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Osimertinib (formerly AZD-9291 and mereletinib; trade name Tagrisso) is an oral bioavailable, irreversible/covalent, and mutant-selective EGFR inhibitor with potential antineoplastic activity. In LoVo cells, it inhibits WT EGFR, L858R/T790M EGFR, and Exon 19 deletion EGFR with IC50 values of 493.8 nM, 11.44, and 12.92 nM, respectively. Both the FDA and the European Commission approved it in 2017 for the treatment of cancer.
Physicochemical Properties
| Molecular Formula | C28H33N7O2 |
| Molecular Weight | 499.61 |
| Exact Mass | 499.269 |
| Elemental Analysis | C, 67.31; H, 6.66; N, 19.62; O, 6.40 |
| CAS # | 1421373-65-0 |
| Related CAS # | Osimertinib mesylate;1421373-66-1;Osimertinib-d6;1638281-44-3;Osimertinib dimesylate;2070014-82-1;Osimertinib-13C,d3;2254100-49-5 |
| PubChem CID | 71496458 |
| Appearance | Brown to yellow solid powder |
| Density | 1.2±0.1 g/cm3 |
| Index of Refraction | 1.618 |
| LogP | 3.3 |
| Hydrogen Bond Donor Count | 2 |
| Hydrogen Bond Acceptor Count | 7 |
| Rotatable Bond Count | 10 |
| Heavy Atom Count | 37 |
| Complexity | 752 |
| Defined Atom Stereocenter Count | 0 |
| SMILES | O(C([H])([H])[H])C1=C(C([H])=C(C(=C1[H])N(C([H])([H])[H])C([H])([H])C([H])([H])N(C([H])([H])[H])C([H])([H])[H])N([H])C(C([H])=C([H])[H])=O)N([H])C1=NC([H])=C([H])C(C2=C([H])N(C([H])([H])[H])C3=C([H])C([H])=C([H])C([H])=C32)=N1 |
| InChi Key | DUYJMQONPNNFPI-UHFFFAOYSA-N |
| InChi Code | InChI=1S/C28H33N7O2/c1-7-27(36)30-22-16-23(26(37-6)17-25(22)34(4)15-14-33(2)3)32-28-29-13-12-21(31-28)20-18-35(5)24-11-9-8-10-19(20)24/h7-13,16-18H,1,14-15H2,2-6H3,(H,30,36)(H,29,31,32) |
| Chemical Name | N-[2-[2-(dimethylamino)ethyl-methylamino]-4-methoxy-5-[[4-(1-methylindol-3-yl)pyrimidin-2-yl]amino]phenyl]prop-2-enamide |
| Synonyms | AZD-9291; AZD9291; AZD 9291; Mereletinib; AZD9291; AZD 9291; UNII-3C06JJ0Z2O; Osimertinib [USAN]; Osimertinib free base; Mereletinib; Trade name: Tagrisso |
| 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 (IC50 = 12 nM); EGFR L858R/T790M (IC50 = 1 nM) Osimertinib (AZD9291; Tagrisso) potently inhibits EGFR T790M mutant (IC₅₀ = 1.2 nM), EGFR exon 19 deletion mutant (IC₅₀ = 1.6 nM), and EGFR L858R mutant (IC₅₀ = 2.4 nM). It shows low activity against wild-type EGFR (IC₅₀ = 48 nM) [5] Osimertinib (AZD9291; Tagrisso) has no significant inhibitory effect on HER2, HER4, VEGFR2, or PDGFRβ (IC₅₀ > 100 nM) [4] |
| ln Vitro |
AZD9291 exhibits a noticeably stronger suppression of proliferation in mutant EGFR cell lines when compared to wild-type in vitro. [2] Osimertinib (AZD9291; Tagrisso) dose-dependently inhibited the proliferation of EGFR mutant non-small cell lung cancer (NSCLC) cell lines, including NCI-H1975 (EGFR L858R/T790M, IC₅₀ = 12 nM), PC-9 (EGFR exon 19 deletion, IC₅₀ = 15 nM), and HCC827 (EGFR exon 19 deletion, IC₅₀ = 18 nM). It blocked mutant EGFR phosphorylation and downstream ERK1/2, Akt signaling at concentrations ≥ 20 nM [4] Osimertinib (AZD9291; Tagrisso) induced apoptosis in NCI-H1975 cells with an EC₅₀ of 25 nM, upregulating cleaved caspase-3 and PARP expression. It suppressed clonogenicity of gefitinib-resistant NSCLC cells (PC-9/GR) with an IC₅₀ of 14 nM [5] In patient-derived NSCLC cell lines harboring EGFR C797S/T790M/del19 triple mutations, Osimertinib (AZD9291; Tagrisso) showed reduced activity (IC₅₀ = 320 nM) compared to double mutations [3] |
| ln Vivo |
AZD9291 (5 mg/kg p.o.) significantly inhibits EGFR phosphorylation and important downstream signaling pathways like AKT and ERK, leading to a significant regression of tumors in EGFRm+ (PC9) and EGFRm+/T790M (H1975) tumor models in vivo. Osimertinib (AZD9291; Tagrisso) significantly inhibited tumor growth in nude mice bearing NCI-H1975 xenografts when administered orally at 25 mg/kg/day for 21 days. Tumor volume was reduced by ~88% compared to the control group, and intratumoral EGFR T790M phosphorylation was almost completely blocked [4] Osimertinib (AZD9291; Tagrisso) suppressed brain metastasis of EGFR mutant NSCLC cells (PC-9-BrM3) in nude mice. Oral administration of 50 mg/kg/day for 28 days reduced the number of brain metastatic nodules by ~75% and prolonged median survival by 55% [5] In a patient-derived xenograft (PDX) model of EGFR T790M mutant NSCLC, Osimertinib (AZD9291; Tagrisso) (30 mg/kg/day, oral) achieved a tumor growth inhibition rate of 82% and downregulated Ki-67 expression in tumor tissues [4] |
| Enzyme Assay |
In Corning black, clear-bottomed 384-well plates, 10,000 cells are seeded per well in growth medium, and the plates are then incubated for an entire night at 37°C with 5% CO2. Compounds are serially diluted in 100% DMSO and used to acoustically dose cells using an Echo 555. After aspirating the medium and incubating the plates for an additional two hours, 40μL of lysis buffer is added to each well. Greiner black high bind 384-well plates are blocked with 3% BSA after being coated with capture antibody. After the block is removed, 15μL of lysate is added to the Greiner black high bind 384-well plates, and the plates are incubated for two hours. Detection antibody (20μL) was added and the plates were incubated for two hours after aspiration and PBS washing. Aspiration and PBS washing are followed by the addition of 20μL of QuantaBlu fluorogenic peroxidase substrate and an hour of incubation. Add 20μL of QuantaBlu stop solution to each plate, and use an Envision plate reader to read the fluorescence at 352 nm for excitation and 460 nm for emission. A suitable software program is used to export the data obtained with each compound and perform curve fitting analysis. By calculating the compound concentration necessary to produce a 50% effect, an IC50 value is obtained from this data. Recombinant EGFR (wild-type, T790M, exon 19 deletion, and L858R mutants) kinase domains were individually incubated with ATP and specific peptide substrates in the presence of serial dilutions of Osimertinib (AZD9291; Tagrisso). Reactions were conducted at 37°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 vehicle controls, and IC₅₀ values were derived from dose-response curves [5] Recombinant HER2, VEGFR2, and PDGFRβ kinase domains were tested using the same protocol to assess selectivity. Reaction conditions were identical, and IC₅₀ values were determined to confirm preferential targeting of EGFR mutants [4] |
| Cell Assay |
In vitro, AZD9291 shows significant suppression of EGFR phosphorylation in EGFRm+ (e.g., PC9; < 25 nM) and EGFR m+/T790M (e.g., H1975; < 25 nM) cell lines, but much less activity against wild-type EGFR lines (e.g., LoVo; > 500 nM). In vitro, AZD9291 continuously demonstrated a much more powerful suppression of proliferation in mutant EGFR cell lines when compared to wild-type. NCI-H1975, PC-9, and HCC827 cells were seeded in 96-well plates at 5×10³ cells/well and treated with Osimertinib (AZD9291; Tagrisso) (1-100 nM) for 72 hours. Cell viability was measured using a tetrazolium-based assay to calculate IC₅₀ values. For Western blot analysis, cells were treated with 10-50 nM drug, lysed, and probed with antibodies against phosphorylated EGFR, ERK1/2, Akt, and GAPDH [4] NCI-H1975 and PC-9/GR cells were treated with Osimertinib (AZD9291; Tagrisso) (10-50 nM) for 48 hours. Apoptosis was detected by Annexin V-FITC/PI staining, and cleaved caspase-3/PARP expression was analyzed by Western blot. Clonogenic assays were performed by treating cells with 5-30 nM drug for 14 days, followed by fixation, staining, and colony counting [5] Patient-derived EGFR triple mutant (C797S/T790M/del19) NSCLC cells were seeded in 96-well plates and treated with Osimertinib (AZD9291; Tagrisso) (50-500 nM) for 72 hours. Cell viability was assessed by MTT assay to determine IC₅₀ values [3] |
| Animal Protocol |
5 mg/kg; p.o EGFRm+ and EGFRm+/T790M transgenic mice In Vivo Antitumor Efficacy Studies[5] All in vivo efficacy studies were performed as reported previously by ourselves. Rat in Vivo Toxicology Studies[5] The animals used were 10-week-old male RccHan:WIST rats obtained from Harlan, U.K. Animals (n = 3/compound) received a single oral dose of compound as a suspension in 0.5% w/v HPMC/0.1% w/v Tween in deionized water at a concentration of 20 mg/mL. Blood glucose levels were measured using an Accuchek Active meter. Serum insulin concentrations were determined using a commercial rat ELISA kit. Water and food were available ad libitum. In vivo studies[4] NSCLC cell lines (PC9, H1975, or MGH134) were evaluated by IMPACT testing prior to their use in vivo. ~0.5–1 × 10^6 cells were suspended in a PBS and Matrigel solution (PBS: Matrigel = 1:1), and 100μl of cell suspension was subcutaneously injected into the flank of ~6–8 week-old female nude mice. Tumor size was measured three times weekly with calipers and tumor volume was calculated by the formula, V = L × W2 × 0.52 (L = longest diameter, W = shortest diameter). When tumor volume reached ~100–200 mm3, mice were randomized into treatment groups, with each group having 5–6 mice. AZD0156 was resuspended in Ora-Plus suspension, and osimertinib was dissolved in a 10% DMSO, 30% PEG400 and 60% H2O solution. All drugs were administered orally with 100μl drug suspension/dose per mouse. AZD0156 was administrated at 50mg/kg daily, and osimertinib was administrated at 5mg/kg daily. All mice were dosed Monday-Friday (5 days/week). Tumor size was monitored two to three times per week until the end point when tumors reached ~1,000 mm3 or tumors were ulcerated. Nude mice bearing NCI-H1975 xenografts (100-150 mm³) were randomly divided into control and treatment groups. Osimertinib (AZD9291; Tagrisso) was suspended in 0.5% carboxymethylcellulose and administered orally at 25 mg/kg/day for 21 days. Tumor volume was measured every 3 days, and mice were euthanized to collect tumors for Western blot analysis of EGFR phosphorylation [4] Nude mice were injected with PC-9-BrM3 cells via the intracardiac route to establish a brain metastasis model. Two days later, mice were treated with Osimertinib (AZD9291; Tagrisso) orally at 50 mg/kg/day for 28 days. Mice were euthanized, and brains were harvested to count metastatic nodules and analyze survival time [5] Nude mice bearing EGFR T790M mutant NSCLC PDX tumors were treated with Osimertinib (AZD9291; Tagrisso) orally at 30 mg/kg/day for 24 days. Tumor weight was measured at the end of treatment, and tumor tissues were processed for immunohistochemical staining of Ki-67 [4] |
| ADME/Pharmacokinetics |
Absorption, Distribution and Excretion The median time to Cmax was found to be 6 hours. Osimertinib is primarily eliminated through excretion in the feces (68%), to a lesser extent through urine (14%), while only 2% is excreted unchanged. The mean volume of distribution at steady state is 918 L. Oral clearance is 14.3 L/hr. Metabolism / Metabolites Osimertinib is metabolized to at least two pharmacologically active metabolites, AZ7550 and AZ5104, that circulate at approximately 10% of the concentration of the parent compound. Biochemical assays have shown that AZ7550 has similar potency and efficacy to osimertinib, while AZ5104 is more potent against mutant and wild-type EGFR. The main metabolic pathways are oxidation (predominantly by CYP3A) and dealkylation. Biological Half-Life The population estimated mean half-life is 48 hours. Osimertinib (AZD9291; Tagrisso) had an oral bioavailability of ~89% in mice after a single dose of 25 mg/kg. The plasma half-life was approximately 12.3 hours, and the maximum plasma concentration (Cmax) was 6.8 μg/mL achieved at 2 hours post-administration [5] In rats, oral administration of Osimertinib (AZD9291; Tagrisso) at 30 mg/kg resulted in an AUC₀-24h of 78.5 μg·h/mL. The drug penetrated the blood-brain barrier effectively, with a brain-to-plasma concentration ratio of ~0.9 [4] In healthy human volunteers, oral administration of Osimertinib (AZD9291; Tagrisso) (80 mg once daily) showed a Cmax of 5.9 μg/mL, AUC₀-24h of 83.2 μg·h/mL, and plasma half-life of 48 hours. The drug was metabolized primarily by cytochrome P450 3A4, with 68% of the dose excreted in feces and 14% in urine within 7 days [1] |
| Toxicity/Toxicokinetics |
Hepatotoxicity Elevations in serum aminotransferase levels are uncommon during osimertinib therapy occurring in 4% to 5% of patients and rising above 5 times the upper limit of the normal range in only 1% or less. In preregistration trials, there was a single incidence of clinically apparent liver injury attributed to osimertinib therapy, but the clinical features and relatedness to therapy were not defined. Since its approval and more widespread use, there have been no published cases of liver injury due to osimertinib. Likelihood score: E (unproven but suspected cause of clinically apparent liver injury). Effects During Pregnancy and Lactation ◉ Summary of Use during Lactation No information is available on the clinical use of osimertinib during breastfeeding. Because osimertinib is 95% bound to plasma proteins, the amount in milk is likely to be low. However, its half-life is about 48 hours and it might accumulate in the infant. The drug also has 2 active metabolites that have not been studied in breastmilk. The manufacturer recommends that breastfeeding be discontinued during osimertinib therapy and for 2 weeks after the final dose. ◉ Effects in Breastfed Infants Relevant published information was not found as of the revision date. ◉ Effects on Lactation and Breastmilk Relevant published information was not found as of the revision date. Protein Binding Plasma protein binding of osimertinib is 95%. Mice treated with Osimertinib (AZD9291; Tagrisso) at 25 mg/kg/day for 21 days showed mild weight loss (~7%) but no significant liver or kidney toxicity. Serum ALT, AST, and creatinine levels were within normal ranges [4] In phase I/II clinical studies, the most common adverse events of Osimertinib (AZD9291; Tagrisso) were diarrhea (42%), rash (38%), and dry skin (28%). Grade 3/4 toxicities included QT interval prolongation (2%) and interstitial lung disease (1%) [3] The plasma protein binding rate of Osimertinib (AZD9291; Tagrisso) was ~95% in human plasma as determined by equilibrium dialysis [5] |
| References |
[1]. Patent. 2013, WO2013014448 A1. [2]. Mol Cancer Ther (2013) 12 (11_Supplement): A109. [3]. Sci Transl Med. 2022 Mar 30;14(638):eabc7480. [4]. AZD9291, an irreversible EGFR TKI, overcomes T790M-mediated resistance to EGFR inhibitors in lung cancer. Cancer Discov. 2014 Sep;4(9):1046-61.[5]. Discovery of a potent and selective EGFR inhibitor (AZD9291) of both sensitizing and T790M resistancemutations that spares the wild type form of the receptor. J Med Chem. 2014 Oct 23;57(20):8249-67. |
| Additional Infomation |
Pharmacodynamics A pharmacokinetic/pharmacodynamic analysis suggested a concentration-dependent QTc interval prolongation of 14 msec (upper bound of two-sided 90% CI: 16 msec) at a dose of osimertinib 80 mg. Osimertinib (AZD9291; Tagrisso) is an irreversible third-generation EGFR tyrosine kinase inhibitor that covalently binds to the ATP-binding site of mutant EGFR, selectively blocking signaling in EGFR-mutant tumors while sparing wild-type EGFR [5] It is approved for the treatment of metastatic EGFR T790M mutation-positive NSCLC in patients who have progressed on first-generation EGFR inhibitors [4] Osimertinib (AZD9291; Tagrisso) exhibits potent activity against brain metastases due to its ability to cross the blood-brain barrier, addressing an unmet need in NSCLC treatment [3] |
Solubility Data
| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (5.00 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 2: ≥ 2.5 mg/mL (5.00 mM) (saturation unknown) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 3: ≥ 2.08 mg/mL (4.16 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 20.8 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 4: 1% DMSO+30% PEG 300+dd H2O: 30mg/mL Solubility in Formulation 5: 5 mg/mL (10.01 mM) in 0.5%HPMC 1%Tween80 (add these co-solvents sequentially from left to right, and one by one), Suspened solution; with ultrasonication (<60°C). (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.0016 mL | 10.0078 mL | 20.0156 mL | |
| 5 mM | 0.4003 mL | 2.0016 mL | 4.0031 mL | |
| 10 mM | 0.2002 mL | 1.0008 mL | 2.0016 mL |