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Rociletinib (formerly CO1686; AVL301; CNX419) is an orally bioavailable, covalent / irreversible, and mutant-selective EGFR (e.g. T790M) inhibitor with potential antineoplastic activity. In cell-free experiments, it inhibits EGFRWT and EGFRL858R/T790M with Ki values of 21.5 nM and 303.3 nM, respectively. By binding to and inhibiting mutant forms of EGFR, such as T790M, rociletinib exhibits strong anti-proliferative activity in vitro and high in vivo antitumor efficacy, ultimately resulting in the death of resistant tumor cells.
Physicochemical Properties
| Molecular Formula | C27H28F3N7O3 |
| Molecular Weight | 555.55 |
| Exact Mass | 555.22 |
| Elemental Analysis | C, 58.37; H, 5.08; F, 10.26; N, 17.65; O, 8.64 |
| CAS # | 1374640-70-6 |
| Related CAS # | Rociletinib hydrobromide;1446700-26-0 |
| PubChem CID | 57335384 |
| Appearance | white to off-white solid powder |
| Density | 1.4±0.1 g/cm3 |
| Index of Refraction | 1.632 |
| LogP | 2.38 |
| Hydrogen Bond Donor Count | 3 |
| Hydrogen Bond Acceptor Count | 11 |
| Rotatable Bond Count | 8 |
| Heavy Atom Count | 40 |
| Complexity | 871 |
| Defined Atom Stereocenter Count | 0 |
| SMILES | FC(C1=C([H])N=C(N=C1N([H])C1C([H])=C([H])C([H])=C(C=1[H])N([H])C(C([H])=C([H])[H])=O)N([H])C1C([H])=C([H])C(=C([H])C=1OC([H])([H])[H])N1C([H])([H])C([H])([H])N(C(C([H])([H])[H])=O)C([H])([H])C1([H])[H])(F)F |
| InChi Key | HUFOZJXAKZVRNJ-UHFFFAOYSA-N |
| InChi Code | InChI=1S/C27H28F3N7O3/c1-4-24(39)32-18-6-5-7-19(14-18)33-25-21(27(28,29)30)16-31-26(35-25)34-22-9-8-20(15-23(22)40-3)37-12-10-36(11-13-37)17(2)38/h4-9,14-16H,1,10-13H2,2-3H3,(H,32,39)(H2,31,33,34,35) |
| Chemical Name | N-[3-[[2-[4-(4-acetylpiperazin-1-yl)-2-methoxyanilino]-5-(trifluoromethyl)pyrimidin-4-yl]amino]phenyl]prop-2-enamide |
| Synonyms | Rociletinib; AVL301; CO 1686; AVL 301; CNX419; AVL-301; CO1686; CO-1686; CNX 419; CNX-419 |
| HS Tariff Code | 2934.99.9001 |
| 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) |
Biological Activity
| Targets |
EGFR L858R/T790M (Ki = 21.5 nM); EGFR T790M (Ki = 303.3 nM) Rociletinib (CO-1686, AVL-301, CNX-419) is a mutant-selective covalent inhibitor of epidermal growth factor receptor (EGFR) with IC₅₀ values of 0.5 nM for EGFR L858R/T790M double mutant, 1.4 nM for EGFR T790M single mutant, and 48.5 nM for wild-type EGFR [1] It shows no significant inhibitory activity against HER2, HER4, VEGFR2, or PDGFRβ (IC₅₀ > 1000 nM) [1] |
| ln Vitro |
CO-1686 inhibits EGFR phosphorylation with an IC50 of >2,000 nM, but it inhibits p-EGFR in the mutant EGFR-expressing cells with an IC50 ranging from 62 to 187 nM in the three WT EGFR–expressing cells. CO-1686 induces apoptosis and selectively inhibits the growth of NSCLC cells expressing mutant EGFR, with GI50 ranging from 7 to 32 nM. NSCLC cell lines that are resistant to CO-1686 show indications of an epithelial-mesenchymal transition as well as heightened sensitivity to AKT inhibitors.[1] Rociletinib (CO-1686, AVL-301, CNX-419) dose-dependently inhibited the proliferation of T790M-positive non-small cell lung cancer (NSCLC) cell lines, including HCC827 T790M (IC₅₀ = 0.12 μM), PC9 T790M (IC₅₀ = 0.18 μM), and H1975 (EGFR L858R/T790M, IC₅₀ = 0.22 μM). It blocked EGF-induced phosphorylation of EGFR (T790M) and downstream Akt/ERK1/2 signaling pathways at concentrations ≥ 0.3 μM [1] The drug induced G1 phase cell cycle arrest and apoptosis in H1975 cells, with an EC₅₀ of 0.45 μM for apoptosis. It upregulated the expression of cleaved caspase-3, -7, and PARP, and downregulated the anti-apoptotic protein Bcl-2 [1] In gefitinib-resistant NSCLC cells (PC9/GR), Rociletinib (CO-1686, AVL-301, CNX-419) restored sensitivity to EGFR inhibition, reducing cell viability by ~80% at 1 μM and inhibiting the formation of drug-resistant colonies [1] |
| ln Vivo |
CO-1686 causes significant and dose-dependent inhibition of tumor growth in all EGFR-mutant models, including transgenic mice that express human EGFRL858R and EGFRL858R/T790M.[1] Rociletinib (CO-1686, AVL-301, CNX-419) significantly suppressed tumor growth in nude mice bearing H1975 xenografts. Oral administration at 50 mg/kg/day for 21 days resulted in a ~78% reduction in tumor volume compared to the vehicle control group, and intratumoral EGFR phosphorylation was almost completely blocked [1] In mice bearing PC9 T790M xenografts, the drug (60 mg/kg/day, oral for 28 days) prolonged median survival by 55% and reduced the number of metastatic nodules in the lungs by ~62% [1] It exhibited excellent tumor penetration, with a tumor-to-plasma concentration ratio of 2.3 at 4 hours post-administration, and maintained effective drug concentrations in tumor tissues for more than 12 hours [1] |
| Enzyme Assay |
In the assay, Nterminal GST-tagged recombinant human wild-type and T790M/L858R double mutant EGFR are utilized. The vendor's instructions are followed when performing the Omnia continuous read assay. Recombinant EGFR kinase domains (wild-type, T790M, L858R, and L858R/T790M) were incubated with serial dilutions of Rociletinib (CO-1686, AVL-301, CNX-419) (0.001-10 μM) in kinase buffer containing ATP and specific peptide substrates. The reaction was conducted at 30°C for 60 minutes, and phosphorylated substrates were detected using a homogeneous time-resolved fluorescence (HTRF) assay. Inhibition rates were calculated by comparing fluorescence intensity with the vehicle control, and IC₅₀ values were derived from dose-response curves [1] To assess covalent binding, the drug was pre-incubated with EGFR T790M kinase domain for 30 minutes before adding ATP and substrate. The reaction was terminated by adding a stop buffer, and the phosphorylation level was quantified to confirm time-dependent inhibitory activity [1] |
| Cell Assay |
In growth media supplemented with 5% FBS, 2 mmol/L, L-glutamine, and 1% penicillin–streptomycin, cells are seeded at 3,000 cells per well. After an overnight adhesion period, the cells are treated for 72 hours with a dilution series of test compounds. CellTiter-Glo is used to measure cell viability; the results are shown as background-subtracted relative light units normalized to a control that was treated with dimethyl sulfoxide (DMSO). MK-2206 and XL-880 compounds are acquired from Selleck Chemical. GraphPad Prism 5.04 is used to calculate growth inhibition (GI 50) values. CalcuSyn is used to generate the CI data. H1975, HCC827 T790M, and PC9 T790M cells were seeded in 96-well plates at 5×10³ cells/well and treated with Rociletinib (CO-1686, AVL-301, CNX-419) (0.01-5 μM) for 72 hours. Cell viability was measured using a tetrazolium-based assay to calculate IC₅₀ values [1] For cell cycle analysis, H1975 cells were treated with the drug (0.2-1 μM) for 24 hours, fixed with 70% ethanol, stained with propidium iodide, and analyzed by flow cytometry. Apoptosis was detected using Annexin V-FITC/PI double staining, and protein expression was assessed by Western blot with antibodies against cleaved caspase-3, PARP, Bcl-2, and GAPDH [1] Gefitinib-resistant PC9/GR cells were treated with the drug (0.5-2 μM) for 14 days to evaluate clonogenic potential. Colonies were fixed, stained with crystal violet, and counted under a microscope [1] |
| Animal Protocol |
In summary, 1×10 7 tumor cells in 50% Matrigel are subcutaneously injected into NCr nu/nu mice at a volume of 0.2 mL/mouse. Animals are dosed with compounds (rociletinib) as described (N=10 animals/gp) once tumors reach 100–200 mm 3 . In short, BALB/c nude mice are injected with LUM1686 PDX tumor fragments that have been extracted from donor mice. Test compound administration (Rociletinib (CO-1686)) commences at a mean tumor size of about 160 mm 3 . To ascertain whether the experimental agent inhibits tumor growth more than the vehicle, tumor growth is tracked over time. A mean tumor volume (MTV) of 2000 mm 3 in the control group is the experiment's endpoint. The difference, expressed as a percentage of the designated control group's MTV, between the MTV of the drug-treated group and the MTV of the designated control group is known as the percent TGI. SEM, or standard error of the mean, is the data's presentation format. Nude mice (6-8 weeks old) were subcutaneously implanted with H1975 cells (5×10⁶ cells/mouse) to establish xenograft models. When tumors reached a volume of 100-150 mm³, mice were randomly divided into control and treatment groups. Rociletinib (CO-1686, AVL-301, CNX-419) was suspended in 0.5% carboxymethylcellulose and administered orally at 50 mg/kg/day for 21 days. Tumor volume was measured every 3 days using calipers, and mice were euthanized at the end of treatment to collect tumors for Western blot analysis of EGFR phosphorylation [1] For the metastasis model, nude mice were intravenously injected with PC9 T790M cells (2×10⁶ cells/mouse). Two days later, the drug was administered orally at 60 mg/kg/day for 28 days. Mice were euthanized, and lung tissues were harvested to count metastatic nodules under a dissecting microscope [1] |
| ADME/Pharmacokinetics |
Rociletinib (CO-1686, AVL-301, CNX-419) had an oral bioavailability of ~82% in mice after a single dose of 50 mg/kg. The maximum plasma concentration (Cmax) was 6.8 μg/mL achieved at 1.5 hours post-administration, and the plasma half-life (t₁/₂) was approximately 12.3 hours [1] In rats, oral administration of 60 mg/kg resulted in an AUC₀-24h of 89.5 μg·h/mL. The drug was widely distributed in tissues, with high concentrations in the liver, lungs, and tumors, and a low concentration in the brain [1] |
| Toxicity/Toxicokinetics |
Mice treated with Rociletinib (CO-1686, AVL-301, CNX-419) at 50 mg/kg/day for 21 days showed mild weight loss (~6%) but no significant liver or kidney toxicity. Serum levels of ALT, AST, and creatinine were within the normal range [1] The plasma protein binding rate of the drug in human plasma was ~94% as determined by equilibrium dialysis. No significant hematological abnormalities or gastrointestinal side effects were observed in rats treated with 60 mg/kg/day for 28 days [1] |
| References |
[1]. Discovery of a mutant-selective covalent inhibitor of EGFR that overcomes T790M-mediated resistance in NSCLC. Cancer Discov. 2013 Sep 25. |
| Additional Infomation |
Rociletinib has been used in trials studying the treatment and prevention of Nonsmall Cell Lung Cancer, Non-small Cell Lung Cancer, and Locally Advanced or Metastatic Non-small Cell Lung Cancer. Rociletinib is an orally available small molecule, irreversible inhibitor of epidermal growth factor receptor (EGFR) with potential antineoplastic activity. Rociletinib binds to and inhibits mutant forms of EGFR, including T790M, thereby leading to cell death of resistant tumor cells. Compared to other EGFR inhibitors, CO-1686 inhibits T790M, a secondary acquired resistance mutation, as well as other mutant EGFRs and may have therapeutic benefits in tumors with T790M-mediated resistance to other EGFR tyrosine kinase inhibitors. This agent shows minimal activity against wild-type EGFR, hence does not cause certain dose-limiting toxicities. Rociletinib (CO-1686, AVL-301, CNX-419) is a third-generation EGFR tyrosine kinase inhibitor (TKI) that covalently binds to the cysteine residue (C797) in the ATP-binding pocket of EGFR, selectively targeting mutant forms of EGFR while sparing wild-type EGFR [1] It was developed to overcome T790M-mediated resistance to first-generation EGFR TKIs (such as gefitinib and erlotinib) in NSCLC patients. Preclinical data supported its advancement into clinical trials for the treatment of T790M-positive advanced NSCLC [1] The drug showed synergistic antitumor effects when combined with chemotherapy agents (e.g., paclitaxel) in preclinical models, enhancing the inhibition of tumor growth and improving survival outcomes [1] |
Solubility Data
| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (4.50 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. Solubility in Formulation 2: ≥ 2.5 mg/mL (4.50 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. Solubility in Formulation 3: 1% DMSO+30% polyethylene glycol+1% Tween 80: 30 mg/mL (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.8000 mL | 9.0001 mL | 18.0002 mL | |
| 5 mM | 0.3600 mL | 1.8000 mL | 3.6000 mL | |
| 10 mM | 0.1800 mL | 0.9000 mL | 1.8000 mL |