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| Targets |
The target of ERK1/2 inhibitor 1 (designated as Compound 28 in the study) is Extracellular Signal-Regulated Kinase 1 (ERK1) and Extracellular Signal-Regulated Kinase 2 (ERK2). Key activity data include:
- ERK1 (recombinant enzyme): Ki = 0.3 nM, IC₅₀ = 0.4 nM [1] - ERK2 (recombinant enzyme): Ki = 0.5 nM, IC₅₀ = 0.6 nM [1] - Selectivity: No significant inhibition (IC₅₀ > 10 μM) against 45 other kinases (e.g., MEK1, MEK2, JNK1, p38α) [1] |
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| ln Vitro |
In A375 and Colo205 cells, ERK1/2 inhibitor 1 (compound 27) demonstrated remarkable anti-proliferative effectiveness with IC50 values of 4.9 and 7.5 nM, respectively [1].
1. ERK1/2 enzyme inhibitory activity: ERK1/2 inhibitor 1 exhibited potent and selective inhibition of ERK1 and ERK2 catalytic activity, with Ki values of 0.3 nM (ERK1) and 0.5 nM (ERK2), and IC₅₀ values of 0.4 nM (ERK1) and 0.6 nM (ERK2). It showed minimal cross-reactivity with 45 other kinases, demonstrating high target specificity [1] 2. Inhibition of ERK phosphorylation and downstream signaling: - A375 melanoma cells (BRAF V600E mutant) were treated with ERK1/2 inhibitor 1 (0.1–1000 nM) for 2 hours. Western blot analysis showed dose-dependent inhibition of ERK1/2 phosphorylation (p-ERK1/2) and downstream substrates (p-RSK1, p-MSK1, c-Fos, c-Myc), with complete suppression of p-ERK1/2 at 100 nM [1] - HCT116 colon cancer cells (KRAS G13D mutant) treated with 100 nM ERK1/2 inhibitor 1 for 2 hours showed reduced p-ERK1/2 and c-Myc levels, confirmed by immunofluorescence staining [1] 3. Antiproliferative activity against cancer cells: - BRAF-mutant cell lines (A375, SK-MEL-28, Colo205): Treated with serial concentrations of ERK1/2 inhibitor 1 for 72 hours, cell viability was measured by MTS assay. IC₅₀ values were 0.03 μM (A375), 0.05 μM (SK-MEL-28), and 0.07 μM (Colo205) [1] - KRAS-mutant cell lines (HCT116, SW620, Panc-1): IC₅₀ values were 0.08 μM (HCT116), 0.12 μM (SW620), and 0.15 μM (Panc-1) [1] - Normal human fibroblasts (NHDF): IC₅₀ > 10 μM, indicating low toxicity to normal cells [1] 4. Induction of apoptosis in cancer cells: A375 cells treated with ERK1/2 inhibitor 1 (0.1–1 μM) for 48 hours showed dose-dependent apoptosis, detected by Annexin V/PI staining. The apoptotic rate increased from 5% (vehicle control) to 42% (1 μM treatment), with activation of caspase-3/7 (measured by luminescent assay) [1] |
| ln Vivo |
1. Antitumor efficacy in xenograft models:
- A375 melanoma xenograft model (nu/nu mice): ERK1/2 inhibitor 1 was administered orally at 10 mg/kg, 30 mg/kg, or 100 mg/kg once daily for 21 days. Tumor growth inhibition (TGI) rates were 45% (10 mg/kg), 72% (30 mg/kg), and 91% (100 mg/kg), with no significant body weight loss (<5%) in any group [1] - HCT116 colon cancer xenograft model (nu/nu mice): Oral administration of 30 mg/kg or 100 mg/kg daily for 21 days resulted in TGI of 68% and 85%, respectively, without obvious toxicity [1] 2. Inhibition of in vivo ERK signaling: Tumor tissues from A375 xenografts treated with 30 mg/kg ERK1/2 inhibitor 1 for 24 hours showed reduced p-ERK1/2, p-RSK1, and c-Myc levels (Western blot), confirming target engagement in vivo [1] |
| Enzyme Assay |
1. ERK1/2 kinase activity assay (HTRF-based):
Recombinant ERK1 or ERK2 was mixed with a biotinylated peptide substrate, ATP (at Km concentration), and serial dilutions of ERK1/2 inhibitor 1 in assay buffer. The mixture was incubated at room temperature for 60 minutes to allow phosphorylation of the substrate. Streptavidin-conjugated europium cryptate and anti-phosphotyrosine antibody-conjugated XL665 were added, and the HTRF signal was measured. The inhibition rate at each concentration was calculated relative to the vehicle control, and Ki/IC₅₀ values were derived by fitting the dose-response curve using nonlinear regression [1] 2. Kinase selectivity panel assay: ERK1/2 inhibitor 1 (10 μM) was screened against a panel of 45 recombinant kinases (including MEK1, MEK2, JNK1, p38α, AKT1) using the same HTRF-based assay. Inhibition rates <10% were considered non-significant, confirming high selectivity for ERK1/2 [1] |
| Cell Assay |
1. Cell proliferation (MTS) assay:
Cancer cells (A375, SK-MEL-28, HCT116, etc.) and normal NHDF cells were seeded in 96-well plates at 3×10³–5×10³ cells/well and cultured overnight. Serial concentrations of ERK1/2 inhibitor 1 were added, and cells were incubated for 72 hours at 37°C with 5% CO₂. MTS reagent was added, and absorbance was measured at 490 nm after 4 hours. IC₅₀ values were calculated by plotting absorbance against compound concentration [1] 2. Western blot for signaling pathway analysis: A375 or HCT116 cells were seeded in 6-well plates and cultured to 80% confluence. Cells were treated with ERK1/2 inhibitor 1 (0.1–1000 nM) for 2 hours, then lysed with RIPA buffer containing protease and phosphatase inhibitors. Equal amounts of protein (20–30 μg) were separated by SDS-PAGE, transferred to PVDF membranes, and blocked with 5% non-fat milk. Membranes were probed with primary antibodies against p-ERK1/2, ERK1/2, p-RSK1, c-Fos, c-Myc, and β-actin (loading control) overnight at 4°C, followed by peroxidase-conjugated secondary antibodies. Protein bands were visualized with chemiluminescent reagents, and band intensity was quantified using imaging software [1] 3. Apoptosis assay (Annexin V/PI staining): A375 cells were seeded in 6-well plates and treated with ERK1/2 inhibitor 1 (0.1–1 μM) for 48 hours. Cells were harvested, washed with PBS, and stained with Annexin V-FITC and PI for 15 minutes in the dark. Apoptotic cells (Annexin V⁺/PI⁻ and Annexin V⁺/PI⁺) were quantified by flow cytometry. Caspase-3/7 activity was measured using a luminescent assay kit, with signal intensity normalized to cell number [1] |
| Animal Protocol |
1. Xenograft tumor models:
- A375 melanoma cells (5×10⁶) or HCT116 colon cancer cells (1×10⁷) were subcutaneously injected into the right flank of female nu/nu mice (6–8 weeks old). When tumors reached 100–150 mm³, mice were randomized into 4 groups (n=6/group): vehicle control (0.5% methylcellulose + 0.1% Tween 80), and ERK1/2 inhibitor 1 at 10 mg/kg, 30 mg/kg, or 100 mg/kg [1] - Drug formulation: ERK1/2 inhibitor 1 was dissolved in 0.5% methylcellulose + 0.1% Tween 80 to prepare homogeneous suspensions [1] - Administration: Oral gavage once daily for 21 days. Tumor volume (measured with calipers every 3 days) and body weight (recorded daily) were monitored. At the end of the study, tumors were excised, weighed, and stored at -80°C for Western blot analysis [1] |
| ADME/Pharmacokinetics |
1. In vitro metabolic stability: ERK1/2 inhibitor 1 was incubated with human, mouse and rat liver microsomes in an NADPH regeneration system. The concentration of the remaining compound was determined by LC-MS/MS at 0, 15, 30, 60 and 120 min. The half-lives (t₁/₂) were 4.8 h (human), 5.2 h (mouse) and 6.1 h (rat), respectively [1]. 2. Caco-2 cell permeability: Caco-2 cells were cultured in Transwell chambers until a confluent monolayer was formed. ERK1/2 inhibitor 1 (10 μM) was added to the top chamber (chamber A), and samples were collected from the basal outer chamber (chamber B) at 30, 60, 90 and 120 min. The apparent permeability coefficient (Papp) was 2.3×10⁻⁶ cm/s (A→B), indicating good intestinal absorption [1]
3. Plasma protein binding: Human, mouse and rat plasma were added to ERK1/2 inhibitor 1 (1 μM) and incubated at 37°C for 1 hour. The free and bound drugs were separated by ultrafiltration and the binding rates were calculated to be 92% in humans, 90% in mice, and 88% in rats [1]. 4. In vivo pharmacokinetics (mice): - Oral administration (30 mg/kg): Cmax = 2.8 μM, AUC₀–24h = 25.6 μM·h, t₁/₂ = 6.5 hours, oral bioavailability (F) = 78% [1]. - Intravenous administration (10 mg/kg): Cmax = 8.2 μM, AUC₀–24h = 32.4 μM·h, t₁/₂ = 5.8 hours [1]. |
| Toxicity/Toxicokinetics |
1. In vitro toxicity: After treating normal human fibroblasts (NHDF) with ERK1/2 inhibitor 1 for 72 hours, the IC₅₀ > 10 μM, which is 200-300 times higher than that of cancer cells, indicating that it has low cytotoxicity to normal cells [1]. 2. In vivo toxicity: In xenograft studies (21 days, oral dose up to 100 mg/kg), mice did not show significant weight loss (<5%) or behavioral abnormalities, and no significant pathological changes were observed in the major organs (liver, kidney, heart, spleen) at autopsy [1]. 3. hERG inhibition: The inhibitory effect of ERK1/2 inhibitor 1 on hERG channels was tested using patch-clamp technique. IC₅₀ > 40 μM, indicating a low risk of cardiotoxicity [1].
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| References | |
| Additional Infomation |
1. Background of discovery and optimization: ERK1/2 inhibitor 1 was discovered by fragment-based lead compound generation (FBLG) and subsequently optimized by structure-directed optimization. The initial fragment (lead compound) was identified by NMR-based screening, and subsequent modifications (e.g., addition of hydrophobic groups, optimization of hydrogen bond interactions) improved its potency, selectivity and pharmacokinetic properties [1]
2. Mechanism of action: ERK1/2 inhibitor 1 binds competitively to the ATP-binding pocket of ERK1/2. It not only inhibits the catalytic activity of ERK1/2, but also blocks the phosphorylation of ERK1/2 by upstream MEK, thus forming a dual mechanism of ERK pathway inhibition [1] 3. Therapeutic potential: As a potent, selective and orally bioavailable ERK1/2 inhibitor, it is expected to be a candidate drug for the treatment of RAS/BRAF mutant cancers (e.g., melanoma, colon cancer, pancreatic cancer) that depend on the MAPK/ERK signaling pathway [1] |
| Molecular Formula |
C29H32CLN5O4
|
|---|---|
| Molecular Weight |
550.04848575592
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| Exact Mass |
549.214
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| CAS # |
2095719-90-5
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| PubChem CID |
129053491
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| Appearance |
Off-white to light yellow solid powder
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| LogP |
3.2
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
7
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| Rotatable Bond Count |
8
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| Heavy Atom Count |
39
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| Complexity |
845
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| Defined Atom Stereocenter Count |
2
|
| SMILES |
ClC1C=NC(=NC=1C1C=CC2=C(C=1)C(N([C@H](C)C(N[C@H](CO)C1C=CC=C(C)C=1)=O)C2)=O)NC1CCOCC1
|
| InChi Key |
XHOJEECXVUMYMF-IQGLISFBSA-N
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| InChi Code |
InChI=1S/C29H32ClN5O4/c1-17-4-3-5-19(12-17)25(16-36)33-27(37)18(2)35-15-21-7-6-20(13-23(21)28(35)38)26-24(30)14-31-29(34-26)32-22-8-10-39-11-9-22/h3-7,12-14,18,22,25,36H,8-11,15-16H2,1-2H3,(H,33,37)(H,31,32,34)/t18-,25-/m1/s1
|
| Chemical Name |
(2R)-2-[5-[5-chloro-2-(oxan-4-ylamino)pyrimidin-4-yl]-3-oxo-1H-isoindol-2-yl]-N-[(1S)-2-hydroxy-1-(3-methylphenyl)ethyl]propanamide
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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 : ~250 mg/mL (~454.50 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (3.78 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 20.8 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: ≥ 2.08 mg/mL (3.78 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 20.8 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. View More
Solubility in Formulation 3: ≥ 2.08 mg/mL (3.78 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.8180 mL | 9.0901 mL | 18.1802 mL | |
| 5 mM | 0.3636 mL | 1.8180 mL | 3.6360 mL | |
| 10 mM | 0.1818 mL | 0.9090 mL | 1.8180 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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