Novel Potent Selective Orally Active S1P5 Receptor Antagonists

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.

Sphingosine-1-phosphate (S1P, Figure 1) signals through cell surface GPCR receptors S1PRs (S1P1–5) and plays important roles in cell survival, trafficking, growth, and differentiation in many cell types, including cells of the immune, cardiovascular, and central nervous (CNS) systems.¹⁻⁴ S1P1 internalization has been well documented in the trafficking of B and T cells by preventing both from leaving the lymph nodes and entering the lymphatic circulation.⁵ The U.S. Food and Drug Administration approved drug FTY720 induces S1P1 internalization, and S1P signaling has become an interesting therapeutic target.⁶ However, the role and contribution of S1P5 to 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

Figure 1.

S1P5 agonists and antagonist.

It has been reported that S1P5 is ubiquitously expressed in the cells of CNS and particularly highly expressed in the mature oligodendrocytes relative to other S1PRs and that it plays an 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 pharmacodynamic 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 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 compound was shown to demonstrate unique activity on blocking natural killer (NK) cell migration in vitro.¹⁶

Screening an internal lipid mimetic library by using a calcium mobilization S1P1–5 assay,¹⁷ we found a 4-methylnaphthalen-1-yl)methyl amino acid analogue 1 with moderate S1P5 antagonist activity (406 nM) without S1P1 activity (>5000 nM). A modification to the tail portion led to compound 3 with 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 (K_i (S1P5) = 1.4 nM and K_i (S1P1) > 5 μM). Dosing of compound 3 (5 mg/kg) in mice led to good exposure (AUC = 2723 h·ng/mL, T_1/2 = 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.

Table 1. S1P1-5 Activity of 3-Aminopropanoic Acid Analogues.

^aCa²⁺ mobilization assay EC₅₀, see ref (17).

^bAntagonist.

^cK_i = 1.4 nM in the S1P5 ³³P binding assay.

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

Scheme 1. General Synthetic Scheme of Amino Acid Analogues.

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 (K_i (S1P5) = 4.4 nM, and K_i (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 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).

Table 2. S1P1–5 Activity of Different Amino Acid Analogues.

^aCa²⁺ mobilization assay EC₅₀.

^bAntagonist.

^cK_i = 4.4 nM in the S1P5 ³³P binding assay.

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 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 (K_i = 0.3 nM). However, 28 showed high clearance in rat PK.

Table 3. S1P1-5 Activity of Different Tail Analogues.

^aCa²⁺ mobilization assay EC₅₀.

^bAntagonist.

^cK_i = 0.3 nM in the S1P5 ³³P binding assay.

^dHigh 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 P_app (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).

Table 4. Single Dose Pharmacokinetic Parameters of 15 in Rats^a.

compd	route	dose (mg/kg)	Cl (mL/min/kg)	Vss (L/kg)	t1/2 (h)	AUC0–24h (ng/mL·h)	Cmax (ng/mL)	Tmax (h)	F (%)
15d	IVb	1	12.2 ± 2.7	4.2 ± 0.5	4.6 ± 1.6	1416 ± 356
	POc	5			4.5 ± 0.5	6515 ± 23	947 ± 44	0.75 ± 0	92

^aMean ± SD (n = 3).

^bFormulation: solution in 1:1:1:7 EtOH:PEG400:Solutol:water.

^cFormulation: Suspension in 15% HPCD.

^dPlasma/brain ratio at 2/7/24 h = 0.67/0.66/0.83.

Figure 2.

Plasma and total brain concentration after 1 mg/kg IV dosing of compound 15 in rats.

Compound 15 was further tested on the migratory response of mouse spleen lymphocytes toward S1P. Compound 15 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.

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.

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 with the phenotype of the mice harboring genetic deficiency in S1P5.²⁰

Glossary

Abbreviations

CL

clearance

CNS

central nervous system

HPCD

hydroxypropyl-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.

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.

Supporting Information Available

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)

Author Contributions

All authors have given approval to the final version of the manuscript.

The authors declare no competing financial interest.