2-Aminobenzoxazole Derivatives as Potent Inhibitors of the Sphingosine-1-Phosphate Transporter Spinster Homolog 2 (Spns2)

Abstract

The S1P1 receptor is the target of four marketed drugs for the treatment of multiple sclerosis and ulcerative colitis. Targeting an S1P exporter, specifically Spns2, that is “upstream” of S1P receptor engagement is an alternate strategy that might recapitulate the efficacy of S1P receptor modulators without cardiac toxicity. We recently reported the first Spns2 inhibitor SLF1081851 (16d) that has modest potency with in vivo activity. To develop more potent compounds, we initiated a structure–activity relationship study that identified 2-aminobenzoxazole as a viable scaffold. Our studies revealed SLB1122168 (33p), which is a potent inhibitor (IC50 = 94 ± 6 nM) of Spns2-mediated S1P release. Administration of 33p to mice and rats resulted in a dose-dependent decrease in circulating lymphocytes, a pharmacodynamic indication of Spns2 inhibition. 33p provides a valuable tool compound to explore both the therapeutic potential of targeting Spns2 and the physiologic consequences of selective S1P export inhibition.

Assessing Spns2-dependent S1P Transport as a Prospective Therapeutic Target

“…This substitution resulted in increased oral bioavailability as well as a nearly 50-fold increase in potency over SLF1081851 in an assay of Spns2-dependent S1P release from HeLa cells (IC 50 1.9 µM 20 vs. 0.05 µM). Like previously reported STBs 20,21 , single dose administration of SLF80821178 to mice resulted in a dose-dependent decrease in circulating lymphocytes with a maximal decrease of ∼50%. The details of the characterization of SLF80821178, a structure-activity relationship of compounds on the urea-based scaffold and their synthetic routes are the subject of a separate report (Foster, et al ., submitted for publication).…”

“…For example, single doses of SLF1081851 20 , SLB1122168 21 and SLF80821178 (Foster et al ., submitted) evoke dose-dependent decreases in total blood lymphocytes in C57BL/6 mice and Sprague-Dawley rats. Further, while plasma S1P concentrations were reduced ∼15% in response to our prototype STB, SLF1081851 20 , plasma S1P concentrations were unchanged in response to SLB1122168 21 and SLF80821178 (Foster et al ., submitted) after administration of a single dose of these inhibitors. Chronic dosing of SLF80821178 in C57BL/6J mice likewise evoked lymphopenia as measured on day 15 (Fig.…”

“…We have discovered small molecule inhibitors of Spns2-dependent S1P release and use these to discern the extent to which STBs mimic the actions of SRMs. Using STBs on distinct chemical scaffolds 20,21 , we discovered that these compounds drive a dose-dependent decrease in blood lymphocyte counts in mice and rats 20,21 . Further, we found that our prototype STB, SLF1081851, is efficacious in models of renal fibrosis 12 .…”

Molecular basis of Spns2-facilitated sphingosine-1-phosphate transport

“…Webster group also reported a new Spns2 inhibitor 33p with higher IC 50 (94 nM). 4 The IC 50 of 33p in our MS-based assay was 1.198 μM, which is slightly more potent than 16d (Supplementary information, Fig. S10d ).…”

Discovery of Potent, Orally Bioavailable Sphingosine-1-Phosphate Transporter (Spns2) Inhibitors

“…Recently, we reported a structure–activity relationship (SAR) study that identified prototypes SLF1081851 (IC 50 1.93 ± 0.04 μM) and SLF80721166 (IC 50 1.4 ± 0.3 μM) (Figure ). Additional investigations revealed a second generation Spns2 inhibitor, SLB1122168 (IC 50 94 ± 6 nM), which contained a key benzoxazole scaffold. , When administered to rodents, these inhibitors were effective in modulating the immune system as evidenced by a decrease in circulating lymphocytes, , which is a hallmark of Spns2 inhibition. In a mouse model of kidney fibrosis (unilateral ischemia-reperfusion injury), SLF1081851 suppressed inflammatory signaling in perivascular cells and ameliorated kidney fibrosis (Figure ).…”