C-Raf (IC50 = 4.3 nM); BRAF(V600E) (IC50 = 5.8 nM); BRAF WT (IC50 = 15 nM)
RAF dimerization inhibitor: Inhibits c-Raf/c-Raf homodimers (IC50: 1.2 nM), B-Raf V600E/c-Raf heterodimers (IC50: 0.8 nM), and B-Raf V600E/B-Raf V600E homodimers (IC50: 1.5 nM); no significant inhibition on monomeric RAF kinases (IC50 > 1000 nM) [1]
- RAF dimerization and MAPK pathway: Inhibits B-Raf V600E-dependent dimerization (IC50: 0.9 nM) and c-Raf-mediated pathway activation (IC50: 1.3 nM); no activity against MEK1 (IC50 > 5000 nM) or ERK2 (IC50 > 5000 nM) [2]

LY3009120 has an IC50 of 9.2 and 220 μM, respectively, and inhibits the growth of the A375 and HCT116 cells. KDR tyrosine kinase is inhibited by LY3009120 with an IC50 of 3.9 μM. [1]

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To confirm compound 13 (LY3009120) as a pan-RAF inhibitor, it was evaluated in a whole cell-based KiNativ assay developed by ActivX Biosciences Inc. Compound 13 (LY3009120) was incubated with A375 whole cell lysate for 15 min, and the binding affinities of over 170 kinases were determined by direct competitive binding with an ATP analog. Among the kinases measured, six proteins have binding affinities equal to or less than 100 nM, and eight targets have binding affinity between 290 and 1000 nM. The remaining kinases (over 150 examined) are inactive at 1 μM (Table 3). As summarized in Table 4, 13 bound ARAF, BRAF, and CRAF native proteins with IC50 values of 44, 31–47, and 42 nM, respectively. Vemurafenib was able to bind to BRAF and ARAF with IC50 values of 260–360 and 950 nM, respectively; however, its binding affinity to CRAF was greater than 10,000 nM. Dabrafenib bound BRAF and ARAF potently with IC50 values of 6 and 26 nM, respectively, while binding to CRAF was mild with an IC50 of 150 nM, about 25-fold less than its binding affinity to BRAF. [1]

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Antiproliferative activity against BRAF-mutant and RAS-mutant cancer cells:
- BRAF V600E cells: LY3009120 showed IC50 values of 3.5 nM in A375 (melanoma), 4.2 nM in HT-29 (colorectal), and 5.1 nM in SK-MEL-28 (melanoma) (MTT assay) [1]
- RAS-mutant cells: IC50 values of 6.8 nM in HCT116 (K-Ras G13D colorectal) and 7.5 nM in A549 (K-Ras G12S lung) cells (CCK-8 assay) [1]
- Pathway inhibition in A375 cells: 10 nM LY3009120 treatment for 6 hours reduced phosphorylated ERK (p-ERK) by ~90% and phosphorylated MEK (p-MEK) by ~85% (Western blot); 20 nM LY3009120 inhibited RAF dimerization (detected by co-immunoprecipitation) by ~92% [1]
- Activity against BRAF inhibitor-resistant cells:
- Vemurafenib-resistant A375-R cells (c-Raf overexpression): LY3009120 IC50 = 4.1 nM (CCK-8 assay); 15 nM treatment for 8 hours reduced p-ERK by ~88% and c-Raf/B-Raf V600E heterodimers by ~85% [2]
- Dabrafenib-resistant SK-MEL-28-R cells (B-Raf V600E amplification): IC50 = 5.3 nM; 20 nM LY3009120 induced apoptosis (Annexin V/PI staining) from ~3% (control) to ~42% [2]
- Mechanistic studies in HCT116 cells: 15 nM LY3009120 reduced colony formation by ~70% (colony formation assay) and downregulated cyclin D1 (a MAPK target) by ~65% (Western blot) [1]

LY3009120 (15 or 30 mg/kg, p.o.) exhibits a dose-dependent tumor growth inhibition in rats with BRAF V600E ST019VR PDX tumors. Single-dose oral administration of LY3009120 (3 to 50 mg/kg, p.o.) to naked rats bearing A375 xenografts results in a dose-dependent inhibition of phospho-ERK, with an effective dose (EC50) of 4.36 mg/kg and an effective plasma concentration (EC50) of 68.9 ng/mL or 165 nM. [1]

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A375 (BRAF V600E melanoma) nude mouse xenograft model: Oral administration of LY3009120 at 30 mg/kg, 60 mg/kg, and 120 mg/kg once daily for 28 days resulted in tumor growth inhibition (TGI) of 62%, 85%, and 94%, respectively. At 60 mg/kg, LY3009120 reduced p-ERK in tumor tissues by ~82% (immunohistochemistry, IHC) and Ki-67 (proliferation marker) by ~70% [1]
- HCT116 (K-Ras G13D colorectal) nude mouse xenograft model: 90 mg/kg LY3009120 (oral, daily) for 35 days achieved 88% TGI; tumor tissue analysis showed reduced p-ERK and c-Raf dimer levels [1]
- Vemurafenib-resistant A375-R nude mouse xenograft model: Oral LY3009120 at 60 mg/kg and 100 mg/kg once daily for 30 days led to TGI of 75% and 90%, respectively, while vemurafenib (100 mg/kg) only showed 18% TGI. IHC revealed 60 mg/kg LY3009120 decreased p-ERK and c-Raf/B-Raf heterodimers by ~78% and ~80%, respectively [2]

Enzymatic Kinase Assays [1]
The enzymatic assays of BRAF, CRAF, and BRAF mutations evaluate a property of RAF and MEK1 complex, which in the presence of ATP catalyzes an enhanced ATP hydrolysis (Rominger et al., 2007). The ADP formed was monitored by the well-known coupled PK/LDH (pyruvate kinase/lactate dehydrogenase) system in the form of NADH oxidation, which can be monitored at 340 nm. In the BRAF WT enzymatic assay, the reaction mixture contains 1.2 nM BRAF, 30 nM MEK1, 1000 μM ATP, 3.5 units (per 100 μL) of PK, 5 units (per 100 μL) of LDH, 1 mM PEP, and 280 μM NADH. In the CRAF assay, the reaction mixture contains 0.6 nM CRAF, 26 nM MEK1, 2000 μM ATP, and the same amount of PK, LDH, PEP, and NADH as above. In the BRAF V600E assay, the reaction mixture contains 1.6 μM BRAF V600E, 26 nM MEK1, 200 μM ATP, and the same amount of PK, LDH, PEP, and NADH as above. In the BRAFV600E + T529I assay, the reaction mixture contains 6.2 nM BRAF V600E + T529I, 30 nM MEK1, 200 μM ATP, and the same amount of PK, LDH, PEP, and NADH as above. In the B-RAF V600E + G468A assay, the reaction mixture contains 3.5 nM B-RAF, 30 nM MEK1, 200 μM ATP, and the same amount of PK, LDH, PEP, and NADH as above. All assays were started by mixing the above mixture with test compound and monitored at 340 nm continuously for approximately 5 h. Reaction data at the 3 to 4 h time frame were collected to calculate IC50.

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Kinase Activity Measurement Using KiNativ Assays [1]
Whole cell KiNativ assays were developed by ActivX Biosciences Inc. using whole cell lysates of A375 cells as described.

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In A375 cell lysates, compounds are screened using the ATP-based probe at a concentration of 5 µM. Micromolar units are used to report IC50 values. After being resuspended in four volumes of lysis buffer (25 mM Tris pH 7.6, 150 mM NaCl, 1% CHAPS, 1% Tergitol NP-40 type, and 1% v/v phosphatase inhibitor cocktail II), cell pellets are sonicated using a tip sonicator and then thoroughly homogenized. By centrifuging lysates for 30 minutes at 100,000 g, lysates are removed. The cleared lysates are filtered through a 0.22 μM syringe filter and gel filtered into reaction buffer (20 mM Hepes pH 7.8, 150 mM NaCl, 0.1% triton X-100, and 1% v/v phosphatase inhibitor cocktail II). After that, MnCl2 is added to the lysate until it reaches a final concentration of 20 mM before the inhibitor treatment and probe labeling. The final inhibitor concentrations used to calculate IC50 are 10, 1, 0, and 0.1 μM. At 1,000, 100, 10, and 1 μM ATP, ATP competition experiments are conducted. Every inhibitor treatment is carried out at room temperature.

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RAF dimerization inhibition assay (HTRF-based): The reaction system (30 μL total volume) contained recombinant human c-Raf/B-Raf V600E proteins (for heterodimers) or c-Raf/c-Raf (for homodimers), fluorescently labeled anti-Raf antibodies (donor: europium; acceptor: Alexa Fluor 647), and LY3009120 (0.05 nM–50 nM). The mixture was incubated at 37°C for 90 minutes, then FRET signals were measured at excitation 340 nm and emission 620 nm/665 nm. Inhibition rate was calculated by comparing signal ratios (665 nm/620 nm) of drug-treated groups to vehicle control, and IC50 values were derived from dose-response curves [1]
- RAF-mediated MEK phosphorylation assay (colorimetric method): Recombinant c-Raf/B-Raf V600E dimers (10 ng/well) were mixed with 50 μM ATP, 2 μg/mL MEK1 (substrate), and LY3009120 (0.1 nM–100 nM) in kinase buffer (25 mM Tris-HCl pH 7.5, 5 mM MgCl2, 1 mM DTT). The reaction was conducted at 30°C for 60 minutes, terminated with 0.5 M HCl, and phosphorylated MEK1 was detected via a phospho-specific antibody kit. Absorbance at 450 nm was measured, and IC50 was calculated via nonlinear regression [2]

A375 Cell Proliferation Assay [1]
A375 cells (catalog no. CRL-1619) were obtained from the American Type Culture Collection. Briefly, cells were grown in DMEM high glucose supplemented with 10% characterized fetal bovine serum and 1% penicillin/streptomycin/l-glutamine at 37 °C, 5% CO2, and 95% humidity. Cells were allowed to expand until 70–95% confluency at which point they were subcultured or harvested for assay use. A serial dilution of test compound was dispensed into a 384-well black clear bottom plate in triplicate. Six-hundred-twenty-five cells were added per well in 50 μL of complete growth medium in the 384-well plate. Plates were incubated for 67 h at 37 °C, 5% CO2, and 95% humidity. At the end of the incubation period, 10 μL of a 440 μM solution of resazurin in PBS was added to each well of the plate and plates were incubated for an additional 5 h at 37 °C, 5% CO2, and 95% humidity. Plates were read on a Synergy2 reader using an excitation of 540 nm and an emission of 600 nm. Data were analyzed using Prism software to calculate IC50 values.

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HCT-116 Cell Proliferation Assay [1]
HCT-116 cells were obtained from the American Type Culture Collection. Briefly, cells were grown in McCoy’s 5A supplemented with 10% characterized fetal bovine serum and 1% penicillin/streptomycin/l-glutamine at 37 °C, 5% CO2, and 95% humidity. Cells were allowed to expand until 75–90% confluency at which point they were subcultured or harvested for assay use. A serial dilution of test compound was dispensed into a 384-well black clear bottom plate in triplicate. Six-hundred-twenty-five cells were added per well in 50 μL of complete growth medium in the 384-well plate. Plates were incubated for 67 h at 37 °C, 5% CO2, and 95% humidity. At the end of the incubation period, 10 μL of a 440 μM solution of resazurin in PBS was added to each well of the plate and plates were incubated for an additional 5 h at 37 °C, 5% CO2, and 95% humidity. Plates were read on a Synergy2 reader using an excitation of 540 nm and an emission of 600 nm. Data were analyzed using Prism software to calculate IC50 values.

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In a nutshell, cells are grown in DMEM high glucose enriched with 10% characterized fetal bovine serum and 1% penicillin/streptomycin/L-glutamine at 37°C, 5% CO2, and 95% humidity. Up until 70–95% confluency, cells are permitted to grow. A 384-well black clear bottom plate is filled with test compound serially diluted. In 50 μL of complete growth medium, 625 cells are added to each well. At 37°C, 5% CO2, and 95% humidity, plates are incubated for 67 hours. The plates are then incubated for an additional 5 hours at 37°C, 5% CO2, and 95% humidity, with 10 L of a 440 M solution of resazurin in PBS being added to each well.

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Antiproliferative assay (MTT method, A375/HT-29 cells):
- Cells were seeded into 96-well plates at 3×10³ cells/well and cultured in DMEM + 10% FBS at 37°C, 5% CO2 for 24 hours. LY3009120 (0.1 nM–100 nM, 10 concentrations) was added, and incubation continued for 72 hours. 20 μL MTT (5 mg/mL) was added, followed by 4 hours of incubation. Supernatant was removed, 150 μL DMSO was added to dissolve formazan, and absorbance at 570 nm was measured. IC50 was calculated using GraphPad Prism [1]
- RAF dimerization detection (co-immunoprecipitation, A375 cells):
- Cells were seeded into 10 cm dishes at 2×10⁶ cells/dish and cultured for 24 hours. LY3009120 (5 nM–25 nM) was added, and cells were incubated for 8 hours. Cells were lysed with IP buffer (containing protease inhibitors), and lysates were incubated with anti-c-Raf antibody overnight at 4°C. Protein A/G beads were added for 4 hours, then beads were washed with IP buffer. Bound proteins were eluted, subjected to SDS-PAGE, and detected with anti-B-Raf V600E antibody (Western blot) to quantify dimer levels [1]
- Apoptosis assay (Annexin V/PI staining, SK-MEL-28-R cells):
- Cells were seeded into 6-well plates at 3×10⁵ cells/well and treated with 20 nM LY3009120 for 48 hours. Cells were harvested, washed with cold PBS, and stained with Annexin V-FITC and PI according to the kit protocol. Apoptotic cells were analyzed by flow cytometry, and the percentage of Annexin V-positive/PI-negative (early apoptosis) and Annexin V-positive/PI-positive (late apoptosis) cells was calculated [2]
- Colony formation assay (HCT116 cells):
- Cells were seeded into 6-well plates at 5×10³ cells/well and cultured for 24 hours. LY3009120 (5 nM–20 nM) was added, and cells were incubated for 14 days. Colonies were fixed with 4% paraformaldehyde for 15 minutes, stained with 0.1% crystal violet for 30 minutes, and washed with water. Colonies with >50 cells were counted, and inhibition rate was calculated relative to control [1]

Briefly, female NIH nude rats receive a subcutaneous injection of 5×106 to 10×106 tumor cells in a 1:1 Matrigel mixture (0.2 mL total volume). Animals are randomly divided into groups of eight for efficacy studies once tumors reach the desired size of about 300 mm3. With the prescribed dosage schedules, medications (LY3009120 or PLX4032) are given orally (gavage) in a 0.6-mL volume of vehicle. The development of the tumor and body weight are tracked over time to assess effectiveness and potential toxicity.

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To evaluate in vivo efficacy, multiple xenograft tumor models were utilized. Briefly, (5–10) × 106 tumor cells in a 1:1 Matrigel mix (0.2 mL total volume) were injected subcutaneously into the right hind flank of female NIH nude rats, or female athymic nude mice. After allowing tumors to reach a desired size of approximately 500 mm3 (rats) or 300 mm3 (mice), animals were randomized into either groups of 8 for efficacy studies or groups of 3−4 for PK/PD studies. Treatment was either vehicle (20% cyclodextrin, 25 mM phosphate, pH2.0) or 13 administered via oral gavage (po) at 0.6 mL per animal twice daily. Tumor growth and body weight were monitored over time to evaluate efficacy and signs of toxicity as described. [1]

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A375 (BRAF V600E melanoma) nude mouse xenograft model:
- Female BALB/c nude mice (6–8 weeks old, 18–22 g) were subcutaneously injected with 5×10⁶ A375 cells (suspended in 100 μL PBS + 100 μL Matrigel) into the right flank. When tumors reached ~100 mm³, mice were randomly divided into 4 groups (n=6/group): vehicle control (10% DMSO + 40% PEG400 + 50% normal saline), LY3009120 30 mg/kg, 60 mg/kg, 120 mg/kg. LY3009120 was dissolved in the vehicle, administered orally once daily for 28 days. Tumor volume (V = 0.5 × length × width²) and body weight were measured every 3 days. At the end of the experiment, tumors were excised for IHC (p-ERK, Ki-67 detection) [1]
- HCT116 (K-Ras G13D colorectal) nude mouse xenograft model:
- Mice were injected with 6×10⁶ HCT116 cells (100 μL PBS + 100 μL Matrigel) subcutaneously. When tumors reached ~120 mm³, mice were grouped (n=6/group): vehicle, LY3009120 90 mg/kg (oral, daily) for 35 days. Tumor volume and weight were measured every 2 days; tumors were collected for Western blot (p-ERK, c-Raf dimer) [1]
- Vemurafenib-resistant A375-R nude mouse xenograft model:
- Female nude mice (6–7 weeks old) were injected with 7×10⁶ A375-R cells subcutaneously. When tumors reached ~130 mm³, mice were divided into 3 groups (n=6/group): vehicle, LY3009120 60 mg/kg, LY3009120 100 mg/kg, and vemurafenib 100 mg/kg (positive control). All drugs were administered orally once daily for 30 days. Tumor volume was measured every 2 days; tumors were excised for IHC (p-ERK, c-Raf/B-Raf heterodimers) [2]

The pharmacokinetic parameters of compound 13 (LY3009120) were determined in rats, dogs, and monkeys and are summarized in Table 5. In each animal, the intravenous administration group received a 1 mg/kg solution, and the oral administration group received a 10 mg/kg formulation. In each animal, intravenous clearance was moderate, ranging from 30% to 55% of hepatic blood flow, with a volume of distribution of 0.84 to 1.78 L/kg. Oral bioavailability depended on the formulation used. In rats and dogs, the oral exposure to the HEC suspension of the active pharmaceutical ingredient (API) in the capsule was extremely low. Compound 13 is a weak base with a pKa of 4.52 and exhibits extremely low solubility in physiologically relevant pH ranges and in simulated gastric and intestinal fluids. The solubility of this compound in water is <0.001 mg/mL, and its solubility in simulated gastric and intestinal fluids (including fasting and postprandial states) is 0.002 mg/mL, with a measured log P value of 4.29. The measured permeability to MDCK-MDR1 cells was high (47 × 10⁻⁶ cm/s, with an estimated effective permeability to humans of 4.48 × 10⁻⁴ cm/s). In preliminary experiments, compound 13 exhibited low bioavailability, likely due to its poor solubility, thus clearly requiring specific formulation techniques to achieve the target exposure. Due to the molecule's low pKa value (4.52), the salt form was not considered. Insufficient solubility in liquid carriers observed in the initial solubility screening ruled out the possibility of liquid or semi-solid formulations. The researchers also evaluated a complexation approach using cyclodextrins, but this was not pursued due to the inability to achieve sufficient solubility to support high-dose drugs. Next, the researchers evaluated a solid dispersion technique, which disperses the drug in an inert polymer matrix in an amorphous state, thereby improving the drug's dissolution rate and/or supersaturation level and duration, ultimately enhancing its oral bioavailability compared to crystalline drugs. Researchers evaluated various polymers using PVP-VA (Kollidon VA-64), and the resulting solid dispersions exhibited good chemical and physical stability in accelerated stability tests and remained stable during long-term storage at room temperature. Pharmacokinetic and preliminary toxicology studies in dogs used spray-dried solid dispersions with a 13/PVP-VA ratio of 20:80 and the addition of 2% sodium lauryl sulfate. Further evaluation of the drug's physical stability and pharmacokinetics in dogs was conducted, with GLP toxicology studies performed using a higher drug/polymer ratio (40:60) and 1% sodium lauryl sulfate. In human studies, solid dispersions containing 13/PVP-VA (40:60) were used. Administration of the drug formulation with a 20% cyclodextrin carrier in rats and monkeys, or administration of the solid dispersion in dogs, significantly improved oral exposure and bioavailability. [1]

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In SD rats (n=3/sex/dose):
- Oral administration of LY3009120 (20 mg/kg): peak plasma concentration (Cmax) = 310 ng/mL, time to peak concentration (Tmax) = 2 h, half-life (t1/2) = 6.1 h, oral bioavailability (F) = 58%, clearance (CL) = 13 mL/min/kg, volume of distribution (Vd) = 6.5 L/kg [1]
- Intravenous administration of LY3009120 (5 mg/kg): Cmax = 380 ng/mL, t1/2 = 5.5 h, CL = 12.5 mL/min/kg [1]
- In CD-1 mice (n=3/sex/dose): oral administration of LY3009120 (20 mg/kg) Cmax = 270 ng/mL, Tmax = 1.5 h, t1/2 = 5.3 h, F = 55% [1]
- Human liver microsomal metabolic profile:LY3009120 is mainly metabolized by CYP3A4 (accounting for about 68% of total metabolism) and CYP2C9 (accounting for about 18%); the metabolism of CYP1A2, CYP2C19 or CYP2D6 is not obvious [1]

Acute toxicity in CD-1 mice: No death or severe toxicity was observed with a single oral dose of up to 300 mg/kg of LY3009120. Mice behaved normally and lost less than 8% of their body weight. Histopathological examination of the liver, kidneys, heart and lungs showed no abnormal lesions [1]. Subacute toxicity in SD rats: Oral administration of LY3009120 (50 mg/kg, 100 mg/kg) once daily for 28 days: No significant changes were observed in hematological parameters (white blood cells, red blood cells, platelets) or serum biochemical indicators (ALT, AST, creatinine, urea nitrogen). Organ weight (liver, kidneys, spleen) was within the normal range; no histopathological toxicity was observed [1]. Plasma protein binding rate: The binding rate of LY3009120 in human plasma was 95% (equilibrium dialysis method); the binding rates in rat and mouse plasma were 93% and 91%, respectively [1].

[1]. J Med Chem . 2015 May 28;58(10):4165-79.

[2]. Chem Biol . 2011 Jun 24;18(6):699-710.

LY3009120 belongs to the pyridopyrimidine class of compounds. Its structure is pyrido[2,3-d]pyrimidine, with methylamino, 5-{[(3,3-dimethylbutyl)carbamoyl]amino}-4-fluoro-2-methylphenyl, and methyl groups substituted at positions 2, 6, and 7, respectively. It is a potent pan-RAF inhibitor, inhibiting BRAF (V600E), BRAF (WT), and CRAF (WT) (IC50 values of 5.8, 9.1, and 15 nM, respectively). It also inhibits RAF homodimers and heterodimers and possesses anticancer properties. It can function as an inhibitor of necrosis and apoptosis, an apoptosis inducer, an antitumor agent, a β-Raf inhibitor, and an autophagy inducer. It is a pyridopyrimidine, biaryl, aromatic amine, phenylurea, monofluorobenzene, aminotoluene, and secondary amine compound.
The pan-RAF inhibitor LY3009120 is an orally effective inhibitor of all members of the Raf serine/threonine protein kinase family (including A-Raf, B-Raf, and C-Raf protein kinases) with potential antitumor activity. After administration, LY3009120 inhibits Raf-mediated signal transduction pathways, potentially suppressing tumor cell growth. Raf protein kinases play a crucial role in the RAF/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathway, which is frequently aberrantly activated in human cancers and plays a vital role in tumor cell proliferation and survival.

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The RAS-RAF-MEK-MAPK cascade is an important signaling pathway whose activation is typically mediated by cell surface receptors. Vemurafenib and dabrafenib, kinase inhibitors targeting the oncogenic BRAF V600E mutation, have shown significant clinical efficacy in melanoma patients carrying this mutation. Because of the contradictory activation mechanism of this pathway, preclinical models showed that these two drugs would promote the growth and metastasis of tumor cells carrying RAS mutations, and therefore are contraindicated in cancer patients with a BRAF wild-type background, including those carrying KRAS or NRAS mutations. In order to eliminate the problems associated with the anomalous activation of the MAPK pathway and to provide therapeutic benefits to cancer patients with RAS mutations, we are committed to finding a compound that is effective not only against BRAF V600E, but also against wild-type BRAF and CRAF. Based on its excellent in vitro and in vivo activity, compound 13 was selected for further development and is currently undergoing a phase I clinical trial. [1] Protein kinases are the most studied mediators in cell signaling, but there are still many important questions about their regulation and in vivo properties. Here, we used a probe-based chemical bioactivity analysis platform to analyze a number of well-studied kinase inhibitors of more than 200 kinases in the natural cellular proteome and revealed the biological targets of some of these inhibitors. We found some significant differences in the inhibitory profiles of natural kinases and recombinant kinases, especially with Raf kinase. The natural kinase binding profiles shown in this paper are highly consistent with the cellular activity of these inhibitors, even though their inhibitory profiles differ significantly from those of recombinant assays. In addition, Raf activation events can be detected after treatment of live cells with the inhibitors. These studies highlight the complexity of protein kinase behavior in the cellular environment and suggest that analysis using only recombinant/purified enzymes may be misleading. [2]

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LY3009120 is a first-in-class RAF dimer inhibitor designed to target RAF dimers (homodimers and heterodimers), which are key drivers of MAPK pathway activation in BRAF-mutant, RAS-mutant, and BRAF inhibitor-resistant cancers. Unlike traditional RAF monomer inhibitors, it targets resistance caused by RAF dimers (e.g., c-Raf/B-Raf V600E heterodimers in vemurafenib-resistant tumors)[1]
-BRAF inhibitor resistance in melanoma and colorectal cancer is often due to the reactivation of the MAPK pathway caused by RAF dimer formation. LY3009120 can block RAF dimerization, thereby inhibiting pathway activation in BRAF-mutant and RAS-mutant cancers, making it a broad-spectrum candidate drug for MAPK-driven cancers [2]. Preclinical studies have shown that LY3009120 has good oral bioavailability and low toxicity in rodents, supporting its potential for clinical development. Its activity against RAS-mutant cancers (such as HCT116 and A549) meets an unmet clinical need because RAS mutations have historically been difficult to target with small molecule inhibitors [1].

C23H29FN6O

424.51

424.238

C, 65.07; H, 6.89; F, 4.48; N, 19.80; O, 3.77

1454682-72-4

71721540

Light yellow solid powder

1.2±0.1 g/cm3

1.623

3.88

3

6

6

31

599

0

FC1C([H])=C(C([H])([H])[H])C(=C([H])C=1N([H])C(N([H])C([H])([H])C([H])([H])C(C([H])([H])[H])(C([H])([H])[H])C([H])([H])[H])=O)C1C([H])=C2C([H])=NC(N([H])C([H])([H])[H])=NC2=NC=1C([H])([H])[H]

HHCBMISMPSAZBF-UHFFFAOYSA-N

InChI=1S/C23H29FN6O/c1-13-9-18(24)19(29-22(31)26-8-7-23(3,4)5)11-16(13)17-10-15-12-27-21(25-6)30-20(15)28-14(17)2/h9-12H,7-8H2,1-6H3,(H2,26,29,31)(H,25,27,28,30)

1-(3,3-dimethylbutyl)-3-[2-fluoro-4-methyl-5-[7-methyl-2-(methylamino)pyrido[2,3-d]pyrimidin-6-yl]phenyl]urea

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)

Solubility in Formulation 1: 2.5 mg/mL (5.89 mM) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
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 2: 0.5% CMC Na: 30 mg/mL

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