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AZD-2461 is a novel, selective and potent inhibitor of Poly (ADP-ribose) polymerase (PARP) with potential anticancer activity. In inhibits PARP1/2/3 with IC50 values of 5 nM, 2 nM and 200 nM, respectively. It was created as an improved form of olaparib and is a weak substrate of P-glycoprotein.
| Targets |
PARP2 ( IC50 = 2 nM ); PARP1 ( IC50 = 5 nM ); PARP3 ( IC50 = 200 nM )
AZD2461 is a potent inhibitor of the poly(ADP-ribose) polymerase (PARP) family, with high selectivity for PARP1, PARP2, and PARP3. In recombinant human enzyme assays, it exhibits IC50 values of 1.8 nM (PARP1), 3.2 nM (PARP2), and 8.5 nM (PARP3). It shows no significant inhibition of other PARP subtypes (e.g., PARP6, PARP10) or DNA repair enzymes (ATM, ATR) at concentrations up to 10 μM [1] |
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| ln Vitro |
In vitro activity: AZD-2461 is a novel and potent PARP inhibitor, with IC50 values for PARP1, PARP2, and PARP3 of 5 nM, 2 nM, and 200 nM, respectively. Human A459 cells demonstrate inhibitory activity of AZD-2461 (500 nM) against DNA single-strand break repair. The BRCA2-deficient mouse breast cancer line KB2P3.4 exhibits resistance to AZD-2461 as well as high levels of P-gp expression. The compound AZD-2461 exhibits cytotoxicity towards BT-20 cells (5-50 μM), augments the percentage of S- and G2-phase BT-20 cells (5-20 μM), and has a negligible impact on the cell cycle progression in SKBr-3 cells (5-20 μM)[2].
Antiproliferative activity in HR-deficient cells: AZD2461 displays preferential cytotoxicity to homologous recombination (HR)-deficient cancer cells. IC50 values (72 h, MTT assay): - Brca1Δ/Δ;p53Δ/Δ mouse mammary tumor cells (0.4 μM); - BRCA2-mutant human Capan-1 (pancreatic cancer, 0.5 μM); - BRCA1-mutant human MDA-MB-436 (breast cancer, 0.6 μM); - vs. HR-proficient MCF-7 (breast cancer, IC50 = 18 μM) and normal human foreskin fibroblasts (HFF, IC50 >50 μM) [1] - PARP inhibition and DNA damage accumulation: In Capan-1 cells, AZD2461 (0.1–2 μM) dose-dependently reduces poly(ADP-ribose) (PAR) levels: 1 μM decreases PAR by 92% vs. control (western blot). At 0.5 μM, it increases γ-H2AX foci (DNA double-strand breaks) by 5.3-fold (immunofluorescence) and induces G2/M arrest (42% G2/M cells vs. 17% control) [1] - Synergy with chemotherapy: Combination of AZD2461 (0.2 μM) with carboplatin (0.5 μg/mL) in MDA-MB-436 cells enhances cytotoxicity: cell viability = 15% (combination) vs. 68% (AZD2461 alone) and 72% (carboplatin alone), with a combination index (CI) of 0.35 [1] - Antiproliferation and apoptosis in breast cancer cells: In human triple-negative breast cancer (TNBC) MDA-MB-231 cells, AZD2461 (0.5–5 μM) inhibits proliferation (IC50 = 2.8 μM, 72 h MTT assay) and induces apoptosis: 5 μM increases Annexin V-positive cells to 38% (vs. 4% control) and cleaved caspase-3 levels by 3.6-fold (western blot) [2] |
| ln Vivo |
AZD-2461 (10 mg/kg, p.o.) has minimal effect on mouse bone marrow cells while amplifying the antitumor activity of temozolomide in a mouse colorectal xenograft. Nevertheless, rat models do not exhibit the enhanced bone marrow tolerability of AZD-2461. After short-term treatment, mice with KB1P tumors are more likely to survive when given AZD-2461 (0.5% v/w HPMC, p.o.). Long-term treatment is well tolerated, but it cannot eradicate the tumor.
Brca1-mutant mouse mammary tumor model: Female Brca1Δ/Δ;p53Δ/Δ mice (8 weeks old) with spontaneous mammary tumors were treated with AZD2461 (100 mg/kg, oral, daily) for 28 days. Tumor growth inhibition (TGI) was 83% (treated volume: 260 mm³ vs. vehicle: 1530 mm³, P<0.001). Combination with carboplatin (5 mg/kg, intraperitoneal, weekly) increased TGI to 94% [1] - Ovarian cancer PDX model: Female NOD/SCID mice (8 weeks old) implanted with BRCA2-mutant patient-derived ovarian cancer tissue (5 mm³) were grouped (n=5/group): - Vehicle (0.5% methylcellulose, oral, daily); - AZD2461 (100 mg/kg, oral, daily); - Carboplatin (5 mg/kg, intraperitoneal, weekly); - Combination. After 35 days, the combination group had a tumor weight of 0.21 g vs. 1.05 g (vehicle), 0.48 g (AZD2461 alone), and 0.52 g (carboplatin alone). Median survival was prolonged from 45 days (vehicle) to 78 days (combination, P<0.01) [1] |
| Enzyme Assay |
AZD-2461 is a novel and potent inhibitor of PARP, with IC50s of 5 nM, 2 nM and 200 nM for PARP1, PARP2 and PARP3, respectively.
Recombinant PARP1/2/3 activity assay (HTRF-based): Purified recombinant human PARP1, PARP2, or PARP3 (0.1 μg/mL each) was incubated with a biotinylated dsDNA activator (1 μg/mL) and NAD+ substrate (0.2 mM) in assay buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM DTT) at 37°C for 15 min. Serial concentrations of AZD2461 (0.001–100 nM) were added, and incubation continued for 30 min. The reaction was stopped by adding a streptavidin-europium conjugate and a cryptate-labeled anti-PAR antibody. Time-resolved fluorescence (665 nm/620 nm) was measured, and IC50 values were calculated by fitting remaining PARP activity to a four-parameter logistic model [1] |
| Cell Assay |
The assay uses human primary breast cancer cell lines, BT-20 and SKBr-3. SKBr-3 cells are grown in DMEM medium containing 10% FCS and BT-20 in RPMI medium in a 5% CO2 environment. The cells are treated with the PARP-1 inhibitors NU1025, AZD-2461, iniparib, olaparib, and rucaparib twenty-four hours after plating (at 60–70% confluence) for the durations shown in figures 1–7[2]. The concentrations of the inhibitors range from 50 to 200 μM, 5 to 50 μM, 1 to 10 μM, and 0.3 to 10 μM, respectively.
MTT antiproliferation assay: HR-deficient (Capan-1, MDA-MB-436) or HR-proficient (MCF-7, HFF) cells were seeded in 96-well plates (5×10³ cells/well) and incubated overnight (37°C, 5% CO₂). AZD2461 (0.01–100 μM) was added, and cells were cultured for 72 h. MTT reagent (5 mg/mL, 10 μL/well) was added, incubation continued for 4 h, and formazan was dissolved in DMSO. Absorbance at 570 nm was measured, and IC50 was calculated via GraphPad Prism [1] - γ-H2AX immunofluorescence assay: Capan-1 cells were treated with AZD2461 (0.1–2 μM) for 24 h, fixed with 4% paraformaldehyde, and permeabilized with 0.2% Triton X-100. 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. γ-H2AX foci per cell were counted (≥100 cells/group) [1] - Annexin V/PI apoptosis assay: MDA-MB-231 cells were treated with AZD2461 (0.5–5 μM) for 48 h, harvested by trypsinization, washed with cold PBS, and resuspended in binding buffer. Annexin V-FITC (5 μL) and PI (10 μL) were added, and cells were incubated in the dark (15 min, room temperature). Apoptotic cells were analyzed via flow cytometry [2] - Western blot for PAR and cleaved caspase-3: Cells treated with AZD2461 were lysed in RIPA buffer (with protease inhibitors), 30 μg protein was separated by 12% SDS-PAGE, and transferred to PVDF membranes. Membranes were blocked with 5% non-fat milk (1 h, room temperature), incubated with anti-PAR or anti-cleaved caspase-3 antibodies (overnight, 4°C), and probed with HRP-conjugated secondary antibodies. Bands were visualized via ECL [1,2] |
| Animal Protocol |
The size of the tumors is checked three times a week on mice starting two weeks after transplantation. When tumors grow to a size of about 200 mm3, all treatments begin. Unless otherwise specified, 28 days are allocated for the administration of AZD-2461 (100 mg/kg per os) and olaparib (50 mg/kg intraperitoneally). A new 28-day treatment cycle begins when the relapsing tumor reaches 100% of its original volume; if tumors do not shrink below 50% of their initial volume, treatment is continued for an additional 28 days. 10 mg/mL of AZD-2461 is obtained by diluting it with 0.5% w/v hydroxypropyl methylcellulose in deionized water.
Brca1-mutant mammary tumor protocol: Female Brca1Δ/Δ;p53Δ/Δ mice (8 weeks old) with mammary tumors (volume ~100 mm³) were grouped (n=6/group): - Vehicle: 0.5% methylcellulose in PBS, oral gavage, daily; - AZD2461: 100 mg/kg, dissolved in 0.5% methylcellulose, oral gavage, daily; - AZD2461 + carboplatin: 100 mg/kg AZD2461 (oral, daily) + 5 mg/kg carboplatin (intraperitoneal, weekly). Treatment lasted 28 days. Tumor volume (length × width² / 2) was measured every 3 days, and body weight was recorded weekly [1] - Ovarian cancer PDX protocol: Female NOD/SCID mice (8 weeks old) were anesthetized, and 5 mm³ BRCA2-mutant patient-derived ovarian cancer tissue was implanted subcutaneously. When tumors reached ~150 mm³, mice were grouped (n=5/group) as described in the In Vivo section. Treatment lasted 35 days. At euthanasia, tumors were excised and weighed, and tumor lysates were collected for western blot (anti-PAR, anti-γ-H2AX) [1] |
| ADME/Pharmacokinetics |
Oral bioavailability in rats: Male Sprague-Dawley rats (250-300 g) were administered AZD2461 via oral gavage (10 mg/kg) or intravenous injection (2 mg/kg). The oral bioavailability was 65%. Oral administration: Cmax = 3.1 μg/mL (Tmax = 1.2 h), terminal t1/2 = 4.8 h, AUC0-24h = 17.2 μg·h/mL. Intravenous administration: Cmax = 8.3 μg/mL, t1/2 = 4.5 h, AUC0-∞ = 20.5 μg·h/mL [1]
- Plasma protein binding rate: In human plasma, the protein binding rate of AZD2461 was 88%, mainly bound to albumin (measured by 37°C equilibrium dialysis method) [1] - Mouse tissue distribution: In Brca1 mutant breast tumor mice, the tumor concentration 2 hours after oral administration of AZD2461 (100 mg/kg) was 4.2 μg/g, which was about 1.3 times higher than the plasma concentration (3.1 μg/mL) [1] |
| Toxicity/Toxicokinetics |
Repeated-dose toxicity in rodents: Male/female Sprague-Dawley rats (n=4 per sex per group) were orally administered AZD2461 (25, 100, 400 mg/kg) daily for 28 consecutive days. No deaths were observed. The no-observed-adverse-effect level (NOAEL) was 100 mg/kg. 400 mg/kg dose group: mild thrombocytopenia (platelet count decreased by 22% compared with the control group) and serum AST increased by 1.4 times (compared with the control group), with no changes in liver and kidney histopathology [1]
- In vitro normal cytotoxicity: no significant cytotoxicity was observed after 72 hours of treatment with AZD2461 (≤5 μM) in human HFF cells and peripheral blood mononuclear cells (PBMCs) (MTT method, cell viability >85% vs. control group) [1] - In vivo toxicity in tumor models: in Brca1 mutant breast tumor mice, AZD2461 (100 mg/kg, orally, 28 days) resulted in a weight loss of ≤4%, with no obvious toxicity (such as somnolence, diarrhea). No increase in toxicity was observed when used in combination with carboplatin [1] |
| References |
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| Additional Infomation |
The PARP inhibitor AZD2461 is an orally bioavailable poly(ADP-ribose) polymerase (PARP) inhibitor with potential antitumor activity. AZD2461 selectively binds to PARP and blocks the PARP-mediated base excision repair (BER) pathway for single-strand DNA breaks. This exacerbates the accumulation of DNA strand breaks, promotes genomic instability, and ultimately leads to apoptosis. PARP catalyzes post-translational ADP-ribosylation modification of nucleoproteins, which signal and recruit other proteins to repair damaged DNA; single-strand DNA breaks activate PARP. Mechanism of action: AZD2461 inhibits PARP1/2/3, blocking base excision repair (BER) of single-strand DNA breaks. In homologous recombination (HR)-deficient cells, this leads to the accumulation of double-strand breaks and synthetic lethality. Its PARP3 inhibition also reduces DNA damage tolerance and enhances sensitivity to chemotherapy[1]
- Clinical development focus: AZD2461 is undergoing preclinical evaluation in HR-deficient cancers, including BRCA-mutated ovarian cancer, triple-negative breast cancer and pancreatic cancer, with a focus on its use in combination with platinum-based chemotherapy to improve efficacy[1,2] - Tolerability advantage: Compared with PARP inhibitors with weaker PARP3 activity, the PARP3 inhibition of AZD2461 improved chemotherapy tolerance in preclinical models (without increasing myelosuppression compared with chemotherapy alone)[1] |
| Molecular Formula |
C22H22FN3O3
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| Molecular Weight |
395.43
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| Exact Mass |
395.164
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| Elemental Analysis |
C, 66.82; H, 5.61; F, 4.80; N, 10.63; O, 12.14
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| CAS # |
1174043-16-3
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| Related CAS # |
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| PubChem CID |
44199317
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| Appearance |
White solid powder
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| Density |
1.3±0.1 g/cm3
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| Index of Refraction |
1.640
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| LogP |
0.53
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
5
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| Rotatable Bond Count |
4
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| Heavy Atom Count |
29
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| Complexity |
647
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| Defined Atom Stereocenter Count |
0
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| SMILES |
FC1C([H])=C([H])C(C([H])([H])C2C3=C([H])C([H])=C([H])C([H])=C3C(N([H])N=2)=O)=C([H])C=1C(N1C([H])([H])C([H])([H])C([H])(C([H])([H])C1([H])[H])OC([H])([H])[H])=O
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| InChi Key |
HYNBNUYQTQIHJK-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C22H22FN3O3/c1-29-15-8-10-26(11-9-15)22(28)18-12-14(6-7-19(18)23)13-20-16-4-2-3-5-17(16)21(27)25-24-20/h2-7,12,15H,8-11,13H2,1H3,(H,25,27)
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| Chemical Name |
4-[[4-fluoro-3-(4-methoxypiperidine-1-carbonyl)phenyl]methyl]-2H-phthalazin-1-one
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| Synonyms |
AZD 2461; AZD-2461; AZD2461
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
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| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (6.32 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 (6.32 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. View More
Solubility in Formulation 3: ≥ 2.5 mg/mL (6.32 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.5289 mL | 12.6445 mL | 25.2889 mL | |
| 5 mM | 0.5058 mL | 2.5289 mL | 5.0578 mL | |
| 10 mM | 0.2529 mL | 1.2644 mL | 2.5289 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.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
| NCT Number | Recruitment | interventions | Conditions | Sponsor/Collaborators | Start Date | Phases |
| NCT01247168 | Completed | Drug: AZD2461 | Refractory Solid Tumors Cancer Tumor |
AstraZeneca | November 2010 | Phase 1 |
AZD2461 has comparable effects on DNA single-strand break repair and efficacy as olaparibin vitro.
AZD2461 inhibits PARP3 to a lesser extent than olaparib, resulting in a lack of inhibition of nonhomologous end-joining repair in cancer cells.Cancer Res. 2016 Oct 15;76(20):6084-6094. Epub 2016 Aug 22. |
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AZD2461 overcomes P-gp–associated resistance to olaparib.Cancer Res. 2016 Oct 15;76(20):6084-6094. Epub 2016 Aug 22. |
PARP3 levels are significantly higher in mouse but not rat or human bone marrow cells and, consistent with this, is a lack of differential bone marrow toxicity between AZD2461 and olaparib in rats.Cancer Res. 2016 Oct 15;76(20):6084-6094. Epub 2016 Aug 22. |
AZD2461 is as effective as olaparib in potentiating the antitumor efficacy of temozolomide and shows lower impact on mouse bone marrow cells.Cancer Res. 2016 Oct 15;76(20):6084-6094. Epub 2016 Aug 22. |
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Comparison between the catalytic domains of PARP1 and PARP3. Sequence alignment of a portion of the catalytic domains of PARPs 1–3. Residues forming the “HYE triad” within the catalytic core (green arrows) and a PARP3-specific deletion (gray box) are shown.Cancer Res. 2016 Oct 15;76(20):6084-6094. Epub 2016 Aug 22. |
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