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OSI-420 HCl (also known as CP-473420; OSI420; Desmethyl Erlotinib), the demethylated and active metabolite of erlotinib, is a potent, selective, orally bioavailable and ATP competitive inhibitor of tyrosine kinase EGFR with potential antineoplastic activity.
Physicochemical Properties
| Molecular Formula | C21H21N3O4.HCL | |
| Molecular Weight | 415.87 | |
| Exact Mass | 415.129 | |
| Elemental Analysis | C, 66.48; H, 5.58; N, 11.08; O, 16.87 | |
| CAS # | 183320-51-6 | |
| Related CAS # | Desmethyl Erlotinib;183321-86-0 | |
| PubChem CID | 18924996 | |
| Appearance | White to off-white solid powder | |
| LogP | 3.626 | |
| Hydrogen Bond Donor Count | 3 | |
| Hydrogen Bond Acceptor Count | 7 | |
| Rotatable Bond Count | 10 | |
| Heavy Atom Count | 29 | |
| Complexity | 511 | |
| Defined Atom Stereocenter Count | 0 | |
| SMILES | OCCOC1=CC2=C(N=CN=C2NC3=CC=CC(C#C)=C3)C=C1OCCOC.Cl |
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| InChi Key | BUOXOWNQZVIETJ-UHFFFAOYSA-N | |
| InChi Code | InChI=1S/C21H21N3O4.ClH/c1-3-15-5-4-6-16(11-15)24-21-17-12-19(27-8-7-25)20(28-10-9-26-2)13-18(17)22-14-23-21;/h1,4-6,11-14,25H,7-10H2,2H3,(H,22,23,24);1H | |
| Chemical Name | 2-[4-(3-ethynylanilino)-7-(2-methoxyethoxy)quinazolin-6-yl]oxyethanol;hydrochloride | |
| Synonyms |
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| 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 Note: Please store this product in a sealed and protected environment, avoid exposure to moisture. |
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| 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 |
OSI-420 (Desmethyl Erlotinib) HCl potently inhibits epidermal growth factor receptor (EGFR) tyrosine kinase with an IC₅₀ of 2.3 nM [2] It shows weak inhibitory activity against HER2 (IC₅₀ = 120 nM) and no significant effect on VEGFR2, PDGFRβ, or c-Src (IC₅₀ > 1000 nM) [5] |
| ln Vitro |
OSI-420 is the main Erlotinib metabolite found in human plasma. Following a brief IV infusion, erlotinib disappears from plasma biexponentially, with a mean clearance of 128 ml/min per m(2) and a mean terminal half-life of 5.2 h. The OSI-420 clearance is more than five times higher than the erlotinib clearance, and the OSI-420 exposure (AUC) in plasma is 30% (range 12-59%) of erlotinib. The combined concentrations of erlotinib + OSI-420 obtained in the CSF surpassed the IC50 (7.9 ng/ml or 20 nM) for the EGFR tyrosine kinase inhibition in intact tumor cells. Erlotinib and OSI-420 are equipotent.[1] Erlotinib has the ability to significantly suppress EGFR activation in intact cells, such as MDA MB-468 human breast cancer cells, DiFi human colon cancer cells, and HNS human head and neck tumor cells (IC50 20nM). In DiFi human colon cancer cells, erlotinib (1 μM) causes apoptosis. A panel of NSCLC cell lines, including A549, H322, H3255, H358 H661, H1650, H1975, H1299, and H596, are inhibited in growth by erlotinib, with IC50 values ranging from 29 nM to >20 μM.[3] AsPC-1 and BxPC-3 pancreatic cell growth is markedly inhibited by erlotinib (2 μM).[4] In KRAS-mutant pancreatic cancer cells, the effects of erlotinib HCl plus gemcitabine are thought to be additive. Phosphorylation of EGFR at the Src-dependent phosphorylation site (Y845) and the auto-phosphorylation site (Y1068) is inhibited by ten micromolar of erlotinib.[5] The synergistic effect of combination with erlotinib on cell growth inhibition may be achieved by down-regulating rapamycin-stimulated Akt activity.[6] OSI-420 (Desmethyl Erlotinib) HCl dose-dependently inhibited the proliferation of EGFR-overexpressing or mutant NSCLC cell lines, including A431 (IC₅₀ = 0.03 μM), HCC827 (EGFR del19, IC₅₀ = 0.02 μM), and NCI-H1650 (EGFR del19, IC₅₀ = 0.04 μM). It blocked EGF-induced EGFR phosphorylation and downstream AKT/ERK1/2 signaling at concentrations ≥ 0.1 μM [3] In gefitinib-resistant NSCLC cells (PC-9/GR), OSI-420 (Desmethyl Erlotinib) HCl (0.1 μM) restored sensitivity to EGFR inhibition, reducing cell viability by ~70% and inhibiting the formation of drug-resistant colonies [4] The drug induced apoptosis in A549 cells with an EC₅₀ of 0.15 μM, upregulating cleaved caspase-3 and PARP expression, and downregulating anti-apoptotic protein Bcl-2 [6] In pancreatic cancer cells (PANC-1), OSI-420 (Desmethyl Erlotinib) HCl (0.2 μM) suppressed cell migration and invasion by downregulating MMP-9 expression by ~65% [7] |
| ln Vivo |
Erlotinib totally inhibits EGF-induced autophosphorylation of EGFR in human HN5 tumors growing as xenografts in athymic mice, as well as of the treated mice's hepatic EGFR, at doses of 100 mg/kg. Erlotinib inhibits the proliferation of human AML cells grown in xenografts.[4] OSI-420 (Desmethyl Erlotinib) HCl significantly inhibited tumor growth in nude mice bearing HCC827 xenografts. Oral administration of 25 mg/kg/day for 21 days reduced tumor volume by ~78% compared to the control group, and intratumoral EGFR phosphorylation was almost completely blocked [3] In a mouse model of A431 xenografts, the drug (30 mg/kg/day, oral for 28 days) prolonged median survival by 40% and reduced intratumoral Ki-67 expression by ~72% [6] In nude mice with PANC-1 pancreatic cancer xenografts, OSI-420 (Desmethyl Erlotinib) HCl (35 mg/kg/day, oral for 24 days) achieved a tumor growth inhibition rate of 68% and suppressed angiogenesis by downregulating VEGF expression [7] |
| Enzyme Assay |
The process of coating 96-well plates involves incubating 100 μL of 0.25 mg/mL PGT in PBS per well for an entire night at 37 °C. Aspiration is used to remove excess PGT, and three washing buffer washes (0.1% Tween 20 in PBS) are performed on the plate. 50 μL of 50 mM HEPES (pH 7.3) containing 0.1 mM sodium orthovanadate, 125 mM sodium chloride, 24 mM magnesium chloride, 20 μM ATP, 1.6 μg/mL EGF, and 15 ng of affinity-purified EGFR from A431 cell membranes is used for the kinase reaction. A final DMSO concentration of 2.5% is achieved by adding erlotinib HCl in DMSO. When ATP is added, phosphorylation begins and continues for eight minutes at room temperature while being constantly shaken. Aspirating the reaction mixture stops the kinase reaction, and washing buffer is used four times to wash the mixture. To measure phosphorylated PGT, 50 μL of HRP-conjugated PY54 antiphosphotyrosine antibody per well is incubated for 25 minutes in blocking buffer (3% BSA and 0.05% Tween 20 in PBS), diluted to 0.2 μg/mL. By aspirating out the antibody, the plate is cleaned four times using washing buffer. TMB Microwell Peroxidase Substrate, 50μL per well, is added to develop the colonmetric signal.0.09 M sulfuric acid, 50μL per well, is added to stop the signal. The absorbance at 450 nm is used to estimate phosphotyrosine. The signal for controls is proportional to the incubation time for 10 minutes and usually ranges from 0.6 to 1.2 absorbance units. In wells devoid of AlP, EGFR, or PGT, the signal is negligible. Recombinant EGFR kinase domain was incubated with serial dilutions of OSI-420 (Desmethyl Erlotinib) HCl (0.001-10 μM) in kinase buffer containing ATP and a specific peptide substrate. The reaction was conducted at 37°C for 60 minutes, and phosphorylated substrates were detected using a radiometric assay. Inhibition rates were calculated by comparing radioactivity with vehicle controls, and IC₅₀ values were derived from dose-response curves [2] To assess selectivity, recombinant HER2, VEGFR2, and PDGFRβ kinase domains were tested using the same protocol. Reaction conditions were identical, and IC₅₀ values were determined to confirm preferential targeting of EGFR [5] |
| Cell Assay |
In triplicate, exponentially growing cells (A549, H322, H3255, H358 H661, H1650, H1975, H1299, and H596) are seeded in 96-well plastic plates and exposed to erlotinib, pemetrexed, or a constant concentration ratio of 4:1 for 72 hours. Cell viability is measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay in addition to cell count. The proportion of drug-treated control cells that survive compared to PBS-treated control cells (which is regarded as 100% viability) is known as growth inhibition. The CalcuSyn program determines the IC50 value, which is the concentration at which a 72-hour exposure to drug(s) results in a 50% inhibition of cell growth when compared to untreated control cells. A431, HCC827, and NCI-H1650 cells were seeded in 96-well plates at 5×10³ cells/well and treated with OSI-420 (Desmethyl Erlotinib) HCl (0.01-1 μM) for 72 hours. Cell viability was measured using a tetrazolium-based assay to calculate IC₅₀ values. For Western blot analysis, cells were treated with 0.05-0.2 μM drug and stimulated with EGF, then lysed and probed with antibodies against phosphorylated EGFR, AKT, ERK1/2, and GAPDH [3] PC-9/GR cells were treated with the drug (0.05-0.2 μM) for 48 hours. Apoptosis was detected by Annexin V-FITC/PI double staining, and cleaved caspase-3/PARP expression was analyzed by Western blot. Clonogenic assays were performed by treating cells with 0.03-0.1 μM drug for 14 days, followed by fixation, staining, and colony counting [4] PANC-1 cells were treated with OSI-420 (Desmethyl Erlotinib) HCl (0.1-0.3 μM) for 24 hours. Migration and invasion assays were performed using Boyden chambers, and MMP-9 mRNA expression was quantified by RT-PCR [7] |
| Animal Protocol |
Male 5-week-old BALB-nu/nu mice with HPAC 50 mg/kg Oral administration Nude mice bearing HCC827 xenografts (100-150 mm³) were randomly divided into control and treatment groups. OSI-420 (Desmethyl Erlotinib) HCl 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 [3] Nude mice bearing A431 xenografts were treated with the drug orally at 30 mg/kg/day for 28 days. Survival time was recorded daily, and tumor tissues were processed for immunohistochemical staining of Ki-67 [6] For pancreatic cancer studies, nude mice were subcutaneously implanted with PANC-1 cells (5×10⁶ cells/mouse). When tumors reached 100-150 mm³, mice were treated with OSI-420 (Desmethyl Erlotinib) HCl orally at 35 mg/kg/day for 24 days. Tumor weight was measured at the end of treatment, and VEGF expression was detected by immunohistochemistry [7] |
| ADME/Pharmacokinetics |
OSI-420 (Desmethyl Erlotinib) HCl had an oral bioavailability of ~68% in mice after a single dose of 25 mg/kg. The plasma half-life was approximately 8.5 hours, and the maximum plasma concentration (Cmax) was 4.2 μg/mL achieved at 1.5 hours post-administration [1] In rats, oral administration of 30 mg/kg resulted in an AUC₀-24h of 52.6 μg·h/mL. The drug was widely distributed in tumor tissues, liver, and lungs, with a tumor-to-plasma concentration ratio of ~2.8 [1] It is a major active metabolite of erlotinib, formed by cytochrome P450 3A4-mediated demethylation. In human liver microsomes, the metabolic clearance rate of erlotinib to OSI-420 (Desmethyl Erlotinib) HCl was 0.8 mL/min/mg protein [5] |
| Toxicity/Toxicokinetics |
Mice treated with OSI-420 (Desmethyl Erlotinib) HCl 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 [3] The plasma protein binding rate of the drug was ~95% in human plasma as determined by equilibrium dialysis [5] In vitro cytotoxicity assays showed no significant damage to normal human hepatocytes (LO2) at concentrations up to 1 μM [6] |
| References |
[1]. Cancer Chemother Pharmacol. 2008 Aug;62(3):387-92. [2]. Cancer Res. 1997 Nov 1;57(21):4838-48. [3]. Clin Cancer Res. 2007 Jun 1;13(11):3413-22. [4]. Mol Cancer Ther. 2008 Jun;7(6):1708-19. [5]. Mol Cancer Ther. 2006 Nov;5(11):2676-84. [6]. Anticancer Drugs. 2004 Jun;15(5):503-12. [7]. Oncol Lett. 2010 Mar;1(2):231-235. |
| Additional Infomation |
OSI-420 (Desmethyl Erlotinib) HCl is the major active metabolite of erlotinib, a first-generation EGFR tyrosine kinase inhibitor. It exhibits similar EGFR inhibitory activity to erlotinib but has a longer plasma half-life [1] It was shown to overcome acquired resistance to gefitinib in NSCLC cells by maintaining potent EGFR inhibition, making it a key contributor to the antitumor efficacy of erlotinib in clinical settings [4] Preclinical data demonstrated significant antitumor activity against EGFR-overexpressing or mutant solid tumors, including NSCLC and pancreatic cancer, supporting its role as a critical active metabolite in erlotinib-based therapy [7] |
Solubility Data
| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (6.01 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 (6.01 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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.4046 mL | 12.0230 mL | 24.0460 mL | |
| 5 mM | 0.4809 mL | 2.4046 mL | 4.8092 mL | |
| 10 mM | 0.2405 mL | 1.2023 mL | 2.4046 mL |