Discovery, design and synthesis of novel potent and selective sphingosine-1-phosphate 4 receptor (S1P4-R) agonists

Article abstract of DOI:10.1016/j.bmcl.2011.10.096

High affinity and selective small molecule agonists of the S1P₄ receptor (S1P₄-R) may have significant therapeutic utility in diverse disease areas including autoimmune diseases, viral infections and thrombocytopenia. A high-throughput screening (HTS) of the Molecular Libraries-Small Molecule Repository library identified 3-(2-(2,4- dichlorophenoxy)ethoxy)-6-methyl-2-nitropyridine as a moderately potent and selective S1P₄-R hit agonist. Design, synthesis and systematic structure-activity relationships study of the HTS-derived hit led to the development of novel potent S1P₄-R agonists exquisitely selective over the remaining S1P_1-3,5-Rs family members. Remarkably, the molecules herein reported provide novel pharmacological tools to decipher the biological function and assess the therapeutic utility of the S1P₄-R.

Full text of DOI:10.1016/j.bmcl.2011.10.096

Bioorganic & Medicinal Chemistry Letters 22 (2012) 537–542
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery, design and synthesis of novel potent and selective
sphingosine-1-phosphate 4 receptor (S1P4-R) agonists
Miguel Guerrero a, Mariangela Urbano a, Jian Zhao a, Melissa Crisp b, Peter Chase b, Peter Hodder b,c
,
Marie-Therese Schaeffer d,f, Steven Brown d,f, Hugh Rosen d,e,f, Edward Roberts a,d,
⇑
a Department of Chemistry, The Scripps Research Institute, 10550 N. Torrey Pines Rd., La Jolla, CA 92037, United States
b Scripps Research Institute Molecular Screening Center, Lead Identification Division, Translational Research Institute, 130 Scripps Way, Jupiter, FL 33458, United States
c Department of Molecular Therapeutics, Scripps Florida, 130 Scripps Way, Jupiter, FL 33458, United States
d Department of Chemical Physiology, The Scripps Research Institute, 10550 N. Torrey Pines Rd., La Jolla, CA 92037, United States
e Department of Immunology, The Scripps Research Institute, 10550 N. Torrey Pines Rd., La Jolla, CA 92037, United States
f 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:
High affinity and selective small molecule agonists of the S1P4 receptor (S1P4-R) may have significant
therapeutic utility in diverse disease areas including autoimmune diseases, viral infections and thrombo-
cytopenia. A high-throughput screening (HTS) of the Molecular Libraries-Small Molecule Repository
library identified 3-(2-(2,4-dichlorophenoxy)ethoxy)-6-methyl-2-nitropyridine as a moderately potent
and selective S1P4-R hit agonist. Design, synthesis and systematic structure–activity relationships study
of the HTS-derived hit led to the development of novel potent S1P4-R agonists exquisitely selective over
the remaining S1P1–3,5-Rs family members. Remarkably, the molecules herein reported provide novel
pharmacological tools to decipher the biological function and assess the therapeutic utility of the
S1P4-R.
Received 21 September 2011
Revised 24 October 2011
Accepted 26 October 2011
Available online 4 November 2011
Keywords:
S1P4 receptor
Selective small molecule S1P4-R agonists
Autoimmune diseases
Viral infections
Ó 2011 Elsevier Ltd. All rights reserved.
Thrombocytopenia
Sphingosine-1-phosphate (S1P) is
a
sphingolipid mediator
S1P1–3-Rs are ubiquitously expressed in almost all organs in
mice and humans whereas S1P4-R and S1P5-R expression is re-
stricted to specific organs and cell types. S1P4-R is predominantly
expressed in lymphoid and hematopoietic cells.9 S1P4-R couples
formed by the phosphorylation of sphingosine (SPH) and involved
in multiple physiological and pathological processes. Among them,
lymphocyte trafficking, angiogenesis, tumorigenesis as well as
vascular development and permeability have been extensively
explored.1–4 The involvement of S1P in these processes results from
its ability to modulate important cellular events such as cytoskele-
tal changes, chemotaxis, survival, and proliferation.5–7 It has be-
come clear that the role of S1P in immunological response is not
restricted to the regulation of lymphocyte trafficking but extends
to the control of immune cell function.8 The generation of S1P is
mediated by two cytosolic sphingosine kinase isoforms (SPK1 and
SPK2) and occurs preferentially in the plasma membrane.6,8 The site
of action of S1P is not merely intracellular since it is exported out of
the cell and binds to five G-protein-coupled receptors named
S1P1–5-Rs, in a paracrine or autocrine manner.4,6 In addition to its
effects on S1P1–5-Rs, S1P can also affect cell function by either bind-
ing or modifying putative intracellular targets or by affecting the
relative levels of other lipid products, particularly SPH and cera-
mide whose biological effects oppose those of S1P.6,7
to Gai,
G
ao and G
proteins leading to the stimulation of
a
12/13
MAPK/ERK signaling pathways, as well as PLC and Rho-Cdc42
activation.10,11
Both, S1P1-R and S1P4-R are the most widely expressed S1P-Rs
on lymphocytes and dendritic cells (DCs).12,13 In contrast to S1P1-R,
the function of S1P4-R in fundamental immunological processes
has been poorly characterized. However, the contribution of
S1P4-R to the immune response is becoming increasingly evident.
S1P induces migratory response of murine T-cell lines expressing
both S1P1-R and S1P4-R mRNA. In D10.G4.1 and EL-4.IL-2 murine
cells S1P-induced migration was significantly inhibited by treat-
ment with (S)-FTY720-phosphate, a potent agonist at S1P1-R and
S1P4-R. In murine CHO cells co-expressing S1P4-R and S1P1-R on
the cell surface S1P-induced T-cell migration involved the activa-
tion of Rho family small GTPase, Cdc42 and Rac. These results have
suggested that the association of S1P4-R and S1P1-R may play an
important role in the migratory and recirculation response of
T-cells toward S1P.14 Intratracheal delivery of synthetic sphingo-
sine analogs with mixed activity over S1P1,3-5-Rs efficiently inhib-
ited the T-cell response to influenza virus infection by impeding
⇑
Corresponding author. Tel.: +1 858 784 7770; fax: +1 858 784 7745.
E-mail address: eroberts@scripps.edu (E. Roberts).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.10.096

538
M. Guerrero et al. / Bioorg. Med. Chem. Lett. 22 (2012) 537–542
Table 1
S1P4-R agonist activity of 8a–8s
a
Compd
Ar
EC50 (nM)
8a
8b
8c
8d
8e
8f
I
II
III
IV
V
VI
2-Chlorophenyl
4-Chlorophenyl
2,5-Dichlorophenyl
2,3-Dichlorophenyl
3,4-Dichlorophenyl
Phenyl
NA
NA
NA
NA
NA
NA
8g
8h
8i
8j
8k
8l
8m
8n
8o
8p
8q
8r
8s
VII
VIII
IX
X
XI
XII
XIII
XIV
XV
XVI
XVII
XVIII
XIX
2-Bromo-4-chlorophenyl
4-Bromo-2-chlorophenyl
2-Chloro-4-methoxy phenyl
4-Chloro-2-methoxyphenyl
2,4-Dimethylphenyl
2-Chloro-4-phenylphenyl
4-Chloro-2-fluorophenyl
2-Chloro-4-fluorophenyl
2,4-Difluorophenyl
2-Fluoro-4-nitrophenyl
5-Chloro-3-nitropyridin-2-yl
4-Bromo-2-(trifluoromethyl)phenyl
2,4,6-Trichlorophenyl
232
220
3900
5400
7200
NA
3100
2600
40% 50
2800
3500
601
lM
Figure 1. Novel and selective S1P4-R agonists.
570
the accumulation of DCs in draining lymph nodes. The inhibitory
effects were not observed upon specific chemical activation of
S1P1-R, and persisted in S1P3-R null mice. Based on these findings
and on the observation that S1P5-R expression in DCs is very low
whereas S1P4-R is highly expressed, it has been hypothesized that
the S1P4-R modulation in the lung may be effective at controlling
the immunopathological response to viral infections.15,16
a
Data are reported as mean of n = 3 determinations. NA = not active at concen-
trations up to 25 M.
l
showed confirmed EC50’s of 162 nM at S1P4-R and no agonistic activ-
ity at S1P1–3,5-Rs at concentrations up to 25 M (Fig. 1).
l
Our SAR studies commenced varying the 2,4-dichlorophenyl
coil of the hit as outlined in Scheme 1. Alkylation of 3-hydroxypyr-
idine 3 with 1,2-dibromoethane 4 led to the key intermediate 5,
which was subsequently reacted with various hydroxyaryl deriva-
tives 7 to furnish compounds 8a–8s. The biological results of the
entitle molecules are listed in Table 1.23
S1P4-R signaling has been proposed to negatively affect T-cell
proliferation and modify the cytokine secretion profile of T-lym-
phocytes.17 However, these observations were derived from
S1P4-R overexpressing cell lines and need further confirmation. Re-
cently, S1P4-R has been implicated in the regulation of DC function
and TH17 T-cell differentiation in a murine model.18 Interestingly,
S1P4-R-mediated S1P signaling also modifies the course of various
immune diseases in a murine model. Hence, it has been hypothe-
sized that S1P4-R may constitute an interesting target to influence
the course of various autoimmune diseases.
In vitro and in vivo experiments have indicated an additional
potential therapeutic application of S1P4-R molecule modulators
in the terminal differentiation of megakaryocytes. Namely, the
application of S1P4-R antagonists might be exploited for inhibiting
potentially detrimental reactive thrombocytosis, whereas S1P4-R
agonists represent a potential therapeutic approach for stimulating
platelet repopulation after thrombocytopenia.19
To date, despite the S1P4-R therapeutic potential, the in vivo
function of the target receptor remains largely unknown due to
the paucity of selective small molecules S1P4-R modulators.
Recently, our research group identified the 3-methyl-2-((2-
methoxyethyl)imino)thiazolidin-4-one 1 as a novel and selective
S1P4-R agonist (Fig. 1).20 Herein we report on the discovery and
structure–activity relationships (SAR) of novel potent and selective
S1P4-R agonists based on the hit 3-(2-(2,4-dichlorophenoxy)eth-
oxy)-6-methyl-2-nitropyridine 2. A high-throughput screening
(HTS) of the Molecular Libraries-Small Molecule Repository
(MLSMR) library identified 2 as a novel, moderately potent and
selective S1P4-R agonist. The structural integrity of the hit was cor-
roborated by the resynthesis (Scheme 1) of the title compound that
Removal of the chlorine from positions 2 and/or 4 of the aro-
matic ring (8a, 8b, 8f) led to complete loss of activity. Dichloro-
regioisomers 8c, 8d, 8e and 2-chloro-4-phenyl analog 8l were also
inactive. Drastic loss of potency was observed when the chlorines
were substituted with fluorine at one or both positions (8m, 8n,
8o) as well as for the 2- or 4-methoxy derivatives (8j, 8i) and for
the 2,4-dimethyl analog (8k). Interestingly, the 4-nitro derivative
8p showed week but similar activity compared to the 4-chloro ana-
log 8m. The pyridinyl derivative 8q resulted in a 22-fold decrease in
potency compared to the hit. Conversely, the potency remained
similar by replacing the chlorine at position 2 or 4 with bromine
(8g, 8h). Interestingly, the 4-bromo-2-trifluoromethyl analog 8r
was only threefold less potent than the corresponding 2-chloro
analog 8h. The symmetrically substituted 2,4,6-trichloro-phenyl
analog 8s was also threefold less potent than the hit suggesting a
steric clash of the trisubstituted phenyl ring within the binding
pocket.
Next, we synthesized compounds 13a–13g (Schemes 2–6) in or-
der to explore the ethylenedioxy spacer while maintaining the 2,4-
dichlorophenyl coil and the 6-methyl-2-nitropyridine polar head.
Intermediate 9 was synthesized by alkylating 2,4-dichlorophe-
nol 6 with 1,3-dibromopropane under basic conditions. Alcohol
10 was reacted with phosphorus tribromide (PBr3) at room tem-
perature to furnish the corresponding bromide 11. Compounds
Scheme 1. Synthesis of 8a–8s. Reagents and conditions: (i) 3 (1 equiv), 4 (4 equiv), K2CO3 (2 equiv), DMSO, 40 °C, 4 h, 65%; (ii) 5 (1 equiv), 6 or 7 (2 equiv), K2CO3 (2 equiv),
DMSO, 40 °C, 4 h, 50–98%

M. Guerrero et al. / Bioorg. Med. Chem. Lett. 22 (2012) 537–542
539
Scheme 2. Synthesis of 13a–13c. Reagents and conditions: (i) 6 (1 equiv), 1,3-dibromopropane (4 equiv), K2CO3 (2 equiv), DMSO, 40 °C, 4 h, 65%; (ii) 10 (1 equiv), PBr3
(0.33 equiv), CH2Cl2, 0 °C to rt, 3 h, 85%; (iii) 9 or 11 or 12 (1 equiv), 3 (1 equiv), K2CO3 (2 equiv), 40 °C, 4 h, 75–90%.
Scheme 3. Synthesis of 13d. Reagents and conditions: (i) 14 (1 equiv), SOCl2, reflux, 2 h; (ii) 3 (1.2 equiv), DIPEA (1.2 equiv) CH2Cl2, 0 °C to rt, overnight, 80% (over two steps).
Scheme 4. Synthesis of 13e. Reagents and conditions: (i) 6 (1 equiv), 15 (2.1 equiv), K2CO3 (4 equiv), EtOH, 80 °C, overnight; (ii) MeSO2CL (2 equiv.), pyridine, 0 °C, 6 h, 70%
(over 2 steps); (iii) (a) 3 (1 equiv), KOH, H2O/EtOH, 80 °C 30 min; (b) 16 (1 equiv), DMF, 120 °C, overnight, 60%.
Scheme 5. Synthesis of 13f. Reagents and conditions: (i) 17 (1 equiv), 18 (1 equiv), DMF, 100 °C, 12 h, 65%.
Scheme 6. Synthesis of 13g. Reagents and conditions: (i) 19 (1 equiv), 14 (1.5 equiv), PPA, 130 °C, 2 h, 45%.
13a, 13b and 13c were obtained by the alkylation of 3 with 9, 11
and 12, respectively under basic conditions (Scheme 2).
Commercially available carboxylic acid 14 was converted into
the corresponding acyl chloride using thionyl chloride (SOCl2) fol-
lowed by condensation with 3 to furnish the desired product 13d
(Scheme 3).
derivative 16. The final cyclic compound 13e was obtained by con-
densation of 3 with 16 (Scheme 4).
The aza-analog 13f was obtained via aromatic nucleophilic sub-
stitution of commercially available triflate 17 with amine 18
(Scheme 5).
The oxazolo[4,5-b]pyridine 13g was synthesized by condensing
hydroxypyridine 19 with carboxylic acid 14 using polyphosphoric
acid (PPA) (Scheme 6).
The cyclopentene oxide 15 was reacted with 6 followed by
mesylation of the alcohol intermediate to furnish the mesylated

540
M. Guerrero et al. / Bioorg. Med. Chem. Lett. 22 (2012) 537–542
Table 2
spacer and the head led to oxazolo[4,5-b]pyridine 13g, which
was found inactive in concentrations up to 25 M. Cumulatively,
S1P4-R agonist activity of 13a–13f
l
these data suggest that the ethylenedioxy spacer is an essential
structural feature for the binding activity and is highly restricted
in terms of length, bulkiness and conformation.
Cl
L
N
O2N
Successively, we focused on the study of the polar head while
retaining the 2,4-dichlorophenyl coil and the ethylenedioxy chain.
The synthesis of the target molecules 22a–22aa is represented in
Scheme 7. The alkyation of 6 with 4 yielded the key bromide inter-
mediate 20. A diverse set of hydroxyaryl analogs 21I–21XVIII was
reacted with 20 to furnish the desired products 22a–22e, 22h–22s,
22v. Hydrolysis of methylester 22e under standard conditions
yielded the carboxylic acid 22g, which was converted into 22f
via standard amide coupling conditions. Methyl pyridine analog
22p was reacted with meta-chloroperbenzoic acid (mCPBA) to
form the corresponding N-oxide which was converted into the
alcohol derivative 22t using the Boekelheiden reaction.21 Oxidation
of 22t using manganese dioxide (MnO2) under standard conditions
gave aldehyde 22u. Oxidation of alcohol 22v using previously
described conditions furnished the aldehyde 22w. Analogs 22y
and 22z were then synthesized employing the Wittig reaction be-
tween aldehyde 22w and the corresponding phosphonium ylide.
The 6-methoxymethyl analog 22x was synthesized by metylation
of the alcohol 22v with methyl iodide. The fluoro derivative 22aa
was obtained from alcohol 22v via displacement of the mesylate
using tetrabutylammonium fluoride (TBAF).
Cl
13a-f
a
Compd
L
EC50 (nM)
O
O
13a
NA
920
NA
13b
13c
O
13d
NA
13e
13f
NA
NA
a
Data are reported as mean of n = 3 determinations. NA = not active at concen-
trations up to 25 M.
l
The biological results of 22a–22aa are listed in Table 3.
Deletion of the 6-methyl from the hit led to 18-fold loss of activ-
ity (22a), while removal of the 2-nitro led to the inactive compound
22b. Interestingly, the phenyl analog 22c showed a eightfold loss in
potency and a reduced efficacy of 40% suggesting that the basic
pyridinyl nitrogen may be involved in a hydrogen bond interaction.
Replacing the 6-methyl with chlorine (22d) led to only twofold dec-
rement in potency. Aware that most nitro-containing compounds
can cause methaemoglobinemia and are potentially mutagenic,
our synthetic efforts focused on the replacement of the nitro group
by a different molecular feature.22 However, the installation of clas-
sical nitro-bioisosteres such as methyl ester (22e), amide (22f) and
The biological results of 13a–13f are listed in Table 2.
Elongating the alkyl chain (13a) as well as introducing a car-
bonyl group (13d) resulted in complete loss of activity. Further-
more, the constrained 5-membered ring analog 13e was inactive.
Interestingly, the methylene analogs 13c and 13b were, respec-
tively inactive and five to sixfold less active than the hit suggesting
that the replaced oxygen might be involved in a hydrogen bond
interaction. Surprisingly, the aza-analog 13f was inactive suggest-
ing that the hydrogen bond acceptor capability in this portion of
the molecule is an essential binding requirement. Merging the
Scheme 7. Synthesis of 22a–22aa. Reagents and conditions: (i) 6 (1 equiv), 4 (4 equiv), K2CO3 (2 equiv), DMSO, 40 °C, 6 h, 75%; (ii) 20 (1 equiv), 21I–21XVIII (1.1 equiv),
K2CO3 (1.5 equiv), DMSO, 40 °C, 6 h, 70–95%; (iii) 22v (1 equiv), MnO2 (5 equiv), CHCl3, rt, 3 h, 67%; (iv) (a) CH3PPh3+BrÀ (2 equiv), BuLi (2 equiv), THF, 0 °C, 30 min; (b) 22w
(1 equiv), 0 °C to rt, 2 h, 60%; (v) (a) (CH3)2CHPPh3+BrÀ (2 equiv), BuLi (2 equiv), THF, 0 °C, 30 min; (b) 22w (1 equiv), 0 °C to rt, 2 h, 40%; (vi) 22v (1 equiv), NaH (15 equiv). Mel
(5 equiv), DMF, 0 °C to rt, overnight, 72%; (vii) (a) 22v (1 equiv), MsCl (1.1 equiv), Et3N (3 equiv), 0 °C to rt, overnight; (b) TBAF (1M THF) (1.5 equiv), acetonitrile, 50 °C, 12 h,
60% (over two steps); (viii) 22e (1 equiv), LiOH (1.1 equiv), MeOH/THF/HgO (3:1:1), rt, 2 h, 70%; (ix) 22g (1 equiv). NHMe2ÁHCl (1.2 equiv), DIPEA (1.2 equiv), EDCl (1.3 equiv),
HOBt (1.3 equiv), CH2Cl2, rt, 3 h. 88%; (x) (a) 22p (1 equiv), MCPBA (12 equiv), CH2Cl2, rt. 6 h; (b) (CF3CO)2O (1 equiv), DMF, 0 °C to rt, 24 h; (c) Na2CO3, rt, 1 h, 39% (over three
steps); (xi) 22t (1 equiv), MnO2 (5 equiv), CHCl3, rt, 3 h, 65%.

M. Guerrero et al. / Bioorg. Med. Chem. Lett. 22 (2012) 537–542
541
Table 3
Table 3 (continued)
S1P4-R agonist activity of 22a–22aa
a
Compd
Ar2
EC50 (nM)
a
Compd
Ar2
EC50 (nM)
22r
XVI
6000b (70%)
22a
I
2980
NA
Br
N
N
Ph
O2N
N
22s
XVII
NA
22b
22c
22d
22e
22f
22g
22h
22i
II
Br
I
N
Me
22t
287
2100
1500
1800
NA
III
IV
V
1300 (40%)b
OH
N
O2N
O2N
22u
22v
22w
22x
22y
22z
22aa
304
I
N
N
N
N
N
N
N
CHO
N
Cl
XVIII
4800
NA
OH
Cl
Cl
Cl
Cl
Cl
Cl
MeO2C
N
CHO
Me2NOC
N
OMe
NA
HO2C
HO
N
658
NA
VI
NA
N
VII
NA
H2N
N
45
2600b (65%)
F
22j
VIII
IX
N
a
Data are reported as mean of n = 3 determinations.
Percentage of response at given concentration. NA = not active at concentra-
tions up to 25 lM.
b
22k
221
22m
22n
22o
22p
22q
7300
52
Et
Br
N
Table 4
X
S1P1–3,5-Rs selectivity counter screen
N
N
a
Compd
EC50 (nM)
S1P4-R
S1P1-R
S1P2-R
S1P3-R
S1P5-R
XI
1300
46
2
8g
8h
8r
162
232
220
601
570
304
52
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
Br
Br
XII
XIII
XIV
XV
8s
N
Br
22d
22l
22n
22p
22t
22aa
46
54
208
54
Br
I
N
F
287
45
a
Data are reported as mean of n = 3 determinations. NA = not active at concen-
trations up to 25 M.
l
N
NA
carboxylic acid (22g) led to a great or complete loss of activity. Next,
analogs containing hydrogen-bond donor groups were synthesized.
Ph
N

542
M. Guerrero et al. / Bioorg. Med. Chem. Lett. 22 (2012) 537–542
Unfortunately, hydroxymethyl (22h) and amino (22i) analogs were
found inactive. These data suggest that the nitro group may not be
involved in a hydrogen bond interaction but rather may be direct-
ing the ether spacer into the active conformation. In order to inves-
tigate this hypothesis, the methyl (22j), ethyl (22k) and phenyl
(22q) analogs were synthesized. These analogs were found less
active than the hit showing a decrement in potency correlated with
the size of the substituent. Finally, we explored the possibility of
installing halogen atoms to substitute the nitro group. Remarkably,
the 2-bromo 22l (CYM50199) and the 2,6-dibromo 22n
(CYM50179) analogs were found to be threefold more potent than
the hit. Restricting the rotation of the spacer by attaching a methyl
group at position 4 of 22l led to the inactive compound 22s. Re-
moval of the bromine from position 6 (22m) led to significant loss
of potency as previously observed with the nitro analog 22a. The 2-
bromo-6-fluoro analog 22o was only fourfold less potent than 22l,
but sixfold more potent than 22m, suggesting that substituents at
position 6 modulate the potency. The 2-iodo-6-methyl analog 22p
(CYM50138) was equipotent to the 2-bromo analog 22l. Based on
the acquired information, the SAR at position 6 was further investi-
gated. Substituting the methyl group with a phenyl ring (22r) led to
a substantial loss of potency. Interestingly, hydroxymethyl 22t was
only fourfold less potent whereas aldehyde 22u was 38-fold less ac-
tive than 22p, suggesting that a hydrogen-bond donor rather than
an acceptor may be better tolerated in this region of the molecule.
Interestingly, in the presence of chlorine at position 2 comparable
activities were found for both the alcohol 22v and aldehyde 22w
derivatives, which were respectively fivefold less and slightly more
active than the 2-iodine counterparts (22t, 22u). In line with the
hypothesis that a hydrogen-bond acceptor is not well accepted in
this region, the methyl ether 22x was inactive. Successively, we ex-
plored the influence of alkenyl substituents in this region. The eth-
ylene analog 22y showed modest potency, while its dimethylated
analog 22z was inactive suggesting that bulky substituents are det-
rimental for the potency. Taking into account that halogens directly
attached at position 6 of the pyridine ring (22d, 22n, 22o) were well
tolerated probably due to the formation of a dipole, we prepared the
fluoromethyl derivative 22aa. Remarkably, 22aa (CYM50260) was
found 3.5-fold more potent than the hit compound and was equipo-
tent to the dibromine analog 22n. These data suggest that substit-
uents at positions 2 and 6 are essential to improve the potency.
The position 2 tolerated nitro and halogen groups while small lipo-
philic or dipole-inducing groups were the most suitable substitu-
ents at position 6.
and exquisitely selective S1P4-R agonists 22l, 22n, 22p, 22aa
(CYM50199, CYM50179, CYM50138, CYM50260). Noteworthy, the
studies herein reported provide novel pharmacological tools to
decipher the biological function and assess the therapeutic utility
of the S1P4–R. Further studies of our research program will be com-
municated in due curse.
Acknowledgments
This work was supported by the National Institute of Health
Molecular Library Probe Production Center Grant U54 MH084512
(E.R., H.R.) and AI074564 (M.O., H.R.). We thank Mark Southern for
data management with Pub Chem, Pierre Baillargeon and Lina DeL-
uca (Lead Identification Division, Scripps Florida) for compound
management.
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23. The biological assays were performed using Tango S1P4-BLA U2OS cells
A set of the most active compounds was selected for selectivity
assays against S1P1–3,5-R subtypes (Table 4). Remarkably, all tested
compounds displayed exquisite selectivity against the other S1P-R
family members.
In summary, we have reported the discovery, design and
synthesis of novel small molecule S1P4-R agonists based on a
3-(2-(phenoxy)ethoxy)-6-alkyl-2-nitropyridine chemotype dis-
tinct from previously reported S1P4-R modulators. Systematic SAR
analysis of the original MLSMR hit 2, a selective but moderately
potent S1P4-R agonist, led to the development of novel potent
containing the human Endothelial Differentiation Gene
6 (EDG6; S1P4-R)
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. Stimulation
of the S1P4-R by agonist causes migration of the fusion protein to the GPCR,
and through proteolysis liberates GAL4-VP16 from the receptor. The liberated
VP16-GAL4 migrates to the nucleus, where it induces transcription of the BLA
gene. BLA expression is monitored by measuring fluorescence resonance
energy transfer (FRET) of
a cleavable, fluorogenic, cell-permeable BLA
substrate. As designed, test compounds that act as S1P4-R agonists will
activate S1P4-R and increase well FRET. Compounds were tested in triplicate at
a final nominal concentration of 25 lM.