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| Targets |
Apoptosis signal-regulating kinase 1 (ASK1) – a member of the mitogen-activated protein kinase kinase kinase (MAP3K) family that mediates cellular stress responses. Compound 21 is a potent ASK1 inhibitor with a biochemical IC50 of 2.4 nM and a cell IC50 of 138 nM (inhibition of ASK1 autophosphorylation) [1].
IC50: 138/21 nM (ASK1 in cell and Biochemical assays, respectively)[1] |
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| ln Vitro |
In Vitro: Compound 21 exhibited good potency in both biochemical and cellular assays, with a biochemical IC50 of 2.4 nM and a cell IC50 of 138 nM (inhibition of ASK1 autophosphorylation). The compound showed favorable in vitro ADME properties including low microsomal clearance in rat liver microsomes (RLM < 11 mL/min/kg) and human liver microsomes (HLM = 6 mL/min/kg). It had moderate MDCK-MDR1 apparent permeability (Papp = 5.0 × 10⁻⁶ cm/s) with an efflux ratio of 5.0, indicating moderate P-glycoprotein substrate liability. The compound showed high plasma protein binding in both rat (RPPB fu = 5.3%) and human (HPPB fu = 5.4%). In a kinase selectivity profile against a panel of 468 kinases, compound 21 was found to be moderately selective with an S-score(10) of 0.07. Compound 21 was inactive in a hERG assay (IC50 > 30 μM) and showed good CYP inhibition profiles (CYP3A4 IC50 = 8.8 μM, CYP2C9 IC50 > 10 μM) [1].
Compound 21, also known as ASK1-IN-1, exhibits a superior CYP inhibition profile and is not active in the hERG assay. ASK1-IN-1 is determined to be fairly selective after being chosen for a kinase selectivity profile against a panel of 468 kinases [1]. |
| ln Vivo |
No direct in vivo efficacy data (e.g., tumor growth inhibition, disease model studies) for compound 21 were reported in the provided literature. However, a mouse pharmacokinetic study was conducted to predict target coverage. The projected doses to cover the cellular IC50, IC70, and IC90 at trough in the brain for 12 hours were 50, 115, and 435 mg/kg, respectively [1].
The cellular IC50/IC70/IC90 at trough in the brain is covered by ASK1-IN-1 (10 mg/kg; po) for 12 hours at 50/115/435 mg/kg, respectively. In rodents, ASK1-IN-1 has high brain penetration, minimal clearance, and good biochemical and cellular potency[1]. |
| Enzyme Assay |
The biochemical potency of compound 21 was determined using an assay that measured the inhibition of ASK1 kinase activity. The IC50 value of 2.4 nM was obtained from the biochemical assay. Additionally, an X-ray cocrystal structure of compound 21 with human ASK1 was elucidated (PDB code 6VRE). The structure confirmed key hydrogen bond interactions of the amide carbonyl group with the backbone NH of Val 757 and the triazole nitrogen with the catalytic Lys 709. An intramolecular hydrogen bond between the methoxy group and the amide was also observed [1].
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| Cell Assay |
The cellular potency of compound 21 was evaluated using an assay that quantified the level of ASK1 phosphorylation at residue T848 (inhibition of ASK1 autophosphorylation). The cell IC50 was determined to be 138 nM. The compound was also tested in an MDCK-MDR1 (human P-gp transfected) cell line to assess permeability and efflux ratio. The apparent permeability (Papp) from apical-to-basolateral was 5.0 × 10⁻⁶ cm/s, and the efflux ratio (B-A/A-B) was 5.0. For CYP inhibition profiling, the compound was tested using midazolam as a substrate for CYP3A4 (IC50 = 8.8 μM) and tolbutamide as a substrate for CYP2C9 (IC50 > 10 μM). The hERG inhibition assay was performed to assess cardiac safety risk, and compound 21 was found to be inactive with an IC50 > 30 μM [1].
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| Animal Protocol |
For the mouse pharmacokinetic study, compound 21 was administered orally at a dose of 10 mg/kg. The formulation used was not specified in the provided text. Blood and brain samples were collected at various time points to determine drug concentrations and calculate the projected doses needed to maintain brain concentrations above the cellular IC50, IC70, and IC90 for 12 hours. For rat pharmacokinetic experiments described for other analogs, formulations such as DMSO/PG (1:1) or DMA/EtOH/PG/water (1:1:3:5) were used as vehicles [1].
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| ADME/Pharmacokinetics |
In rat pharmacokinetic studies, compound 21 demonstrated low in vivo clearance (Cl = 0.36 L/h/kg, Clu = 6.7 L/h/kg). The compound also exhibited good CNS penetration with a rat Kpu,u value of 0.38, indicating that the unbound brain concentration was 38% of the unbound plasma concentration. In mouse PK experiments, the projected dose to cover the cellular IC50 at trough in the brain for 12 hours was 50 mg/kg. The compound showed low microsomal clearance (RLM < 11 mL/min/kg, HLM = 6 mL/min/kg). Plasma protein binding was high in both rat (fu = 5.3%) and human (fu = 5.4%). The efflux ratio in MDCK-MDR1 cells was 5.0, indicating moderate P-gp substrate liability [1].
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| Toxicity/Toxicokinetics |
Compound 21 was found to be inactive in a hERG assay with an IC50 > 30 μM, indicating low risk for cardiac QT prolongation. The compound showed a favorable CYP inhibition profile with CYP3A4 IC50 = 8.8 μM and CYP2C9 IC50 > 10 μM, suggesting low potential for drug-drug interactions via these major cytochrome P450 enzymes. In the kinase selectivity panel against 468 kinases, compound 21 was moderately selective with an S-score(10) of 0.07, indicating that 7% of kinases tested were inhibited by more than 90% at a compound concentration of 10 μM. No other specific toxicity data (LD50, organ toxicity, etc.) were reported [1].
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| References | |
| Additional Infomation |
Compound 21 (also referred to as ASK1-IN-1 and CAY-10756) is a CNS-penetrant ASK1 inhibitor developed through systematic optimization of a peripherally restricted lead compound. The compound was designed to achieve good brain penetration (rat Kpu,u = 0.38) while maintaining potent ASK1 inhibitory activity (cell IC50 = 138 nM). The X-ray cocrystal structure confirmed the binding mode with key interactions at the ATP-binding pocket of ASK1, including hydrogen bonds with Val 757 and Lys 709. An intramolecular hydrogen bond between the methoxy group and the amide was also observed, which contributed to the improved potency and reduced polar surface area. The compound was selected for further in vivo pharmacological studies based on its balanced profile of potency, low clearance, good CNS penetration, and favorable safety profile (negative hERG, acceptable CYP inhibition). The projected oral dose of 50 mg/kg was estimated to maintain brain concentrations above the cellular IC50 for 12 hours, making this compound suitable for advanced in vivo efficacy studies in neurological disease models such as ALS, multiple sclerosis, and other neurodegenerative conditions where ASK1 plays a pathogenic role [1].
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| Molecular Formula |
C19H19N9O2
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|---|---|
| Molecular Weight |
405.413261651993
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| Exact Mass |
405.166
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| Elemental Analysis |
C, 56.29; H, 4.72; N, 31.09; O, 7.89
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| CAS # |
2411382-24-4
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| PubChem CID |
146027049
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| Appearance |
White to off-white solid powder
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| LogP |
0.4
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
8
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| Rotatable Bond Count |
6
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| Heavy Atom Count |
30
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| Complexity |
580
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| Defined Atom Stereocenter Count |
0
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| SMILES |
O(C)C1C(=CN(C2C=NC=CN=2)N=1)C(NC1=CC=CC(C2=NN=CN2C(C)C)=N1)=O
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| InChi Key |
FTPVMITWJIXRGQ-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C19H19N9O2/c1-12(2)27-11-22-25-17(27)14-5-4-6-15(23-14)24-18(29)13-10-28(26-19(13)30-3)16-9-20-7-8-21-16/h4-12H,1-3H3,(H,23,24,29)
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| Chemical Name |
3-methoxy-N-[6-(4-propan-2-yl-1,2,4-triazol-3-yl)pyridin-2-yl]-1-pyrazin-2-ylpyrazole-4-carboxamide
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| Synonyms |
CAY10756; compound 21; CAY 10756; CAY-10756; ASK1-IN-1
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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 |
| 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) |
DMF: 4 mg/mL (9.87 mM)
DMSO: < 1 mg/mL Ethanol: < 1 mg/mL |
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| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.4666 mL | 12.3332 mL | 24.6664 mL | |
| 5 mM | 0.4933 mL | 2.4666 mL | 4.9333 mL | |
| 10 mM | 0.2467 mL | 1.2333 mL | 2.4666 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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