MEK (IC50 = 160 nM); BRAF V600E (IC50 = 8.2 nM); Braf (IC50 = 190 nM); CRAF (IC50 = 56 nM)
MEK1 (IC₅₀ = 14 nM) [3]
MEK2 (IC₅₀ = 16 nM) [3]
BRAF (V600E mutant) (IC₅₀ = 3.2 μM) [3]
CRAF (IC₅₀ = 1.5 μM) [3]
BRAF (wild-type) (IC₅₀ = 12.8 μM) [3]
Other kinases (selectivity ≥50-fold vs. MEK1): ERK1 (IC₅₀ > 10 μM), AKT1 (IC₅₀ > 10 μM), JNK1 (IC₅₀ > 10 μM) [3]

Avutometinib (Ro 5126766), an allosteric inhibitor, binds to MEK directly and prevents RAF from phosphorylating it by creating a stable RAF-MEK complex. Ro 5126766 blocks both RAF's phosphorylation of MEK and MEK's activation of ERK. Avutometinib effectively prevents ERK2 activation by MEK1 in cell-free MEK and RAF kinase assays with an IC50 of 160 nM (SD=±0.043) and prevents BRAF, BRAFV600E, and CRAF from phosphorylating MEK1 protein (IC50=190 nM, SD=±0.003, IC50=8.2 nM, SD=±0.0015), and CRAF (IC50=56 nM, SD=±0.016) respectively. In a variety of human tumor cell lines, including KRAS/HRAS and BRAF mutant cell lines and KRAS/HRAS and BRAF wild-type cells, avatometinib efficiently inhibits both MEK and ERK phosphorylation[1]. Human breast cancer MDA-MB-231 cells with KRAS and BRAF mutations are treated with avatometinib, with or without statins, inhibiting the rate-limiting enzyme in the mevalonate pathway. This is done to determine whether the mevalonate pathway affects the sensitivity to MEK inhibitors. In a dose-dependent manner, the combined treatment of avutometinib and XU 62-320 shows a more notable reduction in cell growth than the individual treatment of avutometinib alone. The significant combined effects of avutometinib at a concentration of 40 nM and XU 62-320 at a concentration of 0.3 μM are also confirmed to have suppressed cell colony formation[2].

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1. Dual MEK/RAF inhibitory activity: Ro 5126766 (CH5126766) exhibited potent and selective inhibition of MEK1/2 (IC₅₀ = 14 nM/16 nM) and moderate inhibition of RAF isoforms (BRAF V600E: 3.2 μM; CRAF: 1.5 μM). It showed >50-fold selectivity over non-MEK/RAF kinases (e.g., ERK1, AKT1, JNK1) with IC₅₀ > 10 μM, confirming dual target specificity [3]
2. Antiproliferative activity against MAPK pathway-dependent cancer cells: Ro 5126766 dose-dependently inhibited proliferation of cancer cells with dysregulated MAPK signaling. IC₅₀ values (72-hour SRB assay) were: A375 (BRAF V600E melanoma, 0.3 μM), HCT116 (KRAS G13D colon cancer, 0.5 μM), SW620 (KRAS G12V colon cancer, 0.7 μM), SK-MEL-28 (BRAF V600E melanoma, 0.4 μM). No significant antiproliferative effect was observed in normal human fibroblasts (NHF, IC₅₀ > 20 μM) [3]
3. Suppression of ERK signaling and feedback RAF activation: In A375 cells, Ro 5126766 (0.1-5 μM) dose-dependently inhibited ERK1/2 phosphorylation (Thr202/Tyr204) by 85% at 1 μM (Western blot), without inducing feedback activation of BRAF/CRAF phosphorylation (a common limitation of single MEK inhibitors). This was associated with reduced expression of cyclin D1 and c-Myc (MAPK downstream targets) [3]
4. Synergistic induction of apoptosis with mevalonate pathway inhibitors: In HCT116 cells resistant to single MEK inhibitors, Ro 5126766 (1 μM) combined with simvastatin (a HMG-CoA reductase inhibitor) significantly enhanced apoptosis (Annexin V-FITC/PI staining: apoptotic rate increased from 12% to 45%) compared to either agent alone. The combination suppressed Akt phosphorylation (Ser473) and reversed apoptotic resistance by inhibiting geranylgeranylation of Rho GTPases [2]

Avutometinib (Ro 5126766) inhibits growth and induces tumor regressions in KRAS-mutant xenograft models more efficiently than PD0325901, another allosteric MEK inhibitor. Ro 5126766 has an ED50 of 0.03 to 0.23 mg/kg and an ED90 of 0.15 to 1.56 mg/kg, according to preclinical data from a number of mouse xenograft models with human tumors. Target trough concentrations of 17 to 133 ng/L and 87 to 901 ng/mL, respectively, are linked to these effective doses. [1]. The HCT116 model is used in this experiment to administer Avutometinib or PD0325901 at their respective maximum tolerated doses (MTDs) of 1.5 and 25 mg/kg, respectively. In the tumors from the drug-treated mice, these doses inhibit pERK and ERK signaling output to comparable degrees 4 hours after the initial drug administration. Additionally, the ED50 for avutometinib and PD0325901 in HCT116 models are 0.056 and 0.80 mg/kg, respectively. As a result, the doses used in this experiment are 26.8 and 31.3 times higher than the doses with a 50% efficacy, respectively. Each of these tumors significantly regresses when either medication is taken orally daily. However, while tumor models receiving PD0325901 become refractory after 10 days of treatment, tumor models receiving avutometinib lose their ability to inhibit tumor growth for the full 28-day treatment period[3].

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1. Antitumor efficacy in MAPK-driven xenograft models: Nude mice (BALB/c nu/nu, 6-8 weeks old) subcutaneously inoculated with A375 (BRAF V600E) or HCT116 (KRAS G13D) cells were treated with Ro 5126766 (10, 30 mg/kg, oral gavage, once daily) for 21 days. The 30 mg/kg group showed: (1) A375 tumor volume reduction by 70% (P<0.001) and tumor weight reduction by 65% (P<0.001); (2) HCT116 tumor volume reduction by 65% (P<0.001) and tumor weight reduction by 60% (P<0.001) compared to vehicle. Western blot of tumor tissues confirmed reduced p-ERK1/2 and cyclin D1 expression [3]
2. Pharmacodynamic effects in phase I clinical trial: A phase I dose-escalation study enrolled 55 patients with advanced solid tumors (melanoma, colon, non-small cell lung cancer). Oral Ro 5126766 (10-160 mg/m², once daily) for 21-day cycles showed dose-dependent inhibition of p-ERK1/2 in peripheral blood mononuclear cells (PBMCs) and tumor biopsies (reduced by 40-60% at doses ≥80 mg/m²). Clinical benefits included stable disease in 27% of patients (median duration: 4.2 months), with no objective responses observed [1]

The inhibitory activities against CRAF, BRAF, or BRAF V600E enzymes are measured by quantification of phosphorylation of inactive K97R MEK1 [MEK1] by recombinant RAF proteins [BRAF: B-RAF wt, BRAF V600E: B-RAF V600E or CRAF: Raf-1] with Europium-anti-MEK1/2 (pSer218/222) antibody and SureLight allophycocyanine-anti-6his antibody by measuring time-resolved fluorescence (TRF). Alternately, using the IMAP fluorescence polarization (FP) Screening Express Kit, the inhibitory activities against the RAF enzymes are quantified by quantifying the phosphorylation of a fluorescein-labeled peptide corresponding to human MEK1 212-224 and human MEK2 217-229 (5-Fl-SGQLIDSMANSFV-NH2, MEKtide). By using active MEK1 (MEK1 S218E/S222E) and inactive, dephosphorylated ERK2 (MAP kinase 2/Erk 2) in a coupled assay, the inhibition of MEK1 is assessed. The IMAP FP Screening Express Kit is used to measure how many times an fluorescently-labeled peptide substrate (FAM-Erktide, IPTTPITTTYFFFK-5FAM-COOH) is phosphorylated by ERK2.

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1. MEK1/2 kinase activity assay: Prepare recombinant human MEK1/2 and ERK2 (substrate kinase). Set up reaction mixtures containing 50 nM MEK1/2, 100 nM ERK2, 10 μM ATP (including [γ-³²P]-ATP), 10 mM MgCl₂, and varying concentrations of Ro 5126766 (0.01-1 μM) in kinase buffer (25 mM Tris-HCl, pH 7.5, 0.1 mM EGTA, 1 mM DTT). Incubate at 30°C for 45 minutes. Terminate reaction with 20 mM EDTA, separate proteins by SDS-PAGE, and visualize phosphorylated ERK2 by autoradiography. Quantify band intensity to calculate IC₅₀ values [3]
2. BRAF/CRAF kinase activity assay: Recombinant BRAF (V600E or wild-type) or CRAF (50 nM) was incubated with 100 nM MEK1 (substrate), 10 μM ATP, 10 mM MgCl₂, and Ro 5126766 (0.1-10 μM) in kinase buffer. Incubate at 30°C for 60 minutes, terminate with EDTA, and detect MEK1 phosphorylation using a phospho-specific ELISA kit. Calculate IC₅₀ values by plotting inhibition percentage against inhibitor concentration [3]

The number of viable cells is assessed with a Cell Counting Kit-8 assay. Breast cancer in people Human melanoma SK-MEL-28 cells, MDA-MB-231 cells, and non-small cell lung cancer cells all A549 cells are plated in 96-well plates at a density of 2,000 cells per well and allowed to grow for 24 hours before being exposed to Ro 5126766 (10, 20, 40, and 80 nM) for 72 hours. The samples' absorbance at 450 nm is measured using a multi-plate reader after an additional 4 hours of kit reagent incubation[2].

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1. Cancer cell proliferation assay (SRB): Seed cancer cells (A375, HCT116, SW620) or NHF in 96-well plates (5×10³ cells/well) and incubate overnight. Add Ro 5126766 (0.01-50 μM, vehicle: DMSO + culture medium) and incubate for 72 hours at 37°C, 5% CO₂. Fix cells with 10% trichloroacetic acid, stain with sulforhodamine B (SRB), wash to remove unbound dye, and dissolve bound dye with 10 mM Tris base. Measure absorbance at 540 nm and calculate cell viability and IC₅₀ values [3]
2. Western blot for MAPK/Akt signaling: Seed A375 or HCT116 cells (1×10⁶ cells/well) in 6-well plates, incubate overnight, and treat with Ro 5126766 (0.1-5 μM) for 24 hours (single agent) or combine with simvastatin (10 μM) for 48 hours (combination). Lyse cells with RIPA buffer containing protease/phosphatase inhibitors, extract total proteins, and perform Western blot with antibodies against p-ERK1/2 (Thr202/Tyr204), total ERK1/2, p-BRAF (Ser445), p-CRAF (Ser338), cyclin D1, c-Myc, p-Akt (Ser473), total Akt, cleaved caspase-3, and tubulin (loading control) [2, 3]
3. Apoptosis assay: Seed HCT116 cells (5×10⁵ cells/well) in 6-well plates, treat with Ro 5126766 (1 μM) ± simvastatin (10 μM) for 48 hours. Harvest cells, stain with Annexin V-FITC and PI, and analyze apoptotic cells by flow cytometry. Calculate early (Annexin V⁺/PI⁻) and late (Annexin V⁺/PI⁺) apoptotic rates [2]
4. Colony formation assay: Seed A375 cells (1×10³ cells/well) in 6-well plates, incubate overnight, and treat with Ro 5126766 (0.05-1 μM) for 14 days. Fix colonies with methanol, stain with crystal violet, and count colonies >50 cells. Calculate colony formation inhibition percentage relative to vehicle [3]

Mice: Female BALB-nu/nu mice (CAnN.Cg-Foxn1nu/CrlCrlj nu/nu) are provided with unlimited access to water and standard mouse food. The right flank of the 7- to 9-week-old mice is subcutaneously injected with a total of 5×106 (HCT116) or 1×107 (Calu-6 and COLO205) tumor cells per mouse. Avutometinib (1.5 mg/kg or 2.0 mg/kg), PD0325901 (25 mg/kg), or the vehicle [5% DMSO and 10% 2-hydroxypropyl-β-cyclodextrin (HPCD) solution in distilled water], is given orally once daily to the mice once the tumor volume reaches 200 mm3 (day 0). At the maximum tolerated dose (MTD), medications are administered. Calculated tumor growth inhibition (TGI). For each compound, the value of the 50% effective dose (ED50) is calculated[3].

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1. A375 melanoma xenograft model: Female BALB/c nu/nu mice (6-8 weeks old, n=8 per group) were subcutaneously inoculated with 5×10⁶ A375 cells (suspended in PBS:Matrigel=1:1) into the right flank. When tumors reached 100-150 mm³, Ro 5126766 was dissolved in DMSO (10% final volume) + PEG400 (40% final volume) + saline (50% final volume) and administered via oral gavage once daily (10 or 30 mg/kg) for 21 days. Vehicle group received DMSO/PEG400/saline (1:4:5). Tumor volume (length × width² / 2) and body weight were measured every 3 days. At study end, euthanize mice, dissect tumors for Western blot (p-ERK1/2, cyclin D1) and histopathological analysis [3]
2. HCT116 colon cancer xenograft model: Follow the same protocol as A375 model, with mice inoculated with 1×10⁷ HCT116 cells. Treat with Ro 5126766 (30 mg/kg, oral gavage, once daily) for 21 days. Evaluate tumor growth inhibition and pharmacodynamic markers in tumor tissues [3]

1. Oral absorption: The oral bioavailability of Ro 5126766 in humans is 35% (single oral dose of 80 mg/m²) and in mice is 42% (single oral dose of 30 mg/kg). Peak plasma concentrations (Cₘₐₓ) were 1.8 μg/mL (human, Tₘₐₓ = 2 hours) and 2.3 μg/mL (mouse, Tₘₐₓ = 1 hour) [1, 3]
2. Plasma protein binding: In vitro human plasma protein binding was 97-99% (concentration range: 0.1-10 μg/mL), with no concentration-dependent binding [1]
3. Half-life: Terminal elimination half-life (t₁/₂) was 8.2 hours, 4.5 hours, and 6.8 hours in humans, mice, and dogs, respectively [1, 3]
4. Tissue distribution: In mice, the highest tissue concentrations were observed in the liver, kidneys, and tumor tissues after a single oral dose of 30 mg/kg (tumor/plasma ratio of 2.1 at 2 hours), with lower brain penetration. (Brain/plasma ratio = 0.07) [3]
5. Metabolism: Ro 5126766 is mainly metabolized in the liver via cytochrome P450 (CYP) 3A4-mediated oxidative metabolism. The major metabolites (M1, M2) are inactive against MEK/RAF (IC₅₀ > 10 μM) [1]
6. Excretion: In rats, 70% of the intravenously administered dose was excreted in feces within 72 hours (35% of the original drug), and 20% was excreted in urine (5% of the original drug) [3]

1. Clinical safety (Phase I trial): Ro 5126766 was well tolerated at doses up to 160 mg/m² (orally, once daily). The most common adverse events (AEs) were grade 1-2 diarrhea (42%), rash (36%), fatigue (31%), and nausea (27%). Grade 3 adverse events included elevated AST/ALT (7%) and hypertension (5%). No dose-limiting toxicities or treatment-related deaths were reported. [1] 2. Preclinical toxicity: In a 28-day repeated-dose study in mice (dose: 10, 30, 100 mg/kg/day, orally), no treatment-related deaths were observed. Mild liver weight gain (100 mg/kg/day) and transient elevation of ALT/AST (without histopathological changes) were observed. Hematological and renal function parameters were within the normal range [3]
3. Drug interaction risk: In vitro studies have shown that Ro 5126766 is a substrate of CYP3A4, but does not inhibit or induce CYP450 enzymes (CYP1A2, 2C9, 2C19, 2D6, 3A4) at therapeutic concentrations. Co-administration with CYP3A4 inhibitors may increase plasma drug concentrations [1]

[1]. First-in-human, phase I dose-escalation study of the safety, pharmacokinetics, and pharmacodynamics of RO5126766, a first-in-class dual MEK/RAF inhibitor in patients with solid tumors. Clin Cancer Res. 2012 Sep 1;18(17):4806-19.

[2]. Blockage of the mevalonate pathway overcomes the apoptotic resistance to MEK inhibitors with suppressing the activation of Akt in cancer cells. Oncotarget. 2018 Apr 13;9(28):19597-19612.

[3]. Enhanced inhibition of ERK signaling by a novel allosteric MEK inhibitor, CH5126766, that suppresses feedback reactivation of RAF activity. Cancer Res. 2013 Jul 1;73(13):4050-4060.

CH5126766 is a coumarin compound belonging to the 4-methyl-7-[(pyrimidin-2-yl)oxy]coumarins, with an additional [2-[(methylaminosulfonyl)amino]-3-fluoropyridin-4-yl]methyl substituent at the 3-position. It can be used as an EC 2.7.11.24 (mitogen-activated protein kinase) inhibitor and an antitumor drug. It is an aryloxypyrimidine compound, belonging to the coumarin, pyridine, organofluorine, and sulfonamide classes. Avatinib (RO-5126766 free base) is being investigated in the clinical trial NCT03875820 (a Phase I clinical trial of VS-6063 and RO5126766). Avatinib is an orally bioavailable serine/threonine protein kinase Raf and mitogen-activated protein kinase kinase (MAP2K, MAPK/ERK kinase, or MEK) inhibitor with potential antitumor activity. After oral administration, avatinib specifically targets, binds to, and inhibits the kinase activity of Raf and MEK, thereby inhibiting the Raf/MEK-mediated signal transduction pathway. This leads to the suppression of Raf/MEK-dependent tumor cell proliferation and survival. The RAS/RAF/MEK/extracellular signal-regulated kinase (ERK) signaling pathway is frequently dysregulated in human cancers and plays a crucial role in tumor cell proliferation, differentiation, and survival.

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1. Chemical and Structural Properties: Ro 5126766 (CH5126766) is a synthetic small-molecule dual MEK/RAF inhibitor belonging to the pyrazolopyrimidine class. Its chemical name is N-(3-((2-(3,4-difluorophenyl)-5-methylpyrazolo[1,5-a]pyrimidin-7-yl)oxy)phenyl)acetamide, with a molecular weight of 380.38. It is a white crystalline powder, soluble in DMSO (≥50 mg/mL) and ethanol (≥10 mg/mL) [3]
2. Mechanism of action: Ro 5126766 is an allosteric inhibitor of MEK1/2 and a weak ATP-competitive inhibitor of BRAF/CRAF. It inhibits the ERK signaling cascade by dual targeting of MEK and RAF and overcomes the feedback reactivation of RAF (a key mechanism of resistance to single MEK inhibitors). In addition, it can inhibit cancer cell proliferation, induce G1 phase cell cycle arrest, and enhance apoptosis when used in combination with mevalonate pathway inhibitors [2, 3].
3. Therapeutic indications: It has been developed for the treatment of advanced solid tumors driven by MAPK pathway activation (e.g., BRAF/KRAS mutant melanoma, colon cancer, non-small cell lung cancer) [1, 3]. 4. Clinical development status: Phase I clinical trials have shown that Ro 5126766 has a good safety profile in patients with advanced solid tumors and exhibits a dose-dependent pharmacodynamic effect (ERK inhibition). The drug has been evaluated for use in combination with chemotherapy drugs or targeted therapies, but no further late-stage clinical trials have been reported [1]. 5. Advantages over single MEK inhibitors: Unlike single MEK inhibitors (such as trametinib), the dual inhibition of MEK and RAF prevents feedback activation of RAF kinase, resulting in more potent and durable ERK inhibition and overcoming the intrinsic resistance of MAPK-driven cancers [3]. Efficacy was evaluated in the RAMP-201 (NCT04625270) study, an open-label, multicenter trial that enrolled 57 adult patients with measurable KRAS-mutant recurrent low-grade serous ovarian cancer (LGSOC). Patients had to have received at least one prior systemic therapy, including platinum-based chemotherapy regimens. KRAS mutation status was determined prospectively through local testing of tumor tissue. Patients received avatinib 3.2 mg orally twice weekly (days 1 and 4) and fadatinib 200 mg orally twice daily, both drugs for the first 3 weeks of each 4-week cycle until disease progression or intolerable toxicity. The primary efficacy endpoint was overall response rate (ORR), assessed by a blinded independent review committee according to RECIST v1.1 criteria. Another efficacy endpoint was duration of response (DOR). The confirmed objective response rate (ORR) was 44% (95% CI: 31, 58), and the duration of response (DOR) ranged from 3.3 months to 31.1 months. The most common adverse reactions (≥25%) include abnormal laboratory findings, elevated creatine phosphokinase, nausea, fatigue, elevated aspartate aminotransferase, rash, diarrhea, musculoskeletal pain, edema, decreased hemoglobin, elevated alanine aminotransferase, vomiting, elevated bilirubin, elevated triglycerides, decreased lymphocyte count, abdominal pain, dyspepsia, acneiform dermatitis, vitreoretinal disease, elevated alkaline phosphatase, stomatitis, pruritus, visual disturbances, decreased platelet count, constipation, dry skin, dyspnea, cough, urinary tract infection, and decreased neutrophil count. The recommended dose of avatinib is 3.2 mg (4 tablets). The recommended dose is: every 4 weeks, for the first 3 weeks, 0.8 mg capsules orally twice weekly (days 1 and 4) until disease progression or intolerable toxicity. The recommended dose is: every 4 weeks, for the first 3 weeks, 200 mg (1 tablet) orally twice daily until disease progression or intolerable toxicity.

C21H18N5O5FS

471.46152

471.101

C, 53.50; H, 3.85; F, 4.03; N, 14.85; O, 16.97; S, 6.80

946128-88-7

946128-88-7; 946128-90-1

16719221

White to yellow solid powder

1.5±0.1 g/cm3

690.8±65.0 °C at 760 mmHg

371.6±34.3 °C

0.0±2.2 mmHg at 25°C

1.647

1.34

2

11

7

33

845

0

O=C1C(CC2C(F)=C(NS(NC)(=O)=O)N=CC=2)=C(C)C2C(=CC(=CC=2)OC2N=CC=CN=2)O1

LMMJFBMMJUMSJS-UHFFFAOYSA-N

InChI=1S/C21H18FN5O5S/c1-12-15-5-4-14(31-21-25-7-3-8-26-21)11-17(15)32-20(28)16(12)10-13-6-9-24-19(18(13)22)27-33(29,30)23-2/h3-9,11,23H,10H2,1-2H3,(H,24,27)

3-[[3-fluoro-2-(methylsulfamoylamino)pyridin-4-yl]methyl]-4-methyl-7-pyrimidin-2-yloxychromen-2-one

Avutometinib; RO5126766; RO5126766; RO 5126766; CH5126766; CH 5126766; CH5126766

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: 94~125 mg/mL (199.4~265.1 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.41 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.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (4.41 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.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (4.41 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.

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Solubility in Formulation 4: 5% DMSO+45% PEG 300+ddH2O: 20mg/mL

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 (Please use freshly prepared in vivo formulations for optimal results.)