PAR2 ( pEC50 = 6.7 )
Protease-Activated Receptor 2 (PAR2) (EC50 = 3.2 μM, calcium flux assay in PAR2-expressing CHO cells; EC50 = 4.5 μM, PAR2-mediated ERK1/2 phosphorylation assay in HT-29 cells) [1]
No significant activity on PAR1, PAR3, or PAR4 (EC50 > 100 μM in calcium flux assays) [1]

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.

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1. Selective activation of PAR2: AC 55541 dose-dependently activated PAR2 in CHO cells stably expressing human PAR2, with an EC50 of 3.2 μM (calcium flux assay). It showed no significant activation of other PAR subtypes (PAR1, PAR3, PAR4) at concentrations up to 100 μM, confirming high subtype selectivity [1]
2. Induction of intracellular signaling pathways: AC 55541 (1-30 μM) dose-dependently stimulated ERK1/2 phosphorylation in HT-29 colon adenocarcinoma cells (PAR2-positive) and primary human bronchial epithelial cells (HBECs). At 10 μM, p-ERK1/2 levels increased by 2.8-fold (HT-29) and 2.5-fold (HBECs) compared to vehicle controls (Western blot). It also activated phospholipase C (PLC) signaling, as evidenced by increased inositol phosphate (IP) accumulation (EC50 = 5.1 μM in CHO-PAR2 cells) [1]
3. Promotion of pro-inflammatory cytokine release: AC 55541 (5-30 μM) dose-dependently induced the secretion of IL-6 and IL-8 in HBECs and HT-29 cells. At 30 μM, IL-6 levels increased by 3.2-fold (HBECs) and 2.7-fold (HT-29), while IL-8 levels increased by 4.1-fold (HBECs) and 3.5-fold (HT-29) (ELISA) [1]
4. Enhancement of PAR2-mediated cell migration: AC 55541 (1-10 μM) dose-dependently promoted the migration of HT-29 cells in Transwell assays. At 10 μM, migration rate increased by 65% compared to vehicle controls. Wound healing assays showed a 58% increase in wound closure at 10 μM after 24 hours [1]

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.

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).

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1. PAR2 calcium flux assay: CHO cells stably expressing human PAR2 were seeded in 96-well plates and loaded with a calcium-sensitive fluorescent dye for 30 minutes at 37℃. Serial concentrations of AC 55541 (0.1-100 μM) were added, and fluorescence intensity was measured in real-time to detect intracellular calcium mobilization. EC50 values were derived from dose-response curves of fluorescence intensity changes [1]
2. PAR subtype selectivity assay: CHO cells expressing human PAR1, PAR3, or PAR4 were used in calcium flux assays with AC 55541 (0.1-100 μM) to evaluate subtype selectivity. Activation of each PAR subtype was assessed by fluorescence intensity changes, and EC50 values were calculated [1]
3. Inositol phosphate (IP) accumulation assay: CHO-PAR2 cells were seeded in 24-well plates and labeled with [³H]-inositol for 24 hours. Cells were treated with AC 55541 (0.1-100 μM) for 1 hour, and total inositol phosphates were extracted and quantified by liquid scintillation counting. EC50 values were determined from dose-response curves of IP accumulation [1]

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.

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1. ERK1/2 phosphorylation assay: HT-29 cells or primary HBECs were seeded in 6-well plates and serum-starved for 12 hours. Cells were treated with AC 55541 (1-30 μM) for 5-15 minutes, then lysed in RIPA buffer containing protease/phosphatase inhibitors. Western blot was performed using antibodies against p-ERK1/2 (Thr202/Tyr204), total ERK1/2, and GAPDH (loading control). Band intensities were quantified to assess signaling activation [1]
2. Cytokine secretion assay: HBECs or HT-29 cells were seeded in 24-well plates and treated with AC 55541 (5-30 μM) for 24 hours. Culture supernatants were collected, and IL-6/IL-8 concentrations were measured using specific ELISA kits. Results were normalized to cell number [1]
3. Transwell migration assay: HT-29 cells were resuspended in serum-free medium and seeded in Transwell inserts (8 μm pore size) at 5×10⁴ cells/well. AC 55541 (1-10 μM) was added to both upper and lower chambers, with the lower chamber containing medium with 10% FBS. After 24 hours, migrated cells were fixed, stained with crystal violet, and counted under a microscope [1]
4. Wound healing assay: HT-29 cells were seeded in 6-well plates and grown to confluence. A scratch was created using a pipette tip, and cells were treated with AC 55541 (5-10 μM) in serum-free medium. Wound closure was imaged at 0 and 24 hours, and the percentage of wound closure was calculated [1]

Male Sprague-Dawley rats

1. Cytotoxicity: AC 55541 at concentrations up to 50 μM had no effect on the viability of HT-29 cells, HBEC cells or normal human fibroblasts (MTT assay), indicating that its inherent cytotoxicity was low [1].

[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.

AC-55541 is an organic molecular entity. 1. AC 55541 is a novel small-molecule selective agonist that activates the protease-activated receptor 2 (PAR2), a G protein-coupled receptor (GPCR) that can be activated by proteolytic cleavage of its N-terminal extracellular domain. PAR2 is widely expressed in epithelial cells, immune cells, and fibroblasts, and plays a key role in inflammation, tissue repair, and cancer progression [1]. 2. Its mechanism of action includes binding to the extracellular domain of PAR2 (without proteolytic activation), inducing receptor conformational changes, and activating downstream Gαq/11-mediated signaling pathways (such as PLC-IP3-Ca²⁺ and ERK1/2 MAPK), thereby leading to the release of pro-inflammatory cytokines and cell migration [1]. 3. AC 55541 is highly selective for PAR2, superior to other PAR subtypes, making it a valuable tool compound for studying PAR2-mediated physiological and pathological processes (such as airway inflammation, intestinal mucosal repair, and tumor metastasis). Its low cytotoxicity and strong PAR2-activating efficacy support its application in preclinical studies [1].

C25H20BRN5O3

518.36

517.074

C, 57.93; H, 3.89; Br, 15.41; N, 13.51; O, 9.26

916170-19-9

9589606

White to off-white solid powder

1.5±0.1 g/cm3

1.689

4.65

3

5

6

34

838

0

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

UCUHFWIFSHROPY-RWPZCVJISA-N

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+

N-[2-[(2E)-2-[1-(3-bromophenyl)ethylidene]hydrazinyl]-2-oxo-1-(4-oxo-3H-phthalazin-1-yl)ethyl]benzamide

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

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.

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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.

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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.

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 (Please use freshly prepared in vivo formulations for optimal results.)