide coupling with 4 yielded the desired furan
33 (Scheme 7).
Synthesis of the regioisomer 37 involved a selective Suzuki cou-
pling between dibromo furan 34 and boronic acid 16 followed by
methylation through Stille coupling,21 hydrolysis of ester 36, and
coupling of the acyl chloride derivative with aniline 4 (Scheme 8).
Finally, new chemical space was explored by replacing the furan
ring with phenyl and pyridine as potential 6-membered ring bio-
isosteres. The synthesis is depicted in Scheme 9. Suzuki coupling
of phenylboronic acids (3 or 16) with the appropriate bromo phe-
nyl (38 or 39) or pyridine 40 derivatives afforded the correspond-
ing biaryl esters 41–43. Successively, ester hydrolysis, acyl chloride
formation and amide coupling with aniline 4 furnished the desired
products 47 (CYM50367), 48, 49.
The S1P4 functional antagonist activity of the aforementioned
compounds is listed in Table 1. Surprisingly, the 2,5-disubstituted
oxadiazole 9 was inactive. Similarly, the N-methyl-pyrrole 14
was 98-fold less potent than 1b. Conversely, the thiophene was
found to be a suitable bioisoster of the furan ring, with the 2,5-
disubstituted thiophene analog 19 displaying similar potency to
the hit and 1b. Furthermore, both regioisomers 23 and 28 were
3-fold less potent than 1b. Trisubstituted furan regioisomers 33
and 37 were respectively 3- and 14-fold less potent than 1b; the
loss of potency was attributed to unfavorable steric interactions
of the methyl group with the receptor or resulting conformational
modification of the molecule. Particularly, it was hypothesized that
the methyl in 37 forces the biaryl C–C bond angle into a less active
anti-coplanar conformation. Geometrically similar molecules 9, 14,
19, 23, and 28, having the amide group e and the ring A arranged in
a 1,3 substitution pattern on the heteroaromatic central system d,
displayed very different potencies suggesting that electronic ef-
fects are essential for the S1P4 antagonist activity. Interestingly,
the 1,3-disubstituted phenyl derivative 47 showed similar potency
to 19, 1a, and 1b, whereas the pyridine analog 49 was inactive. The
lack of activity of oxadiazole 9 and pyridine 49 suggested that
As regioisomers of 1b, 5-aryl furan-3-carboxamide 23 and 4-
aryl furan-2-carboxamide 28 were synthesized in order to eluci-
date the electronic requirements of the 5-membered ring d, while
keeping the pendant regions A and C geometrically similar to 1a
and 1b. The synthesis of 23 is depicted in Scheme 5. Esterification
of 20 followed by Suzuki coupling with boronic acid 16 afforded
22. Subsequent ester hydrolysis, formation of acyl chloride and
coupling with 4 provided the desired compound 23.
The 4-arylfuran-2-carboxamide 28 was synthesized as shown in
Scheme 6. Selective debromination of 24 followed by esterification
and Suzuki coupling afforded the 4-arylfuran-2-methyl ester 27.
Synthesis of 28 was finally achieved by ester hydrolysis of 27, for-
mation of acyl chloride and subsequent coupling with aniline 4.
Successively, a methyl group was inserted either on position
3 or 4 of the furan ring in order to evaluate the steric require-
ments of region B as well as conformational restrictions on the
pendant regions A and C. Trisubstituted furans 33 and 37 were
synthesized as illustrated in Schemes 7 and 8. Bromination of
furan 29, followed by Suzuki coupling with boronic acid 16
afforded ester 31. Ester hydrolysis followed by acyl chloride

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M. Urbano et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5470–5474
heterocycles with basic lone pares at the ring d are detrimental to
the potency. In agreement with the SAR trend within the furan
derivatives, the methylated phenyl analog 48 was 32-fold less po-
tent than the corresponding homolog 47, supporting the hypothe-
sis that a methyl group in the ring d ortho to the biaryl C–C bond is
detrimental to the activity.
The most active compounds 19 and 47 were selected for func-
tional assays at S1P1–3,5 subtypes (Table 2). Notably, both com-
pounds displayed an exquisite selectivity for the S1P4 receptor
versus the other receptor family subtypes.
References and notes
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new chemotypes which may address potential metabolic/toxicity
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20. 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 beta-
arrestin/TEV protease fusion protein and a beta-lactamase (BLA) reporter gene
under the control of a UAS response element. BLA expression is monitored by
Acknowledgments
This work was supported by the National Institute of Health
Molecular Library Probe Production Center grant U54 MH084512
(ER, HR), and by AI074564 (Michael Oldstone, PI). We thank Mark
Southern for data management with Pub Chem.
measuring fluorescence resonance energy transfer (FRET) of
fluorogenic, cell-permeable BLA substrate.
a cleavable,
21. Bach, T.; Krüger, L. Eur. J. Org. Chem. 1999, 1999, 2045.