ML-161 is a novel and potent allosteric inhibitor of PAR1 (proteinase-activated receptor 1) on platelets with IC50 of 0.26 μM. ML161 exhibits a different selectivity with the reported ALK inhibitor crizotinib. ML161 treatment was found to inhibit the activation of thrombin-induced platelets in a dose-dependent manner through the detection of P-selectin expression in human platelets. ML161 demonstrated a significant suppression of P-selectin expression in a dose-dependent manner with an EC50 value of 0.3μM when tested with granule secretion. Additionally, it prevented SFLLRN-induced thrombus formation. With an IC50 value of 0.26 µM, ML-161 inhibits P-selectin'ssurfaceexpression that is induced by the peptide SFLLRN. When thrombin or a PAR1 peptide agonist were used to cause platelet aggregation, ML161 prevented it, but not when using multiple other platelet agonists.

Physicochemical Properties

Molecular Formula	C17H17BRN2O2
Molecular Weight	361.23
Exact Mass	360.047
Elemental Analysis	C, 56.52; H, 4.74; Br, 22.12; N, 7.75; O, 8.86
CAS #	423735-93-7
Related CAS #	423735-93-7
PubChem CID	1048267
Appearance	White to off-white solid powder
Density	1.4±0.1 g/cm3
Boiling Point	465.7±30.0 °C at 760 mmHg
Flash Point	235.4±24.6 °C
Vapour Pressure	0.0±1.2 mmHg at 25°C
Index of Refraction	1.656
LogP	3.45
Hydrogen Bond Donor Count	2
Hydrogen Bond Acceptor Count	2
Rotatable Bond Count	5
Heavy Atom Count	22
Complexity	389
Defined Atom Stereocenter Count	0
SMILES	BrC1=C([H])C([H])=C([H])C([H])=C1C(N([H])C1=C([H])C([H])=C([H])C(=C1[H])N([H])C(C([H])([H])C([H])([H])C([H])([H])[H])=O)=O
InChi Key	DFOVLSMXPWPCFH-UHFFFAOYSA-N
InChi Code	InChI=1S/C17H17BrN2O2/c1-2-6-16(21)19-12-7-5-8-13(11-12)20-17(22)14-9-3-4-10-15(14)18/h3-5,7-11H,2,6H2,1H3,(H,19,21)(H,20,22)
Chemical Name	2-bromo-N-[3-(butanoylamino)phenyl]benzamide
Synonyms	ML-161; ML 161; ML161; AG-670; AG 670; AG670; CID-1048267; CID1048267; CID 1048267
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	PAR1 ( IC50 = 0.26 μM )
ln Vitro	In vitro activity: Parmodulin 2 (ML161; 10 µM; for 30 minutes) blocks proinflammatory signaling in endothelial HUVECs cells[3].
ln Vivo	Parmodulin 2 (ML161; 5 mg/kg; IV) significantly prevents platelet thrombus formation, with aa 73% reduction in AUC (area under the curve)[2]. Parmodulin 2 does not increase bleeding time; instead, it prevents platelet thrombus formation in vivo. Platelet aggregation is selectively inhibited by Parmodulin 2 via the α2A-adrenergic receptor and Par1[2].
Enzyme Assay	In order to determine possible targets of hit compounds, a high-throughput screen of the NIH-MLSMR compound collection and a number of secondary assays were used to find a 1,3-diaminobenzene scaffold that targets protease-activated receptor 1 (PAR1). We hereby present further studies of the structure-activity relationship (SAR) that characterize the conditions for activity at PAR1 and pinpoint analogues that are stable in plasma that exhibit nanomolar inhibition of PAR1-mediated platelet activation. Compound 4 was declared as a probe (ML161) with the NIH Molecular Libraries Program. This substance prevented platelet aggregation brought on by thrombin or a PAR1 peptide agonist, but not by a number of other platelet agonists. According to preliminary research, ML161 is an allosteric inhibitor of PAR1. The identification of antithrombotics with an enhanced safety profile may benefit from these findings.
Animal Protocol	C57BL/6J wild type mice 5 mg/kg (Pharmacokinetic Analysis) IV
References	[1]. Characterization of Protease-Activated Receptor (PAR) ligands: Parmodulins are reversible allosteric inhibitors of PAR1-driven calcium mobilization in endothelial cells. Bioorg Med Chem. 2018 May 15;26(9):2514-2529. [2]. Allosteric inhibition of protease activated receptor 1: a new antiplatelet therapy. [3]. Parmodulins inhibit thrombus formation without inducing endothelial injury caused by vorapaxar. Blood. 2015 Mar 19;125(12):1976-85.
Additional Infomation	2-bromo-N-[3-(1-oxobutylamino)phenyl]benzamide is a member of benzamides.

Solubility Data

Solubility (In Vitro)

DMSO: 72~100 mg/mL (199.3~276.8 mM) Water: <1 mg/mL Ethanol: <1 mg/mL

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.92 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 (6.92 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication. 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 (6.92 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	2.7683 mL	13.8416 mL	27.6832 mL
	5 mM	0.5537 mL	2.7683 mL	5.5366 mL
	10 mM	0.2768 mL	1.3842 mL	2.7683 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.