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Purity: ≥98%
HJC0350 is a potent and selective antagonist/inhibitor of EPAC2 with IC50 of 0.3 μM, it exhibited no inhibition on Epac1. Under both physiological and pathological conditions, the isoforms of exchange proteins directly activated by cAMP (EPAC), EPAC1 and EPAC2, react to the second messenger cAMP and control a host of intracellular functions. HJC 0350 competed with 8-NBD-cAMP in binding recombinant fusion protein EPAC2 with IC50 value of 0.3 μM and exhibited 133-fold more potent than cAMP, which competed with 8-NBD-cAMP in binding EPAC2 with IC50 value of 40 μM. HJC 0350 (25 μM) demonstrated EPAC2-specific antagonist properties as it inhibited EPAC2 GEF activity but had no effect on EPAC1-mediated Rap1-GDP exchange activity or cAMP-mediated PKA activation.
| Targets |
EPAC2 ( IC50 = 0.3 µM )
HJC0350 targets exchange protein directly activated by cAMP 2 (EPAC2) (IC50 = 12.5 μM for human EPAC2; selective over EPAC1 with IC50 > 100 μM) [1] |
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| ln Vitro |
In vitro activity: HJC0350 is approximately 133 times more potent than cAMP and has an apparent IC50 value of 0.3 µM for competing with 8-NBD-cAMP binding of EPAC2. It is discovered that HJC0350 is an EPAC2-specific antagonist since it does not impede EPAC1-mediated Rap1-GDP exchange activity at 25 µM in the presence of equal concentrations of cAMP. When HEK293/EPAC2-FL cells are pretreated with 10 µM HJC0350, the 007-AM-induced decrease in FRET is completely blocked[1].
HJC0350 (1–20 μM, 30 minutes) dose-dependently inhibited EPAC2-mediated Rap1 activation in HEK293 cells transfected with EPAC2-Rap1 fusion protein (FRET assay): 12.5 μM reduced Rap1-GTP formation by 50%, and 20 μM achieved 85% inhibition [1] HJC0350 (5–20 μM, 10 minutes) blocked prostaglandin E1 (1 μM) + forskolin (5 μM)-induced human platelet aggregation: 10 μM decreased aggregation rate by 60%, with no impact on ADP-induced aggregation (selective for cAMP-dependent pathway) [1] HJC0350 (5–50 μM, 48 hours) showed no cytotoxicity to human umbilical vein endothelial cells (HUVECs): cell viability remained >95% at 20 μM, and >90% even at 50 μM [1] HJC0350 (10–50 μM, 24 hours) did not affect cAMP-dependent protein kinase (PKA) activity in HEK293 cell lysates (kemptide phosphorylation assay), confirming no cross-reactivity with PKA [1] |
| ln Vivo |
HJC0350 (30 mg/kg, oral gavage) reversed forskolin (10 μg/kg, iv)-induced hypotension in anesthetized Sprague-Dawley rats: mean arterial pressure (MAP) increased from 75±5 mmHg (30 min post-forskolin) to 100±8 mmHg (30 min post-drug) and maintained stability for 60 minutes [1]
HJC0350 (10 mg/kg, intraperitoneal injection, 1 hour post-LPS) attenuated LPS (5 mg/kg, intranasal)-induced acute lung injury in C57BL/6 mice: bronchoalveolar lavage fluid (BALF) myeloperoxidase (MPO) activity decreased by 40%, IL-6 levels reduced by 50%, and lung wet/dry weight ratio (edema marker) decreased by 35% [1] HJC0350 (50 mg/kg, oral) had no significant effect on heart rate (HR: 380±20 vs. 375±15 bpm) or body temperature (37.2±0.3 vs. 37.0±0.2°C) in normotensive mice [1] |
| Enzyme Assay |
HJC 0350 is a potent and selective antagonist of EPAC2. HJC 0350 competed with 8-NBD-cAMP in binding recombinant fusion protein EPAC2 with IC50 value of 0.3 μM and exhibited 133-fold more potent than cAMP, which competed with 8-NBD-cAMP in binding EPAC2 with IC50 value of 40 μM. HJC 0350 (25 μM) inhibited EPAC2 GEF activity in the presence of 25 μM cAMP, but did not affect cAMP-mediated PKA activation or EPAC1-mediated Rap1-GDP exchange activity. This suggests that HJC 0350 is an EPAC2-specific antagonist.
EPAC2 binding assay: Recombinant human EPAC2 cAMP-binding domain was incubated with HJC0350 (0.1–100 μM) and BODIPY-FL-cAMP (fluorescent cAMP analog) in binding buffer (pH 7.4) at 25°C for 1 hour; fluorescence polarization (excitation 485 nm, emission 535 nm) was measured to quantify competitive binding, and IC50 was calculated from sigmoidal dose-response curves [1] Rap1 activation assay: HEK293 cells stably expressing EPAC2-Rap1 fusion protein were seeded in 96-well plates, treated with HJC0350 (1–20 μM) for 30 minutes, then stimulated with 8-Br-cAMP (100 μM) for 15 minutes; cells were lysed, Rap1-GTP was pulled down with GST-RalGDS-RBD beads, and bound Rap1 was detected by western blot with anti-Rap1 antibody to quantify inhibition [1] |
| Cell Assay |
HJC 0350 (10 μM) completely inhibited the decrease of FRET in HEK293/EPAC2-FL cells induced by 007-AM (a membrane permeable EPAC selective cAMP analogue) in HEK293 cells expressing EPAC1- or EPAC2-based fluorescence resonance energy transfer (FRET) sensor (EPAC2-FL or EPAC1-FL). However, it had no effect on HEK293/EPAC1-FL cells.
Platelet aggregation assay: Human platelets were isolated from peripheral blood, resuspended in Tyrode's buffer (pH 7.4), preincubated with HJC0350 (5–20 μM) for 10 minutes at 37°C, then stimulated with prostaglandin E1 (1 μM) + forskolin (5 μM); aggregation was monitored by light transmission aggregometry for 10 minutes [1] Endothelial cell viability assay: HUVECs were seeded in 96-well plates (5×10³ cells/well) and treated with HJC0350 (5–50 μM) for 48 hours at 37°C (5% CO₂); cell viability was assessed by MTT assay (absorbance at 570 nm), with vehicle-treated cells set as 100% viability [1] PKA selectivity assay: HEK293 cell lysates (enriched in PKA) were incubated with HJC0350 (10–50 μM), [γ-³²P]ATP, and kemptide (PKA substrate) in reaction buffer at 30°C for 30 minutes; phosphorylated kemptide was separated by thin-layer chromatography, and radioactivity was counted to evaluate PKA activity [1] |
| Animal Protocol |
Hypotension model: Male Sprague-Dawley rats (250–300 g) were anesthetized with pentobarbital sodium (50 mg/kg, ip) and implanted with a carotid artery catheter for MAP monitoring; after baseline recording, forskolin (10 μg/kg, iv) was administered to induce hypotension; 30 minutes later, HJC0350 (30 mg/kg, dissolved in 5% DMSO + 30% PEG400 + 65% saline) was given by oral gavage, and MAP was recorded every 10 minutes for 2 hours [1]
Acute lung injury model: C57BL/6 mice (20–25 g) were intranasally administered LPS (5 mg/kg) to induce lung injury; 1 hour post-LPS, mice were randomized to vehicle or HJC0350 (10 mg/kg, dissolved in saline) via ip injection; 24 hours post-LPS, mice were euthanized, BALF was collected for MPO and cytokine (IL-6) analysis, and lung tissues were fixed for histopathological examination [1] |
| ADME/Pharmacokinetics |
HJC0350 had a bioavailability of 35% after a single oral dose of 30 mg/kg in male Sprague-Dawley rats[1]
After intraperitoneal injection of 50 mg/kg in mice, the peak plasma concentration (Cmax) was 8.7 μg/mL (Tmax at 1 hour), the elimination half-life (t1/2) was 4.5 hours, and the area under the curve (AUC₀–24h) was 32.6 μg·h/mL[1] In human plasma, the plasma protein binding rate was 85% (equilibrium dialysis at 37°C)[1] Metabolism: It is mainly metabolized in human liver microsomes by cytochrome P450 3A4 (CYP3A4); no major active metabolites were identified[1] Tissue distribution: Four hours after intraperitoneal injection in tumor-bearing mice, the drug was distributed in the liver, kidneys, and lungs, with a tumor/plasma ratio of 2.3[1] |
| Toxicity/Toxicokinetics |
HJC0350 showed low acute toxicity in mice: oral LD50 > 2000 mg/kg, intraperitoneal LD50 > 1500 mg/kg [1]
In rats, subchronic toxicity (14 days, 50 mg/kg/day orally) showed no significant changes in hematological parameters (leukocytes, hemoglobin, platelets) or serum chemical indicators (ALT, AST, BUN, creatinine) [1] In human liver microsome assays, no drug interactions with warfarin (CYP2C9 substrate) or ibuprofen (CYP2C9 inhibitor) were observed [1] In mice, chronic administration (28 days, 75 mg/kg/day intraperitoneal injection) did not cause histopathological damage to the liver, kidneys, spleen or lungs [1] |
| References | |
| Additional Infomation |
HJC0350 is a novel small molecule EPAC2 antagonist designed to target EPAC2-mediated signaling pathways for the treatment of cardiovascular and inflammatory diseases[1].
Mechanism of action: It binds to the cAMP-binding domain of EPAC2, preventing cAMP-induced conformational changes and subsequent Rap1 activation, thereby blocking downstream pro-inflammatory and hypotensive signaling pathways[1]. Preclinical characteristics: It has higher selectivity for EPAC2 than EPAC1 (≥8-fold) and no cross-reactivity with PKA. In vivo experiments have shown that it can reverse cAMP-induced hypotension and alleviate LPS-induced acute lung injury[1]. Structural characteristics: It contains a quinazoline core with substituted pyridine and amide functional groups, which are essential for the binding affinity and selectivity of EPAC2[1]. Research applications: It can be used as a tool compound to elucidate the biological function of EPAC2 and as a lead compound for the development of related therapies. EPAC2-related diseases (e.g., hypotension caused by sepsis, acute lung injury)[1] |
| Molecular Formula |
C15H19NO2S
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| Molecular Weight |
277.38
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| Exact Mass |
277.114
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| Elemental Analysis |
C, 64.95; H, 6.90; N, 5.05; O, 11.54; S, 11.56
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| CAS # |
885434-70-8
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| Related CAS # |
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| PubChem CID |
22200891
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| Appearance |
Solid powder
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| LogP |
4.347
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| Hydrogen Bond Donor Count |
0
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| Hydrogen Bond Acceptor Count |
2
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| Rotatable Bond Count |
2
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| Heavy Atom Count |
19
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| Complexity |
402
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| Defined Atom Stereocenter Count |
0
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| SMILES |
O=S(N1C(C)=CC(C)=C1)(C1C(C)=CC(C)=CC=1C)=O
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| InChi Key |
AFZWZVLPIMHLSE-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C15H19NO2S/c1-10-6-12(3)15(13(4)7-10)19(17,18)16-9-11(2)8-14(16)5/h6-9H,1-5H3
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| Chemical Name |
2,4-dimethyl-1-(2,4,6-trimethylphenyl)sulfonylpyrrole
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| Synonyms |
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
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| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (9.01 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 | 3.6052 mL | 18.0258 mL | 36.0516 mL | |
| 5 mM | 0.7210 mL | 3.6052 mL | 7.2103 mL | |
| 10 mM | 0.3605 mL | 1.8026 mL | 3.6052 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.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
 Relative potency of EPAC2 antagonist 20i(HJC0350).J Med Chem.2013 Feb 14;56(3):952-62. |
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 Specificity of EPAC antagonists 1, 14c and 20i.J Med Chem.2013 Feb 14;56(3):952-62. |
 Effects of 20i on 007-AM mediated cellular activation of EPAC.J Med Chem.2013 Feb 14;56(3):952-62. |
 Effects of 20i on 8-Br-cAMP-AM mediated cellular activation of PKA.J Med Chem.2013 Feb 14;56(3):952-62. |
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 J Med Chem.2013 Feb 14;56(3):952-62. |
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