AG-14361 328543-09-5_PARP_DNA Damage_Signaling Pathways_Products

AG14361 (AG-14361) is a novel, potent and selective inhibitor of poly (ADP-ribose) polymerase-1 (PARP1) with potential anticancer activity. In a cell-free assay, it inhibits PARP1 with a Ki of less than 5 nM. It also exhibits strong in vivo antitumor efficacy and strong antiproliferative activity against a variety of cancer cells.

Physicochemical Properties

Molecular Formula

C19H20N4O

Molecular Weight

320.39

Exact Mass

320.163

Elemental Analysis

C, 71.23; H, 6.29; N, 17.49; O, 4.99

CAS #

328543-09-5

Related CAS #

328543-09-5

PubChem CID

9840076

Appearance

White to off-white solid powder

Density

1.3±0.1 g/cm3

Index of Refraction

1.676

LogP

1.92

Hydrogen Bond Donor Count

Hydrogen Bond Acceptor Count

Rotatable Bond Count

Heavy Atom Count

Complexity

460

Defined Atom Stereocenter Count

SMILES

O=C1NCCN2C3=C1C=CC=C3N=C2C4=CC=C(C=C4)CN(C)C

InChi Key

SEKJSSBJKFLZIT-UHFFFAOYSA-N

InChi Code

InChI=1S/C19H20N4O/c1-22(2)12-13-6-8-14(9-7-13)18-21-16-5-3-4-15-17(16)23(18)11-10-20-19(15)24/h3-9H,10-12H2,1-2H3,(H,20,24)

Chemical Name

2-[4-[(dimethylamino)methyl]phenyl]-1,3,10-triazatricyclo[6.4.1.04,13]trideca-2,4,6,8(13)-tetraen-9-one

Synonyms

AG 14361; AG14361; AG-14361

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 = 0.5 nM )

ln Vitro

AG14361 is a minimum of 1000-fold more potent than benzamides. In permeabilized SW620 cells, the IC50 for AG14361 is 29 nM, whereas in intact SW620 cells, it is 14 nM. The tricyclic ring system of AG14361, as revealed by crystallographic analysis of the compound bound to the chicken PARP-1 catalytic domain, is situated in a pocket made up of amino acid residues Trp861, His862, Gly863, Tyr896, Phe897, Ala898, Lys903, Ser904, Tyr907, and Glu988. AG14361 establishes significant hydrogen bonds with Gly863, Ser904, and Glu988, as well as a water-mediated hydrogen bond. Since maximal PARP-1 inhibition is observed at much lower concentrations (≤1 μM) than the GI50, AG14361-induced growth inhibition cannot be attributed to PARP-1-related effects. At 0.4 μM, AG14361 has no effect on the growth or gene expression of cancer cells. However, it increases the antiproliferative activity of topotecan and temozolomide and inhibits 73% of LoVo cells' ability to recover from potentially fatal γ-radiation damage. In addition, microarray analysis demonstrates that 0.4 μM AG14361 does not significantly change gene expression. The expression of the 6800 genes remains unchanged in A549 cells exposed to 0.4 μM AG14361 for a duration of 17 hours. Thus, it can be concluded that the cellular effects of this low concentration of AG14361 are specific to PARP-1 inhibition because, despite inhibiting cellular PARP-1 activity by more than 85%, 0.4 μM AG14361 essentially does not change gene expression or cell proliferation. Gene expression is impacted by higher, growth-inhibitory concentrations of AG14361; however, since PARP-/- and PARP-1+/+ cells are equally affected in terms of cell proliferation, these effects are unlikely to be related to PARP-1 inhibition. Lower concentrations of AG14361 are found in the brain, but it is quickly absorbed into the bloodstream and distributed to the tumor and liver. In both xenograft models, the tissue-to-plasma concentration ratio shows that AG14361 is sustained in tumor tissue over time, with tumor concentrations (≥15 μM for 2 hours) higher than those needed to inhibit PARP-1 activity in vitro.[1] AG14361 overcomes temozolomide resistance by increasing temozolomide activity in all MMR-proficient cells (1.5–3.3-fold), but it is more successful in MMR-deficient cells (3.7–5.2-fold potentiation). On the other hand, benzylguanine is ineffective in MMR-deficient cells and only boosts the effectiveness of temozolomide in MMR-proficient cells.[2] AG14361 amplifies the cytotoxic and growth-inhibitory properties of topoisomerase I poisons. The DNA single-strand breaks caused by camptothecin are more persistent when AG14361 is present.[3]

ln Vivo

The administration of AG14361 prior to radiation therapy has a statistically significant effect on the sensitivity of mice harboring LoVo xenografts to radiation therapy. In xenografts, AG14361 statistically significantly increases blood flow, which may enhance medication delivery to tumor xenografts. Nontoxic dosages of AG14361 cause a 2- to 3-fold delay in LoVo xenograft growth when induced by irinotecan, x-irradiation, or temozolomide in vivo. The tumor growth delay was increased from 3 days to 9 days and from 10 days to 15 days by AG14361 at 5 mg/kg and 15 mg/kg, respectively, when coadministration of AG14361 with temozolomide was observed. This increase in temozolomide activity against LoVo xenografts was statistically significant. AG14361 and temozolomide together result in the full remission of SW620 xenograft tumors. Following intraperitoneal injection of AG14361 (10 mg/kg), PARP-1 activity in SW620 xenografts is inhibited by more than 75% for at least 4 hours, as determined by pharmacodynamic assay. This is consistent with the concentration of AG14361 remaining in the tumor.[1]

Enzyme Assay

Full-length recombinant human PARP-1 activity is assessed in a reaction mixture containing activated calf thymus DNA (10 μg/mL) at 25 °C, 500 μM NAD⁺ plus [³²P]NAD⁺ (0.1–0.3 μCi per reaction mixture), and 20 nM PARP-1. The reaction is stopped after 4 minutes by adding ice-cold 10% (wt/vol) trichloroacetic acid. A PhosphorImager is used to quantify the reaction product [³²P]ADP-ribose that is integrated into acid-insoluble material after it has been deposited onto Whatman GF/C glass fiber filters using a Bio-Dot microfiltration device. Inhibition of PARP-1 activity by AG14361 at 0–600 nM is measured, and the K_i for AG14361 is calculated by nonlinear regression analysis.

Cell Assay

The luciferase-coupled ATP quantization assay of metabolically active cells in a 96-well plate is used in the cell viability assay along with MTT. One well of a 24-well plate is plated with one to two × 10⁴ cells for MTT. The target medications (AG14361) are dissolved in DMSO at different concentrations and subsequently added to the cells in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum. Additionally, each well receives an addition of the IC50 concentration of AG14361. The addition to medium results in a final DMSO concentration of 0.1%. The medium is removed after 48 hours, and each well receives 0.3 mL of 0.1% MTT in phosphate-buffered saline (PBS). The MTT solution is removed and 0.8 mL of 2-propanol is added after 30 minutes of incubation in a 37°C CO2 incubator. A plate reader is used to measure the OD560 following 30 minutes of shaking. Every time point is platented three times.

Animal Protocol

CD-1 nude mice with palpable, subcutaneous SW620 or LoVo xenografts are given intraperitoneally AG14361 (at 5 or 15 mg/kg) or normal saline (control animals) once a day for five days (five mice per group). In cases where drugs are combined, AG14361 is injected intraperitoneally once a day for five days.This is done either right before the cytotoxic medication (NSC 362856 at 68 mg/kg or CPT-11 at 2.5 mg/kg) is administered or half an hour before applying 2 Gy of x-irradiation locally to the tumor once a day for five days. The tumor volumes are expressed as median relative tumor volume (RTV), which are calculated using the formula a² × b/2, where a is the tumor's width and b its length. These volumes are obtained from two-dimensional caliper measurements. In other words, RTV1 represents the tumor volume on day 0 of treatment, and RTV4 represents the tumor volume four times that on day 0 of treatment. The term "tumor growth delay" refers to the difference between the time to RTV4 in mice receiving medication or radiation therapy and that of control mice (vehicle alone).

References

[1]. J Natl Cancer Inst . 2004 Jan 7;96(1):56-67.

[2]. Clin Cancer Res . 2004 Feb 1;10(3):881-9.

[3]. Clin Cancer Res . 2005 Dec 1;11(23):8449-57.

Additional Infomation

LSM-1988 is a member of benzimidazoles.

Solubility Data

Solubility (In Vitro)

DMSO: 10~12 mg/mL (31.2~37.5 mM)

Water: <1 mg/mL

Ethanol: <1 mg/mL

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 1 mg/mL (3.12 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 10.0 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of 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: ≥ 1 mg/mL (3.12 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 10.0 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: ≥ 1 mg/mL (3.12 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 10.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

Solubility in Formulation 4: 4% DMSO+ddH2O: 2mg/mL

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

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	3.1212 mL	15.6060 mL	31.2120 mL
	5 mM	0.6242 mL	3.1212 mL	6.2424 mL
	10 mM	0.3121 mL	1.5606 mL	3.1212 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.

PeptideDB

AG-14361 328543-09-5

CAS No.: 328543-09-5

Physicochemical Properties

Biological Activity

Solubility Data