C-Raf (IC50 = 4.3 nM); BRAF(V600E) (IC50 = 5.8 nM); BRAF WT (IC50 = 15 nM)

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]

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]

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.

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.

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]

Pharmacokinetic parameters for compound 13 (LY3009120) have been determined in rat, dog, and monkey and are summarized in Table 5. In each species the compound was dosed as a 1 mg/kg solution in the iv arm and a 10 mg/kg formulation in the oral arm. In each species, iv clearance was moderate at 30–55% of hepatic blood flow and volumes of distribution between 0.84 and 1.78 L/kg. The oral bioavailability was dependent on the formulation used. In rat and dog, HEC suspension of API in capsule gave very low oral exposure. Compound 13 is a weak base with a pKa of 4.52, and the solubility is very low across the physiologically relevant pH range, as well as in simulated gastric and intestinal fluids. Solubility in water is <0.001 mg/mL and is 0.002 mg/mL in simulated gastric and intestinal (both fasted and fed) fluids, and the measured log P value was determined to be 4.29. Measured MDCK-MDR1 permeability is high (47 × 10–6 cm/s, with an estimated human effective permeability of 4.48 × 10–4 cm/s). In initial experiments, 13 demonstrated low bioavailability, likely due to its poor solubility, and it became clear this compound would require use of enabling formulation technology to achieve target exposures. Use of a salt form was not considered because of the low pKa (4.52) of the molecule. Insufficient solubility observed in liquid vehicles during preliminary solubility screens ruled out the possibility of liquid or semisolid formulations. Complexation approaches using cyclodextrins were also evaluated but were not pursued because of an inability to achieve a solubility that would support high drug dose. Evaluation of solid dispersion technologies was next pursued where the drug is dispersed in an inert polymeric matrix and rendered in an amorphous form which results in faster dissolution rate and/or higher extent and duration of supersaturation leading to enhanced oral bioavailability relative to the crystalline drug. Various polymers were evaluated with PVP-VA (Kollidon VA-64), resulting in a solid dispersion that was both chemically and physically stable under accelerated stability testing, as well as long-term storage under ambient conditions. Pharmacokinetic studies in dogs and pilot toxicology studies contained spray dried solid dispersion with 13/PVP-VA in ratio of 20:80, with added 2% sodium lauryl sulfate. Further evaluations of physical stability and in vivo pharmacokinetics in dogs were done, followed by GLP toxicology studies using higher drug/polymer ratio (40:60) and 1% added sodium lauryl sulfate. For human studies the solid dispersion containing 13/PVP-VA (40:60) was used. Dosing the molecule as an enabled formulation of 20% cyclodextrin in rat and monkey or solid dispersion in dog led to significantly improved oral exposure and bioavailability. [1]

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

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

LY3009120 is a member of the class of pyridopyrimidines that is pyrido[2,3-d]pyrimidine substituted by methylamino, 5-{[(3,3-dimethylbutyl)carbamoyl]amino}-4-fluoro-2-methylphenyl, and methyl groups at positions 2, 6 and 7, respectively. It is a potent pan RAF inhibitor which inhibits BRAF(V600E), BRAF(WT) and CRAF(WT) (IC50 = 5.8, 9.1 and 15 nM, respectively). It also inhibits RAF homo- and heterodimers and exhibits anti-cancer properties. It has a role as a necroptosis inhibitor, an apoptosis inducer, an antineoplastic agent, a B-Raf inhibitor and an autophagy inducer. It is a pyridopyrimidine, a biaryl, an aromatic amine, a member of phenylureas, a member of monofluorobenzenes, an aminotoluene and a secondary amino compound.
Pan-RAF Inhibitor LY3009120 is an orally available inhibitor of all members of the serine/threonine protein kinase Raf family, including A-Raf, B-Raf and C-Raf protein kinases, with potential antineoplastic activity. Upon administration, pan-RAF kinase inhibitor LY3009120 inhibits Raf-mediated signal transduction pathways, which may inhibit tumor cell growth. Raf protein kinases play a key role in the RAF/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathway, which is often dysregulated in human cancers and plays a key role in tumor cell proliferation and survival.

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The RAS-RAF-MEK-MAPK cascade is an essential signaling pathway, with activation typically mediated through cell surface receptors. The kinase inhibitors vemurafenib and dabrafenib, which target oncogenic BRAF V600E, have shown significant clinical efficacy in melanoma patients harboring this mutation. Because of paradoxical pathway activation, both agents were demonstrated to promote growth and metastasis of tumor cells with RAS mutations in preclinical models and are contraindicated for treatment of cancer patients with BRAF WT background, including patients with KRAS or NRAS mutations. In order to eliminate the issues associated with paradoxical MAPK pathway activation and to provide therapeutic benefit to patients with RAS mutant cancers, we sought to identify a compound not only active against BRAF V600E but also wild type BRAF and CRAF. On the basis of its superior in vitro and in vivo profile, compound 13 was selected for further development and is currently being evaluated in phase I clinical studies.[1]

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Protein kinases are intensely studied mediators of cellular signaling, yet important questions remain regarding their regulation and in vivo properties. Here, we use a probe-based chemoprotemics platform to profile several well studied kinase inhibitors against >200 kinases in native cell proteomes and reveal biological targets for some of these inhibitors. Several striking differences were identified between native and recombinant kinase inhibitory profiles, in particular, for the Raf kinases. The native kinase binding profiles presented here closely mirror the cellular activity of these inhibitors, even when the inhibition profiles differ dramatically from recombinant assay results. Additionally, Raf activation events could be detected on live cell treatment with inhibitors. These studies highlight the complexities of protein kinase behavior in the cellular context and demonstrate that profiling with only recombinant/purified enzymes can be misleading.[2]

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.)