Vps34-IN-2 is a novel and potent inhibitor of Vps34 (IC50s = 2 and 82 nM for enzymatic and cellular assay, respectively).

Physicochemical Properties

Molecular Formula	C18H25F3N4O3
Molecular Weight	402.411314725876
Exact Mass	402.187
CAS #	1523404-29-6
PubChem CID	72709284
Appearance	White to off-white solid powder
Density	1.4±0.1 g/cm3
Boiling Point	466.4±55.0 °C at 760 mmHg
Flash Point	235.9±31.5 °C
Vapour Pressure	0.0±1.2 mmHg at 25°C
Index of Refraction	1.581
LogP	1.73
Hydrogen Bond Donor Count	0
Hydrogen Bond Acceptor Count	8
Rotatable Bond Count	4
Heavy Atom Count	28
Complexity	705
Defined Atom Stereocenter Count	2
SMILES	C[C@@H]1COCCN1C2=CC(=O)N3CC[C@H](N(C3=N2)CC(=O)C(C)C)C(F)(F)F
InChi Key	ITCIAOUZMPREOO-OCCSQVGLSA-N
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
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
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

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].
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].
Cell Assay	Cells are cultured in RPMI-1640 medium with 10% fetal bovine serum. Cells are lysed by sonication in a detergent containing lysis buffer and cleared by centrifugation, and the resulting supernatant is collected for compound treatment. Final protein concentration of lysates is 4 mg/mL. An amount of 5 μL of Vps34-IN-2 (Compound 31) is added from 100× stock solutions in DMSO to 445 μL of lysate in duplicate. An amount of 5 μL of DMSO is added to 445 μL of lysate in quadruplicate for controls. After 15 min incubation, 5 μL of a 100× aqueous solution of the ATP probe I is added to each sample (final concentration of ATP probe I is 0.5 μM). After 5 min, 50 μL of a 10× aqueous solution of the ATP probe II is added to each sample (final concentration of ATP probe II is 20 μM). All samples are then incubated for an additional 10 min[1].
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 to 400 mm3, mice are treated with vehicle (98% PEG200/2% PS80) or a single dose 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 with Vps34-IN-2 are sacrificed at each time point; tumor tissues are harvested for immunohistochemistry (IHC) analysis and plasma samples are collected to determine the concentration of Vps34-IN-2[1].
References	[1]. Discovery of (2S)-8-[(3R)-3-methylmorpholin-4-yl]-1-(3-methyl-2-oxobutyl)-2-(trifluoromethyl)-3,4-dihydro-2H-pyrimido[1,2-a]pyrimidin-6-one: a novel potent and selective inhibitor of Vps34 for the treatment of solid tumors. J Med Chem. [2]. Genome-wide CRISPR screening identifies TMEM106B as a proviral host factor for SARS-CoV-2. Nat Genet. 2021 Mar 8.

Solubility Data

Solubility (In Vitro)

Ethanol : ~50 mg/mL (~124.25 mM)

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. 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. 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 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	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.