CYM-5541 (also known as ML249) is a potent, selective, full, and allosteric agonist of the S1P3 (Sphingosine 1-phosphate) receptor with EC50 in the range of 72 to 132 nM. S1P is a lysophospholipid signaling molecule that activates G protein-coupled receptors for S1P, specifically S1P(1) through S1P(5), to regulate key biological processes, such as lymphocyte trafficking and vascular development. Phe263 is an important gate-keeper residue for the affinity and efficacy of an allosteric site that was identified using CYM-5541. This ligand is nonpolar, and the unique allosteric hydrophobic pocket allows CYM-5541's S1P(3) selectivity within the remarkably similar S1P receptor family.

Physicochemical Properties

Molecular Formula	C19H28N2O2
Molecular Weight	316.45
Exact Mass	316.215
Elemental Analysis	C, 72.12; H, 8.92; N, 8.85; O, 10.11
CAS #	945128-26-7
Related CAS #	945128-26-7
PubChem CID	17253208
Appearance	White to off-white solid powder
Density	1.1±0.1 g/cm3
Boiling Point	503.1±38.0 °C at 760 mmHg
Flash Point	258.1±26.8 °C
Vapour Pressure	0.0±1.3 mmHg at 25°C
Index of Refraction	1.559
LogP	3.47
Hydrogen Bond Donor Count	0
Hydrogen Bond Acceptor Count	3
Rotatable Bond Count	4
Heavy Atom Count	23
Complexity	394
Defined Atom Stereocenter Count	0
SMILES	O=C(N(C1CCCCC1)C1CCCCC1)C1C=C(C2CC2)ON=1
InChi Key	NDKGACIWVAOUQH-UHFFFAOYSA-N
InChi Code	InChI=1S/C19H28N2O2/c22-19(17-13-18(23-20-17)14-11-12-14)21(15-7-3-1-4-8-15)16-9-5-2-6-10-16/h13-16H,1-12H2
Chemical Name	N,N-dicyclohexyl-5-cyclopropyl-1,2-oxazole-3-carboxamide
Synonyms	ML249; ML 249; ML-249; CYM-5541; CYM 5541; CYM5541
HS Tariff Code	2934.99.9001
Storage	Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month
Shipping Condition	Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Biological Activity

Targets	S1P3 ( EC50: between 72 and 132 nM )
ln Vitro	In vitro activity: CYM-5541 is a complete agonist, able to phosphorylate ERK to the highest extent seen with S1P. CYM-5541 demonstrates remarkable selectivity over other S1P receptor subtypes, including S1P1 EC50>10 μM, S1P2 EC50>50 μM, S1P4 EC50>50 μM, and S1P5 EC50>25 μM, with an EC50 ranging from 72 to 132 nM. In the Ricerca profiling panel of 55 GPCRs, ion channels, and transporters, CYM-5541 likewise demonstrates selectivity over a broad panel of protein targets with no discernible activities. Our identification of an allosteric site where F263 is a crucial gate-keeper residue for its efficacy and affinity was made possible by CYM-5541. CYM-5541's S1P3 selectivity could be explained by its unique allosteric hydrophobic pocket[1].
ln Vivo
Enzyme Assay	For four hours, WT or mutant S1P3-expressing Jump-In TI CHO-K cells are serum-starved. Following this, they are incubated for 30 minutes at 4 °C in the binding buffer, which contains 20 mM Tris-HCl (pH 7.5), 100 mM NaCl, 15 mM NaF, 0.5 mM EDTA, 1 mM Na2VO4, 0.5% fatty acid-free bovine serum albumin, and a protease inhibitor mixture containing 0.1 nM [33P]S1P and increasing concentrations of S1P, SPM-242, or CYM-5541. Cold binding buffer is used to wash the cells three times. Liquid scintillation counting is the method used to measure cell-bound radioactivity after the cells have been lysed with 0.5% SDS. In order to reference the level of [33P]S1P bound to each cell line (WT or mutant) in the absence of a competing ligand as 100% for its own cell line, the raw data is normalized[1].
Cell Assay	The pcDNA3.1 plasmid encoding the triple HA-tagged N-terminal S1P3 fusion protein was acquired from cDNA.org. Overlapping PCR mutagenesis was used to create S1P3 receptor mutants, and sequences were checked before use. The Gateway cloning vectors (pDONR 221, pJTI R4 DEST, and pJTI R4 Int), enzymes (BP clonase II and LR clonase), and Jump-InTM TI™ CHO-K parental cells were acquired from Invitrogen Corp. The BP recombination reaction was first used to clone triple HA-tagged WT and mutant S1P3 into entry clones. In order to create a pJTI R4 EXP retargeting expression vector, they were retargeted into a suitable pJTI R4 DEST vector. For retargeting, both the pJTI R4 EXP S1P1 and the pJTI R4 Int vector were transfected into Jump-In TI CHO-K cells. Retargeted Jump-In TI cells were selected using blasticidin at 10 μg/mL for four weeks after the cells had expanded.
Animal Protocol
References	[1]. Novel selective allosteric and bitopic ligands for the S1P(3) receptor. ACS Chem Biol. 2012 Dec 21;7(12):1975-83.

Solubility Data

Solubility (In Vitro)

DMSO: 25~32 mg/mL (79.0~101.1 mM) Water: N/A Ethanol: ~63 mg/mL

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.5 mg/mL (7.90 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: ≥ 2.5 mg/mL (7.90 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution. Solubility in Formulation 3: ≥ 2.5 mg/mL (7.90 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	3.1601 mL	15.8003 mL	31.6006 mL
	5 mM	0.6320 mL	3.1601 mL	6.3201 mL
	10 mM	0.3160 mL	1.5800 mL	3.1601 mL

*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.