A-966492 is a selective and orally bioavailable PARP [poly(ADP-ribose) polymerase] inhibitor with potential anticancer activity. It inhibits PARP1 and PARP2 with Kis of 1 nM and 1.5 nM, respectively. In a B16-F10 melanoma model, it also showed exceptional in vivo antitumor efficacy when combined with temozolomide.

Physicochemical Properties

Molecular Formula	C18H17FN4O
Molecular Weight	324.35
Exact Mass	324.138
Elemental Analysis	C, 66.65; H, 5.28; F, 5.86; N, 17.27; O, 4.93
CAS #	934162-61-5
Related CAS #	934162-61-5
PubChem CID	16666333
Appearance	White solid powder
Density	1.3±0.1 g/cm3
Boiling Point	605.5±65.0 °C at 760 mmHg
Flash Point	320.0±34.3 °C
Vapour Pressure	0.0±1.7 mmHg at 25°C
Index of Refraction	1.665
LogP	0.96
Hydrogen Bond Donor Count	3
Hydrogen Bond Acceptor Count	4
Rotatable Bond Count	3
Heavy Atom Count	24
Complexity	476
Defined Atom Stereocenter Count	1
SMILES	C(C1C=CC=C2N=C(C3C=CC([C@H]4NCCC4)=CC=3F)NC=12)(=O)N
InChi Key	AHIVQGOUBLVTCB-AWEZNQCLSA-N
InChi Code	InChI=1S/C18H17FN4O/c19-13-9-10(14-5-2-8-21-14)6-7-11(13)18-22-15-4-1-3-12(17(20)24)16(15)23-18/h1,3-4,6-7,9,14,21H,2,5,8H2,(H2,20,24)(H,22,23)/t14-/m0/s1
Chemical Name	2-[2-fluoro-4-[(2S)-pyrrolidin-2-yl]phenyl]-1H-benzimidazole-4-carboxamide
Synonyms	A-966492; A966492; A 966492
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	PARP-1 ( Ki = 1 nM ); PARP-2 ( Ki = 1.5 nM )
ln Vitro	In vitro activity: A-966492 is one of the most potent PARP inhibitors. With a Ki of 1 nM and an EC50 of 1 nM in a whole cell assay, A-966492 demonstrates remarkable potency against the PARP-1 enzyme. In a dose-dependent way, A-966492 greatly increases the effectiveness of TMZ. Furthermore, A-966492 is able to penetrate the blood-brain barrier, be orally bioavailable in a variety of species, and seem to diffuse into tumor tissue. A-966492 is a structurally diverse analogue of benzimidazoles that shows promise and is undergoing preclinical characterization [1].
ln Vivo	A-966492 shows good in vivo efficacy as a single agent and in combination with carboplatin in an MX-1 breast cancer xenograft model, as well as in a B16F10 subcutaneous murine melanoma model when combined with temozolomide. A-966492 also has good pharmacological characteristics and has shown promise as a single agent in an MX-1 tumor model lacking BRCA1, as well as in vivo efficacy in preclinical mouse tumor models when combined with TMZ and carboplatin. A-966492 is characterized in Sprague-Dawley rats, beagle dogs, and cynomolgus monkeys. In these animals, A-966492 exhibits 34-72% oral bioavailabilities and 1.7-1.9 hour half-lives. A-966492 significantly increases the in vivo efficacy of TMZ in a murine B16F10 syngeneic melanoma model; the combination groups with A-966492 exhibit greater efficacy[1].
Enzyme Assay	The buffer used for the enzyme assay contains 50 mM Tris, pH 8.0, 1 mM dithiothreitol (DTT), and 4 mM magnesium chloride. 1.5 μM [3H]-NAD+ (1.6 μCi/mmol), 200 nM biotinylated histone H1, 200 nM slDNA, and 1 nM or 4 nM PARP-2 enzyme are the ingredients of the PARP reaction. Autoreactions in 100 μL volumes in white 96-well plates are performed using SPA bead-based detection. 50 μL of the 2X NAD+ substrate mixture is added to 50 μL of the 2× enzyme mixture, which contains DNA and PARP, to start the reaction. The addition of 150 μL of 1.5 mM benzamide (approximately 1×103-fold over its IC50) stops these reactions. The stopped reaction mixtures are put in amounts of 170 μL to Flash Plates coated with streptavidin, incubated for an hour, and then counted using a TopCount microplate scintillation counter. Inhibition curves at varied substrate concentrations are used to calculate ki data.
Cell Assay	In a 96-well plate, C41 cells are exposed to A-966492 for 30 minutes. DNA damage with 1 mM H2O2 for 10 minutes activates PARP. After one ice-cold wash in phosphate-buffered saline (PBS), cells are fixed for ten minutes at -20°C using prechilled methanol/acetone (7:3). Plates are allowed to air dry before being rehydrated with PBS and blocked for 30 minutes at room temperature using 5% nonfat dry milk in PBS-Tween (0.05%) as the blocking solution. Goat antimouse fluorescein 5(6)-isothiocyanate (FITC)-coupled antibody (1:50) and 1 μg/mL 40,6-diamidino-2-phenylindole (DAPI) are then added to the blocking solution and the cells are incubated for 60 minutes at room temperature. After washing the cells five times with PBS-Tween20, the cells are again incubated for 60 minutes with anti-PAR antibody 10H (1:50) in blocking solution. Following five PBS-Tween20 washes, the analysis is carried out using an fmax Fluorescence Microplate Reader that is configured to read at either the FITC or DAPI excitation and emission wavelengths. Cell numbers are used to normalize PARP activity (FITC signal) using DAPI.
Animal Protocol	On study day 0, a 0.2 cc volume of a 1:10 dilution of tumor brei in 45% Spinner MEM and 45% Matrigel is subcutaneously injected into the flank of female SCID mice. After allowing tumors to reach the desired size, therapy groups are assigned (N=10 mice/group). Day 14 is when PARP inhibitor therapy starts, and Day 16 is when cisplatin treatment begins. Tumor volumes are computed by serially measuring each tumor's dimensions with calibrated microcalipers at different times after the tumor is injected.
References	[1]. Optimization of phenyl-substituted benzimidazole carboxamide poly(ADP-ribose) polymerase inhibitors: identification of (S)-2-(2-fluoro-4-(pyrrolidin-2-yl)phenyl)-1H-benzimidazole-4-carboxamide (A-966492), a highly potent and efficacious inhibitor. J Med Chem . 2010 Apr 22;53(8):3142-53.

Solubility Data

Solubility (In Vitro)

DMSO: 33~64 mg/mL (101.7~197.3 mM) Water: <1 mg/mL Ethanol: <1 mg/mL

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.5 mg/mL (7.71 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 (7.71 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 (7.71 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	3.0831 mL	15.4154 mL	30.8309 mL
	5 mM	0.6166 mL	3.0831 mL	6.1662 mL
	10 mM	0.3083 mL	1.5415 mL	3.0831 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.