Novel Potent Selective Orally Active S1P5 Receptor Antagonists

Article abstract of DOI:10.1021/acsmedchemlett.0c00631

S1P5 is one of the five sphingosine-1-phosphate (S1P) receptors which play important roles in immune and CNS cell homeostasis, growth, and differentiation. Little is known about the effect of modulation of S1P5 due to the lack of S1P5 specific modulators with suitable druglike properties. Here we describe the discovery and optimization of a novel series of potent selective S1P5 antagonists and the identification of an orally active brain-penetrant tool compound 15.

Full text of DOI:10.1021/acsmedchemlett.0c00631

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Novel Potent Selective Orally Active S1P5 Receptor Antagonists
Bin Ma,* Kevin M. Guckian, Xiao-gao Liu, Chunhua Yang, Bing Li, Robert Scannevin,
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Michael Mingueneau, Annabelle Drouillard, and Thierry Walzer
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ABSTRACT: S1P5 is one of the five sphingosine-1-phosphate
(S1P) receptors which play important roles in immune and CNS
cell homeostasis, growth, and differentiation. Little is known about
the effect of modulation of S1P5 due to the lack of S1P5 specific
modulators with suitable druglike properties. Here we describe the
discovery and optimization of a novel series of potent selective
S1P5 antagonists and the identification of an orally active brain-
penetrant tool compound 15.
KEYWORDS: S1P5, GPCR, antagonist, selectivity, brain penetration
oligodendrocytes relative to other S1PRs and that it plays an
phingosine-1-phosphate (S1P, Figure 1) signals through cell
Ssurface GPCR receptors S1PRs (S1P1−5) and plays important role in cell survival of mature oligodendrocytes.9,10 It
is also reported that S1P5 activation on brain endothelial cells
enhances barrier integrity and reduces transendothelial
migration of monocytes in vitro.11 Nevertheless, little more is
known about the effect of modulation of S1P5 due to the lack of
suitable tool compounds.
There are several nonspecific S1P1 agonists that also bind to
S1P5 including FTY720-P (Figure 1), BAF312, ONO-4641,12
and AbbVie’s A-971432.13 Novartis reported a selective S1P5
agonist,14 but it was proved to have insufficient pharmacody-
namic properties for in vivo target validation.13,15 So far, to the
best of our knowledge, there are no S1P5 specific antagonists
reported.
Here we report the discovery and optimization of a novel
Figure 1. S1P5 agonists and antagonist.
series of potent selective S1P5 antagonists without S1P1−4
activity, together with the identification of a brain-penetrable
orally active tool compound 15. In a recent publication, this
important roles in cell survival, trafficking, growth, and
differentiation in many cell types, including cells of the immune,
compound was shown to demonstrate unique activity on
blocking natural killer (NK) cell migration in vitro.16
cardiovascular, and central nervous (CNS) systems.1−4 S1P1
Screening an internal lipid mimetic library by using a calcium
mobilization S1P1−5 assay,17 we found a 4-methylnaphthalen-
internalization has been well documented in the trafficking of B
and T cells by preventing both from leaving the lymph nodes and
1-yl)methyl amino acid analogue 1 with moderate S1P5
entering the lymphatic circulation.5 The U.S. Food and Drug
antagonist activity (406 nM) without S1P1 activity (>5000
nM). A modification to the tail portion led to compound 3 with
Administration approved drug FTY720 induces S1P1 internal-
ization, and S1P signaling has become an interesting therapeutic
target.6 However, the role and contribution of S1P5 to
Received: November 29, 2020
Accepted: January 13, 2021
Published: January 15, 2021
pharmacological effects of FTY720 or the next generation of
S1PR signaling modulators such as Ozanimod remain unclear,
despite reports suggesting the ability of these molecules to
modulate oligodendrocyte biology via S1P5.7,8
It has been reported that S1P5 is ubiquitously expressed in the
cells of CNS and particularly highly expressed in the mature
https://dx.doi.org/10.1021/acsmedchemlett.0c00631
© 2021 American Chemical Society
ACS Med. Chem. Lett. 2021, 12, 351−355
351

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Table 1. S1P1-5 Activity of 3-Aminopropanoic Acid Analogues
a
b
c
Ca2+ mobilization assay EC50, see ref 17. Antagonist. Ki = 1.4 nM in the S1P5 33P binding assay.
Scheme 1. General Synthetic Scheme of Amino Acid
Analogues
Figure 2. Plasma and total brain concentration after 1 mg/kg IV dosing
of compound 15 in rats.
much improved S1P5 activity (0.1 nM) while maintaining
selectivity over other S1P receptor family members (Table 1). A
33P -S1P radiolabel binding assay confirmed the observed S1P5
selectivity over S1P1 of compound 3 (Ki (S1P5) = 1.4 nM and Ki
(S1P1) > 5 μM). Dosing of compound 3 (5 mg/kg) in mice led
to good exposure (AUC = 2723 h·ng/mL, T1/2 = 3h) and did not
lead to any lymphopenia which is consistent with lack of S1P1
activity. Encouraged by this result, we decided to further explore
the structure−activity relationship (SAR) based on this scaffold.
The chemistry was started by making the literature compound
4-methylnaphthalen-2-ol,18 followed by alkylation and SnCl4
mediated aldehyde formation, with subsequent reductive
amination and hydrolysis as exemplified by the synthesis of
compound 5 (Scheme 1).19
A small library based on varying the amino acid head portion
of compound 3 was generated (Table 2). All analogues lack
S1P1 and S1P2 activity. However, their activity on S1P3, S1P4,
and S1P5 varies significantly depending on the secondary amine.
Compounds 10−16 generally lack S1P3 and S1P4 activity, with
the piperidine headgroup (15) being a clear standout as the
most potent S1P5 antagonist (EC50 = 0.1 nM) with no activity
on S1P1−4. The observed S1P5 selectivity over S1P1 of
compound 15 was confirmed with a second S1P1/S1P5 binding
assay (Ki (S1P5) = 4.4 nM, and Ki (S1P1) > 5 μM).
Replacement of the core 4-Me group of 3 with an iodide led
to the complete loss of S1P5 activity of compound 4. Further
Figure 3. Chemotaxis assays of mouse spleen naive T cells (A) or NK
cells (B) toward S1P. Cells were pretreated for 1 h with FTY720 or 15
at the indicated concentration in the top chamber. Results show the
migration relative to control condition (no inhibitor). N = 3
experiments. **P < 0.01; ***P < 0.001.
modification of the 4-Me position on compound 15 resulted in a
sharp SAR with CF3, I, Cl, and H replacement leading to
significant loss of S1P5 activity without affecting S1P1−4
activity (compounds 17−20).
With the optimized 4-carboxy piperidine of compound 15 in
place, the cyclohexyl tail was further explored (Table 3).
Replacing the 4-trans-t-butyl group of 15 with bulkier trans-t-
amyl led to loss of activity, and further expansion to the trans-
phenyl completely abolished the S1P5 activity. cis-4-Substituted
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Table 2. S1P1−5 Activity of Different Amino Acid Analogues
a
b
c
Ca2+ mobilization assay EC50. Antagonist. Ki = 4.4 nM in the S1P5 33P binding assay.
analogues (compound 23−25) did not show much S1P5
activity, with the unsubstituted cyclohexyl analogue 26 giving
only moderate activity (EC50 (S1P5) = 236 nM). Quite
interestingly, the spiro analogues (28) showed the best potency
on S1P5 (EC50 (S1P5) = 0.03 nM), which was confirmed with
the S1P5 binding assay (Ki = 0.3 nM). However, 28 showed high
clearance in rat PK.
Compound 15 was further profiled and showed high plasma
protein binding with 0.60% free fraction in human. It had decent
permeability with Papp (A-B, 10−6 cm/s) = 9.1 and efflux ratio =
1.84 in Caco-2 cells. IC50’s at CYP isoforms (3A4, 1A2, 2C19,
2C9, 2D6) were all greater than 10 μM. The in vitro hepatocyte
stability was favorable with Qh% = 30% in rat and 49% in
human; therefore, 15 was tested in rat PK. At 5 mg/kg oral
dosing, 15 demonstrated good exposure (AUC = 6510 ng/mL·
h) and excellent oral bioavailability (F = 92%), with a half-life of
4.5 h (Table 4). And in IV dosing, it showed CL = 12.2 mL/
min/kg (%Qh = 22%). Compound 15 also showed good brain
penetration with a plasma/brain ratio of 0.67 at 2 h, 0.66 at 7 h,
and 0.83 at 24 h (Figure 2).
Compound 15 was further tested on the migratory response
of mouse spleen lymphocytes toward S1P. Compound 15
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https://dx.doi.org/10.1021/acsmedchemlett.0c00631
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Table 3. S1P1-5 Activity of Different Tail Analogues
a
b
c
d
Ca2+ mobilization assay EC50. Antagonist. Ki = 0.3 nM in the S1P5 33P binding assay. High clearance in rat PK.
a
Table 4. Single Dose Pharmacokinetic Parameters of 15 in Rats
compd
route
dose (mg/kg)
1
Cl (mL/min/kg)
12.2 2.7
V
ss (L/kg)
t
1/2 (h)
AUC0−24h (ng/mL·h)
C
max (ng/mL)
947 44
T
max (h)
F (%)
92
d
b
15
IV
4.2 0.5
4.6 1.6
4.5 0.5
1416 356
6515 23
c
PO
5
0.75
0
a
b
c
d
Mean SD (n = 3). Formulation: solution in 1:1:1:7 EtOH:PEG400:Solutol:water. Formulation: Suspension in 15% HPCD. Plasma/brain
ratio at 2/7/24 h = 0.67/0.66/0.83.
prevented NK cell migration toward S1P, with an IC50 of 553
nM, while it had no effect on T cell migration in contrast to
FTY720 which blocked T cell but not NK cell migration (Figure
3). Of note, at the 100 nM concentration, compound 15 tended
to increase NK cell migration to S1P, an effect that was not seen
with higher doses. Further studies will be needed to understand
what causes this biphasic response to the compound in the
experimental setting. Nevertheless, as FTY720-P is an agonist on
S1P1,3,4,5 (EC50 = 4, 27, 22, 0.36 nM) except S1P2 (EC50 > 5
μM), the S1P5 specific antagonist effect of compound 15 is
remarkable.
with the phenotype of the mice harboring genetic deficiency in
S1P5.20
ASSOCIATED CONTENT
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* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acsmedchemlett.0c00631.
Experimental details for synthetic procedures and
analytical data for key compounds and assay conditions
(PDF)
In summary, we have discovered a series of S1P5 specific
antagonists with excellent potency and identified a brain-
penetrable tool, compound 15, with good oral bioavailability.
Compound 15 demonstrated an inhibitory effect on NK cell
migration toward S1P but not on T cells. This is in agreement
AUTHOR INFORMATION
■
Corresponding Author
Bin Ma − Medicinal Chemistry, Biogen Inc, Cambridge,
Massachusetts 02142, United States; orcid.org/0000-
354
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mouse cerebellar cortex by sphingosine kinase. J. Comp. Neurol. 2004,
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0002-3913-0545; Phone: 617-914-4955; Email: bin.ma@
biogen.com
(4) Kono, M.; Allende, M. L.; Proia, R. L. Sphingosine-1-phosphate
regulation of mammalian development. Biochim. Biophys. Acta, Mol. Cell
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Authors
Kevin M. Guckian − Medicinal Chemistry, Biogen Inc,
Cambridge, Massachusetts 02142, United States
Xiao-gao Liu − Medicinal Chemistry, Biogen Inc, Cambridge,
Massachusetts 02142, United States
Chunhua Yang − Research, Biogen Inc, Cambridge,
Massachusetts 02142, United States
Bing Li − Research, Biogen Inc, Cambridge, Massachusetts
02142, United States
Robert Scannevin − Research, Biogen Inc, Cambridge,
Massachusetts 02142, United States
(5) Hla, T.; Brinkmann, V. Sphingosine 1-phosphate (S1P):
physiology and the effects of S1P receptor modulation. Neurology
2011, 76 (8 Suppl 3), S3−S8.
(6) David, O. J.; Kovarik, J. M.; Schmouder, R. L. Clinical
pharmacokinetics of fingolimod. Clin. Pharmacokinet. 2012, 51, 15−28.
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Antel, J. P. FTY720 modulates human oligodendrocyte progenitor
process extension and survival. Ann. Neurol. 2008, 63 (1), 61−71.
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H.; Roberts, E.; Boehm, M. F.; Peach, R. J. Ozanimod (RPC1063) is a
potent sphingosine-1-phosphate receptor-1 (S1P1) and receptor-5
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̈
Michael Mingueneau − Research, Biogen Inc, Cambridge,
Massachusetts 02142, United States
Annabelle Drouillard − Université Lyon 1, Lyon 69007,
France; International Center for Infectiology Research, Lyon
69007, France
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Thierry Walzer − Université Lyon 1, Lyon 69007, France;
International Center for Infectiology Research, Lyon 69007,
France
Complete contact information is available at:
https://pubs.acs.org/10.1021/acsmedchemlett.0c00631
Biography
Bin Ma is senior scientist of medicinal chemistry at Biogen. Bin received
BS and MS degrees from Lanzhou University and a PhD degree in
organic chemistry from Boston University. Bin gained his postdoc
training at Harvard University in Professor Kishi’s labs. In 2007, Bin
moved to Biogen and started his industrial career. Bin contributed
multiple development candidates in multiple therapeutic areas at
Biogen and has extensive experience on target validation, hit ID, lead
optimization, candidate selection, and preclinical development and has
served as leader for chemistry teams and project teams. Bin’s interests
span the broad scope of drug discovery for neurodegenerative diseases.
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971432, an orally bioavailable selective sphingosine-1-phosphate
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degenerative disorders. J. Med. Chem. 2015, 58, 9154−9170.
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Guerini, D.; Mir, A. K.; Orain, D.; Osinde, M.; Picard, A.; Dubois, C.;
Tasdelen, E.; Haessig, S. Design and synthesis of selective and potent
orally active S1P5 agonists. ChemMedChem 2010, 5, 1693−1696.
(15) Miron, V. E.; Hall, J. A.; Kennedy, T. E.; Soliven, B.; Antel, J. P.
Cyclical and dose-dependent responses of adult human mature
oligodendrocytes to Fingolimod. Am. J. Pathol. 2008, 173, 1143−1152.
Author Contributions
All authors have given approval to the final version of the
manuscript.
Notes
The authors declare no competing financial interest.
(16) Drouillard, A.; Mathieu, A. L.; Marca̧ is, A.; Belot, A.; Viel, S.;
Mingueneau, M.; Guckian, K.; Walzer, T. S1PR5 is essential for human
NK cell migration towards sphingosine-1 phosphate. J. Allergy Clin.
Immunol. 2018, 141 (6), 2265−2268.
ACKNOWLEDGMENTS
The authors thank the DMPK group for the support on
compound profiling and PK studies.
■
(17) Ma, B.; Guckian, K. M.; Lin, E. Y.-S.; Lee, W.-C.; Scott, D.;
Kumaravel, G.; Macdonald, T. L.; Lynch, K. R.; Black, C.; Chollate, S.;
Hahm, K.; Hetu, G.; Jin, P.; Luo, Y.; Rohde, E.; Rossomando, A.;
Scannevin, R.; Wang, J.; Yang, C. Stereochemistry activity relationship
of orally active tetralin S1P agonist prodrugs. Bioorg. Med. Chem. Lett.
2010, 20, 2264−2269.
ABBREVIATIONS
■
CL, clearance; CNS, central nervous system; HPCD, hydrox-
ypropyl-beta-cyclodextrin; IV, intravenous; NK, natural killer;
PK, pharmacokinetics; Qh, normalized clearance based on
hepatic blood flow; S1PR, sphingosine 1-phosphate receptor;
SAR, structure−activity relationship.
(18) Essiz, M.; Guillaumet, G.; Brunet, J. J.; Caubere, P. Arynic
condensations of ketone enolates. 15. New synthetic applications of the
condensation of α,β-unsaturated ketone enolates on benzyne. J. Org.
Chem. 1980, 45 (2), 240−246.
(19) Guckian, K.; Kumaravel, G.; Liu, X.; Ma, B.; Peng, H. Preparation
of naphthalene compounds as S1P receptor modulating agents for
therapy. PCT/WO 2014/120764, 1−96.
(20) Jenne, C. N.; Enders, A.; Rivera, R.; Watson, S. R.; Bankovich, A.
J.; Pereira, J. P.; Xu, Y.; Roots, C. M.; Beilke, J. N.; Banerjee, A.; Reiner,
S. L.; Miller, S. A.; Weinmann, A. S.; Goodnow, C. C.; Lanier, L. L.;
Cyster, J. G.; Chun, J. T-bet-dependent S1P5 expression in NK cells
promotes egress from lymph nodes and bone marrow. J. Exp. Med.
2009, 206 (11), 2469−2481.
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