JW-55 (JW55) is a potent and selective small molecule inhibitor of β-catenin signaling pathway with anticancer activity. It inhibits the β-catenin destruction complex regulators tankyrase 1 and tankyrase 2 (TNKS1/2) PARP domains. TNKS1/2 auto-PARsylation is reduced in vitro by JW 55, with IC50 values of 1.9 μM and 830 nM, respectively. When JW55 inhibited the poly(ADP-ribosyl)ation activity of TNKS1/2, AXIN2, a component of the β-catenin destruction complex, stabilized. This was followed by an increase in the degradation of β-catenin. In colon carcinoma cells with mutations in either the β-catenin allele or the APC (adenomatous polyposis coli) locus, JW55 inhibited canonical Wnt signaling in a dose-dependent manner. Furthermore, JW55 inhibited the formation of polyposis in conditional APC mutant mice induced by tamoxifen and the axis duplication induced by XWnt8 in Xenopus embryos.

Physicochemical Properties

Molecular Formula	C₂₅H₂₆N₂O₅
Molecular Weight	434.48
Exact Mass	434.184
Elemental Analysis	C, 69.11; H, 6.03; N, 6.45; O, 18.41
CAS #	664993-53-7
Related CAS #	664993-53-7
PubChem CID	2946601
Appearance	White solid powder
Density	1.2±0.1 g/cm3
Boiling Point	572.6±50.0 °C at 760 mmHg
Flash Point	300.1±30.1 °C
Vapour Pressure	0.0±1.6 mmHg at 25°C
Index of Refraction	1.601
LogP	3.54
Hydrogen Bond Donor Count	2
Hydrogen Bond Acceptor Count	5
Rotatable Bond Count	7
Heavy Atom Count	32
Complexity	620
Defined Atom Stereocenter Count	0
SMILES	O1C([H])([H])C([H])([H])C(C2C([H])=C([H])C(=C([H])C=2[H])OC([H])([H])[H])(C([H])([H])N([H])C(C2C([H])=C([H])C(=C([H])C=2[H])N([H])C(C2=C([H])C([H])=C([H])O2)=O)=O)C([H])([H])C1([H])[H]
InChi Key	ZJZWZIXSGNFWQQ-UHFFFAOYSA-N
InChi Code	InChI=1S/C25H26N2O5/c1-30-21-10-6-19(7-11-21)25(12-15-31-16-13-25)17-26-23(28)18-4-8-20(9-5-18)27-24(29)22-3-2-14-32-22/h2-11,14H,12-13,15-17H2,1H3,(H,26,28)(H,27,29)
Chemical Name	N-[4-[[4-(4-methoxyphenyl)oxan-4-yl]methylcarbamoyl]phenyl]furan-2-carboxamide
Synonyms	JW-55; JW55; JW 55
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	TNKS2 ( IC50 = 0.83 μM ); TNKS1 ( IC50 = 1.9 μM )
ln Vitro	In vitro activity: JW55 suppresses the TNKS1/2 PARP domain, stabilizing AXIN2 and accelerating β-catenin degradation in the process. With an IC50 value of 470 nmol/L, JW55 inhibits Wnt3a-induced HEK293 cells that have a transiently transfected ST-Luc (SuperTop-luciferase) reporter. JW55 has an IC50 value of 1.9 μmol/L and 830 nmol/L, respectively, which decreases the auto-PARsylation of TNKS1/2 in vitro. In vitro, the CRC cell line SW480 exhibits decreased cell-cycle progression, proliferation, and colony formation when JW55-mediated canonical Wnt signaling inhibition is applied[1].
ln Vivo	JW55 inhibits the formation of polyposis in conditional APC mutant mice induced by tamoxifen and axis duplication in Xenopus embryos[1].
Enzyme Assay	JW 55, also known as JW55, is a strong and specific inhibitor of the Wnt pathway. JW55 exhibits inhibition of Wnt3a-induced HEK293 cells with an IC50 value of 470 nM, when the cells contain a transiently transfected ST-Luc (SuperTop-luciferase) reporter. JW55 exhibits efficacy within the concentration range of 1 to 5 μM in SW480 cells and 0.01 to 5 μM in HCT-15 cells. JW55 exhibits efficacy within the concentration range of 1 to 5 μM in SW480 cells and 0.01 to 5 μM in HCT-15 cells.
Cell Assay	In 96-well plates, 1,000 SW480 or RKO cells are seeded. The following day, solutions containing 0.1% DMSO or 10 μM JW55 for RKO cells and 0.1% DMSO or 10, 5, or 1 μM JW55 for SW480 cells are substituted for the original cell culture medium. Every sample has at least six duplicates. Within a cell culture incubator, the plate is incubated in an IncuCyte. Every two hours, pictures are taken to track proliferation.
Animal Protocol	Mice: Seven 12-week-old female ApcCKO/CKO/Lgr5-CreERT2 mice receive intraperitoneal injections of tamoxifen at a dose of 25 mg/kg, diluted in a 1:4 ratio of ethanol to corn oil. The mice are treated with JW55 (100 mg/kg) or a vehicle (DMSO) after being randomly assigned to two groups. The next day, daily oral applications were started, and they lasted for three weeks. The body weight of the mice is measured twice a week. Following the mice's sacrifice, the intestines are removed, cleaned in PBS, and fixed in formaldehyde (10% solution (v/v) in PBS). 10% paraformaldehyde (PFA)/PBS solution containing 1% methylene blue is used to stain the small intestines. Hematoxylin and eosin is used to stain small ileum Swiss-rolls that have been embedded in paraffin sections.
References	[1]. A novel tankyrase inhibitor decreases canonical Wnt signaling in colon carcinoma cells and reduces tumor growth in conditional APC mutant mice. Cancer Res. 2012 Jun 1;72(11):2822-32.
Additional Infomation	N-[4-[[[4-(4-methoxyphenyl)-4-oxanyl]methylamino]-oxomethyl]phenyl]-2-furancarboxamide is a member of furans and an aromatic amide.

Solubility Data

Solubility (In Vitro)

DMSO: ~10 mM Water: <1 mg/mL Ethanol: 4 mg/mL

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.75 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 (5.75 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 (5.75 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	2.3016 mL	11.5080 mL	23.0160 mL
	5 mM	0.4603 mL	2.3016 mL	4.6032 mL
	10 mM	0.2302 mL	1.1508 mL	2.3016 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.