Stenoparib (formerly E-7449; 2X-121) is a novel, potent and orally bioavailable dual inhibitor of PARP1/2 [nuclear enzymes poly (ADP-ribose) polymerase] and tankyrases (TNKS1/2) with anticancer activity. Utilizing 32P-NAD+ as the substrate, it inhibits PARP1, PARP2, TNKS1, and TNKS2 with IC50s of 2.0, 1.0, ∼50, and ∼50 nM, respectively. A mechanism that has been demonstrated to increase cytotoxicity, E7449 also traps PARP1 onto damaged DNA while suppressing the enzymatic activity of PARP. Treating cells with defective DNA repair pathways other than homologous recombination made them susceptible to E7449. BRCA-deficient xenografts showed strong antitumor activity in response to chemotherapy, which was enhanced by E7449. In colon cancer cell lines, E7449 also suppressed Wnt/β-catenin signaling, most likely by inhibiting TNKS. In line with this theory, E7449 caused β-catenin to become destabilized and significantly changed the expression of Wnt target genes by stabilizing the axin and TNKS proteins. Notably, E7449 inhibited Wnt signaling-mediated hair growth. E7449 showed a pharmacodynamic effect on Wnt target genes in tumors, but, as is common with selective TNKS inhibitors, it lacked single agent antitumor activity in vivo. MEK inhibition was combined with E7449 to increase its antitumor activity. The absence of toxicity was especially noteworthy, especially in relation to the lack of intestinal toxicity observed with other TNKS inhibitors. E7449 is a new dual inhibitor of PARP1/2 and TNKS1/2 that has the advantage of specifically targeting tumors that are dependent on Wnt/β-catenin signaling. E7449 is presently in the early stages of clinical research.

Physicochemical Properties

Molecular Formula	C18H15N5O
Molecular Weight	317.34
Exact Mass	317.127
Elemental Analysis	C, 68.13; H, 4.76; N, 22.07; O, 5.04
CAS #	1140964-99-3
Related CAS #	1140964-99-3
PubChem CID	135565981
Appearance	Light yellow to yellow solid powder
Density	1.4±0.1 g/cm3
Boiling Point	381.4±34.0 °C at 760 mmHg
Flash Point	184.5±25.7 °C
Vapour Pressure	0.0±0.9 mmHg at 25°C
Index of Refraction	1.731
LogP	1.12
Hydrogen Bond Donor Count	2
Hydrogen Bond Acceptor Count	3
Rotatable Bond Count	2
Heavy Atom Count	24
Complexity	586
Defined Atom Stereocenter Count	0
SMILES	O=C1C2C([H])=C([H])C([H])=C3C=2C(=NN1[H])N=C(C([H])([H])N1C([H])([H])C2=C([H])C([H])=C([H])C([H])=C2C1([H])[H])N3[H]
InChi Key	JLFSBHQQXIAQEC-UHFFFAOYSA-N
InChi Code	InChI=1S/C18H15N5O/c24-18-13-6-3-7-14-16(13)17(21-22-18)20-15(19-14)10-23-8-11-4-1-2-5-12(11)9-23/h1-7H,8-10H2,(H,22,24)(H,19,20,21)
Chemical Name	11-(1,3-dihydroisoindol-2-ylmethyl)-2,3,10,12-tetrazatricyclo[7.3.1.05,13]trideca-1,5(13),6,8,11-pentaen-4-one
Synonyms	Stenoparib; E7449; 2X-121; E 7449; 2X 121; E-7449; 2X121
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	PARP2 ( IC50 = 1 nM ); PARP1 ( IC50 = 2 nM ); TNKS1 ( IC50 = 50 nM ); TNKS2 ( IC50 = 50 nM )
ln Vitro	Stenoparib is a strong inhibitor of PARP1 and PARP2, as well as TNKS1 and TNKS2. It uses 32P-NAD+ as substrate and has IC50 values of 2.0, 1.0, ∼50, and ∼50 nM for PARP1, PARP2, TNKS1, and TNKS2, respectively. There are no discernible inhibitory effects of stenoparib on PARP3 or PARPs 6–16. Stenoparib affects DNA repair pathways other than homologous recombination (HR) by binding to damaged DNA and trapping PARP1. Cells lacking in BRCA1 and 2, CtIP, and Rad54—components of the HR pathway—are most effectively suppressed by tenoparib. In SW480 cells, stenoparib (10 μM) inhibits Wnt signaling[1].
ln Vivo	Stenoparib moderately suppresses the growth of tumors at a dose of 100 mg/kg and dramatically increases the inhibition in the mouse melanoma B16-F10 isograft model when oral doses of 10, 30, and 100 mg/kg are combined with temozolomide (TMZ). In a BRCA mutant xenograft model, stenoparib (30 or 100 mg/kg, p.o.) inhibits PARP, exhibits anti-tumor activity, and is well-tolerated with no apparent body weight loss or fatalities. In C57BL/6 mice, tenoparib (30, 100, or 300 mg/kg, p.o.) inhibits Wnt signaling and dose-dependently suppresses hair regrowth. Breast tumors originally isolated from Wnt1 (int-1) transgenic mice demonstrate antitumor activity when stenoparib (100 mg/kg, p.o.) is combined with MEK inhibitor[1].
Enzyme Assay	In summary, cells are lysed for 20 minutes on ice in cell lysis buffer (CLB: 50 mM HEPES, pH 7.4, 150 mM NaCl, 1 mM MgCl2, 1 mM EGTA, 1 mM DTT, 1% TritonX-100, 1 μg/mL leupeptin, aprotinin, pepstatin, PMSF) after being washed three times in ice-cold PBS within 24 to 48 hours after transfection. Lysates undergo a 30-minute ultracentrifugation process at 100,000 g. After lysates are cleared, they are incubated with pre-bound protein A magnetic beads and anti-GFP antibody (3E6) for one hour at 4°C. Following a 1-minute soak in CLB, beads are then washed for 3 minutes in CLB containing 1 M NaCl and 1 minute in PARP reaction buffer (PRB; 50 mM Tris, pH 7.5, 50 mM NaCl, 0.5 mM DTT, 0.1% TritonX-100, 1 μg/mL leupeptin, aprotinin, and pepstatin). For 30 minutes at 25°C, NAD+ incorporation reactions are carried out in PRB containing 10 μM NAD+ supplemented with 32P-NAD+ at a 1:20 ratio. NAD+ incorporation is carried out at a 1:5 ratio for one hour at 25°C for PARPs with low incorporation signals (PARP4, 5a, and 16). After that, the beads are once again suspended in Laemmli sample buffer, heated for ten minutes to 65°C, the beads are extracted with a magnet, and the supernatant is spotted onto Whatman paper. Phosphorimaging is used to analyze samples[1].
Cell Assay	The 32 isogenic DT40 cell lines, each lacking a different DNA repair gene, are used as subjects for proliferation experiments. For two to three days (approximately eight cell cycles), cells are seeded and incubated with test compounds at different concentrations. Version 5.02 of the GraphPad Prism 5 software is used to compute IC50 values and evaluate cell growth using XTT. A minimum of three independent experiments are carried out, each carried out in duplicate. T47D, MDA-MB-157, MDA-MB-231, MDA-MB-468, MDA-MB-453, BT-20, and Hs578T are human breast cancer cell lines that are used. Cells are cultured and analyzed in RPMI 1640 or DMEM medium containing 10% FBS for cell line panel assays. Cells are plated in 96-well plates at a low density for proliferation assays. After adding tenoparib at different concentrations, the plates are incubated for eight days, during which time the compound and medium are replaced on day four. Utilizing the CellTiter-Glo cell viability assay, cell growth is evaluated. At least three independent experiments are carried out, and each experiment is carried out in duplicate[1].
Animal Protocol	Combination of Temozolomide (TMZ) and B16-F10 isograft model: B16-F10 cells (2 × 105) are subcutaneously injected into female C57BL/6 mice. One day after vaccination, medication treatment is started, with randomization based on body weight. Both TMZ and stenoparib are administered orally once daily and are both formulated in 0.5% methyl cellulose. Every day from day 1 to day 5, 50 mg/kg of TMZ is given either alone or in combination. Daily doses of 10, 30, and 100 mg/kg of tenoparib in combination with TMZ, as well as 100 mg/kg as a single agent, are given to patients on days 1 through 7. 0.5% methyl cellulose in water is the vehicle used to treat the control group. Animals receiving the combination are given TMZ after all animals have received their dose of stenoparib or the vehicle[1].
References	[1]. E7449: A dual inhibitor of PARP1/2 and tankyrase1/2 inhibits growth of DNA repair deficient tumors and antagonizes Wnt signaling. Oncotarget. 2015 Dec 1;6(38):41307-23.
Additional Infomation	2X-121 is under investigation in clinical trial NCT03562832 (Investigation of Anti-tumour Effect and Tolerability of the PARP Inhibitor 2X-121 in Patients With Metastatic Breast Cancer Selected by the 2X-121 DRP). PARP/Tankyrase Inhibitor 2X-121 is an orally available small molecule inhibitor of the nuclear enzymes poly (ADP-ribose) polymerase (PARP) 1 and 2, with potential antineoplastic activity. Upon administration, E7449 selectively binds to PARP 1 and 2, thereby preventing the repair of damaged DNA via the base excision repair (BER) pathway. This agent enhances the accumulation of single and double strand DNA breaks and promotes genomic instability eventually leading to apoptosis. PARP 1/2 inhibitor E7449 may enhance the cytotoxicity of DNA-damaging agents and of radiotherapy. PARP catalyzes post-translational ADP-ribosylation of nuclear proteins that signal and recruit other proteins to repair damaged DNA.

Solubility Data

Solubility (In Vitro)

DMSO: ≥ 5mg/mL Water: N/A Ethanol: N/A

Solubility (In Vivo)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples. Injection Formulations (e.g. IP/IV/IM/SC) Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] *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. Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline) Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline) Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline) Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil) Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Oral Formulations Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). Oral Formulation 3: Dissolved in PEG400 Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose Oral Formulation 6: Mixing with food powders Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	3.1512 mL	15.7560 mL	31.5119 mL
	5 mM	0.6302 mL	3.1512 mL	6.3024 mL
	10 mM	0.3151 mL	1.5756 mL	3.1512 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.