CB-5083 is a novel, potent, selective, first in class and orally bioavailable p97 AAA ATPase inhibitor with potential anticancer activity. Its IC50 value for p97 AAA ATPase inhibition is 11 nM. In hematological and solid models, CB-5083 exhibits antitumor activity and disturbs cellular protein homeostasis. After just one administration, CB-5083 causes poly-ubiquitin to accumulate quickly and persistently in tumor xenografts. Additionally, CB-5083 seems to be more potent than the proteasome inhibitors that are currently on the market, which supports the idea of targeting p97 and protein homeostasis in cancer treatment.

Physicochemical Properties

Molecular Formula	C24H23N5O2
Molecular Weight	413.47
Exact Mass	413.185
Elemental Analysis	C, 69.72; H, 5.61; N, 16.94; O, 7.74
CAS #	1542705-92-9
Related CAS #	1542705-92-9
PubChem CID	73051434
Appearance	White to off-white solid powder
Density	1.4±0.1 g/cm3
Boiling Point	760.5±70.0 °C at 760 mmHg
Flash Point	413.8±35.7 °C
Vapour Pressure	0.0±2.6 mmHg at 25°C
Index of Refraction	1.710
LogP	2.61
Hydrogen Bond Donor Count	2
Hydrogen Bond Acceptor Count	5
Rotatable Bond Count	5
Heavy Atom Count	31
Complexity	627
Defined Atom Stereocenter Count	0
SMILES	O1C([H])([H])C([H])([H])C2=C(C(N([H])C([H])([H])C3C([H])=C([H])C([H])=C([H])C=3[H])=NC(=N2)N2C(C([H])([H])[H])=C([H])C3=C(C(N([H])[H])=O)C([H])=C([H])C([H])=C23)C1([H])[H]
InChi Key	RDALZZCKQFLGJP-UHFFFAOYSA-N
InChi Code	InChI=1S/C24H23N5O2/c1-15-12-18-17(22(25)30)8-5-9-21(18)29(15)24-27-20-10-11-31-14-19(20)23(28-24)26-13-16-6-3-2-4-7-16/h2-9,12H,10-11,13-14H2,1H3,(H2,25,30)(H,26,27,28)
Chemical Name	1-[4-(benzylamino)-7,8-dihydro-5H-pyrano[4,3-d]pyrimidin-2-yl]-2-methylindole-4-carboxamide
Synonyms	CB5083; CB-5083; CB 5083
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

Targets	p97 ( IC50 = 11 nM )
ln Vitro	In vitro activity: CB-5083 kills tumor cells with an IC50 of 680 nM in A549 cells by causing significant accumulation of K48 poly-ubiquitinated protein and CHOP, as well as mRNA reduction.[1]
ln Vivo	CB-5083 (75 mg/kg, p.o.) significantly inhibits tumor growth in mice containing human HCT 116 colon tumor xenografts. CB-5083 (100 mg/kg, p.o.) also significantly inhibits the growth of tumors in mice harboring established human AMO-1 multiple myeloma and A549 lung carcinoma tumor xenografts.[1]
Enzyme Assay	Starting at 10 μM, compounds are diluted in DMSO using a 3-fold 10-point serial dilution method. The assay is carried out in a 384-well plate, with duplicates of each compound concentration point in each row representing a single dilution series. To initiate the reaction, 20 μM ATP and 20 nM p97 hexameric enzyme are added in a total volume of 5 μL. Once the plate is well mixed in an orbital shaker, it is sealed and incubated for 15 minutes at 37 °C. Compound dilution, ATP addition, and enzyme addition are carried out with the Freedom Evo's automated liquid handling. As directed by the manufacturer, add ADP Glo reagents 1 and 2. As the reaction's end point, the luminescence is measured using an Envision plate reader. The luminescence values of each compound are fitted to a four-parameter sigmoidal curve in order to determine its IC50.
Cell Assay	The ATCC recommends that A549 and other tumor cell lines be cultured. Stem cells are grown in 384-well plates with a clear bottom and a black or white color. Compound is applied to cells in well duplicates using a 10-point dose titration. The cell viability of the white plates is measured by adding CellTiter-Glo after a 72-hour treatment. Finding IC50 concentrations involves fitting luminosity values to a four-parameter sigmoidal curve.
Animal Protocol	Suspended in 0.5% methylcellulose in water; 75 mg/kg, qd; oral administration Nu/Nu nude female mice bearing human HCT 116 colon tumor xenografts
References	[1]. J Med Chem . 2015 Dec 24;58(24):9480-97.
Additional Infomation	p97 Inhibitor CB-5083 is an orally bioavailable inhibitor of valosin-containing protein (VCP) p97, with potential antineoplastic activity. Upon oral administration, CB-5083 specifically binds to and inhibits the activity of p97. This prevents ubiquitin-dependent protein degradation and causes cellular accumulation of poly-ubiquitinated proteins. The inhibition of endoplasmic reticulum (ER)-associated protein degradation activates the ER-dependent stress response pathway, and leads to both an induction of apoptosis and inhibition of cell proliferation in susceptible tumor cells. p97, a type II AAA ATPase, plays a key role in cellular protein homeostasis. Its overexpression in many tumor cell types is associated with increased tumor cell proliferation and survival.

Solubility Data

Solubility (In Vitro)

DMSO: 83~100 mg/mL (200.7~241.9 mM) Water: <1 mg/mL Ethanol: ~13 mg/mL (~31.4 mM)

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.05 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 (6.05 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. Solubility in Formulation 3: ≥ 2.5 mg/mL (6.05 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. Solubility in Formulation 4: 0.5%CMC Na +5% Tween 80 : 30mg/mL Solubility in Formulation 5: 10 mg/mL (24.19 mM) in 50% PEG300 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O 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	2.4186 mL	12.0928 mL	24.1856 mL
	5 mM	0.4837 mL	2.4186 mL	4.8371 mL
	10 mM	0.2419 mL	1.2093 mL	2.4186 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.