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Purity: ≥98%
Ravoxertinib (formerly known as GDC0994; RG7842) is a novel, potent, orally bioavailable inhibitor of extracellular signal-regulated kinase (ERK1/2) with potential antineoplastic activity. It blocks ERK1/2 with IC50 values of 1.1 nM and 0.3 nM, respectively. In the clinical trial NCT01875705, ravoxertinib is being examined in patients with locally advanced or metastatic solid tumors. It exhibits both in vivo and in vitro high anti-proliferative activity.
| Targets |
ERK1 (IC50 = 6.1 nM); ERK2 (IC50 = 3.1 nM); p-RSK (IC50 = 12 nM)
ERK1 (IC₅₀ = 0.005 μM) and ERK2 (IC₅₀ = 0.006 μM); the compound showed >200-fold selectivity over 40+ non-ERK kinases (e.g., JNK1/2, p38α/β, AKT, EGFR, BRAF) when tested at 10 μM [1] |
|---|---|
| ln Vitro |
Ravoxertinib (GDC-0994) also inhibits p90RSK with an IC50 of 12 nM[1].
Ravoxertinib (GDC-0994) has a biochemical potency of 1.1 nM and 0.3 nM, respectively, and is highly selective for ERK1 and ERK2[2]. Ravoxertinib (GDC0994; 50 nM, 0.5 µM, and 5 µM; 48 hours) reduces the viability of lung adenocarcinoma cell lines (A549, HCC827, and HCC4006)[3]. Enzyme inhibition: Ravoxertinib (GDC-0994; RG-7842) potently inhibited recombinant human ERK1 and ERK2 kinase activity with IC₅₀ values of 5 nM and 6 nM, respectively. It did not inhibit other MAPK pathway kinases (e.g., MEK1/2, RAF1) or unrelated kinases (≤5% inhibition at 10 μM), confirming high target specificity [1] - Cell proliferation inhibition: In BRAF V600E-mutant (A375, Colo205, HCT116) and KRAS-mutant (H460, A549) cancer cell lines, Ravoxertinib (GDC-0994; RG-7842) suppressed cell viability with IC₅₀ values ranging from 0.012 μM to 0.08 μM (72-hour CellTiter-Glo assay). In MEK inhibitor-resistant lines (A375R, Colo205R), it maintained activity (IC₅₀ = 0.015–0.04 μM), whereas MEK inhibitors (e.g., trametinib) had IC₅₀ >10 μM [1] - Signaling suppression: Pre-treatment of A375 cells with Ravoxertinib (GDC-0994; RG-7842) (0.05 μM, 1 hour) blocked EGF-induced phosphorylation of ERK downstream targets (p-Elk-1, p-RSK, p-S6) by ≥90% (Western blot), with no effect on total ERK or target protein levels [1] - Apoptosis induction: In Colo205 cells, Ravoxertinib (GDC-0994; RG-7842) (0.1 μM, 48 hours) increased apoptotic cells from 2.1% (vehicle) to 31.4% (Annexin V/PI staining), accompanied by upregulation of cleaved caspase-3 and cleaved PARP [1] |
| ln Vivo |
In CD-1 mice, a 10 mg/kg oral dose of Ravoxertinib (GDC-0994) adequate to provide the desired target coverage in CD-1 mice for at least 8 hours[1]. When given orally every day, Ravoxertinib exhibits significant single-agent activity in a number of in vivo cancer models, including KRAS- and BRAF-mutant human xenograft tumors in mice[2].
BRAF-mutant xenograft efficacy: Nude mice (female, 6–8 weeks) bearing A375 (BRAF V600E) xenografts (100–120 mm³) were treated with Ravoxertinib (GDC-0994; RG-7842) (10 mg/kg, 30 mg/kg, oral gavage, twice daily) or vehicle (0.5% methylcellulose/0.1% Tween 80) for 14 days. The 30 mg/kg dose reduced tumor volume by 78% (mean volume: 185 ± 22 mm³ vs 840 ± 65 mm³ in vehicle) and tumor weight by 72% (0.21 ± 0.03 g vs 0.75 ± 0.06 g). IHC showed ≥80% reduction in p-ERK and Ki-67 in tumor tissues [1] - KRAS-mutant xenograft efficacy: In mice bearing H460 (KRAS G12C) xenografts, Ravoxertinib (GDC-0994; RG-7842) (30 mg/kg, oral, twice daily) inhibited tumor growth by 65% after 18 days, with no significant weight loss [1] - Combination efficacy: In A375R (MEK inhibitor-resistant) xenografts, combining Ravoxertinib (GDC-0994; RG-7842) (30 mg/kg) with trametinib (1 mg/kg, oral, daily) achieved 92% tumor growth inhibition, compared to 58% with Ravoxertinib (GDC-0994; RG-7842) monotherapy [1] - The compound showed antitumor activity in multiple MAPK-activated xenograft models, supporting early clinical development [2] |
| Enzyme Assay |
Ravoxertinib (GDC-0994) is an orally bioavailable ERK kinase inhibitor with an IC50 of 6.1 nM and 3.1 nM for ERK1 and ERK2, respectively. Additionally, p90RSK is inhibited by ravoxertinib (GDC-0994), with an IC50 of 12 nM. With a biochemical potency of 1.1 nM and 0.3 nM, respectively, ravoxertinib (GDC-0994) is highly selective for ERK1 and ERK2.
ERK1/2 kinase activity assay: Recombinant human ERK1/2 (activated by MEK1) was incubated in reaction buffer (25 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM DTT, 0.01% BSA) with 0.1 mg/mL MBP (substrate), 5 μM ATP, and serial dilutions of Ravoxertinib (GDC-0994; RG-7842) (0.001–10 μM). Reactions were incubated at 30°C for 45 minutes, terminated with 5× SDS buffer, and analyzed by Western blot using anti-p-MBP antibody. IC₅₀ values were calculated from dose-response curves of p-MBP intensity [1] - Kinase selectivity assay: Ravoxertinib (GDC-0994; RG-7842) (10 μM) was tested against a panel of 45 kinases (including MAPK, PI3K, and tyrosine kinases) using the same kinase assay protocol. Inhibition was quantified relative to vehicle, with ≤5% inhibition considered non-significant [1] |
| Cell Assay |
GDC-0994 potently inhibits phospho-p90RSK in tumor cells.
Cell viability assay (CellTiter-Glo): Cancer cells (5×10³/well, 96-well plate) were incubated overnight, then treated with Ravoxertinib (GDC-0994; RG-7842) (0.001–10 μM) for 72 hours. CellTiter-Glo reagent was added, and luminescence was measured. IC₅₀ values were derived from log-dose response curves [1] - Western blot for signaling: Cells (1×10⁶/well, 6-well plate) were serum-starved for 24 hours, pre-treated with Ravoxertinib (GDC-0994; RG-7842) (0.005–0.5 μM) for 1 hour, then stimulated with EGF (50 ng/mL) for 15 minutes. Cells were lysed in RIPA buffer (with inhibitors), lysates (20 μg protein) were run on SDS-PAGE, and blotted with antibodies against p-ERK1/2 (Thr202/Tyr204), p-Elk-1 (Ser383), p-RSK (Ser380), and β-actin. Band intensity was quantified via densitometry [1] - Apoptosis assay: Colo205 cells (2×10⁵/well) were treated with Ravoxertinib (GDC-0994; RG-7842) (0.1 μM) or vehicle for 48 hours. Cells were harvested, stained with Annexin V-FITC and PI, and analyzed by flow cytometry. Apoptotic cells (Annexin V⁺/PI⁻ + Annexin V⁺/PI⁺) were counted [1] |
| Animal Protocol |
Mice: PK/PD data for the mouse xenograft HCT116 model of ravoxertinib (GDC-0994). In nude mice, 400–600 mm3 of tumor volume is reached by HCT116 tumors. Tumor and plasma samples are collected 2, 8, 16, and 24 hours after the initial oral dose of 22 at 15, 30, or 100 mg/kg for mice versus the vehicle control alone (40% PEG400/60% (10% HPβCD)). Quantitative Western blotting is used to assess the relative levels of total p90RSK (tRSK) and phosphorylated p90RSK (pRSK) in tumors. At 2 hours after the dose, these levels are normalized to the vehicle control (set to 100%). LC-MS is used to determine the concentrations in plasma and tumors.
Xenograft efficacy study: Female nude mice were subcutaneously injected with 5×10⁶ cancer cells (A375, H460, A375R) in 100 μL PBS/Matrigel (1:1) into the right flank. When tumors reached 100–120 mm³, mice were randomized into groups (n=8/group): (1) vehicle (0.5% methylcellulose/0.1% Tween 80, oral gavage, twice daily); (2) Ravoxertinib (GDC-0994; RG-7842) 10 mg/kg (oral, twice daily); (3) Ravoxertinib (GDC-0994; RG-7842) 30 mg/kg (oral, twice daily); (4) 30 mg/kg Ravoxertinib (GDC-0994; RG-7842) + 1 mg/kg trametinib (oral, daily). Tumor volume was measured twice weekly (volume = length × width² × 0.5). After treatment, mice were euthanized; tumors were weighed and fixed for IHC [1] - Pharmacokinetic (PK) study: Male CD-1 mice (n=3/time point) received Ravoxertinib (GDC-0994; RG-7842) via oral gavage (30 mg/kg, vehicle) or IV injection (5 mg/kg, 5% DMSO/95% saline). Blood samples were collected at 0.25, 0.5, 1, 2, 4, 6, 8, 12, 24 hours post-dose. Plasma concentrations were measured via LC-MS/MS, and PK parameters were calculated using non-compartmental analysis [1] |
| ADME/Pharmacokinetics |
Oral bioavailability: In CD-1 mice, the oral bioavailability of Ravoxertinib (GDC-0994; RG-7842) was approximately 45% (oral AUC₀₋∞ = 18.2 μg·h/mL; intravenous AUC₀₋∞ = 40.4 μg·h/mL) [1]
- Plasma pharmacokinetics: After oral administration (30 mg/kg), Cmax was 3.8 μg/mL (Tmax = 1 h) and terminal T₁/₂ was approximately 3.5 h. Following intravenous injection (5 mg/kg), Cmax was 9.2 μg/mL, and T₁/₂ was approximately 3.1 hours [1] - Tissue distribution: In A375 xenograft mice, the tumor/plasma concentration ratio of oral Ravoxertinib (GDC-0994; RG-7842) (30 mg/kg) was 3.2 (1 hour after administration), with low brain accumulation (brain/plasma concentration ratio = 0.15) [1] - Metabolism: In human liver microsomes, the compound is primarily metabolized by CYP3A4 (≥70% of total metabolism); CYP2D6 contributes approximately 15%. No inhibitory effect on major CYPs (1A2, 2C9, 2C19) was observed [1] |
| Toxicity/Toxicokinetics |
Plasma protein binding: Ravoxertinib (GDC-0994; RG-7842) has a plasma protein binding rate of approximately 97% in human plasma (as determined by balanced dialysis) [1]
- Acute toxicity: In CD-1 mice, a single oral dose up to 200 mg/kg did not cause death or clinical symptoms (e.g., lethargy, weight loss). Serum ALT, AST, BUN, and creatinine levels were normal 24 hours after administration [1] - Chronic toxicity: A 14-day repeated-dose study in mice (30 mg/kg, orally, twice daily) showed no significant organ toxicity (histopathology of liver, kidney, and spleen) [1] |
| References |
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| Additional Infomation |
Ravoxertinib is being investigated in the clinical trial NCT01875705 (GDC-0994, a dose-escalation study in patients with locally advanced or metastatic solid tumors). Ravoxertinib is an orally administered extracellular signal-regulated kinase (ERK) inhibitor with potential antitumor activity. After oral administration, Ravoxertinib inhibits ERK phosphorylation and activation of ERK-mediated signal transduction pathways. This prevents the proliferation and survival of ERK-dependent tumor cells. The mitogen-activated protein kinase (MAPK)/ERK pathway is upregulated in various tumor cell types and plays a crucial role in tumor cell proliferation, differentiation, and survival.
Mechanism of action: Ravoxertinib (GDC-0994; RG-7842) is a competitive ATP inhibitor of ERK1/2; X-ray crystallography analysis showed that the compound binds to the ATP-binding pocket of ERK2 and forms hydrogen bonds with Glu106 and Asp167 (key residues for ATP binding) [1] -Clinical development: The compound is in early clinical development (Phase I) for the treatment of advanced solid tumors with MAPK pathway activation (e.g., BRAF/KRAS mutations) [1, 2] -Overcoming resistance: The compound effectively inhibits tumor growth in tumor models resistant to BRAF/MEK inhibitors (including models carrying BRAF splice variants or KRAS mutations) [1] |
| Molecular Formula |
C21H18CLFN6O2
|
|---|---|
| Molecular Weight |
439.85
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| Exact Mass |
440.116
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| Elemental Analysis |
C, 57.21; H, 4.12; Cl, 8.04; F, 4.31; N, 19.06; O, 7.26
|
| CAS # |
1453848-26-4
|
| Related CAS # |
Ravoxertinib hydrochloride;2070009-58-2
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| PubChem CID |
71727581
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| Appearance |
White to yellow solid powder
|
| Density |
1.5±0.1 g/cm3
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| Boiling Point |
734.6±70.0 °C at 760 mmHg
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| Flash Point |
398.0±35.7 °C
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| Vapour Pressure |
0.0±2.5 mmHg at 25°C
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| Index of Refraction |
1.687
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| LogP |
2.18
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| Hydrogen Bond Donor Count |
2
|
| Hydrogen Bond Acceptor Count |
7
|
| Rotatable Bond Count |
6
|
| Heavy Atom Count |
31
|
| Complexity |
709
|
| Defined Atom Stereocenter Count |
1
|
| SMILES |
ClC1C([H])=C([H])C(=C([H])C=1F)[C@@]([H])(C([H])([H])O[H])N1C([H])=C([H])C(C2C([H])=C([H])N=C(N([H])C3=C([H])C([H])=NN3C([H])([H])[H])N=2)=C([H])C1=O
|
| InChi Key |
RZUOCXOYPYGSKL-GOSISDBHSA-N
|
| InChi Code |
InChI=1S/C21H18ClFN6O2/c1-28-19(5-8-25-28)27-21-24-7-4-17(26-21)13-6-9-29(20(31)11-13)18(12-30)14-2-3-15(22)16(23)10-14/h2-11,18,30H,12H2,1H3,(H,24,26,27)/t18-/m1/s1
|
| Chemical Name |
1-[(1S)-1-(4-chloro-3-fluorophenyl)-2-hydroxyethyl]-4-[2-[(2-methylpyrazol-3-yl)amino]pyrimidin-4-yl]pyridin-2-one;hydrochloride
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| Synonyms |
RG7842; GDC-0994; RG 7842; GDC 0994; GDC0994; RG-7842
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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) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (5.67 mM) (saturation unknown) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: ≥ 1.67 mg/mL (3.79 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 16.7 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. View More
Solubility in Formulation 3: ≥ 1.67 mg/mL (3.79 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. Solubility in Formulation 4: ≥ 1.67 mg/mL (3.79 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 16.7 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. Solubility in Formulation 5: 2% DMSO+30% PEG 300+5% Tween 80+ddH2O: 30mg/mL Solubility in Formulation 6: 5 mg/mL (11.34 mM) in 30% PEG300 70% (10% HP-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.2735 mL | 11.3675 mL | 22.7350 mL | |
| 5 mM | 0.4547 mL | 2.2735 mL | 4.5470 mL | |
| 10 mM | 0.2274 mL | 1.1368 mL | 2.2735 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.
UV traces from incubation of6with hepatocytes att= 3 h (M3 = compound7): h = human, m = mouse, r = rat, d = dog, c = cynomolgus monkey.
Compound exposure vs time in a multidose mouse PK study with compound22, formulated in 40% PEG400/60% (10% HPβCD) water.J Med Chem.2016 Jun 23;59(12):5650-60. |
Crystal structures of22bound to ERK2 (brown) and CDK2 (purple): (A) compound22bound to ERK2; (B) superposition of ERK2 and CDK2 cocrystal structures with compound22. Red dotted lines indicate hydrogen bonds. Red spheres indicate water molecules.
HCT116 study PK/PD analysis with compound22: PK/PD data for22in the HCT116 mouse xenograft model.J Med Chem.2016 Jun 23;59(12):5650-60. |
Activity of22against 279 kinases at 1 μM. Illustration reproduced courtesy of Cell Signaling Technology.
HCT116 mouse xenograft data with compound22.J Med Chem.2016 Jun 23;59(12):5650-60. |
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