Synthesis and Evaluation of Serinolamide Derivatives as Sphingosine-1-Phosphate-1 (S1P1) Receptor Agonists

Sphingosine-1-phosphate-1 (S1P1) receptor agonists are well-known drugs for treating multiple sclerosis (MS) caused by autoreactive lymphocytes that attack the myelin sheath. Therefore, an effective therapeutic strategy is to reduce the lymphocytes in the blood by inducing S1P1 receptor internalization. We synthesized serinolamide A, a natural product of the sea, and performed S1P1 receptor internalization assay to evaluate functionally antagonistic S1P1 receptor agonist activity. In order to synthesize derivatives with better efficacy than serinolamide A and B, new derivatives were synthesized by introducing the phenyl ring moiety of fingolimod. Among them, compounds 19 and 21 had superior S1P1 agonistic effects to serinolamide. We also confirmed that compound 19 effectively inhibited lymphocyte outflow in peripheral lymphocyte count (PLC) assay.


Evaluation of the Synthesized Serinolamide Derivatives as S1P 1 Receptor Agonists
The ability of the compounds to internalize the S1P 1 receptor from the cell surface was evaluated using a commercially available in vitro assay system to test the functionally antagonistic S1P 1 receptor agonist activity of the synthesized serinolamide derivatives [13][14][15]. The efficacy of the synthetic compounds was expressed as a percentage of maximal efficacy at 1 µM of FTY720, a highly potent S1P 1 agonist. In the S1P 1 receptor internalization assay, compounds 12, 19, and 21 showed more than 80% efficacies at 30 µM. Notably, compound 19 showed good efficacy of 147%. In addition, compounds 4, 6, and 13 showed lower S1P 1 receptor internalization efficacies than other derivatives ( Table 1). The efficacy of compounds 19 and 21, in which a phenyl ring was introduced, was significantly improved compared to those of compounds 4 and 6, in which a phenyl ring was not introduced.  1 agonists, such as fingolimod, are known to induce peripheral lymphopenia by inhibiting S1P 1 -mediated lymphocyte outflow from lymphoid tissues. Lymphopenia caused by these drugs contributes to the therapeutic effect of autoimmune diseases such as multiple sclerosis [6,16]. Therefore, we investigated the effect of compounds to induce lymphopenia in blood by peripheral lymphocyte count (PLC) analysis ( Figure 2). After intravenous administration of compounds 19, 21 (15 mg/kg, 30 mg/kg, maximum solubility concentration) and fingolimod (3 mg/kg) to mice, blood samples were collected by orbital bleed. At this time, there was no visual change compared to the vehicle treatment group containing the same amount of DMSO. As a result of measuring the number of lymphocytes in the blood, the number of lymphocytes started to decrease within 2.5 h after administration. In particular, the number of lymphocytes in the blood of mice administered 19 was significantly decreased during the first 5 h. In contrast to fingolimod, mice treated with 19 and 21 began to recover lymphocyte counts after 5 h. As a result of a single dose administration, peripheral lymphocyte counts in fingolimod-treated mice continued to decrease for 24 h post-dose. In contrast, lymphocyte counts in mice treated with compound 19 or 21 returned to near baseline levels, suggesting that the cardiac toxicity of fingolimod due to its long-term efficacy on lymphocyte reduction can be overcome ( Figure 2) [17]. Collectively, these results suggest that administration of 19 and 21 can inhibit the egress of lymphocytes from lymphoid tissues to peripheral blood, and that lymphopenia can be reversed within 24 h. , 21 (30 mg/kg, n = 10), or positive control fingolimod (3 mg/kg, n = 8). Blood lymphocyte counts were measured before (0 h) and after the administration (2.5 and 5, 24 h). Percentage of the lymphocyte count at the time before administration (0 h) was considered as a baseline (100%). * p < 0.05, *** p < 0.001, **** p < 0.0001 compared to vehicle-treated group (one-way ANOVA with Dunnett's test). Data are presented as mean ± SEM.

General Methods
All chemicals, reagents, and solvents were obtained from commercially available sources as reagent grades without further purification. The yields reported are for purified products and were not optimized. Synthesized compounds were checked by thin-layer chromatography (TLC) and 1 H and 13 C nuclear magnetic resonance (NMR), melting point (MP), high-resolution mass spectrometry (HRMS), and high-performance liquid chromatography (HPLC) analyses. Analytical thin-layer chromatography plates monitored reactions (Merck, Cat No. 1.05715, Darmstadt, Germany) and analyzed by ultraviolet light at 254 nm and 280 nm. The reactions were purified by MPLC (Biotage ® , Isolera ™ one, Uppsala, Sweden). The NMR spectra were recorded at 400 MHz ( 1 H)/100 MHz ( 13 C) using Bruker spectrometers (Billerica, USA.). Chemical shifts (δ) were reported in ppm downfield from tetramethylsilane (TMS). HPLC analysis was performed using a Waters E2695 system (Milford, USA.) equipped with a YMC-Triart C18 /S-5 µm /12 nm/ Lot No.

General Procedure for Amide Coupling Reaction (Method A)
A mixture of carboxylic acid derivatives, EDC, HOBt, and DIPEA, was dissolved in dichloromethane ([C]~0.1 M) and stirred for 20 min at room temperature. Amine derivatives were added into the reaction mixture and stirred overnight to afford serinolamide A. The reaction mixture was diluted with distilled water and extracted with ethyl acetate. The combined organic layer was dried with Na 2 SO 4 and evaporated in vacuo. The obtained residue was purified by column chromatography on SiO 2 .

General Procedure for Boc Deprotection and Deacetylation Reaction (Method B)
To a mixture of NHBoc derivatives in dichloromethane or ethanol ([C]~0.1 M), 4 M HCl was added, and the reaction mixture was stirred at room temperature for 2 h. The reaction mixture was evaporated in vacuo.

General Procedure for the Reductive Amination with Aldehyde (Method C)
A mixture of aldehyde derivatives in methanol and tetrahydrofuran in a ratio of 1:1 ([C]~0.1 M) was added a mixture of amine derivatives with triethylamine. The resulting suspension was stirred at room temperature (0.5 h). Then, sodium cyanoborohydride was added and stirred at room temperature. The reaction mixture was evaporated in vacuo. The product residue was washed with ethyl acetate and distilled water. The combined organic layer was dried with anhydrous Na 2 SO 4 and evaporated in vacuo. The obtained residue was purified by column chromatography on SiO 2 .

S1P 1 Receptor Internalization Assay
The S1P 1 receptor internalization activity of synthesized compounds was evaluated using PathHunter ® EDG1 total GPCR internalization HEK293 cell line (93-0784C1; Discov-erX). The cell lines are engineered to co-express two fragments of β-galactosidase at S1P 1 receptor and endosome, respectively. The endocytosis of receptor leads β-galactosidase to