KRAS G12C (IC50 <0.01 μM)
KRAS G12C mutant. Divarasib specifically targets the mutant KRAS protein with a glycine-to-cysteine substitution at position 12 (G12C). The compound covalently binds to the switch II (SW-II) pocket of KRAS G12C, forming an irreversible bond with the mutant cysteine residue (Cys12). This binding locks KRAS in the inactive GDP-bound conformation, preventing its activation to the GTP-bound state and thereby blocking downstream signaling pathways including MAPK (RAF-MEK-ERK) and PI3K-AKT, which are essential for cancer cell proliferation and survival.

The EC50 of divarasib (Compound 17a) in K-Ras G12C-alkylated HCC1171 cells is 2 nM [2].

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In cell-free biochemical assays, Divarasib directly binds to and inhibits KRAS G12C. The IC50 for KRAS G12C inhibition is less than 0.01 microM (10 nM). In HTRF (Homogeneous Time-Resolved Fluorescence) assays, Divarasib has an IC50 of 2.9 pM. The compound binds covalently to the switch II pocket of KRAS G12C. Binding is specific for the GDP-bound form of KRAS, and the compound does not bind to wild-type KRAS or other KRAS mutants. The binding mechanism is irreversible, characterized by covalent adduct formation with Cys12.

Divarasib (10-100 mg/kg/day; PO for 7 days) lowers the amount of free KRAS G12C [1].

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Divarasib potently inhibits KRAS G12C-mutant cancer cell proliferation in vitro. In NCI-H358 lung cancer cells (KRAS G12C mutant), Divarasib inhibits KRAS-dependent signaling as measured by phosphorylation of ERK (p-ERK) with IC50 values in the low nanomolar range. The compound has an IC50 of 0.32 nM for alkylation of NCI-HCC1171 cells (KRAS G12C mutant). Divarasib effectively blocks MAPK pathway activation in mutant KRAS-dependent cancer cells and induces G1 cell cycle arrest and apoptosis. In contrast, no significant growth inhibition is observed in KRAS wild-type cells or cells with other KRAS mutations.

Preparation of GDC-6036-Bound KRAS G12C [1]
GDC-6036-bound KRAS G12C protein was prepared through alkylation reaction. Briefly, 5 μM of both unlabeled and isotopically labeled (Arg/Lys 13C15N) KRAS G12C proteins were reacted with 20 μM of GDC-6036 in 20 mM HEPE, pH 8, 150 mM NaCl, 5 mM MgCl2, 1 mM EDTA, 1 mM TCEP, and 5 mM of GDP. After overnight incubation at room temperature, both unlabeled and labeled KRAS G12C were transferred to Amicon 3 kDa centrifugal filters for buffer exchange to 10 mM HEPES, 150 mM NaCl, pH 7.4. A 10-μL aliquot was used to assess the extent of GDC-6036 alkylation using a high-resolution accurate-mass mass spectrometry approach on a Q Exactive Plus mass spectrometer as described previously. [1]

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Spike-In Experiments[1]
Free and GDC-6036-bound KRAS G12C proteins were spiked into aliquots of 20 μg total protein (sufficient for 5 replicate runs) from blank tumor tissue lysates with a twofold serial dilution. For the xenograft sample analysis, the blank tissue lysate was prepared from the A549 mouse xenograft model bearing a non-G12C mutant form of KRAS (KRAS G12S). For the human tumor sample analysis, the blank tissue lysate was prepared from a resected human tumor sample that does not harbor the KRAS G12C mutation. Quality control (QC) samples were prepared by mixing free and GDC-6036-bound KRAS G12C standard materials in blank tissue lysates at a ratio of 4:1, 1:1, and 1:4 with the total KRAS G12C protein concentration constant at 5 fmol/μg. Isotopically labeled (Arg/Lys 13C15N) free and GDC-6036-bound KRAS G12C were spiked into each sample at a fixed amount of 125 fmol.

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The inhibition of KRAS G12C by Divarasib is assessed using a biochemical alkylation assay. Purified KRAS G12C protein (GDP-bound) is incubated with varying concentrations of Divarasib (0.001-1000 nM) for 1-4 hours. The reaction is quenched, and protein is denatured. Alkylation of Cys12 is detected by mass spectrometry (LC-MS) to quantify the percentage of protein modified by the inhibitor. Alternatively, a HTRF assay using labeled anti-KRAS antibodies and inhibitors can quantify binding. The IC50 for alkylation is defined as the concentration at which 50% of KRAS G12C is covalently modified.

Core Needle Biopsy Collection and Preservation [1]
Tumor biopsies were collected using 18-gauge biopsy needles at 2, 8, or 24 h after the last dose of GDC-6036. Samples were either snap-frozen in cryovial tubes or embedded in optimal cutting temperature (OCT) compound and stored at −80 °C until further analysis.[1]
Human NSCLC Tumor Samples[1]
Three fresh frozen human NSCLC tumor samples were procured from BioIVT, including two KRAS G12C-positive samples and one KRAS G12C-negative sample. All samples were stored at −80 °C until further analysis.[1]
Sample Preparation from Frozen Tumor Biopsies[1]
Frozen tumor biopsies were transferred to 0.5 mL polypropylene tubes pre-filled with 1.4 mm ceramic beads and 200 μL of ice-cold Pierce lysis buffer supplemented with cOmplete EDTA-free protease inhibitor cocktail Tablet and then homogenized using a Bead Ruptor 24 Cryo Cooling Unit. Tissue lysates were transferred to 1.5 mL Protein LoBind tubes and centrifuged at 17,000 g for 5 min at 4 °C. The protein concentration of the supernatant was determined using a Pierce BCA Protein Assay Kit

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Cell-based assays use NCI-H358 or other KRAS G12C-mutant cancer cell lines. Cells are seeded in 96-well plates and treated with Divarasib at concentrations ranging from 0.001-100 uM for 48-96 hours. Cell viability is measured using CellTiter-Glo (ATP quantitation) or MTT assays. EC50 values for growth inhibition are calculated from dose-response curves. For p-ERK inhibition, cells are treated with Divarasib for 2-6 hours, lysed, and p-ERK levels are measured by Western blotting, AlphaLISA, or HTRF assays. IC50 values for pathway inhibition are determined. Apoptosis is measured by cleaved caspase-3 or Annexin V flow cytometry.

Animal/Disease Models: Female CB-17 SCID (inbred) mice (20-21 weeks old; 24.1 g) human NSCLC NCI-H2030.X1.1 cells [1]
Doses: 10, 25 or 100 mg/kg
Route of Administration: po (oral gavage) daily (QD) for 7 days (Vehicle: 0.5% methylcellulose)
Experimental Results: diminished ratio of free KRAS G12C to internal standard. Dose-dependent target engagement was observed at all time points (2, 8, and 24 hrs (hrs (hours)) after the last dose), with >90% KRAS G12C engagement observed at the highest dose evaluated, 100 mg/kg.

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Female C.B-17 SCID (Inbred) mice that were 20–21 weeks old and weighed an average of 24.1 g were obtained from the Charles River Lab. Human NSCLC NCI-H2030.X1.1 cells were cultured in vitro, harvested during log-phase growth, and resuspended in Hank’s Balanced Salt Solution containing Matrigel (BD Biosciences) at a 1:1 ratio. The cells were then implanted subcutaneously in the right lateral thorax of C.B-17 SCID (Inbred) mice. Each mouse was injected with 10 × 106 cells in a volume of 100 μL. Tumors were monitored until they reached a mean tumor volume of 238–524 mm3. The mice were distributed into 10 groups on the basis of tumor volumes, with n = 4 mice/group. The mean tumor volume across all 10 groups was 363 mm3 at the initiation of dosing with GDC-6036. Mice were given vehicle (100 μL 0.5% methylcellulose), 10, 25, or 100 mg/kg GDC-6036. The vehicle and GDC-6036 were administered by oral gavage (PO) every day (QD) for 7 days in a volume of 100 μL. Tumor volumes were measured at group out (day 0) and following 1 week of dosing (day 7), and the percent change in tumor volume was plotted in GraphPad Prism software. Core needle biopsies were collected on day 7.

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In vivo efficacy of Divarasib has been evaluated in mouse xenograft models. In the PaCa-2 pancreatic cancer mouse xenograft model (KRAS G12C mutant), Divarasib is administered orally once daily at doses ranging from 1 to 100 mg/kg. Tumor volume is measured twice weekly with calipers. Significant tumor growth inhibition (TGI) is observed, with tumor regression at higher doses. Biomarker analysis of resected tumors shows dose-dependent reduction of p-ERK levels. In other KRAS G12C mutant models (e.g., NCI-H358 lung, MIA PaCa-2 pancreatic, SW837 colorectal), Divarasib demonstrates robust antitumor activity with once-daily oral dosing. The compound is well-tolerated at efficacious doses.

Divarasib is orally bioavailable with good absorption following oral administration in humans and preclinical species. In humans, the terminal elimination half-life is approximately 17.6 +/- 2.7 hours, supporting once-daily dosing. The compound is extensively metabolized, and approximately 98% of the radiolabeled dose is recovered in feces (95%) and urine (3%) within 840 hours after dosing. Cmax and AUC increase dose-proportionally across the clinical dose range (50-400 mg). In Phase 1 studies, the PK profile is characterized by moderate-to-high oral bioavailability, low-to-moderate plasma protein binding, and minimal drug-drug interaction potential. Maximum plasma concentrations are reached within 2-4 hours post-dose.

Divarasib has been evaluated in Phase 1 clinical trials (NCT04449874) in patients with advanced or metastatic solid tumors harboring KRAS G12C mutations. The most common adverse events (all grades) include nausea, diarrhea, vomiting, fatigue, and increased alanine aminotransferase. Grade ≥3 treatment-related adverse events occur in a subset of patients, primarily gastrointestinal toxicities and hepatotoxicity. The overall safety profile is manageable with supportive care. Preclinical toxicity studies in animals showed reversible, dose-dependent gastrointestinal and hepatic effects. No unexpected toxicities have emerged. As with other KRAS G12C inhibitors, rash and other dermatologic reactions may also occur.

[1]. Assessment of KRAS G12C Target Engagement by a Covalent Inhibitor in Tumor Biopsies Using an Ultra-Sensitive Immunoaffinity 2D-LC-MS/MS Approach. Anal Chem. 2022 Sep 20;94(37):12927-12933.

[2]. Fused ring compounds. WO2020097537A2.

Divarasib is an orally administered inhibitor of the KRAS oncogenic mutation G12C with potential antitumor activity. After oral administration, Divarasib selectively targets the KRAS G12C mutant and inhibits KRAS G12C mutant-dependent signaling. KRAS is a member of the RAS oncogene family and plays a crucial role in cell signaling, division, and differentiation. KRAS mutations can induce constitutive signal transduction, leading to tumor cell growth, proliferation, invasion, and metastasis.

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Divarasib (GDC-6036) is being developed by Genentech/Roche. As of 2025, the compound is in clinical trials (Phase 1/2) for KRAS G12C-mutant solid tumors, including non-small cell lung cancer (NSCLC), colorectal cancer (CRC), and other solid tumors. Preliminary clinical activity data from Phase 1 studies show objective response rates (ORR) in NSCLC and CRC patients. The compound is not yet approved by any regulatory agency. Preclinical studies demonstrate that Divarasib has improved potency and selectivity compared to earlier-generation KRAS G12C inhibitors. The chemical structure belongs to the quinazoline class. The compound name "Divarasib" is the International Nonproprietary Name (INN).

C29H32CLF4N7O2

622.06

621.22

C, 55.99; H, 5.19; Cl, 5.70; F, 12.22; N, 15.76; O, 5.14

2417987-45-0

2762240-36-6 (adipate); 2417987-45-0 (active atropisomer);2417917-17-8 (atropisomers)

146624881

White to light yellow solid

5.4

1

12

6

43

997

2

C[C@H]1CN(CCN1C2=NC(=NC3=C(C(=C(C=C32)Cl)C4=C(C(=CC(=N4)N)C)C(F)(F)F)F)OC[C@@H]5CCCN5C)C(=O)C=C

ZRBPIAWWRPFDPY-IRXDYDNUSA-N

InChI=1S/C29H32ClF4N7O2/c1-5-21(42)40-9-10-41(16(3)13-40)27-18-12-19(30)22(26-23(29(32,33)34)15(2)11-20(35)36-26)24(31)25(18)37-28(38-27)43-14-17-7-6-8-39(17)4/h5,11-12,16-17H,1,6-10,13-14H2,2-4H3,(H2,35,36)/t16-,17-/m0/s1

1-((S)-4-(7-((R)-6-amino-4-methyl-3-(trifluoromethyl)pyridin-2-yl)-6-chloro-8-fluoro-2-(((S)-1-methylpyrrolidin-2-yl)methoxy)quinazolin-4-yl)-3-methylpiperazin-1-yl)prop-2-en-1-one

GDC-6036; RG6330; GDC 6036; RG-6330; GDC6036; RG 6330

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 : ~100 mg/mL (~160.76 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.02 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 (4.02 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
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: ≥ 2.5 mg/mL (4.02 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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