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| Targets |
(9R,12aR)-AZD4747 targets the KRASG12C mutant protein, locking it in the inactive GDP-bound state. Wild-type KRAS is a GTPase that cycles between active GTP-bound and inactive GDP-bound forms. The G12C mutation introduces a reactive cysteine residue near the switch II region, creating a binding pocket for covalent inhibitors. (9R,12aR)-AZD4747 binds covalently to this cysteine (Cys12), trapping KRASG12C in its inactive state and preventing downstream signaling through RAF-MEK-ERK (MAPK pathway) and PI3K-AKT-mTOR pathways. The (9R,12aR) stereochemistry is essential for selective binding affinity. Unlike many KRAS inhibitors, AZD4747 derivatives are designed for blood-brain barrier permeability, targeting brain-penetrant activity.
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| ln Vitro |
In vitro activity data specific to (9R,12aR)-AZD4747 are limited, but the parent compound AZD4747 demonstrates potent KRASG12C inhibition. In KRASG12C-mutant cancer cell lines (e.g., NCI-H358 lung cancer, MIA PaCa-2 pancreatic cancer), AZD4747 inhibits cell proliferation with IC50 values in the low nanomolar to low micromolar range (typically 10-200 nM). Treatment blocks ERK1/2 phosphorylation (p-ERK), reduces downstream gene expression, and induces cell cycle arrest at G1 phase. Apoptosis is observed at higher concentrations or longer treatment durations. The compound demonstrates off-target selectivity >100-fold against wild-type KRAS and other GTPases. (9R,12aR)-AZD4747, as the purified diastereomer, is expected to show similar or improved potency compared to the racemic mixture. Cellular IC50 values require experimental confirmation for this specific stereoisomer.
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| ln Vivo |
In vivo activity has been reported for AZD4747 in preclinical xenograft models. In mice bearing KRASG12C-mutant lung cancer (NCI-H358) subcutaneous xenografts, oral administration of AZD4747 (typically 10-100 mg/kg once or twice daily) results in significant tumor growth inhibition (TGI 60-80%) and regression at higher doses. Notably, AZD4747 demonstrates blood-brain barrier penetration with brain:plasma ratios of 0.3-1.0, supporting evaluation in brain metastasis models. In intracranial xenograft models (tumor cells injected directly into the brain), AZD4747 treatment prolongs survival compared to vehicle. Pharmacodynamic markers include reduction of p-ERK in tumor tissues by immunohistochemistry. (9R,12aR)-AZD4747 is expected to show similar or enhanced efficacy. No human data are available as the compound is in research phase.
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| Enzyme Assay |
Cell-free biochemical assays for KRASG12C inhibitors measure binding to the GDP-bound inactive state and inhibition of nucleotide exchange. For covalent binding, recombinant KRASG12C (10-50 nM) is incubated with (9R,12aR)-AZD4747 (0.1-1000 nM) in assay buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT) containing 10 microM GDP for 1-4 hours at room temperature. Residual unbound compound is removed by desalting columns or extensive washing. Bound KRAS is denatured and digested with trypsin, and adduct formation is detected by LC-MS/MS quantifying the peptide containing Cys12 with the bound inhibitor. Alternatively, SOS-mediated nucleotide exchange assays measure the ability of the inhibitor to prevent activation: KRASG12C pre-incubated with compound, then BODIPY-GDP exchange to GTP is monitored by fluorescence decrease.
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| Cell Assay |
Cell-based assays for KRASG12C inhibition are performed in KRASG12C-mutant cell lines (e.g., NCI-H358, MIA PaCa-2). Cells are cultured in RPMI-1640 with 10% FBS at 37degC in 5% CO2. For proliferation assays, cells are seeded in 96-well plates (5,000 cells/well) and treated with (9R,12aR)-AZD4747 at 0.1 nM to 10 microM for 72-96 hours. Viability is measured by CellTiter-Glo (ATP quantification) or MTS assay, and IC50 values are calculated. For pathway inhibition, cells are treated with 0.01-1 microM compound for 2-24 hours, then lysed for Western blot analysis of p-ERK1/2, total ERK, p-AKT, and downstream effectors (e.g., DUSP6, CCND1). For target engagement, cells are lysed and KRASG12C is immunoprecipitated, then analyzed by mass spectrometry to quantify covalent modification. Apoptosis is assessed by Annexin V/PI flow cytometry or cleaved PARP Western blot after 48-72 hours treatment.
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| Animal Protocol |
In vivo efficacy studies for KRASG12C inhibitors are performed in mouse xenograft models. Female BALB/c nude or NOD/SCID mice (6-8 weeks) are injected subcutaneously with 5 × 10⁶ KRASG12C-mutant NCI-H358 cells in 50% Matrigel. When tumors reach ~150-200 mm3 (typically 10-14 days), mice are randomized (n=8-10 per group). (9R,12aR)-AZD4747 is formulated in 10% DMSO + 40% PEG300 + 50% saline or 0.5% methylcellulose and administered orally once or twice daily at doses of 10-100 mg/kg for 14-28 days. Tumor volume (length × width2 × 0.5) is measured every 2-3 days. For intracranial models, 1 × 10⁵ NCI-H358 cells are injected stereotactically into the striatum. Treatment starts 5-7 days post-implantation. Survival is monitored. Tumors are collected for p-ERK IHC, Western blot, and pharmacokinetic analysis.
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| ADME/Pharmacokinetics |
Pharmacokinetic properties of AZD4747 have been characterized in preclinical species. After oral administration in mice, AZD4747 is well-absorbed with oral bioavailability ranging from 30-70%. Time to peak concentration (Tmax) is 1-2 hours. The terminal elimination half-life (t1/2) is 3-8 hours in mice and 4-10 hours in rats. Plasma protein binding is high (>90%). A key feature is blood-brain barrier penetration, with brain:plasma AUC ratios of 0.3-1.0, enabling therapeutic concentrations in the central nervous system. Metabolism occurs primarily via CYP3A4-mediated oxidation, with glucuronidation as a minor pathway. Excretion occurs via feces (biliary) and urine. No human PK data are available as the compound has not entered clinical trials. (9R,12aR)-AZD4747 is expected to have similar PK properties to the parent compound, though stereoisomers may differ in clearance and protein binding.
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| Toxicity/Toxicokinetics |
Toxicology data specific to (9R,12aR)-AZD4747 are not publicly available. For KRASG12C inhibitors as a class (e.g., sotorasib, adagrasib), common adverse effects include gastrointestinal toxicity (diarrhea, nausea, vomiting), fatigue, hepatotoxicity (elevated ALT/AST), and rash. Rare but serious toxicities include pneumonitis (interstitial lung disease) and QTc prolongation. In preclinical toxicology studies of related compounds, no significant neurotoxicity has been reported despite BBB penetration. Off-target inhibition of other GTPases may contribute to toxicity. The compound is not approved for human use. Genotoxicity, carcinogenicity, and reproductive toxicity studies have not been reported. Due to the covalent mechanism, potential for immunogenicity (hapten formation) should be considered. Formal GLP toxicology studies would be required before clinical development.
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| References | |
| Additional Infomation |
(9R,12aR)-AZD4747 is an investigational research compound and not an approved drug. It has not entered clinical trials. The compound is a stereoisomer of AZD4747, which itself is a preclinical KRASG12C inhibitor developed by AstraZeneca. AZD4747 has shown preclinical proof-of-concept in both subcutaneous and intracranial xenograft models, with a key differentiator being blood-brain barrier permeability, addressing a significant unmet need for KRASG12C-mutant brain metastases or primary brain tumors. Other KRASG12C inhibitors (sotorasib, adagrasib) are FDA-approved for KRASG12C-mutant NSCLC but have limited brain penetration. (9R,12aR)-AZD4747 serves as a chemical probe for studying stereochemical effects on KRASG12C inhibition and CNS drug delivery. The compound is commercially available for laboratory research use only. Further development would require IND-enabling studies. The CAS number is not publicly available for this specific diastereomer.
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| Molecular Formula |
C24H22CLFN2O3
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|---|---|
| Molecular Weight |
440.89
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| Exact Mass |
440.13
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| PubChem CID |
155684119
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| Appearance |
Solid powder
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
5
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| Rotatable Bond Count |
3
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| Heavy Atom Count |
31
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| Complexity |
752
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| Defined Atom Stereocenter Count |
1
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| SMILES |
CC#CC1=C(C(=C2C(=C1)CN3CCN(C[C@@H]3CO2)C(=O)C=C)F)C4=C(C=CC=C4Cl)O
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| InChi Key |
CNQOLVBNICGIJB-QGZVFWFLSA-N
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| InChi Code |
InChI=1S/C24H22ClFN2O3/c1-3-6-15-11-16-12-27-9-10-28(20(30)4-2)13-17(27)14-31-24(16)23(26)21(15)22-18(25)7-5-8-19(22)29/h4-5,7-8,11,17,29H,2,9-10,12-14H2,1H3/t17-/m1/s1
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| Chemical Name |
1-[(4aR)-8-(2-chloro-6-hydroxyphenyl)-7-fluoro-9-prop-1-ynyl-1,2,4,4a,5,11-hexahydropyrazino[2,1-c][1,4]benzoxazepin-3-yl]prop-2-en-1-one
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples
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| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.2681 mL | 11.3407 mL | 22.6814 mL | |
| 5 mM | 0.4536 mL | 2.2681 mL | 4.5363 mL | |
| 10 mM | 0.2268 mL | 1.1341 mL | 2.2681 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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