NVP-TNKS656 (also called TNKS-656; NVP-TNKS-656; TNKS 656; NVP-TNKS 656) is a selective, and orally bioactive tankyrase inhibitor with potential antitumor activity. It blocks tankyrase/TNKS2 with an IC50 of 6 nM, and shows > 300-fold selectivity against PARP1 and PARP2.

Physicochemical Properties

Molecular Formula	C27H34N4O5
Molecular Weight	494.58
Exact Mass	494.253
Elemental Analysis	C, 65.57; H, 6.93; N, 11.33; O, 16.17
CAS #	1419949-20-4
Related CAS #	1419949-20-4
PubChem CID	136237316
Appearance	White to light yellow solid powder
LogP	2.122
Hydrogen Bond Donor Count	1
Hydrogen Bond Acceptor Count	7
Rotatable Bond Count	9
Heavy Atom Count	36
Complexity	911
Defined Atom Stereocenter Count	0
SMILES	O=C(C([H])([H])N1C([H])([H])C([H])([H])C([H])(C(C2C([H])=C([H])C(=C([H])C=2[H])OC([H])([H])[H])=O)C([H])([H])C1([H])[H])N(C([H])([H])C1=NC2C([H])([H])C([H])([H])OC([H])([H])C=2C(N1[H])=O)C([H])([H])C1([H])C([H])([H])C1([H])[H]
InChi Key	DYGBNAYFDZEYBA-UHFFFAOYSA-N
InChi Code	InChI=1S/C27H34N4O5/c1-35-21-6-4-19(5-7-21)26(33)20-8-11-30(12-9-20)16-25(32)31(14-18-2-3-18)15-24-28-23-10-13-36-17-22(23)27(34)29-24/h4-7,18,20H,2-3,8-17H2,1H3,(H,28,29,34)
Chemical Name	N-(cyclopropylmethyl)-2-[4-(4-methoxybenzoyl)piperidin-1-yl]-N-[(4-oxo-3,5,7,8-tetrahydropyrano[4,3-d]pyrimidin-2-yl)methyl]acetamide
Synonyms	TNKS-656; NVP-TNKS 656; NVPTNKS-656; NVP-TNKS656; TNKS 656; TNKS656
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 = 6 nM ); PARP2 ( IC50 = 32 μM )
ln Vitro	In vitro activity: NVP-TNKS656 exhibits high solubility and low to moderate microsomal ER values in vitro across species.[1] NVP-TNKS656 promotes apoptosis and blocks the Wnt/β-Catenin pathway in PI3K or AKT inhibitor-resistant cells.[2]
ln Vivo	NVP-TNKS656 shows good exposure and moderate oral bioavailability in mice, but low clearance and volume of distribution. NVP-TNKS656 (350 mg/kg, p.o.) stabilizes Axin1 protein and decreases the Wnt/beta-catenin target gene Axin2 mRNA level by 70–80% in athymic nude mice bearing MMTV-Wnt1 tumors.[1] In colorectal cancer PDX models, NVP-TNKS656 suppresses the growth of tumors, reverses such resistance, and lowers nuclear β-catenin. [2]
Enzyme Assay	Nicotinamide is detected by quantitative liquid chromatography/mass spectrometry (LC-MS), which is used to track PARP catalytic activity. 384-well Greiner flat-bottom plates are used for the autoPARsylation reactions, which are carried out at room temperature. The final reaction mixture contains 2.5% DMSO and inhibitors with concentrations ranging from 0.0001 to 18.75 μM. The enzymes PARP1, PARP2, GST-TNKS2P, and GST-TNKS1P are utilized at final concentrations of 5, 5, 5, and 2 nM, respectively. Quantitative liquid chromatography/mass spectrometry (LC-MS), which monitors PARP catalytic activity, is used to identify nicotinamide. 384-well Greiner flat-bottom plates are used for the autoPARsylation reactions, which are carried out at room temperature. The final reaction mixture contains 2.5% DMSO and inhibitors with concentrations ranging from 0.0001 to 18.75 μM. Final concentrations of 5, 5, 5, and 2 nM are used for the enzymes PARP1, PARP2, GST-TNKS2P, and GST-TNKS1P, respectively.
Animal Protocol	Subcutaneously, a 3x3x3 mm3 tumor fragment from an MMTV-Wnt1 tumor-bearing mouse is implanted in athymic female nude mice weighing 19–22 g. At around 250–300 mm3, tumors reach their full size. Mice are given either vehicle (n=3) or TNKS656 (n=18) at a single oral dose. The vehicle mixture is 4% HCl:10% propylene glycol:20% Solutol HS15:60.5% D5W:0.5% NaOH. After dosing (n=3), mice are put to sleep at 0.5, 1, 2, 4, 8, 16, or 24 hours. Blood is then drawn via cardiac puncture and processed for plasma. In order to conduct PD analysis, tumors are removed from mice and frozen at -80°C.
References	[1]. J Med Chem . 2013 Aug 22;56(16):6495-511. [2]. Clin Cancer Res . 2016 Feb 1;22(3):644-56.

Solubility Data

Solubility (In Vitro)

DMSO: ~100 mg/mL (~202.2 mM) Water: <1 mg/mL Ethanol: ~10 mg/mL (~20.2 mM)

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.05 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 (5.05 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 (5.05 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: 5%DMSO+ 40%PEG300+ 5%Tween 80+ 50%ddH2O: 5.0mg/ml (10.11mM)  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	2.0219 mL	10.1096 mL	20.2192 mL
	5 mM	0.4044 mL	2.0219 mL	4.0438 mL
	10 mM	0.2022 mL	1.0110 mL	2.0219 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.