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| Targets |
As an active metabolite of midostaurin, O-Desmethyl Midostaurin is also a multi-targeted ATP-competitive protein kinase inhibitor. It exhibits inhibitory activity against various kinases, including mutant forms of FLT3 and KIT receptor tyrosine kinases implicated in the pathogenesis of acute myeloid leukemia (AML), as well as multiple kinases involved in their downstream signaling pathways such as IGF1R, JAK, LYN, PDK1, RET, SRC, and SYK. Compared to the parent drug, O-Desmethyl Midostaurin retains a similar kinase inhibition profile and potency, though certain kinases (e.g., LCK) are less sensitive to it.
O-Desmethyl Midostaurin retains the multi-kinase inhibitory profile of its parent compound, midostaurin. It targets a broad spectrum of protein kinases, including wild-type and mutant forms of Fms-like tyrosine kinase 3 (FLT3), protein kinase C (PKC), KIT, and vascular endothelial growth factor receptor 2 (VEGFR2). By inhibiting these key signaling nodes, the metabolite disrupts downstream pro-survival and proliferative pathways. Its primary role is as an active circulating metabolite contributing to the overall pharmacological activity of midostaurin. |
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| ln Vitro |
In vitro studies demonstrate that O-Desmethyl Midostaurin exhibits significant inhibitory activity against various protein kinases at 10 µM concentration. It inhibits both serine-threonine and tyrosine protein kinases, with an inhibition profile similar to that of midostaurin, though differences exist for individual kinases: CGP62221 is the most potent inhibitor of PRKG2 but shows weaker activity against LCK. Notably, the introduction of a hydroxyl group in the pyrrole ring (as seen in metabolites e1 and e2) generally reduces kinase inhibitory potency while increasing selectivity for VEGFR2, whereas O-Desmethyl Midostaurin, as a non-hydroxylated metabolite, retains higher inhibitory activity.
Midostaurin demonstrates potent multi-kinase inhibition with IC50 values ranging from 22 to 500 nM against various targets including PKC, VEGFR2, KIT, FLT3, PDGFR, and others. O-Desmethyl Midostaurin effectively inhibits FLT3 signaling, which is frequently constitutively activated in AML cells. This inhibition leads to reduced cell proliferation and increased apoptosis in FLT3-mutated cell lines. The compound also inhibits tyrosine phosphorylation of both FLT3 internal tandem duplication (ITD) and D835Y point mutants in vitro. |
| ln Vivo |
O-Desmethyl Midostaurin is one of the major circulating active components following chronic dosing of midostaurin in vivo. In humans, following a single 50 mg oral dose of [¹⁴C]-midostaurin, the plasma AUC₀-₉₆hr of CGP62221 is 22.5 μg Eq*h/mL, higher than that of the parent drug (18.0 μg Eq*h/mL). Under a 50 mg twice-daily dosing regimen, steady-state plasma trough levels show CGP62221 at 1.48 µM and another metabolite e2 as high as 6.73 µM, indicating significant accumulation of metabolites in vivo. At physiologically relevant doses, these metabolites exhibit substantial kinase inhibitory activity, potentially contributing to the clinical efficacy of midostaurin in AML through complex cooperative effects with the parent drug's kinase activity.
As the active metabolite, O-Desmethyl Midostaurin circulates in the plasma following oral administration of midostaurin. Studies using a plasma inhibitory activity (PIA) assay have demonstrated that the metabolite contributes to the cytotoxic response against FLT3-mutated leukemia cells in vivo. It serves as an effective indicator for monitoring midostaurin metabolism and systemic exposure in living organisms. However, detailed in vivo efficacy studies specifically for the isolated metabolite are less commonly reported than for the parent drug. |
| Enzyme Assay |
Recombinant Kinase Preparation: Express and purify recombinant target kinases (e.g., FLT3, KIT, PKC) using insect cell or E. coli expression systems.
Radiometric Transphosphorylation Assay: Incubate varying concentrations of O-Desmethyl Midostaurin (typically 0.1 nM-10 µM) with recombinant kinase, specific kinase substrate, and [γ-³³P]ATP at room temperature or 30°C for appropriate duration.
Signal Detection: After the reaction, transfer the mixture to phosphocellulose filters, wash to remove unbound free isotopes, and measure bound radioactivity using a scintillation counter.
Data Analysis: Calculate the inhibition rate of kinase activity at each concentration, plot dose-response curves, and calculate IC₅₀ values via non-linear regression analysis.
For in vitro enzyme inhibition assays, recombinant FLT3 kinase domain is incubated with a peptide substrate and varying concentrations of midostaurin or O-Desmethyl Midostaurin in reaction buffer containing ATP. After incubation, the phosphorylation level of the substrate is quantified using a luminescence-based or radiometric method. The half-maximal inhibitory concentration is calculated to determine potency. These assays confirm that both the parent drug and its metabolite effectively inhibit recombinant FLT3 kinase activity. |
| Cell Assay |
Cell Culture: Culture acute myeloid leukemia cell lines harboring FLT3-ITD or FLT3-TKD mutations (e.g., MV4-11, MOLM-13) in RPMI-1640 medium supplemented with 10% fetal bovine serum at 37°C in a 5% CO₂ incubator.
Drug Treatment: Seed cells in the logarithmic growth phase into 96-well plates (1-2×10⁴/well) or 24-well plates (5-10×10⁵/well), treat with various concentrations (0.001-10 µM) of O-Desmethyl Midostaurin with 3 replicate wells per concentration, and incubate for 48-72 hours.
Viability Assay: Measure cell viability using CCK-8 or MTT assay to calculate IC₅₀ values.
Apoptosis Detection: Detect apoptosis rate using Annexin V-FITC/PI double staining by flow cytometry.
Signaling Pathway Analysis: Detect FLT3 phosphorylation levels and activation status of downstream signaling molecules (e.g., STAT5, AKT, ERK) by Western blot.
Data Analysis: Compare cell viability, apoptosis rates, and signaling pathway changes between treatment and control groups.
For cellular inhibition assays, FLT3-mutant AML cell lines such as MV4-11 or Molm-14 are cultured in RPMI-1640 medium supplemented with fetal bovine serum. The cells are exposed to increasing concentrations of O-Desmethyl Midostaurin for 48-72 hours. Cell viability is assessed using a luminescence-based cell viability assay. Additionally, FLT3 phosphorylation status is evaluated via western blotting using a phospho-specific FLT3 antibody to confirm target engagement at the cellular level. |
| Animal Protocol |
Animals & Models: Use immunodeficient mice (e.g., nude or NSG mice) to establish xenograft tumor models via subcutaneous or tail vein injection of FLT3-mutant AML cells (e.g., MV4-11).
Dosing Regimen: When tumors reach approximately 100-200 mm³, randomize animals into control and treatment groups at various doses. O-Desmethyl Midostaurin can be administered via oral gavage or intraperitoneal injection, using vehicles such as a mixture of 10% DMSO, 40% PEG300, 5% Tween-80, and 45% saline.
Efficacy Assessment: Measure tumor volume and body weight 2-3 times weekly, record survival. Euthanize animals at study termination, collect tumor tissues for weighing and pathological analysis.
Target Inhibition Validation: Detect FLT3 phosphorylation levels and downstream signaling molecule changes in tumor tissues by Western blot.
Data Analysis: Compare tumor growth inhibition rates, survival, and molecular target inhibition between treatment and control groups.
For in vivo pharmacodynamic assessment, female athymic nude mice bearing xenograft tumors derived from FLT3-mutant human AML cell lines are treated orally with midostaurin at doses ranging from 10 to 100 mg/kg. Blood samples are collected at various time points post-administration. Plasma inhibitory activity against FLT3 target cells is then measured to evaluate the functional activity of both midostaurin and its metabolite O-Desmethyl Midostaurin circulating in the blood. |
| ADME/Pharmacokinetics |
O-Desmethyl Midostaurin is the primary active metabolite of midostaurin produced via hepatic CYP3A4 metabolism. In humans, following a single 50 mg oral dose of midostaurin, the plasma AUC₀₋₉₆hr of CGP62221 is 22.5 μg Eq*h/mL, higher than that of the parent drug (18.0 μg Eq*h/mL); under a 50 mg twice-daily dosing regimen, the steady-state plasma trough concentration is 1.48 µM. The exposure level of this metabolite indicates significant accumulation in vivo. O-Desmethyl Midostaurin can serve as a biomarker and internal standard for in vivo studies to assess the metabolic profile of midostaurin. This compound has good solubility in DMSO, which is commonly used as a vehicle for in vitro and in vivo experiments.
Following oral administration, midostaurin is metabolized by cytochrome P450 enzymes to produce O-Desmethyl Midostaurin as its primary active metabolite. The parent drug midostaurin has a half-life of approximately 20 hours, and the metabolite is expected to have similar pharmacokinetic properties, including high plasma protein binding and extensive tissue distribution. O-Desmethyl Midostaurin can be measured in plasma as an indicator of in vivo metabolism and exposure. |
| Toxicity/Toxicokinetics |
As an active metabolite of midostaurin, the toxicological profile of O-Desmethyl Midostaurin is related to the parent drug. Midostaurin, as an FDA-approved drug for the treatment of acute myeloid leukemia, has well-studied clinical tolerability. O-Desmethyl Midostaurin exhibits high exposure levels in systemic circulation and contributes to the efficacy of the parent drug. However, this compound itself is not a therapeutic agent but serves as a research tool for pharmacokinetic analysis and drug metabolism studies. This product is intended for scientific research use only and is not suitable for human diagnostic or therapeutic applications. Standard laboratory safety practices should be followed when handling, and the powder should be stored at -20°C in dry, dark conditions.
As a research chemical, the toxicological profile of O-Desmethyl Midostaurin has not been independently characterized. However, its parent compound midostaurin is known to cause adverse effects in clinical use including gastrointestinal disturbances, fatigue, and hematological toxicities such as neutropenia and thrombocytopenia. These effects are related to its mechanism of action as a multi-kinase inhibitor. Detailed toxicity data for the isolated metabolite remain to be established. |
| References | |
| Additional Infomation |
O-Desmethyl Midostaurin (CGP62221) is available as a reference standard for analytical method development and metabolic studies. It functions as an active circulating metabolite contributing to the in vivo efficacy of the FDA-approved drug midostaurin (Rydapt), which is indicated for the treatment of FLT3-mutated acute myeloid leukemia and advanced systemic mastocytosis. The compound is strictly intended for research applications only and not for human therapeutic use.
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| Molecular Formula |
C34H28N4O4
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|---|---|
| Molecular Weight |
556.6105
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| Exact Mass |
556.211
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| Elemental Analysis |
C, 73.37; H, 5.07; N, 10.07; O, 11.50
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| CAS # |
740816-86-8
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| Related CAS # |
O-Desmethyl Midostaurin-d5
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| PubChem CID |
11261445
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| Appearance |
Light yellow to yellow solid powder
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| LogP |
4.3
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
4
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| Rotatable Bond Count |
2
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| Heavy Atom Count |
42
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| Complexity |
1130
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| Defined Atom Stereocenter Count |
4
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| SMILES |
O1[C@]2([H])C([H])([H])[C@]([H])([C@]([H])([C@@]1(C([H])([H])[H])N1C3=C([H])C([H])=C([H])C([H])=C3C3=C4C([H])([H])N([H])C(C4=C4C5=C([H])C([H])=C([H])C([H])=C5N2C4=C13)=O)O[H])N(C(C1C([H])=C([H])C([H])=C([H])C=1[H])=O)C([H])([H])[H]
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| InChi Key |
PXOCRDZEEXVZQC-AFUPZKSLSA-N
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| InChi Code |
InChI=1S/C34H28N4O4/c1-34-31(39)24(36(2)33(41)18-10-4-3-5-11-18)16-25(42-34)37-22-14-8-6-12-19(22)27-28-21(17-35-32(28)40)26-20-13-7-9-15-23(20)38(34)30(26)29(27)37/h3-15,24-25,31,39H,16-17H2,1-2H3,(H,35,40)/t24-,25-,31-,34+/m1/s1
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| Chemical Name |
N-[(2S,3R,4R,6R)-3-hydroxy-2-methyl-16-oxo-29-oxa-1,7,17-triazaoctacyclo[12.12.2.12,6.07,28.08,13.015,19.020,27.021,26]nonacosa-8,10,12,14,19,21,23,25,27-nonaen-4-yl]-N-methylbenzamide
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| Synonyms |
O-Desmethyl Midostaurin; CGP-62221; CGP62221; O-Desmethyl PKC-412; O Desmethyl Midostaurin; 740816-86-8; O-Desmethyl Midostaurin; o-Desmethyl-pkc 412; H3W3QJ5C7G; CGP 62221;
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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 | 1.7966 mL | 8.9830 mL | 17.9659 mL | |
| 5 mM | 0.3593 mL | 1.7966 mL | 3.5932 mL | |
| 10 mM | 0.1797 mL | 0.8983 mL | 1.7966 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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