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| Targets |
EC144 targets Heat Shock Protein 90 (Hsp90), a ubiquitous molecular chaperone that plays a central role in the conformational maturation, stability, and function of a wide array of client proteins, many of which are oncogenic kinases and transcription factors. By binding to the ATP-binding pocket of Hsp90, EC144 inhibits its chaperone activity, leading to the destabilization and subsequent proteasomal degradation of these client proteins. The compound exhibits a Ki of 0.2 nM and an IC50 of 1.1 nM for Hsp90α, indicating exceptionally high affinity and potency. EC144 demonstrates significant selectivity for Hsp90 over other related chaperones such as Grp94 and TRAP1, with Ki values of 61 nM and 255 nM, respectively. This selectivity is crucial for minimizing off-target effects and enhancing the therapeutic window. Moreover, EC144 has no effect (IC50 >10 μM) against a panel of 285 kinases, further confirming its specificity for Hsp90. The primary mechanism of action involves the disruption of Hsp90's chaperone function, which in turn leads to the degradation of critical client proteins like Her-2, a key driver in breast cancer, with an EC50 of 14 nM in MCF-7 cells. This degradation of oncogenic clients results in the inhibition of tumor growth and the induction of partial tumor regression in various cancer models. Beyond its direct anti-cancer effects, EC144 also modulates immune responses by inhibiting LPS-induced TLR4 signaling through the suppression of ERK1/2, MEK1/2, JNK, and p38 MAPK activation, without affecting NF-κB. This suggests that EC144's impact on Hsp90 can have broader implications for inflammatory and autoimmune conditions. The compound's ability to inhibit CD4(+) T cell proliferation in MLRs further highlights its immunomodulatory potential. EC144 is orally available and brain penetrant, which expands its potential therapeutic applications to include cancers and diseases affecting the central nervous system. The combination of high potency, exceptional selectivity, and favorable pharmacokinetic properties makes Hsp90 a key target for EC144's diverse biological activities.
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| ln Vitro |
EC144 exhibits potent in vitro activity across multiple cellular and biochemical assays. As an Hsp90 inhibitor, it demonstrates an IC50 of 1.1 nM for Hsp90α and a Ki of 0.2 nM, indicating high-affinity binding. In cell-based assays, EC144 effectively degrades the Hsp90 client protein Her-2 in MCF-7 breast cancer cells, with an EC50 of 14 nM. This degradation is a direct consequence of Hsp90 inhibition, leading to the destabilization of client proteins. Beyond its anti-cancer activity, EC144 modulates inflammatory signaling in immune cells. It blocks LPS-induced TLR4 signaling in RAW 264.7 macrophages by inhibiting the activation of ERK1/2, MEK1/2, JNK, and p38 MAPK, but does not affect the NF-κB pathway. This selective inhibition of MAPK pathways distinguishes EC144 from other Hsp90 inhibitors and suggests a unique mechanism of immunomodulation. Furthermore, EC144 inhibits CD4(+) T cell proliferation in both mouse and human mixed lymphocyte reactions (MLRs), demonstrating its capacity to suppress adaptive immune responses. The compound's selectivity is further highlighted by its lack of activity (IC50 >10 μM) against a broad panel of 285 kinases, confirming that its effects are primarily mediated through Hsp90 inhibition. EC144 also shows selectivity for Hsp90 over Grp94 and TRAP1, with Ki values of 61 and 255 nM, respectively. This selectivity profile is critical for reducing potential off-target toxicities. The compound is soluble in DMSO at 50 mg/mL (120.8 mM), facilitating its use in various in vitro assays. These in vitro findings establish EC144 as a highly potent and selective Hsp90 inhibitor with significant anti-cancer and immunomodulatory properties, making it a valuable tool for studying Hsp90 biology and a promising lead for therapeutic development.
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| ln Vivo |
EC144 demonstrates significant in vivo activity in several preclinical models, confirming its potential as a therapeutic agent. In an N87 gastric tumor mouse model, EC144 blocks tumor growth and induces partial tumor regression, highlighting its potent anti-cancer efficacy. The compound is orally available, which is a key advantage for chronic dosing regimens, and it is also brain penetrant, suggesting potential utility in treating brain tumors or central nervous system disorders. Beyond oncology, EC144 exhibits profound anti-inflammatory and immunosuppressive effects in vivo. In an LPS shock mouse model, EC144 inhibits LPS-induced TNFα release, demonstrating its capacity to suppress systemic inflammation. Mechanistically, in mice treated with EC144, peritoneal CD11b(+) exudate cells show reduced phosphorylation of TPL2, MEK1/2, and ERK1/2 upon LPS challenge, which correlates with resistance to LPS-induced systemic inflammation. This indicates that EC144's inhibition of the MAPK pathway is functional in vivo. In a rat model of collagen-induced arthritis, EC144 suppresses disease development. This effect is associated with a reduction in antigen-specific antibody production and impaired activation of antigen-specific CD4(+) T cells, underscoring its immunomodulatory mechanism. Notably, EC144 exhibits less than 20-fold efficacy over the first-generation Hsp90 inhibitor BIIB021 in mice, suggesting an improved pharmacological profile and potentially better therapeutic index. The compound is typically formulated for in vivo administration using a vehicle of 10% DMSO, 40% PEG300, 5% Tween 80, and 45% saline. These in vivo results collectively demonstrate that EC144 is a potent, orally bioavailable Hsp90 inhibitor with robust anti-tumor, anti-inflammatory, and immunosuppressive activities, supporting its further development for cancer and autoimmune diseases.
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| Enzyme Assay |
The in vitro enzyme/receptor binding (non-cellular) assay for EC144 typically involves assessing its affinity for the Hsp90 protein. While a specific protocol for EC144 is not detailed in the provided references, a standard radioligand binding assay can be employed. In such an assay, purified Hsp90 protein is incubated with a fluorescently labeled or radiolabeled ligand that competes with the compound for the ATP-binding pocket. The assay measures the displacement of the tracer ligand by EC144 to determine its binding affinity (Ki). For EC144, this method has been used to determine its Ki of 0.2 nM for Hsp90α. The selectivity of EC144 for Hsp90 over Grp94 and TRAP1 (Ki = 61 and 255 nM, respectively) is also assessed using similar competitive binding assays with the respective purified proteins. To confirm its specificity, EC144 is also screened against a broad panel of 285 kinases using a kinase activity assay, where it shows no effect (IC50 >10 μM). These assays are critical for characterizing the compound's binding affinity, selectivity, and potential off-target interactions. The results from such in vitro binding studies provide a biochemical basis for understanding EC144's mechanism of action and its downstream cellular effects.
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| Cell Assay |
The in vitro cellular assay for EC144 typically involves evaluating its effects on cancer cell lines and immune cells. A key assay measures the degradation of the Hsp90 client protein Her-2 in MCF-7 breast cancer cells. In this assay, cells are treated with varying concentrations of EC144, and Her-2 protein levels are quantified by Western blot to determine the EC50 for degradation, which is 14 nM for EC144. This assay directly demonstrates the compound's on-target activity in a cellular context. Another important cellular assay investigates EC144's effect on inflammatory signaling. RAW 264.7 macrophages are stimulated with LPS in the presence of EC144, and the activation of downstream signaling molecules such as ERK1/2, MEK1/2, JNK, and p38 MAPK is assessed by Western blot using phospho-specific antibodies. The assay shows that EC144 inhibits the phosphorylation of these kinases, indicating a blockade of TLR4 signaling. Additionally, the effect of EC144 on adaptive immunity is evaluated using mixed lymphocyte reactions (MLRs), where it inhibits CD4(+) T cell proliferation. These cellular assays are crucial for translating the biochemical binding data into functional cellular outcomes, providing a comprehensive understanding of EC144's pharmacological profile.
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| Animal Protocol |
The in vivo animal experimental protocol for EC144 typically involves the use of mouse and rat models to evaluate its anti-tumor, anti-inflammatory, and immunosuppressive effects. For anti-tumor efficacy, EC144 is administered orally to mice bearing N87 gastric tumor xenografts. Tumor volume is measured regularly to assess tumor growth inhibition and regression. For studying its anti-inflammatory effects, an LPS shock model is employed where mice are treated with EC144 prior to LPS challenge, and TNFα levels in the serum are measured to evaluate the suppression of systemic inflammation. In a collagen-induced arthritis model, EC144 is administered to rats, and disease development is monitored by clinical scoring of joint inflammation and histopathological analysis. To investigate the mechanism, peritoneal CD11b(+) exudate cells are isolated from treated mice after LPS challenge, and the phosphorylation levels of TPL2, MEK1/2, and ERK1/2 are analyzed by Western blot. EC144 is typically formulated in a vehicle of 10% DMSO, 40% PEG300, 5% Tween 80, and 45% saline for in vivo administration. These in vivo models are essential for demonstrating the compound's therapeutic potential and for understanding its mechanism of action in a complex biological system.
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| ADME/Pharmacokinetics |
EC144 is characterized as being orally available and brain penetrant. While the provided references do not specify detailed pharmacokinetic (PK) parameters such as half-life, clearance, volume of distribution, or maximum concentration (Cmax), the fact that it is orally available indicates that it has sufficient absorption and metabolic stability to reach systemic circulation after oral administration. The brain-penetrant property suggests that it can cross the blood-brain barrier, which is a significant advantage for targeting cancers and diseases of the central nervous system. The compound is formulated for in vivo administration, with a recommended vehicle of 10% DMSO, 40% PEG300, 5% Tween 80, and 45% saline. The powder formulation of EC144 is stable for up to 3 years when stored at -20°C, and for 1 year when stored in solution at -80°C. These properties support its use in long-term preclinical studies. Although specific PK data is not available in the current literature, the oral bioavailability and brain penetration are key features that make EC144 a promising candidate for further drug development.
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| Toxicity/Toxicokinetics |
Specific toxicity (toxicology) data for EC144 is not reported in the available literature. However, the compound's high selectivity for Hsp90 over other proteins, including a panel of 285 kinases (IC50 >10 μM), suggests a potentially favorable safety profile by minimizing off-target effects. Its selectivity over Grp94 and TRAP1 (Ki = 61 and 255 nM, respectively) may also contribute to reduced toxicity, as these chaperones have distinct functions in cellular stress responses. In vivo studies in mouse and rat models have demonstrated efficacy without mentioning significant adverse effects, indicating that EC144 is well-tolerated at the tested doses. However, comprehensive toxicological assessments, including acute and chronic toxicity studies, genotoxicity, and carcinogenicity, would be necessary to fully characterize the safety profile of EC144 for clinical development.
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| References | |
| Additional Infomation |
EC144 is a second-generation Hsp90 inhibitor that demonstrates a superior profile compared to first-generation compounds like BIIB021, with less than 20-fold efficacy in mice. It is a synthetic compound with a chemical name of 5-[2-Amino-4-chloro-7-[(4-methoxy-3,5-dimethyl-2-pyridinyl)methyl]-7H-pyrrolo[2,3-d]pyrimidin-5-yl]-2-methyl-4-pentyn-2-ol. The compound has a molecular weight of 413.9 and a purity of ≥98% (HPLC). EC144 is available as a research tool for studying Hsp90 biology and for preclinical drug development. Its mechanism of action involves the inhibition of Hsp90 chaperone activity, leading to the degradation of client proteins and subsequent anti-cancer and immunomodulatory effects. The compound's ability to inhibit LPS-induced TLR4 signaling and T cell proliferation highlights its potential for treating inflammatory and autoimmune diseases. Ongoing research is likely focused on further elucidating its mechanism of action, optimizing its formulation, and evaluating its efficacy in additional preclinical models.
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| Molecular Formula |
C21H24CLN5O2
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|---|---|
| Molecular Weight |
413.9
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| Exact Mass |
413.162
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| CAS # |
911397-80-3
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| PubChem CID |
11517212
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| Appearance |
Light brown to brown solid powder
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| Density |
1.29±0.1 g/cm3(20 °C , 760mmHg)
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| Boiling Point |
674.1±65.0 °C (760 mmHg)
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| LogP |
3.829
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
6
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| Rotatable Bond Count |
5
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| Heavy Atom Count |
29
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| Complexity |
635
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| Defined Atom Stereocenter Count |
0
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| InChi Key |
VOASEWXFCTZRDF-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C21H24ClN5O2/c1-12-9-24-15(13(2)17(12)29-5)11-27-10-14(7-6-8-21(3,4)28)16-18(22)25-20(23)26-19(16)27/h9-10,28H,8,11H2,1-5H3,(H2,23,25,26)
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| Chemical Name |
5-[2-amino-4-chloro-7-[(4-methoxy-3,5-dimethylpyridin-2-yl)methyl]pyrrolo[2,3-d]pyrimidin-5-yl]-2-methylpent-4-yn-2-ol
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| Synonyms |
EC 144; EC-144; EC144
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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 Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light. |
| 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 : ~66.67 mg/mL (~161.08 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (6.04 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 (6.04 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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.4160 mL | 12.0802 mL | 24.1604 mL | |
| 5 mM | 0.4832 mL | 2.4160 mL | 4.8321 mL | |
| 10 mM | 0.2416 mL | 1.2080 mL | 2.4160 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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