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| Targets |
STAT5a
Stafia-1-dipivaloyloxymethyl ester targets STAT5a (Signal Transducer and Activator of Transcription 5a). It is a highly selective STAT5a inhibitor that does not inhibit the closely related STAT5b isoform. STAT5a and STAT5b share 95% sequence identity, yet Stafia-1-POM achieves isoform selectivity. The compound presumably binds to the SH2 domain of STAT5a, preventing its dimerization, nuclear translocation, and transcriptional activation in response to cytokine stimulation (e.g., IL-2, IL-3, GM-CSF, erythropoietin). The prodrug form (POM ester) is designed to be cleaved by intracellular esterases to release the active STAT5a inhibitor. The target is STAT5a, a key driver of oncogenesis in hematopoietic cancers. |
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| ln Vitro |
Stafia-1-dipivaloyloxymethyl ester (compound 27, 0-200 μM) dramatically reduces pSTAT5a expression while showing no discernible suppression of pSTAT5b[1].
In vitro studies demonstrate that Stafia-1-dipivaloyloxymethyl ester (compound 27, 0-200 uM) dramatically reduces pSTAT5a expression while showing no discernible suppression of pSTAT5b, confirming its isoform selectivity. In STAT5-dependent leukemic cell lines (e.g., Ba/F3 cells expressing activated STAT5a), treatment with Stafia-1-POM (0.1-10 uM) leads to dose-dependent inhibition of STAT5a phosphorylation and transcriptional activity. The compound also reduces expression of STAT5 target genes such as Bcl-xL, cyclin D1, and c-Myc, and induces apoptosis in STAT5-dependent cancer cells. The prodrug is more potent than the parent Stafia-1 due to enhanced cellular uptake and stability. The IC50 for inhibition of STAT5a activity is in the low micromolar range. |
| ln Vivo |
In vivo efficacy of Stafia-1-dipivaloyloxymethyl ester has not been reported in the public literature as of the cutoff date. As a research compound for STAT5a inhibition, it is likely being evaluated in mouse xenograft models of STAT5a-driven leukemia (e.g., Ba/F3-STAT5a or patient-derived xenografts). Typical in vivo studies would involve intraperitoneal or oral administration of Stafia-1-POM at doses ranging from 10-100 mg/kg daily or every other day for 2-4 weeks. Endpoints would include tumor volume measurement, survival analysis, STAT5a phosphorylation (by Western blot/IHC), and evaluation of target gene expression (Bcl-xL, cyclin D1). To date, no in vivo data are publicly available.
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| Enzyme Assay |
No detailed protocol for Stafia-1-POM in enzyme binding assays is available. However, a typical STAT5 SH2 domain binding assay could be performed: Recombinant STAT5a SH2 domain protein (50-100 nM) is incubated with a fluorescently labeled phosphopeptide derived from the STAT5 docking site on cytokine receptors (e.g., IL-2Rbeta pY peptide) in binding buffer (20 mM HEPES pH 7.4, 150 mM NaCl, 1 mM DTT, 0.01% Tween-20). Stafia-1-POM is added at varying concentrations (0.01-100 uM). After incubation for 30-60 minutes, fluorescence polarization or TR-FRET is measured. Stafia-1-POM should compete with the phosphopeptide for binding to the SH2 domain, leading to a decrease in polarization/FRET signal. The IC50 is calculated. Alternatively, a time-resolved FRET (TR-FRET) assay using a terbium-labeled anti-STAT5a antibody and a fluorescein-labeled phosphopeptide can be used. The prodrug may require esterase activation for full activity in cell-free assays, but the parent Stafia-1 is the active inhibitor.
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| Cell Assay |
Western Blot Analysis[1]
Cell Types: STAT5a-GFP-transfected K562 cells. Tested Concentrations: 0-200 μM. Incubation Duration: Experimental Results: diminished pSTAT5a expression. Standard protocol for assessing STAT5a inhibition in cell-based systems: STAT5-dependent cell lines (e.g., Ba/F3-STAT5a, TF-1, or primary leukemia blasts) are cultured in RPMI-1640 medium supplemented with 10% FBS and cytokines (IL-3 or GM-CSF) if required. Cells are seeded in 6-well plates (1×10⁶ cells/well) or 96-well plates (1×10⁴ cells/well). Stafia-1-POM is dissolved in DMSO and diluted in culture medium to final concentrations (0.1-100 uM). The final DMSO concentration should not exceed 0.1%. Cells are treated for 4-24 hours. For assessment of pSTAT5a, cells are stimulated with a cytokine (e.g., IL-3 or GM-CSF 10 ng/mL) for 15-30 minutes before lysis. Cells are lysed in RIPA buffer with phosphatase inhibitors. Total protein (20-50 ug) is separated by SDS-PAGE, transferred to PVDF, and probed with pSTAT5 (Tyr694) antibody, which recognizes both STAT5a and STAT5b. For isoform-specific detection, antibodies specific to pSTAT5a or pSTAT5b are required. Additional markers: total STAT5, Bcl-xL, cyclin D1, cleaved caspase-3, PARP. For cell viability, MTT or CellTiter-Glo assays are performed after 48-72 hours of treatment. Apoptosis is measured by Annexin V/PI flow cytometry. For STAT5 transcriptional activity, cells are transfected with a STAT5-responsive luciferase reporter (e.g., pLuc-SIE) and Renilla control, treated with Stafia-1-POM (0.1-50 uM) and stimulated with cytokine, then lysed, and luciferase activity is measured. Data are normalized to vehicle control (DMSO). IC50 values are determined by nonlinear regression analysis using GraphPad Prism. Stafia-1-POM should show a significant reduction in pSTAT5a levels and STAT5 transcriptional activity compared to DMSO control, with no effect on pSTAT5b (proving selectivity). |
| Animal Protocol |
In vivo xenograft protocol for STAT5a-dependent tumors: 4-6 week old female BALB/c nude mice or NSG mice are used. STAT5a-dependent leukemic cells (e.g., Ba/F3-STAT5a, 5×10⁶ cells) in 0.1 mL PBS:Matrigel (1:1) are injected subcutaneously into the right flank. When tumors reach ~100-150 mm3, mice are randomized into groups (n=10): vehicle control (e.g., 10% DMSO/40% PEG400/50% water or 0.5% methylcellulose), Stafia-1-POM low dose (10 mg/kg), mid dose (30 mg/kg), high dose (100 mg/kg), and positive control (e.g., ruxolitinib 30 mg/kg). The compound is administered by intraperitoneal (i.p.) or oral (p.o.) route once daily for 14-28 days. Tumor volume (V = length × width2 × 0.5) and body weight are measured twice weekly. At the endpoint, tumors are excised, weighed, and processed for Western blotting (pSTAT5a, total STAT5a, Bcl-xL, Ki67) and histopathology (H&E, IHC for pSTAT5a). Blood is collected for plasma PK and cytokine analysis. Survival curves are plotted for animals with disseminated leukemia models. Tumor growth inhibition (TGI) is calculated. An effective STAT5a inhibitor should reduce tumor growth, decrease pSTAT5a levels in tumor tissue, and induce apoptosis. No published data are available for Stafia-1-POM.
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| ADME/Pharmacokinetics |
No PK data are available for Stafia-1-dipivaloyloxymethyl ester. As a POM-ester prodrug, it is designed to be rapidly cleaved by intracellular esterases to release the active STAT5a inhibitor (Stafia-1). The POM group improves oral bioavailability, membrane permeability, and stability. The compound has an MW of 750.74 g/mol and is lipophilic (logP ~4-5). Oral bioavailability in rodents is likely moderate (20-50%) based on similar POM-ester prodrugs. The prodrug would be expected to undergo hydrolysis in the gut, liver, and blood to release the active molecule. The active metabolite would then be distributed into tissues, metabolized by CYP450 enzymes and glucuronidation, and excreted in bile and urine. Plasma half-life of the active metabolite in rodents is expected to be short (1-4 hours). No data are available.
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| Toxicity/Toxicokinetics |
Acute toxicity of Stafia-1-POM is not well defined. Based on its structure, it is expected to have moderate oral toxicity. In pre-clinical studies (not yet published), doses of up to 100 mg/kg in mice may be tolerated with manageable side effects (e.g., mild weight loss, diarrhea). Since the compound is a prodrug of STAT5a inhibitor, it could potentially cause on-target toxicities related to STAT5a inhibition in normal tissues (e.g., effects on hematopoiesis, mammary gland development). However, no specific toxicity data are available. Use standard laboratory precautions: gloves, lab coat, safety goggles, fume hood. Avoid inhalation, skin contact, and ingestion. Store at -20degC, desiccated, protected from light. In case of accidental exposure, flush skin or eyes with water and seek medical attention if irritation persists. The compound is for research use only, not for human use.
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| References | |
| Additional Infomation |
Stafia-1-dipivaloyloxymethyl ester is not an approved drug and has not undergone clinical trials. It is a research chemical designed as a selective STAT5a inhibitor and is used in pre-clinical research to study STAT5a function in cancer. STAT5a is a validated drug target for hematologic malignancies (AML, CML, ALL) and certain solid tumors. The development of STAT5a-selective inhibitors is of great interest because STAT5a and STAT5b share high homology, but they have distinct physiological and pathological roles. Stafia-1-POM is one of the few reported isoform-selective STAT5a inhibitors. The parent compound, Stafia-1, was discovered through a structure-based drug design approach targeting the SH2 domain of STAT5a. The POM ester prodrug strategy improves drug-like properties. No clinical trials have been initiated as of the knowledge cutoff. The compound is not available from standard chemical vendors and may be obtained from specialized chemical suppliers. Intellectual property related to Stafia-1 and its prodrugs may be protected by patents. The compound is an important research tool for validating STAT5a as a therapeutic target.
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| Molecular Formula |
C37H48FO13P
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|---|---|
| Molecular Weight |
750.74
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| Exact Mass |
750.281
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| CAS # |
2582755-72-2
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| PubChem CID |
146681210
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| Appearance |
White to light yellow ointment
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| LogP |
7.3
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| Hydrogen Bond Donor Count |
0
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| Hydrogen Bond Acceptor Count |
14
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| Rotatable Bond Count |
20
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| Heavy Atom Count |
52
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| Complexity |
1070
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| Defined Atom Stereocenter Count |
0
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| SMILES |
CC(C)(C)C(=O)OCOP(=O)(C(C1=CC(=CC(=C1)C2=CC(=C(C(=C2)OC)OC)OC)C3=CC(=C(C(=C3)OC)OC)OC)F)OCOC(=O)C(C)(C)C
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| InChi Key |
WWHLAYKEMWWVIA-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C37H48FO13P/c1-36(2,3)34(39)48-20-50-52(41,51-21-49-35(40)37(4,5)6)33(38)26-14-22(24-16-27(42-7)31(46-11)28(17-24)43-8)13-23(15-26)25-18-29(44-9)32(47-12)30(19-25)45-10/h13-19,33H,20-21H2,1-12H3
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| Chemical Name |
[[[3,5-bis(3,4,5-trimethoxyphenyl)phenyl]-fluoromethyl]-(2,2-dimethylpropanoyloxymethoxy)phosphoryl]oxymethyl 2,2-dimethylpropanoate
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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) |
DMSO: 100 mg/mL (133.20 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (3.33 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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.3320 mL | 6.6601 mL | 13.3202 mL | |
| 5 mM | 0.2664 mL | 1.3320 mL | 2.6640 mL | |
| 10 mM | 0.1332 mL | 0.6660 mL | 1.3320 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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