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| Targets |
EHT-5372 targets the DYRK family of kinases, including DYRK1A, DYRK1B, DYRK2, and DYRK3, as well as the closely related CLK kinases (CLK1, CLK2, CLK4) and glycogen synthase kinases GSK-3α and GSK-3β. It is a highly potent and selective inhibitor of these kinases, with IC50 values in the low nanomolar to sub-nanomolar range. The primary targets of interest are DYRK1A and DYRK1B, which are implicated in the pathogenesis of Alzheimer's disease, diabetes, and cancer. DYRK1A is known to phosphorylate Tau protein, a key event in the formation of neurofibrillary tangles, and to regulate the production of amyloid-beta (Aβ). By inhibiting DYRK1A, EHT-5372 reduces Tau phosphorylation and Aβ production. The compound also inhibits GSK-3α and GSK-3β, which are also involved in Tau phosphorylation and glycogen metabolism. EHT-5372 has a noncanonical ATP-competitive binding mode that contributes to its exceptional selectivity, with activity restricted almost exclusively to the DYRK and CLK families among 339 tested kinases. This high selectivity is important for minimizing off-target effects and for studying the specific roles of these kinases. The binding mode has been elucidated by co-crystallization with DYRK2. The compound's potent and selective inhibition of these kinases makes it a valuable tool for studying their biological functions and for developing new therapeutic strategies for diseases such as Alzheimer's disease, diabetes, and cancer.
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| ln Vitro |
Cell survival is always greater than 87% even when EHT 5372 (0.1–10 μM; 24 hours) dose-dependently lowers pS396-Tau levels with an IC50 of 1.7 μM [1]. EHT 5372 has an IC50 of 1.06 μM and decreases Aβ generation in a dose-dependent manner[1].
EHT-5372 demonstrates potent in vitro activity against its target kinases and in cellular models of Alzheimer's disease. As a kinase inhibitor, it shows exceptional potency against DYRK1A (IC50 = 0.22 nM) and DYRK1B (IC50 = 0.28 nM), and also inhibits DYRK2, DYRK3, CLK1, CLK2, CLK4, GSK-3α, and GSK-3β with IC50 values ranging from 7.44 to 221 nM. In cellular assays, EHT-5372 (0.01-1 μM) inhibits the direct phosphorylation of Tau by DYRK1A. This demonstrates its ability to block the activity of DYRK1A in a cellular context. Furthermore, EHT-5372 reduces Aβ production in a dose-dependent manner, with an IC50 of 1.06 μM. This effect is particularly relevant for Alzheimer's disease, where Aβ accumulation is a hallmark pathology. In addition to its effects on Tau and Aβ, EHT-5372 dose-dependently reduces pS396-Tau levels with an IC50 of 1.7 μM while maintaining cell viability over 87%, indicating that it can effectively reduce pathological Tau phosphorylation without causing significant cytotoxicity. The compound's exceptional kinase selectivity, with activity restricted almost exclusively to the DYRK and CLK families among 339 tested kinases, underscores its potential as a targeted therapeutic agent. These in vitro findings establish EHT-5372 as a highly potent and selective inhibitor of DYRK and related kinases with significant activity in Alzheimer's disease-relevant cellular models. |
| ln Vivo |
Specific in vivo activity data for EHT-5372 are not detailed in the provided references. However, given its potent in vitro activity against DYRK1A and its ability to reduce Tau phosphorylation and Aβ production, it is expected to have beneficial effects in animal models of Alzheimer's disease. The compound's high potency and selectivity suggest that it could be effective at relatively low doses, potentially minimizing off-target effects. EHT-5372 is a valuable tool for studying the roles of DYRK and CLK kinases in vivo, and it is being investigated for its potential therapeutic applications in Alzheimer's disease, diabetes, and cancer. Further in vivo studies are needed to fully characterize its pharmacokinetic properties and to evaluate its efficacy in disease models.
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| Enzyme Assay |
The in vitro enzyme/receptor binding (non-cellular) assay for EHT-5372 typically involves assessing its inhibitory activity against a panel of kinases using a biochemical kinase assay. In a typical protocol, purified recombinant kinases (e.g., DYRK1A, DYRK1B, DYRK2, DYRK3, CLK1, CLK2, CLK4, GSK-3α, GSK-3β) are incubated with a peptide substrate, ATP, and varying concentrations of EHT-5372. The phosphorylation of the substrate is measured using a method such as radiolabeled ATP (e.g., 33P-ATP) or a fluorescence-based detection system. The IC50 values are then calculated from the dose-response curves. For EHT-5372, this method has been used to determine its IC50 values of 0.22, 0.28, 10.8, 93.2, 22.8, 88.8, 59.0, 7.44, and 221 nM for DYRK1A, DYRK1B, DYRK2, DYRK3, CLK1, CLK2, CLK4, GSK-3α, and GSK-3β, respectively. To assess its selectivity, EHT-5372 is also screened against a broad panel of kinases, and its binding mode can be studied by co-crystallization with a target kinase such as DYRK2.
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| Cell Assay |
Cell viability assay [1]
Cell Types: HEK293 Cell Tested Concentrations: 0.1, 0.5, µM) inhibits the direct phosphorylation of Tau by DYRK1A [1]. 1, 5, 10 μM Incubation Duration: 24 hrs (hours) Experimental Results: Cell viability remained above 87% under all conditions. Western Blot Analysis[1] Cell Types: HEK293 Cell Tested Concentrations: 0.01, 0.03, 0.1, 0.3, 1 μM Incubation Duration: Experimental Results: Effective and dose-dependent inhibition of Tau phosphorylation at pS396. The in vitro cellular assay for EHT-5372 typically involves evaluating its effects on Tau phosphorylation and Aβ production in cell-based models of Alzheimer's disease. In a typical assay for Tau phosphorylation, cells expressing Tau protein (e.g., HEK293 cells) are treated with varying concentrations of EHT-5372 (0.01-1 μM). After incubation, the cells are lysed, and Tau phosphorylation at specific sites (e.g., Ser396) is assessed by Western blot using phospho-specific antibodies. The results show that EHT-5372 inhibits the direct phosphorylation of Tau by DYRK1A in a concentration-dependent manner. For studying Aβ production, cells that produce amyloid precursor protein (APP) are treated with EHT-5372, and the levels of Aβ in the culture medium are measured by ELISA. EHT-5372 reduces Aβ production with an IC50 of 1.06 μM. The effect of EHT-5372 on cell viability is also assessed using an MTT or similar assay, and it maintains cell viability over 87% at concentrations up to 1.7 μM. These cellular assays are crucial for demonstrating the compound's functional activity in a relevant biological context. |
| Animal Protocol |
The in vivo animal experimental protocol for EHT-5372 would typically involve the use of mouse models of Alzheimer's disease, such as transgenic mice expressing mutant APP and/or Tau. In a typical study, EHT-5372 would be administered orally or intraperitoneally to the mice, and the effects on Tau phosphorylation, Aβ levels, and cognitive function would be assessed. For example, mice could be treated with EHT-5372 for several weeks, and brain tissue would be collected at the end of the study to measure Tau phosphorylation and Aβ levels by Western blot and ELISA. Cognitive function could be assessed using behavioral tests such as the Morris water maze. The compound's pharmacokinetic properties and tolerability would also be evaluated. While such studies are not detailed in the provided references, they would be essential for demonstrating the in vivo efficacy of EHT-5372.
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| ADME/Pharmacokinetics |
Specific pharmacokinetic (PK) data for EHT-5372, such as half-life, clearance, volume of distribution, or bioavailability, are not reported in the available literature. However, the compound's potency and selectivity make it a promising candidate for further development. The compound is formulated as a solid and has a purity of 98.12%. It is soluble in DMSO. The powder formulation is stable for up to 3 years when stored at -20°C, and for 6 months when stored in solution at -80°C. Further studies are needed to fully characterize the PK properties of EHT-5372, including its absorption, distribution, metabolism, and excretion (ADME) profile.
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| Toxicity/Toxicokinetics |
Specific toxicity (toxicology) data for EHT-5372 are not reported in the available literature. However, the compound's high selectivity for the DYRK and CLK kinase families suggests a potentially favorable safety profile by minimizing off-target effects. In cellular assays, EHT-5372 maintains cell viability over 87% at concentrations up to 1.7 μM, indicating low cytotoxicity. Comprehensive toxicological assessments, including acute and chronic toxicity studies, genotoxicity, and carcinogenicity, would be necessary to fully evaluate the safety profile of EHT-5372 for clinical development. The compound is for research use only.
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| References |
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| Additional Infomation |
EHT-5372 is a highly potent and selective inhibitor of the DYRK family of kinases. Its chemical name is not provided in the references, but its molecular formula is C17H11Cl2N5OS, and it has a molecular weight of 404.27. EHT-5372 inhibits DYRK1A (IC50 = 0.22 nM), DYRK1B (IC50 = 0.28 nM), DYRK2 (IC50 = 10.8 nM), DYRK3 (IC50 = 93.2 nM), CLK1 (IC50 = 22.8 nM), CLK2 (IC50 = 88.8 nM), CLK4 (IC50 = 59.0 nM), GSK-3α (IC50 = 7.44 nM), and GSK-3β (IC50 = 221 nM). It has a noncanonical ATP-competitive binding mode and exceptional selectivity, with activity restricted almost exclusively to the DYRK and CLK families. EHT-5372 reduces Tau phosphorylation and Aβ production, making it a promising candidate for Alzheimer's disease research. It is also being studied for diabetes and cancer. The compound is available for research use only.
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| Molecular Formula |
C17H11CL2N5OS
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| Molecular Weight |
404.273139238358
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| Exact Mass |
403.006
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| CAS # |
1425945-63-6
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| PubChem CID |
71529610
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| Appearance |
Off-white to yellow solid powder
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| LogP |
5.4
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
7
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| Rotatable Bond Count |
4
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| Heavy Atom Count |
26
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| Complexity |
529
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| Defined Atom Stereocenter Count |
0
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| SMILES |
ClC1C=C(C=CC=1NC1C2C(=CC=C3C=2SC(C(=N)OC)=N3)N=CN=1)Cl
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| InChi Key |
QSGKPYRFWJINEH-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C17H11Cl2N5OS/c1-25-15(20)17-24-12-5-4-11-13(14(12)26-17)16(22-7-21-11)23-10-3-2-8(18)6-9(10)19/h2-7,20H,1H3,(H,21,22,23)
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| Chemical Name |
methyl 9-(2,4-dichloroanilino)-[1,3]thiazolo[5,4-f]quinazoline-2-carboximidate
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| Synonyms |
EHT5372; EHT 5372; EHT-5372
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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) |
DMSO : ~6.67 mg/mL (~16.50 mM)
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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 | 2.4736 mL | 12.3680 mL | 24.7359 mL | |
| 5 mM | 0.4947 mL | 2.4736 mL | 4.9472 mL | |
| 10 mM | 0.2474 mL | 1.2368 mL | 2.4736 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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