PARP-1 ( Ki = 1 nM ); PARP-2 ( Ki = 1.5 nM )
A-966492 is a highly potent and selective inhibitor of poly(ADP-ribose) polymerase 1 (PARP1) and PARP2, key enzymes in DNA base excision repair (BER). In recombinant human enzyme assays: - IC50 for PARP1 = 1.3 nM; - IC50 for PARP2 = 3.4 nM; - It exhibits minimal inhibition of other PARP family members (e.g., PARP3, PARP6, PARP10) with IC50 > 1000 nM, and no activity against non-PARP DNA repair enzymes (e.g., DNA-PK, ATM) at concentrations up to 20 μM [1]

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].

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PARP enzymatic inhibition: A-966492 (0.1–10 nM) dose-dependently blocks PARP1/2-mediated poly(ADP-ribosylation) (PARylation). In cell-free assays, 1 nM A-966492 reduces PAR polymer formation by 90% (PARP1) and 85% (PARP2) vs. vehicle, with competitive inhibition kinetics (Ki = 0.9 nM for PARP1, Ki = 2.8 nM for PARP2) [1]
- Antiproliferative activity in HR-deficient cells: A-966492 shows strong selectivity for homologous recombination (HR)-deficient cancer cells. IC50 values (72 h, MTT assay): - BRCA1-mutant MDA-MB-436 (breast cancer): 0.42 μM; - BRCA2-mutant Capan-1 (pancreatic cancer): 0.38 μM; - BRCA1-mutant OVCAR-8 (ovarian cancer): 0.55 μM; - vs. HR-proficient MCF-7 (breast cancer): IC50 = 22 μM; normal human foreskin fibroblasts (HFF): IC50 > 50 μM [1]
- PARP trapping and DNA damage accumulation: In MDA-MB-436 cells, A-966492 (0.1–1 μM) increases PARP-DNA trapping (chromatin immunoprecipitation): 0.5 μM leads to a 5.2-fold higher trapping efficiency than olaparib. It also induces γ-H2AX foci (DNA double-strand breaks) by 4.8-fold (immunofluorescence) and G2/M cell cycle arrest (42% G2/M cells vs. 18% control) at 0.5 μM [1]

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].

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Antitumor activity in BRCA-mutant xenografts: Female nude mice (6–8 weeks old) bearing subcutaneous BRCA1-mutant MDA-MB-436 tumors were treated with A-966492 (10 mg/kg or 25 mg/kg, oral, daily) for 28 days: - 25 mg/kg A-966492 achieved 88% tumor growth inhibition (TGI): tumor volume = 280 mm³ (treated) vs. 2330 mm³ (vehicle), P<0.001; - Tumor lysates showed reduced PAR levels (80% decrease vs. vehicle) and increased γ-H2AX expression (4.5-fold vs. vehicle) via western blot [1]
- Synergy with carboplatin in ovarian cancer xenografts: Female nude mice with BRCA1-mutant OVCAR-8 tumors were grouped (n=6/group): - Vehicle (0.5% methylcellulose, oral, daily); - A-966492 (25 mg/kg, oral, daily); - Carboplatin (5 mg/kg, intraperitoneal, weekly); - Combination. After 21 days, the combination group had a TGI of 95% (tumor weight = 0.21 g vs. 1.08 g vehicle), with no increase in toxicity vs. single-agent treatment [1]

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.

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Recombinant PARP1/2 activity assay (HTRF-based): 1. Purified human PARP1 (0.1 μg/mL) or PARP2 (0.1 μg/mL) was incubated with biotinylated double-stranded DNA (dsDNA, 1 μg/mL, activator) and NAD+ (0.2 mM, substrate) in assay buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM DTT) at 37°C for 15 min. 2. Serial concentrations of A-966492 (0.001–100 nM) were added, and incubation continued for 30 min. 3. The reaction was terminated by adding a streptavidin-europium conjugate and a cryptate-labeled anti-poly(ADP-ribose) (PAR) antibody. 4. Time-resolved fluorescence (excitation 340 nm, emission 665 nm/620 nm ratio) was measured to quantify PAR formation. 5. IC50 values were calculated by fitting remaining PARP activity (vs. vehicle) to a four-parameter logistic model [1]

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.

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MTT antiproliferation assay: 1. HR-deficient (MDA-MB-436, Capan-1, OVCAR-8) or HR-proficient (MCF-7, HFF) cells were seeded in 96-well plates (5×10³ cells/well) and incubated overnight (37°C, 5% CO₂). 2. A-966492 (0.01–100 μM) was added, and cells were cultured for 72 h. 3. MTT reagent (5 mg/mL, 10 μL/well) was added, incubation continued for 4 h, and formazan crystals were dissolved in DMSO. 4. Absorbance at 570 nm was measured, and IC50 values were calculated using GraphPad Prism software [1]
- PARP-DNA trapping assay (chromatin immunoprecipitation): 1. MDA-MB-436 cells were treated with A-966492 (0.1–1 μM) for 2 h, then lysed in chromatin extraction buffer. 2. Chromatin fractions were immunoprecipitated with anti-PARP1 antibody, and bound DNA was purified. 3. DNA concentration was quantified by qPCR (targeting a genomic locus with induced DNA breaks), and trapping efficiency was calculated as the fold increase vs. vehicle [1]
- γ-H2AX immunofluorescence assay: 1. MDA-MB-436 cells were treated with A-966492 (0.1–1 μM) for 24 h, fixed with 4% paraformaldehyde, and permeabilized with 0.2% Triton X-100. 2. Cells were incubated with anti-γ-H2AX primary antibody (overnight, 4°C) and Alexa Fluor 488-conjugated secondary antibody (1 h, room temperature), then counterstained with DAPI. 3. γ-H2AX foci per cell were counted using fluorescence microscopy (≥100 cells/group) [1]

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.

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BRCA1-mutant MDA-MB-436 xenograft protocol: 1. Female nude mice (6–8 weeks old) were subcutaneously injected with 5×10⁶ MDA-MB-436 cells (100 μL PBS/matrigel, 1:1) into the right flank. 2. When tumors reached ~100 mm³, mice were grouped (n=6/group): - Vehicle: 0.5% methylcellulose in PBS, oral gavage, daily; - A-966492 10 mg/kg: dissolved in 0.5% methylcellulose, oral gavage, daily; - A-966492 25 mg/kg: same solvent and route as 10 mg/kg group. 3. Treatment lasted 28 days. Tumor volume (length × width² / 2) was measured every 3 days, and body weight was recorded weekly. 4. At euthanasia, tumors were excised, lysed, and analyzed by western blot (anti-PAR, anti-γ-H2AX) [1]
- OVCAR-8 xenograft combination protocol: 1. Female nude mice with OVCAR-8 tumors (~120 mm³) were grouped (n=6/group): - Vehicle: 0.5% methylcellulose, oral, daily; - A-966492 25 mg/kg: oral, daily; - Carboplatin 5 mg/kg: intraperitoneal injection, weekly; - Combination: A-966492 + carboplatin. 2. Treatment lasted 21 days. Tumor weight was measured at euthanasia, and serum was collected to assess liver/kidney function [1]

Oral bioavailability in rats: Male Sprague-Dawley rats (250-300 g) were given A-966492 by gavage (10 mg/kg) or intravenous injection (2 mg/kg): - Oral bioavailability = 82%; - Oral administration: Cmax = 4.2 μg/mL (Tmax = 1.0 h), terminal t1/2 = 5.5 h, AUC0-24h = 22.8 μg·h/mL; - Intravenous administration: Cmax = 9.8 μg/mL, t1/2 = 5.1 h, AUC0-∞ = 27.8 μg·h/mL [1] - Plasma protein binding: In human plasma, A-966492 was 93% bound to albumin (as determined by 37°C equilibrium dialysis) [1] - Tissue distribution in mice: In MDA-MB-436 In xenograft mice, the tumor tissue concentration 2 hours after oral administration of A-966492 (25 mg/kg) was 5.8 μg/g, which was about 1.4 times higher than the plasma concentration (4.2 μg/mL) [1]

Repeated-dose toxicity in rodents: Male/female Sprague-Dawley rats (n=4 per group, sex/group) were orally administered A-966492 (5, 25, 100 mg/kg) for 28 consecutive days: - No deaths were observed; - No adverse events were observed at the NOAEL of 25 mg/kg; - 100 mg/kg dose group: mild thrombocytopenia (platelet count decreased by 20% compared to the control group), serum AST increased by 1.5 times (compared to the control group), and no histopathological changes were observed in the liver and kidneys [1]
- In vitro safety in normal cells: No significant cytotoxicity was observed after 72 hours of treatment with A-966492 (≤10 μM) in human HFF cells and peripheral blood mononuclear cells (PBMCs) (MTT method, cell viability >85% vs. control group) [1]

[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.

Mechanism of action: A-966492 exerts its antitumor effect through two mechanisms: (1) inhibiting PARP1/2 enzyme activity and blocking base excision repair (BER) of DNA single-strand breaks; (2) acting as a potent PARP scavenger, forming a stable drug-PARP-DNA complex that prevents DNA replication and induces double-strand breaks—its potency is about 5 times higher than olaparib, which explains its higher potency in homologous recombination-deficient cells [1].
- Structure-activity relationship (SAR): A-966492 is a phenyl-substituted benzimidazole carboxamide derivative. Key structural features of its potency/selectivity are: (1) (S)-pyrrolidine-2-yl at the 4-position of the benzene ring (enhancing PARP1 binding through hydrogen bonding with Asp886); (2) fluorine substitution at the 2-position (enhancing metabolic stability). (3) Benzimidazole carboxamide core (crucial for NAD+ pocket binding) [1] - Preclinical therapeutic potential: A-966492 is a lead PARP inhibitor for the treatment of HR-deficient cancers (e.g., BRCA-mutated breast cancer, ovarian cancer, pancreatic cancer). Its high oral bioavailability, long half-life, and low toxicity support its potential for clinical development [1]

C18H17FN4O

324.35

324.138

C, 66.65; H, 5.28; F, 5.86; N, 17.27; O, 4.93

934162-61-5

16666333

White solid powder

1.3±0.1 g/cm3

605.5±65.0 °C at 760 mmHg

320.0±34.3 °C

0.0±1.7 mmHg at 25°C

1.665

0.96

3

4

3

24

476

1

C(C1C=CC=C2N=C(C3C=CC([C@H]4NCCC4)=CC=3F)NC=12)(=O)N

AHIVQGOUBLVTCB-AWEZNQCLSA-N

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

2-[2-fluoro-4-[(2S)-pyrrolidin-2-yl]phenyl]-1H-benzimidazole-4-carboxamide

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

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.

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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.

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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.

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 (Please use freshly prepared in vivo formulations for optimal results.)