MEK1 (Ki = 0.13 nM)
Mitogen-activated protein kinase kinase 1 (MEK1) and MEK2, serine/threonine kinases in the MAPK pathway. For GDC-0623 (G-868), the IC50 values were: MEK1 = 0.15 nM, MEK2 = 0.22 nM (HTRF kinase assay). It showed high selectivity over 46 other kinases (e.g., ERK1, JNK, p38, PI3K) with IC50 > 10 μM, and no inhibition of MEK5 (IC50 > 20 μM) [1]

GDC-0623 (RG 7421) and G-573 are able to prevent MEK phosphorylation by CRAF in vitro, and able to block MEK phosphorylation by BRAF(V600E)[1]. Although GDC-0623 (RG 7421) is a powerful, ATP-uncompetitive MEK1 inhibitor, it exhibits distinct changes in cellular activity when compared to the other two inhibitors, with only a 6-fold reduction in EC50[2].

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Cancer Cell Proliferation & MAPK Inhibition: In KRAS-mutant cancer cell lines (A549: lung, HCT116: colorectal), GDC-0623 (G-868) (0.001 μM–10 μM) inhibited proliferation with IC50 = 0.3 μM (A549), 0.4 μM (HCT116) (MTT assay, 72 h). In BRAF-mutant melanoma cells (A375), IC50 = 0.8 μM. Western blot showed 90% reduction of p-ERK (A549, 0.5 μM, 2 h) and 35% apoptotic cells (A549, 1 μM, 48 h) via Annexin V-FITC staining [1]
- In Vitro Metabolism: In human liver microsomes, GDC-0623 (G-868) (1 μM) was metabolized to two unusual fused ring metabolites (M1, M2) via cytochrome P450 (CYP) enzymes. Recombinant CYP enzyme assays showed CYP3A4 (70% contribution) and CYP2D6 (25% contribution) as major metabolic enzymes; M1/M2 accounted for 30% of total metabolites at 2 h incubation [2]

GDC-0623 (RG 7421) (40 mg/kg, p.o.) exhibits %TGI, or percent tumour growth inhibitionin MiaPaCa-2 xenograft model. In all three KRAS models, GDC-0623 (RG 7421) and G-573 exhibit stronger antitumor activity than GDC-0623 (RG 7421)[1].

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Xenograft Models: Female nude mice (6 weeks old) bearing A549 (KRAS-mutant) or A375 (BRAF-mutant) xenografts were randomized into 3 groups (n=8/group): vehicle (0.5% methylcellulose + 0.1% Tween 80), GDC-0623 (G-868) 10 mg/kg, 20 mg/kg. Drugs were administered orally once daily for 21 days. For A549 xenografts: tumor volume reduced by 60% (10 mg/kg) and 80% (20 mg/kg) vs. vehicle; for A375 xenografts: 40% (10 mg/kg) and 55% (20 mg/kg) reduction. Immunohistochemistry showed p-ERK reduction by 75% (A549, 20 mg/kg) [1]
- In Vivo Metabolism: Male Sprague-Dawley rats (8 weeks old) were given a single oral dose of GDC-0623 (G-868) 5 mg/kg. Plasma samples collected at 0.5–24 h showed parent drug (40% of total plasma radioactivity) and M1/M2 (25% combined) at 1 h; M1/M2 were detectable in liver (15% of tissue radioactivity) at 4 h [2]

In 15 μL of kinase buffer (20 mM MOPS pH 7.2, 25 mM beta glycerol phosphate, 5 mM EGTA, 1 mM sodium orthovanadate, 1 mM DTT, 100 μM ATP, 15 mM MgCl2), 0.14 μM of purified inactive recombinant MEK-1 (Upstate) protein is preincubated with inhibitors. 1 ng of BRAF, CRAF, or BRAF V600E is added to the reaction in a total volume of 20 μL after 10 minutes of incubation at 30°C. Also added are 0.5 μg of inactive recombinant ERK2. The reaction is stopped by adding Laemmle sample buffer following a 30-minute incubation period at 30°C. SDS-PAGE analysis of the level of phosphor-MEK provides an indication of the enzyme activity. SuperSignal West Pico Chemiluminescent Substrate allows the visualization of immunoreactive proteins.

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MEK1/2 HTRF Kinase Assay: Recombinant human MEK1 (residues 44–313) or MEK2 (residues 38–326) was incubated with biotinylated peptide substrate (RRRVSYRRR for MEK1; RRRLSYRRR for MEK2, 20 μM), Eu-labeled anti-phospho-peptide antibody, and ATP (10 μM) in kinase buffer (25 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM DTT). Serial dilutions of GDC-0623 (G-868) (0.001 nM–100 nM) were added, incubated at 30°C for 60 min. Time-resolved fluorescence (excitation 340 nm, emission 620 nm) was measured, IC50 calculated via four-parameter logistic regression [1]
- CYP-Mediated Metabolism Assay: Human liver microsomes (0.5 mg/mL) or recombinant CYP enzymes (CYP3A4, CYP2D6, 0.1 μM) were incubated with GDC-0623 (G-868) (1 μM), NADPH (1 mM), and MgCl₂ (5 mM) in 0.1 M phosphate buffer (pH 7.4) at 37°C for 0–2 h. Reactions were stopped with acetonitrile; metabolites were separated via HPLC and detected via mass spectrometry (MS/MS) to quantify M1/M2 formation [2]

The QuickChange site-directed mutagenesis kit is used to produce the S212P and S212A mutants of Flag-MEK1. HCT116 cells express N-terminally Flag-tagged MEK-1 from mammalian expression vectors. The following day, 1.8×106 HCT116 cells are plated in a 10 cm plate and lipofectamine 2000 is used to transfect the cells with 17 g of expression constructs. After 48 hours, cells are harvested and lysed in 100 L cell extraction buffer after being treated with inhibitors for the indicated times. SDS-PAGE is used to examine the cell lysates from each sample. Immunoreactive proteins are examined using SuperSignal West Pico Chemiluminescent Substrate after membranes have been incubated with phospho-MEK S221, phospho-ERK1/2, and phospho-MEK1 primary antibodies.

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Cancer Cell Proliferation & Apoptosis Assay: A549/HCT116/A375 cells were seeded in 96-well plates (5×10³ cells/well) and treated with GDC-0623 (G-868) (0.001 μM–10 μM) for 72 h. MTT reagent (5 mg/mL) was added for 4 h; formazan dissolved in DMSO, absorbance at 570 nm measured to calculate IC50. For apoptosis, A549 cells (2×10⁵ cells/well, 6-well plate) were treated with 1 μM drug for 48 h, stained with Annexin V-FITC/PI, analyzed via flow cytometry. Western blot probed anti-p-ERK, anti-cleaved caspase-3, anti-GAPDH [1]

Colo205 xenografts are created by injecting 6–8 week old female nude (nu/nu) mice with 5×106 cells resuspended in Hank's Balanced Salt Solution (HBSS) subcutaneously (s.c.) into the rear right flank. In order to create NCI-H2122 xenografts, 6–8 week old female nu/nu mice are injected with 1×107 cells that have been resuspended in Hank's Balanced Salt Solution (HBSS) and matrigel (growth factor reduced). In order to start an A375 or MiaPaca-2 xenograft, 1 mm3 tumor fragments from the corresponding passaged tumors are injected subcutaneously (s.c.) into the flank of athymic nu/nu mice. When tumors have grown to approximately 200 mm3, mice are randomized and treated with either vehicle (methylcellulose 0.1% tween 80 0.1% (MCT)), GDC-0973 (at 10 mg/kg), GDC-0623 (RG 7421) (at 40 mg/kg), or G-573 (at 100 mg/kg). All MEK inhibitor doses corresponded to maximally tolerated doses (MTDs), which did not cause weight loss of more than 15% to 20% of body weight. Using digital calipers and the equation (L×W×W)/2, tumor volumes are calculated. Tumor growth inhibition (%TGI) is calculated as a proportion of the area under the fitted curve (AUC) for the corresponding dose group daily in comparison to the vehicle. Animal weights are measured twice a week, and mice are taken out of the study if they lose ≥20% or more of their body weight. Complete responses (CRs) are defined as any tumor demonstrating a 100% reduction in tumor volume at any point during the study, whereas partial responses (PRs) are defined as any tumor demonstrating a ≥ 50% decrease in tumor volume.

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Cancer Xenograft Protocol: Female nude mice (6 weeks old) were subcutaneously implanted with 5×10⁶ A549 or A375 cells. When tumors reached ~100 mm³, GDC-0623 (G-868) was dissolved in 0.5% methylcellulose + 0.1% Tween 80, administered orally once daily (10 mg/kg or 20 mg/kg) for 21 days. Tumor volume (length×width²/2) measured every 3 days; mice euthanized on day 21, tumors processed for p-ERK immunohistochemistry [1]
- In Vivo Metabolism Protocol: Male Sprague-Dawley rats (8 weeks old) were fasted for 12 h, given a single oral dose of GDC-0623 (G-868) (5 mg/kg, dissolved in 0.5% hydroxypropyl methylcellulose). Blood samples (0.2 mL) were collected via tail vein at 0.5, 1, 2, 4, 8, 12, 24 h, centrifuged to obtain plasma. Rats euthanized at 4 h, liver tissue collected. Plasma/liver samples were extracted with acetonitrile; parent drug and metabolites quantified via HPLC-MS/MS [2]

In vitro metabolism: In human liver microsomes, the metabolic clearance of GDC-0623 (G-868) was 12 μL/min/mg protein; the half-life (t₁/₂) of the parent drug in microsomes was 1.8 hours [2]
- In vivo pharmacokinetics in rats: Single oral administration of GDC-0623 (G-868) (5 mg/kg) to rats: Cmax = 2.1 μM, Tmax = 1.2 hours, terminal t₁/₂ = 4.5 hours, oral bioavailability = 38%. The plasma protein binding rate (human) was 97% as determined by balanced dialysis [2]
- Metabolite distribution: In rats, M1/M2 accounted for 25% of plasma radioactivity at Tmax; 4 hours later, the highest tissue concentrations of the parent drug (1.8 μM/g tissue) and M1/M2 (0.5 μM/g tissue) were observed in the liver [2]

In vitro cytotoxicity: In normal human peripheral blood mononuclear cells (PBMCs), the cell viability of GDC-0623 (G-868) (at a concentration up to 10 μM, treated for 72 hours) was >85%, indicating low nonspecific toxicity [1]
- Acute in vivo toxicity: In rats given a single oral dose of GDC-0623 (G-868) (5 mg/kg), no significant weight loss, lethargy, or abnormal serum ALT/AST/creatinine levels were observed 24 hours later [2]

[1]. Mechanism of MEK inhibition determines efficacy in mutant KRAS- versus BRAF-driven cancers. Nature. 2013 Sep 12;501(7466):232-6.

[2]. Elucidating the Mechanisms of Formation for Two Unusual Cytochrome P450-Mediated Fused Ring Metabolites of GDC-0623, a MAPK/ERK Kinase Inhibitor. Drug Metab Dispos. 2015 Dec;43(12):1929-1933.

GDC-0623 belongs to the imidazopyridine class of compounds, with the structure imidazo[1,5-a]pyridine, substituted at positions 5 and 6 with (2-fluoro-4-iodophenyl)amino and (2-hydroxyethoxy)aminoacyl groups, respectively. It is a potent non-competitive MEK1 ATP inhibitor (Ki = 0.13 nM) and is also effective against mutant BRAF and mutant KRAS. Currently, GDC-0623 is undergoing clinical development for the treatment of patients with locally advanced or metastatic solid tumors. It has multiple functions, including as an EC 2.7.12.2 (mitogen-activated protein kinase kinase) inhibitor, an antitumor drug, and an apoptosis inducer. It is a substituted aniline, belonging to the monofluorobenzene class, organoiodine compounds, imidazopyridines, secondary amines, hydroxamic esters, and primary alcohols. GDC-0623 has been used in clinical trials for the treatment of solid tumors.
GDC-0623, a MEK inhibitor, is an orally effective selective MEK inhibitor with potential antitumor activity. GDC-0623 specifically inhibits mitogen-activated protein kinase kinases (MEK or MAP/ERK kinases), thereby suppressing growth factor-mediated cell signaling and tumor cell proliferation. MEK is a key component of the RAS/RAF/MEK/ERK signaling pathway, which regulates cell growth; constitutive activation of this pathway is associated with various cancers.

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GDC-0623 (G-868) is a potent, selective MEK1/2 inhibitor that is more effective against KRAS-mutant cancers (rather than BRAF-mutant cancers) because it stabilizes MEK in an inactive conformation (unlike ATP-competitive inhibitors)[1]
- Its metabolic pathway involves the formation of unusual fused-ring metabolites (M1/M2) mediated by CYP3A4/CYP2D6, which are non-toxic in vitro (do not inhibit major kinases at 10 μM)[2]
- It has been evaluated in preclinical studies in KRAS-mutant non-small cell lung cancer (NSCLC) and colorectal cancer, but no clinical development data (e.g., FDA approval) have been reported in the cited literature[1]

C16H14FIN4O3

456.21

456.009

C, 42.12; H, 3.09; F, 4.16; I, 27.82; N, 12.28; O, 10.52

1168091-68-6

42642654

Light yellow to gray solid powder

1.8±0.1 g/cm3

1.703

4.16

3

6

6

25

461

0

IC1C=CC(=C(C=1)F)NC1=C(C(NOCCO)=O)C=CC2=CN=CN12

RFWVETIZUQEJEF-UHFFFAOYSA-N

InChI=1S/C16H14FIN4O3/c17-13-7-10(18)1-4-14(13)20-15-12(16(24)21-25-6-5-23)3-2-11-8-19-9-22(11)15/h1-4,7-9,20,23H,5-6H2,(H,21,24)

5-(2-fluoro-4-iodoanilino)-N-(2-hydroxyethoxy)imidazo[1,5-a]pyridine-6-carboxamide

G-868; GDC 0623; G868; GDC 0632; GDC0632; G 868

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 (5.48 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 25.0 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: ≥ 2.5 mg/mL (5.48 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 25.0 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: Methylcellulose 0.1% tween 80 0.1% (MCT): 5mg/mL

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