Enpatoran (M5049) HCl is a potent, orally bioactive TLR7/8 inhibitor (antagonist) with IC50s of 11.1 nM and 24.1 nM in HEK293 cells, respectively. Enpatoran HCl is inactive against TLR3, TLR4 and TLR9. Enpatoran HCl blocks both synthetic ligands and natural endogenous RNA ligands. Enpatoran HCl displays good pharmacokinetic properties in vivo. Enpatoran HCl may be utilized in research related to the blockade of innate and adaptive autoimmunity.

Physicochemical Properties

Molecular Formula	C16H16CLF3N4
Molecular Weight	356.77
Exact Mass	356.101
CAS #	2101945-93-9
Related CAS #	Enpatoran;2101938-42-3
PubChem CID	129241087
Appearance	Light yellow to yellow solid powder
Hydrogen Bond Donor Count	2
Hydrogen Bond Acceptor Count	7
Rotatable Bond Count	1
Heavy Atom Count	24
Complexity	472
Defined Atom Stereocenter Count	2
SMILES	C([C@@H]1CN(C2=C3C(N=CC=C3)=C(C#N)C=C2)C[C@@H](C1)N)(F)(F)F.Cl
InChi Key	OHYZGHQWGHDMRP-ZVWHLABXSA-N
InChi Code	InChI=1S/C16H15F3N4.ClH/c17-16(18,19)11-6-12(21)9-23(8-11)14-4-3-10(7-20)15-13(14)2-1-5-22-15;/h1-5,11-12H,6,8-9,21H2;1H/t11-,12+;/m0./s1
Chemical Name	5-[(3R,5S)-3-amino-5-(trifluoromethyl)piperidin-1-yl]quinoline-8-carbonitrile;hydrochloride
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 Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.
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	TLR7 11.1 nM (IC50) TLR8 24.1 nM (IC50)
ln Vitro	With IC50 values ranging from 35 to 45 nM, enpatoran hydrochloride (0.01 nM-10 μM) suppresses the generation of IL-6 induced by all the ligands (miR-122, Let7c RNA, Alu RNA, and R848)[1].
ln Vivo	Prior to receiving R848 (an intraperitoneal injection of 25 µg), mice treated pre-treatment with Enpatoran hydrochloride (oral gavage; 1 mg/kg) exhibit dose-dependent inhibition of IL-6 and IFN-α production[1]. When given orally (1.0 mg/kg for mice, rats, and dogs), enpatoran hydrochloride has a high oral bioavailability (mouse 100%, rat 87%, and dog 84%)[1]. Because of its high plasma clearance (1.4, 1.2, and 0.59 L/h/kg, respectively) and large volumes of distribution (2.7, 8.7, and 5.7 L/kg, respectively) after intravenous administration (1.0 mg/kg for mice, rats, and dogs), enpatoran hydrochloride has moderate half-lives (mouse 1.4, rat 5.0, and dog 13 h)[1].
Animal Protocol	Animal/Disease Models: Female C57BL/6 mice[1] Doses: 0.1 mg/kg and 1 mg/kg Route of Administration: Oral gavage; administered 1 hour prior to R848 challenge Experimental Results: The TLR7/8 agonist R848 stimulated both IFN- α and IL-6 production in mice. Enpatoran hydrochloride diminished IFN-α and IL-6 production stimulated by R848.
References	[1]. Discovery of M5049: A Novel Selective TLR7/8 Inhibitor for Treatment of Autoimmunity. J Pharmacol Exp Ther. 2020 Dec 16;JPET-AR-2020-000275.

Solubility Data

Solubility (In Vitro)

DMSO: 16.67 mg/mL (46.72 mM)

Solubility (In Vivo)

Solubility in Formulation 1: 2 mg/mL (5.61 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.0 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 mg/mL (5.61 mM) 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; with ultrasonication. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.0 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 mg/mL (5.61 mM) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.0 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	2.8029 mL	14.0146 mL	28.0293 mL
	5 mM	0.5606 mL	2.8029 mL	5.6059 mL
	10 mM	0.2803 mL	1.4015 mL	2.8029 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.