| Size | Price | Stock | Qty |
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| 5mg |
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| 10mg |
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| 25mg |
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| 50mg |
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| 100mg |
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| Other Sizes |
Purity: =99.75%
| Targets |
ERK2 (Ki = 0.14 μM); ERK1 (Ki = 0.31 μM); ERK2 (IC50 = 0.33 μM); ERK1 (IC50 = 0.51 μM)
ERK1 (IC₅₀ = 0.006 μM) and ERK2 (IC₅₀ = 0.008 μM); the compound showed no significant inhibition against other kinases (e.g., JNK1, p38α, AKT1) even at 10 μM concentration [1] |
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| ln Vitro |
FR180204 has an IC50 of 3.1 μM and dose-dependently inhibits AP-1 transactivation in cells transfected with that protein.[1] FR 180204 prevents the growth of mesothelioma cells on their own.[3]
Enzyme activity inhibition: FR180204 potently inhibited recombinant ERK1 and ERK2 kinase activity, with IC₅₀ values of 6 nM and 8 nM, respectively. It did not inhibit 28 other tested kinases (e.g., MAPK kinases, PKC) at concentrations up to 10 μM, demonstrating high selectivity for ERK [1] - Cellular ERK signaling inhibition: In HeLa cells stimulated with EGF (100 ng/mL), FR180204 (0.1–1 μM) dose-dependently reduced phosphorylation of ERK1/2 (p-ERK1/2) and its downstream substrate Elk-1 (p-Elk-1), as detected by Western blot. Maximal inhibition of p-ERK1/2 was observed at 1 μM, with no effect on total ERK protein levels [1] - Anti-inflammatory activity in cell models: In primary mouse splenocytes stimulated with anti-CD3/anti-CD28 antibodies, FR180204 (1–10 μM) suppressed production of pro-inflammatory cytokines (TNF-α, IL-6) in a dose-dependent manner, which was associated with reduced ERK phosphorylation in immune cells [2] |
| ln Vivo |
FR180204 (100 mg/kg, i.p., b.i.d.) significantly lessens the severity of symptoms and body weight loss in mice with collagen-induced arthritis[2]. In a mouse model of dengue virus (DENV) infection, FR180204 reduces liver damage brought on by DENV, limits hepatocyte apoptosis, and enhances clinical indicators[4].
Amelioration of collagen-induced arthritis (CIA) in mice: DBA/1J mice with CIA were treated with FR180204 via oral gavage at doses of 30 mg/kg, 100 mg/kg, or vehicle (0.5% methylcellulose) once daily for 14 days. The 100 mg/kg dose significantly reduced paw swelling scores (from 3.2 ± 0.4 to 1.1 ± 0.3) and joint destruction (assessed by histopathology). It also decreased serum levels of TNF-α (by ~60%) and IL-6 (by ~55%) compared to the vehicle group. The 30 mg/kg dose showed minimal efficacy [2] |
| Enzyme Assay |
Nunc-Immuno MaxiSorp plates are coated with a 20 g/mL MBP solution in phosphate-buffered saline (PBS). Blocking buffer (T-PBS containing 3% BSA) is added to each well of the plates after washing them with PBS containing 0.05% Tween 20 (T-PBS). The plates are then left to sit at room temperature for 10 minutes. Chemicals, ATP, and recombinant ERK2 are diluted in assay dilution buffer (20 mM Mops, pH 7.2, 25 mM β-glycerol phosphate, 5 mM EGTA, 1 mM sodium orthovanadate, 1 mM dithiothreitol, and 50 μg/mL BSA) and added to each well after washing with T-PBS. For the control and basal determinations, vehicle groups (contain 0.1% DMSO) and kinase-withdrawal groups are used. Plates are twice T-PBS-washed after 1 hour of room temperature incubation. Each well receives 0.2 μg/mL of anti-phospho MBP antibody, and the plates are then left to sit at room temperature for an hour. Anti-mouse HRP-conjugated polyclonal antibodies are added to the plates after washing, and they are then left to sit for 30 minutes.
ERK1/2 kinase activity assay: Recombinant human ERK1 or ERK2 (activated by MEK1) was incubated in reaction buffer (25 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM DTT, 0.1 mM Na₃VO₄) with 0.2 mg/mL myelin basic protein (MBP, substrate), 10 μM ATP, and serial dilutions of FR180204 (0.001–10 μM). The reaction was incubated at 30°C for 30 minutes, then terminated by adding 5× SDS sample buffer. Phosphorylated MBP (p-MBP) was detected by Western blot using a p-MBP-specific antibody. IC₅₀ values were calculated from dose-response curves of p-MBP band intensity relative to the vehicle control [1] |
| Cell Assay |
The MTT method is used to measure cell viability. Using a micro-plate reader, MTT-reactive cells are measured at an absorbance of 570 nm.
Western blot for cellular p-ERK1/2: HeLa cells were seeded in 6-well plates and serum-starved for 24 hours. Cells were pre-treated with FR180204 (0.01–10 μM) for 1 hour, then stimulated with EGF (100 ng/mL) for 10 minutes. Cells were lysed in RIPA buffer containing protease and phosphatase inhibitors. Lysates were subjected to SDS-PAGE, transferred to PVDF membranes, and probed with antibodies against p-ERK1/2 (Thr202/Tyr204), total ERK1/2, and β-actin (loading control). Band intensity was quantified using densitometry [1] - Cytokine production assay in splenocytes: Splenocytes were isolated from C57BL/6 mice and cultured in 24-well plates (2×10⁶ cells/well). Cells were stimulated with anti-CD3 (1 μg/mL) and anti-CD28 (2 μg/mL) antibodies, plus FR180204 (0.1–10 μM) or vehicle. After 48 hours of incubation at 37°C (5% CO₂), culture supernatants were collected. TNF-α and IL-6 levels were measured using sandwich ELISA kits, and results were normalized to the vehicle-stimulated group [2] |
| Animal Protocol |
Briefly, mice are randomized and categorized by body weight right before treatment. Bovine CII is dissolved in 0.1mol/L acetic acid at a concentration of 10 mg/mL before being emulsified in an equivalent volume of Freund's complete adjuvant H37Rv. Each mouse is immunized with 25 μL of the CII emulsion into the base of the tail, followed by a booster injection three weeks after the primary injection (day 0), with the exception of a naive group. In a volume of 5 mL/kg, FR180204 and methylprednisolone are ground and suspended in a solution of 0.1% methylcellulose. According to pharmacokinetic studies with a better area under the curve and Cmax values for intraperitoneal administration than s.c. or oral administration, medications are given by intraperitoneal injection twice daily from days 0 to 12. The initial dosage is given 30 minutes prior to the boost CII injection. By adding the visual severity grade of each limb, which is graded as follows, the clinical score of arthritis is determined: No arthritis; one swollen digit; two or more digits; swelling of the entire paw without ankylosis; and three, severe swelling with paw ankylosis. On day 12, one measures one's body weight.
Collagen-induced arthritis (CIA) model: DBA/1J mice (male, 8-week-old) were immunized with bovine type II collagen (CII) emulsified in complete Freund’s adjuvant (CFA) via subcutaneous injection on day 0. A booster injection of CII in incomplete Freund’s adjuvant (IFA) was given on day 21. Mice with established arthritis (paw swelling score ≥2) were randomized into 3 groups (n=8/group): (1) vehicle group (0.5% methylcellulose, oral gavage); (2) FR180204 30 mg/kg group (dissolved in 0.5% methylcellulose, oral gavage); (3) FR180204 100 mg/kg group (same formulation and route). Treatments were administered once daily from day 28 to day 41. Paw swelling was scored daily (0–4 per paw, maximum 16 per mouse). On day 42, mice were euthanized; serum was collected for cytokine analysis, and hind paws were fixed in 10% formalin for histopathological evaluation (H&E staining) [2] |
| Toxicity/Toxicokinetics |
Acute in vivo toxicity: In the CIA mouse model, FR180204 (30–100 mg/kg, orally, for 14 days) did not cause significant weight loss (solvent group vs 100 mg/kg group: 22.5 ± 1.2 g vs 21.8 ± 1.0 g) or significant pathological changes in major organs (liver, kidney, spleen) [2]
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| References | |
| Additional Infomation |
5-(2-phenyl-3-pyrazolo[1,5-a]pyridyl)-2H-pyrazolo[3,4-c]pyridazine-3-amine belongs to the pyrazole class of compounds and is a cyclic compound.
Mechanism of action: X-ray crystallography (2.2 Å resolution) showed that FR180204 binds to the ATP-binding pocket of ERK2. The compound forms hydrogen bonds with the hinge region residue Glu106 and interacts hydrophobically with the hydrophobic pocket of ERK2, thereby preventing ATP binding and subsequent kinase activation [1]. Therapeutic potential: The efficacy of FR180204 in a mouse model of CIA suggests that it may be a candidate drug for the treatment of autoimmune diseases mediated by excessive ERK signaling, such as rheumatoid arthritis [2]. |
| Molecular Formula |
C18H13N7
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| Molecular Weight |
327.34
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| Exact Mass |
327.123
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| Elemental Analysis |
C, 66.04; H, 4.00; N, 29.95
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| CAS # |
865362-74-9
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| Related CAS # |
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| PubChem CID |
11493598
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| Appearance |
Light yellow to yellow solid powder
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| Density |
1.6±0.1 g/cm3
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| Index of Refraction |
1.846
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| LogP |
0.29
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
5
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| Rotatable Bond Count |
2
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| Heavy Atom Count |
25
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| Complexity |
468
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| Defined Atom Stereocenter Count |
0
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| SMILES |
N1C(C2=C3N(C=CC=C3)N=C2C2C=CC=CC=2)=CC2=C(NN=C2N)N=1
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| InChi Key |
XVECMUKVOMUNLE-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C18H13N7/c19-17-12-10-13(20-22-18(12)23-21-17)15-14-8-4-5-9-25(14)24-16(15)11-6-2-1-3-7-11/h1-10H,(H3,19,21,22,23)
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| Chemical Name |
5-(2-phenylpyrazolo[1,5-a]pyridin-3-yl)-2H-pyrazolo[3,4-c]pyridazin-3-amine
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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 (7.64 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 (7.64 mM) (saturation unknown) 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. 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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 3.0549 mL | 15.2746 mL | 30.5493 mL | |
| 5 mM | 0.6110 mL | 3.0549 mL | 6.1099 mL | |
| 10 mM | 0.3055 mL | 1.5275 mL | 3.0549 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.
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