AC-55541 is an agonist of protease-activated receptor (PAR) 2. AC-55541 activated PAR2 signaling in cellular proliferation assays, phosphatidylinositol hydrolysis assays, and Ca(2+) mobilization assays, with potencies ranging from 200 to 1000 nM. Rats receiving AC-55541 intraperitoneally had good absorption, with peak plasma concentrations of micromolar. With elimination half-lives of 6.1 hours, AC-55541 remained stable when metabolized by liver microsomes and was exposed to rats for an extended period of time. The application of AC-55541 or AC-264613 intrapaw caused edema and severe, long-lasting thermal hyperalgesia. These effects were totally inhibited by coadministration of a transient receptor potential vanilloid (TRPV) 1 antagonist or a tachykinin 1 (neurokinin 1) receptor antagonist. Hyperalgesia comparable to that seen with local administration of AC-55541 or AC-264613 was produced when the compounds were administered systemically.

Physicochemical Properties

Molecular Formula	C25H20BRN5O3
Molecular Weight	518.36
Exact Mass	517.074
Elemental Analysis	C, 57.93; H, 3.89; Br, 15.41; N, 13.51; O, 9.26
CAS #	916170-19-9
Related CAS #	916170-19-9
PubChem CID	9589606
Appearance	White to off-white solid powder
Density	1.5±0.1 g/cm3
Index of Refraction	1.689
LogP	4.65
Hydrogen Bond Donor Count	3
Hydrogen Bond Acceptor Count	5
Rotatable Bond Count	6
Heavy Atom Count	34
Complexity	838
Defined Atom Stereocenter Count	0
SMILES	C(C1=NNC(=O)C2C=CC=CC1=2)(C(=O)N/N=C(/C1C=CC=C(Br)C=1)\C)NC(C1C=CC=CC=1)=O
InChi Key	UCUHFWIFSHROPY-RWPZCVJISA-N
InChi Code	InChI=1S/C25H20BrN5O3/c1-15(17-10-7-11-18(26)14-17)28-31-25(34)22(27-23(32)16-8-3-2-4-9-16)21-19-12-5-6-13-20(19)24(33)30-29-21/h2-14,22H,1H3,(H,27,32)(H,30,33)(H,31,34)/b28-15+
Chemical Name	N-[2-[(2E)-2-[1-(3-bromophenyl)ethylidene]hydrazinyl]-2-oxo-1-(4-oxo-3H-phthalazin-1-yl)ethyl]benzamide
Synonyms	AC-55541; AC 55541; AC55541
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	PAR2 ( pEC50 = 6.7 )
ln Vitro	In vitro activity: AC-55541 is a protease-activated receptor (PAR) 2 agonist. With potencies ranging from 200 to 1000 nM, AC-55541 triggered PAR2 signaling in tests for cellular proliferation, phosphatidylinositol hydrolysis, and Ca(2+) mobilization. When given intraperitoneally to rats, AC-55541 was well absorbed and reached micromolar peak plasma concentrations. With 6.1-hour elimination half-lives, AC-55541 remained stable when metabolized by liver microsomes and was exposed to rats for an extended period of time. AC-55541 or AC-264613 administered intrapaw produced edema and strong, long-lasting thermal hyperalgesia. These effects were totally blocked by coadministration of either a transient receptor potential vanilloid (TRPV) 1 antagonist or a tachykinin 1 (neurokinin 1) receptor antagonist. Similar levels of hyperalgesia were seen when AC-55541 or AC-264613 were administered systemically as they were when they were administered locally.
ln Vivo	At the time of testing, 150–200 g male Sprague-Dawley rats from Harrison (Indianapolis, IN) were kept in a climate-controlled room with a 12-hour light/dark cycle (lights on at 7:00 AM) and unlimited access to food and water. Before being used, the rats were kept in pairs for at least two days. The National Institutes of Health's Guide for the Care and Use of Laboratory Animals and the International Association for the Study of Pain's policies and recommendations were followed throughout the entire testing process.
Enzyme Assay	The following assays were used to measure phosphatidylinositol (PI) hydrolysis. In order to generate PAR2 WT, 10,000 HEK 293T cells per well were seeded in DMEM (Invitrogen) supplemented with 10% fetal calf serum, 100 U/ml of penicillin, and 100 mg/ml of streptomycin. The cells were then grown in a 37°C humidified environment with 5% CO2. The cells were transfected using the indicated plasmid DNAs (30 ng/well of a 96-well plate) eighteen hours later, following the procedure previously described. The cells were transfected, and after 20 to 24 hours, they were labeled and washed again using DMEM culture medium that contained 0.2 μCi of NET1114 (37 MBq/ml; PerkinElmer Life and Analytical Sciences, Waltham, MA) per well (0.1 ml).
Cell Assay	In summary, 7 × 103 cells were plated in 0.1 ml of media per well of a 96-well plate one day prior to transfection. A 96-well plate was used, and 10 ng of receptor DNA and 30 ng of pSI-β-galactosidase (Promega) were transiently transfected into each well using Polyfect, as directed by the manufacturer. The following day, the medium was switched, and cells and ligands were mixed together in 200 μl/well of DMEM supplemented with 25% Ultraculture synthetic supplement rather than calf serum. The levels of β-galactosidase were measured exactly as described after five days in culture. Before adding 200 μl of PBS supplemented with 3.5 mM O-nitrophenyl-β-d-galactopyranoside and 0.5% Nonidet P-40 (both from Sigma-Aldrich), cells were rinsed with phosphate-buffered saline (PBS), pH 7.4. Plate readers were used to read the plates at 420 nm following a 2-4 hour incubation period.
Animal Protocol	Male Sprague-Dawley rats
References	[1]. Identification and characterization of novel small-molecule protease-activated receptor 2 agonists. J Pharmacol Exp Ther. 2008 Dec;327(3):799-808. [2]. Role of Aspergillus fumigatus in Triggering Protease-Activated Receptor-2 in Airway Epithelial Cellsand Skewing the Cells toward a T-helper 2 Bias. Am J Respir Cell Mol Biol. 2016 Jan;54(1):60-70.
Additional Infomation	AC-55541 is an organic molecular entity.

Solubility Data

Solubility (In Vitro)

DMSO:≥ 51 mg/mL (~98.4 mM) Water: <1 mg/mL Ethanol: N/A

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.82 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 (4.82 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 (4.82 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	1.9292 mL	9.6458 mL	19.2916 mL
	5 mM	0.3858 mL	1.9292 mL	3.8583 mL
	10 mM	0.1929 mL	0.9646 mL	1.9292 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.