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| Targets |
Vps34 (vacuolar protein sorting 34), a class III phosphatidylinositol 3-kinase. Vps34-IN-2 is a potent and selective inhibitor of Vps34 with IC50s of 2 nM in enzymatic assays and 82 nM in GFP-FYVE cellular assays. The compound exhibits selectivity against mTOR (IC50 > 10 microM) and class I PI3Ks with IC50 values of 2.7, 4.5, 2.5, and >10 microM for PI3K alpha, beta, delta, and gamma isoforms, respectively. By inhibiting Vps34, the compound disrupts autophagosome formation and endosomal trafficking.
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| ln Vitro |
When using the GFP-FYVE cellular test and the Vps34 enzymatic assay, Vps34-IN-2 (Compound 31) exhibits IC50s of 2 and 82 nM, respectively. Selectivity against class I PI3Ks (IC50 values of 2.7, 4.5, 2.5, and >10 μM on PI3K α, β, δ, and γ isoforms, respectively) and mTOR (IC50>10 μM) is demonstrated by Vps34-IN-2[1].
In vitro, Vps34-IN-2 displays potent inhibitory activity against Vps34 with an IC50 of 2 nM in enzymatic assays. In the GFP-FYVE cellular assay, which measures the intracellular localization of the FYVE domain (a PI3P-binding domain), the compound exhibits an IC50 of 82 nM. The compound shows excellent selectivity over mTOR and class I PI3K isoforms. Vps34-IN-2 is used as a chemical tool to study the role of Vps34 in autophagy, endocytosis, and other cellular processes. |
| ln Vivo |
Vps34-IN-2 (Compound 31) concentrations can be measured up to 6, 8, and 24 hours (latest sampling time) following intravenous (IV) dosing, depending on the animal. Vps34-IN -2 is quickly absorbed after oral administration (po), with a bioavailability of 85% and maximal plasma concentrations seen at 0.5 hours. At 4 and 8 hours following oral dosage, there are slight concentration returns that are not readily explained. Acute plasma clearance of 2.3 L/h/kg, or 44% of hepatic blood flow in this species, is observed following an intravenous injection of Vps34-IN-2 at a dose of 3 mg/kg. The volume of distribution at steady state is also found to be moderate, and the terminal elimination half-life is short[1].
In vivo studies of Vps34-IN-2 have characterized its pharmacokinetic properties in animal models. Following intravenous administration, compound concentrations are quantifiable up to 6, 8, and 24 hours depending on the animal species. After oral administration, Vps34-IN-2 is rapidly absorbed with maximal plasma concentrations observed at 0.5 hours and a bioavailability of 85%. Slight rebounds in concentrations are observed at 4 and 8 hours after oral dosing with no obvious explanation. After intravenous injection at 3 mg/kg, plasma clearance is moderate (2.3 L/h/kg), corresponding to 44% of hepatic blood flow. |
| Enzyme Assay |
In vitro enzymatic assays for Vps34-IN-2 measure inhibition of Vps34 lipid kinase activity. The assay uses recombinant Vps34 enzyme and phosphatidylinositol as substrate in the presence of ATP. The compound is serially diluted in assay buffer and pre-incubated with the enzyme. The reaction is initiated by adding ATP and lipid substrate, and after incubation, the production of phosphatidylinositol 3-phosphate (PI3P) is measured using either radiometric (33P-ATP) or non-radiometric (HTRF or AlphaScreen) methods. IC50 values are calculated from dose-response curves. Selectivity profiling against other PI3Ks and mTOR is performed using similar assays with appropriate substrates.
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| Cell Assay |
Cells are cultured in RPMI-1640 medium with 10% fetal bovineserum. Cells are lysed by sonication in a detergent containing lysisbuffer and cleared by centrifugation, and the resulting supernatant iscollected for compound treatment. Final protein concentration of lysatesis 4 mg/mL. An amount of 5 μL of Vps34-IN-2 (Compound 31) is added from100× stock solutions in DMSO to 445 μL of lysate in duplicate. Anamount of 5 μL of DMSO is added to 445 μL of lysate in quadruplicate forcontrols. After 15 min incubation, 5 μL of a 100× aqueous solution ofthe ATP probe I is added to each sample (final concentration of ATPprobe I is 0.5 μM). After 5 min, 50 μL of a 10× aqueous solution of theATP probe II is added to each sample (final concentration of ATP probeII is 20 μM). All samples are then incubated for an additional 10min[1].
In vitro cellular assays for Vps34-IN-2 use the GFP-FYVE cellular assay to measure inhibition of Vps34 activity in cells. Cells expressing GFP-FYVE (a fusion protein that binds PI3P) are treated with varying concentrations of the compound. The intracellular localization of GFP-FYVE (which normally binds to PI3P-containing endosomal membranes) is monitored by fluorescence microscopy or high-content imaging. Inhibition of Vps34 reduces PI3P production, causing GFP-FYVE to become diffusely cytoplasmic. The EC50 for this effect is 82 nM. Alternatively, autophagy markers (LC3-II, p62) can be assessed by Western blotting in cells treated with the compound. |
| Animal Protocol |
For PK/PD studies, 3×106 H1299-GFP-FYVE tumor cells with 50%Matrigel are subcutaneously injected on the dorsal side of SCID mice,one tumor per mouse. When xenografted tumors reach a range of ~200 to400 mm3, mice are treated with vehicle (98% PEG200/2% PS80) or a singledose of Vps34-IN-2 (compound 31) at 100 and 50 mg/kg via oral gavage.Three mice treated with vehicle alone and three mice treated withVps34-IN-2 are sacrificed at each time point; tumor tissues areharvested for immunohistochemistry (IHC) analysis and plasma samples arecollected to determine the concentration of Vps34-IN-2[1].
In vivo pharmacokinetic studies of Vps34-IN-2 are conducted in rodents (typically rats or mice). Animals receive the compound via intravenous (3 mg/kg) or oral administration. Blood samples are collected at various time points (0-24 hours), and plasma concentrations are measured by LC-MS/MS. Pharmacokinetic parameters calculated include Cmax, Tmax, AUC, clearance, volume of distribution, half-life, and oral bioavailability. The compound is formulated in appropriate vehicles (e.g., DMSO/PEG400/saline) for administration. Standard animal handling and ethical guidelines are followed. |
| ADME/Pharmacokinetics |
Pharmacokinetics of Vps34-IN-2 have been characterized in preclinical species. After oral administration, the compound is rapidly absorbed with Tmax at 0.5 hours and oral bioavailability of 85%. Following intravenous administration at 3 mg/kg, plasma clearance is moderate (2.3 L/h/kg), corresponding to 44% of hepatic blood flow. The volume of distribution at steady state is moderate, and the terminal elimination half-life is short. The compound is soluble in DMSO (10 mM) and appears as a white to off-white solid. Storage at -20degC is recommended.
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| Toxicity/Toxicokinetics |
Toxicological data for Vps34-IN-2 are limited to preclinical research studies. As a research-use inhibitor, comprehensive toxicity profiles are not extensively documented. The compound is intended for research purposes only and not for human therapeutic use. Standard safety precautions for handling chemical compounds apply. Acute and chronic toxicity studies have not been systematically reported. In vivo studies monitor body weight, clinical signs, and histopathological findings as part of pharmacokinetic and pharmacodynamic evaluations.
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| References |
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| Additional Infomation |
Vps34-IN-2 is a research-grade potent and selective Vps34 inhibitor. Its IUPAC name is (8S)-2-[(3R)-3-methylmorpholin-4-yl]-9-(3-methyl-2-oxobutyl)-8-(trifluoromethyl)-7,8-dihydro-6H-pyrimido[1,2-a]pyrimidin-4-one. Synonyms include (S)-1-(3-Methyl-2-oxobutyl)-8-((R)-3-methylmorpholino)-2-(trifluoromethyl)-3,4-dihydro-1H-pyrimido[1,2-a]pyrimidin-6(2H)-one. Purity is 99%. The compound has an IC50 of 2 nM in Vps34 enzymatic assays and 82 nM in GFP-FYVE cellular assays. It is used as a tool for studying autophagy, cancer, neurodegenerative diseases, and other conditions where autophagy is implicated. Not approved for clinical use.
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| Molecular Formula |
C18H25F3N4O3
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|---|---|
| Molecular Weight |
402.411314725876
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| Exact Mass |
402.187
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| CAS # |
1523404-29-6
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| PubChem CID |
72709284
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| Appearance |
White to off-white solid powder
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| Density |
1.4±0.1 g/cm3
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| Boiling Point |
466.4±55.0 °C at 760 mmHg
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| Flash Point |
235.9±31.5 °C
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| Vapour Pressure |
0.0±1.2 mmHg at 25°C
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| Index of Refraction |
1.581
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| LogP |
1.73
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| Hydrogen Bond Donor Count |
0
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| Hydrogen Bond Acceptor Count |
8
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| Rotatable Bond Count |
4
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| Heavy Atom Count |
28
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| Complexity |
705
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| Defined Atom Stereocenter Count |
2
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| SMILES |
C[C@@H]1COCCN1C2=CC(=O)N3CC[C@H](N(C3=N2)CC(=O)C(C)C)C(F)(F)F
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| InChi Key |
ITCIAOUZMPREOO-OCCSQVGLSA-N
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| InChi Code |
InChI=1S/C18H25F3N4O3/c1-11(2)13(26)9-25-14(18(19,20)21)4-5-24-16(27)8-15(22-17(24)25)23-6-7-28-10-12(23)3/h8,11-12,14H,4-7,9-10H2,1-3H3/t12-,14+/m1/s1
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| Chemical Name |
(8S)-2-[(3R)-3-methylmorpholin-4-yl]-9-(3-methyl-2-oxobutyl)-8-(trifluoromethyl)-7,8-dihydro-6H-pyrimido[1,2-a]pyrimidin-4-one
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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) |
Ethanol : ~50 mg/mL (~124.25 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (6.21 mM) (saturation unknown) in 10% EtOH + 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 EtOH 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 (6.21 mM) (saturation unknown) in 10% EtOH + 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 EtOH 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. View More
Solubility in Formulation 3: ≥ 2.5 mg/mL (6.21 mM) (saturation unknown) in 10% EtOH + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.4850 mL | 12.4251 mL | 24.8503 mL | |
| 5 mM | 0.4970 mL | 2.4850 mL | 4.9701 mL | |
| 10 mM | 0.2485 mL | 1.2425 mL | 2.4850 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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