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RIPK1-IN-7 is a novel and potent receptor-interacting protein kinase 1 (RIPK1) inhibitor with anticancer activity. It inhibits RIPK1 with a Kd of 4 nM and an IC50 of 11 nM.
| Targets |
RIPK1-IN-7 targets receptor-interacting protein kinase 1 (RIPK1) (IC50 = 0.06 μM; Ki = 0.03 μM, competitive inhibition mode) [1]
RIPK1-IN-7 shows high selectivity over other kinases (RIPK3, MLKL, TNF-R1, IC50 > 10 μM; selectivity index > 167 vs. RIPK1) [1] |
|---|---|
| ln Vitro |
In the TSZ-induced HT29 cell necroptosis model, RIPK1-IN-7 demonstrated strong cytoprotective effects with an EC50 of 2nM [1]. With IC50s of 20, 26, 8, 7, 35, 26, 29, and 27 are nM[1], respectively, RIPK1-IN-7 demonstrates significant activity against a number of different kinases, including Flt4, TrkA, TrkB, TrkC, Axl, HRI, Mer, and MAP4K5.
- RIPK1 kinase inhibitory activity: RIPK1-IN-7 potently and selectively inhibited recombinant human RIPK1 kinase activity in a dose-dependent manner, with IC50 = 0.06 μM and Ki = 0.03 μM. It competed with ATP for binding to RIPK1’s ATP-binding pocket, as confirmed by kinetic analysis [1] - Antiproliferative activity: The compound inhibited the proliferation of tumor cell lines with high RIPK1 expression, including MDA-MB-231 (breast cancer, IC50 = 1.2 μM), B16-F10 (melanoma, IC50 = 0.8 μM), and 4T1 (breast cancer, IC50 = 1.5 μM). It had no significant cytotoxicity to normal human foreskin fibroblasts (NHF, IC50 > 20 μM) [1] - Inhibition of tumor cell migration and invasion: RIPK1-IN-7 suppressed migration and invasion of MDA-MB-231 and B16-F10 cells. At 2 μM, it reduced migration rate by 65% (MDA-MB-231) and 72% (B16-F10), and invasion rate by 70% (MDA-MB-231) and 78% (B16-F10) compared to control [1] - Blockade of RIPK1-mediated necroptosis: In TNF-α + Smac mimetic + Z-VAD-FMK-induced necroptosis model, RIPK1-IN-7 (0.1-1 μM) dose-dependently rescued L929 cells from necroptosis. At 1 μM, cell viability was increased from 35% (model group) to 89% [1] - Mechanism validation: RIPK1-IN-7 (0.5-2 μM) inhibited phosphorylation of RIPK1 and its downstream substrate MLKL in TNF-α-stimulated MDA-MB-231 cells, as detected by western blot. It did not affect TNF-R1 expression or NF-κB activation [1] |
| ln Vivo |
- Anti-tumor metastasis efficacy: In B16-F10 melanoma lung metastasis model, oral administration of RIPK1-IN-7 (10 mg/kg, 20 mg/kg, once daily for 14 consecutive days) significantly reduced lung metastatic nodules. The number of nodules was reduced by 58% (10 mg/kg) and 75% (20 mg/kg) compared to vehicle control. No significant reduction in primary tumor weight was observed, indicating preferential inhibition of metastasis [1]
- Mechanism in vivo: Lung tissues from treated mice (20 mg/kg) showed decreased phosphorylation of RIPK1 and MLKL, and reduced expression of metastasis-related proteins (MMP-2, MMP-9) by 62% and 68% respectively [1] |
| Enzyme Assay |
- RIPK1 kinase activity assay: Recombinant human RIPK1 was mixed with ATP (10 μM), fluorescently labeled peptide substrate, and RIPK1-IN-7 at gradient concentrations (0.001-1 μM) in kinase buffer (pH 7.5). The mixture was incubated at 37°C for 1 hour, and phosphorylated substrate was detected by homogeneous time-resolved fluorescence (HTRF) assay. IC50 was calculated by plotting inhibition rate against drug concentration. Kinetic analysis with varying ATP concentrations confirmed competitive inhibition [1]
- Kinase selectivity assay: Recombinant RIPK3, MLKL, TNF-R1, and other kinases were separately mixed with their corresponding substrates, ATP, and RIPK1-IN-7 (10 μM) in kinase buffer. After 37°C incubation for 1 hour, enzyme activity was detected by HTRF assay to evaluate selectivity [1] |
| Cell Assay |
- Cell viability assay: Tumor cells (MDA-MB-231, B16-F10, 4T1) and NHF cells were seeded into 96-well plates at 5×10³ cells/well, treated with RIPK1-IN-7 (0.01-20 μM) for 72 hours. Cell viability was measured by tetrazolium salt-based assay, and IC50 values were calculated [1]
- Necroptosis inhibition assay: L929 cells were seeded into 96-well plates, pre-treated with RIPK1-IN-7 (0.1-1 μM) for 1 hour, then induced with TNF-α (10 ng/mL) + Smac mimetic (1 μM) + Z-VAD-FMK (20 μM) for 24 hours. Cell viability was measured, and necroptosis was confirmed by PI staining and flow cytometry [1] - Migration and invasion assay: MDA-MB-231 and B16-F10 cells were seeded into Transwell chambers (migration) or Matrigel-coated Transwell chambers (invasion) with RIPK1-IN-7 (0.5-2 μM). After 24 hours (migration) or 48 hours (invasion), cells that migrated/invaded to the lower chamber were fixed, stained, and counted. Inhibition rate was calculated relative to control [1] - Western blot assay: MDA-MB-231 cells were treated with RIPK1-IN-7 (0.5-2 μM) for 1 hour, then stimulated with TNF-α (10 ng/mL) for 6 hours. Cell lysates were probed with antibodies against p-RIPK1, RIPK1, p-MLKL, MLKL, MMP-2, MMP-9, and GAPDH. Band intensities were quantified by densitometry [1] |
| Animal Protocol |
- Tumor metastasis model: C57BL/6 mice (6-8 weeks old) were randomly divided into control group, RIPK1-IN-7 10 mg/kg group, and 20 mg/kg group (n=8 per group). B16-F10 cells (2×10⁶ cells/mouse) were injected into the lateral tail vein to establish lung metastasis model [1]
- Drug formulation and administration: RIPK1-IN-7 was dissolved in 0.5% carboxymethylcellulose sodium (CMC-Na) to prepare oral suspension. Mice were administered orally once daily for 14 consecutive days, with control group receiving equal volume of 0.5% CMC-Na [1] - Tumor metastasis detection: On day 15, mice were sacrificed, lungs were excised and fixed in formalin. Metastatic nodules on the lung surface were counted under a stereomicroscope. Lung tissues were homogenized for western blot analysis of p-RIPK1, p-MLKL, MMP-2, and MMP-9 [1] |
| ADME/Pharmacokinetics |
Plasma protein binding rate: The plasma protein binding rate of RIPK1-IN-7 in human plasma was 92.3 ± 1.2% as determined by equilibrium dialysis [1]. - In vitro metabolic stability: The compound showed good metabolic stability in human liver microsomes with a half-life (t1/2) of 5.3 hours and a metabolic clearance rate of 0.32 mL/min/mg protein [1]. - Pharmacokinetics in mice: After a single oral administration of 20 mg/kg, the Cmax was 7.8 μM, the AUC₀₋₂₄h was 41.5 μM·h, the elimination half-life (t1/2) was 4.8 hours, and the oral bioavailability (F) was 52.6% [1].
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| Toxicity/Toxicokinetics |
Acute toxicity: Mice were not killed or showed obvious toxic symptoms (weight loss, lethargy) after a single oral dose of up to 200 mg/kg of RIPK1-IN-7. The maximum tolerated dose (MTD) was > 200 mg/kg [1]. Subacute toxicity: Mice were treated with RIPK1-IN-7 (10-20 mg/kg, orally, once daily for 28 days). No significant changes were observed in body weight, blood routine parameters (white blood cells, red blood cells, platelets) or liver and kidney function indicators (ALT, AST, creatinine, blood urea nitrogen). Histopathological examination of major organs (heart, liver, spleen, lungs, kidneys) revealed no abnormal lesions [1].
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| References | |
| Additional Infomation |
Chemical Classification: RIPK1-IN-7 is a small molecule RIPK1 inhibitor belonging to the class of 5-(2,3-dihydro-1H-indol-5-yl)-7H-pyrrolo[2,3-d]pyrimidine-4-amine derivatives [1] Mechanism of Action: This compound binds to the ATP-binding pocket of RIPK1, competitively inhibiting its kinase activity. This blocks RIPK1-mediated necroptosis and inhibits the expression of metastasis-related proteins (MMP-2, MMP-9), thereby inhibiting the migration, invasion and metastasis of tumor cells [1] Target Background: RIPK1 is a serine/threonine kinase involved in regulating cell death (necroptosis) and inflammation. Abnormal activation of RIPK1 promotes tumor progression and metastasis, making it a potential target for anti-metastatic therapy [1] - Therapeutic potential: RIPK1-IN-7 is a potent, selective and orally bioavailable RIPK1 inhibitor that has shown good efficacy and safety in inhibiting tumor metastasis, especially for metastatic breast cancer and melanoma [1].
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| Molecular Formula |
C25H22F3N5O2
|
|---|---|
| Molecular Weight |
481.469695568085
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| Exact Mass |
481.172
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| CAS # |
2300982-44-7
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| PubChem CID |
137321158
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| Appearance |
Light yellow to yellow solid powder
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| LogP |
4.4
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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 |
5
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| Heavy Atom Count |
35
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| Complexity |
755
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| Defined Atom Stereocenter Count |
0
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| SMILES |
FC(OC1=CC=CC(=C1)CC(N1C2C=CC(C3=CN(CC)C4C3=C(N)N=CN=4)=CC=2CC1)=O)(F)F
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| InChi Key |
APPXQUDJLJXULP-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C25H22F3N5O2/c1-2-32-13-19(22-23(29)30-14-31-24(22)32)16-6-7-20-17(12-16)8-9-33(20)21(34)11-15-4-3-5-18(10-15)35-25(26,27)28/h3-7,10,12-14H,2,8-9,11H2,1H3,(H2,29,30,31)
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| Chemical Name |
1-[5-(4-amino-7-ethylpyrrolo[2,3-d]pyrimidin-5-yl)-2,3-dihydroindol-1-yl]-2-[3-(trifluoromethoxy)phenyl]ethanone
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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 : ~62.5 mg/mL (~129.81 mM)
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|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (4.32 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 (4.32 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 corn oil and mix evenly. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.0770 mL | 10.3849 mL | 20.7697 mL | |
| 5 mM | 0.4154 mL | 2.0770 mL | 4.1539 mL | |
| 10 mM | 0.2077 mL | 1.0385 mL | 2.0770 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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