CRT0044876 (CRT-0044876; NSC 69877, 7-NO2-ICA; NSC-69877; CRT 0044876; NSC69877), an indole analog, is a potent and selective APE1 inhibitor with potential antitumor activity. With an IC50 of about 3 μM, it inhibits APE1.

Physicochemical Properties

Molecular Formula	C9H6N2O4
Molecular Weight	206.15
Exact Mass	206.032
Elemental Analysis	C, 52.44; H, 2.93; N, 13.59; O, 31.04
CAS #	6960-45-8
Related CAS #	6960-45-8
PubChem CID	81409
Appearance	Light yellow to yellow solid powder
Density	1.6±0.1 g/cm3
Boiling Point	520.8±30.0 °C at 760 mmHg
Melting Point	260-261 °C
Flash Point	268.8±24.6 °C
Vapour Pressure	0.0±1.4 mmHg at 25°C
Index of Refraction	1.761
LogP	2.8
Hydrogen Bond Donor Count	2
Hydrogen Bond Acceptor Count	4
Rotatable Bond Count	1
Heavy Atom Count	15
Complexity	288
Defined Atom Stereocenter Count	0
SMILES	OC(=O)C1=CC2=C(N1)C(=CC=C2)[N+]([O-])=O
InChi Key	BIUCOFQROHIAEO-UHFFFAOYSA-N
InChi Code	InChI=1S/C9H6N2O4/c12-9(13)6-4-5-2-1-3-7(11(14)15)8(5)10-6/h1-4,10H,(H,12,13)
Chemical Name	7-nitro-1H-indole-2-carboxylic acid
Synonyms	CRT-0044876; NSC 69877; 7-NO2-ICA; CRT0044876; NSC-69877; CRT 0044876; NSC69877
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	APE1 ( IC50 ~ 3 μM )
ln Vitro	CRT0044876 shows selectivity for the inhibition of exonuclease III family and potently inhibits the 3'-phosphodiesterase, 3'-phosphatase, and AP endonuclease activities of APE1. Several DNA base-targeting compounds with an accumulation of unrepaired AP sites exhibit enhanced cytotoxicity when combined with CRT0044876. [1] Through the inhibition of the BER pathway, CRT0044876, in an acidic tumor microenvironment, increases intracellular reactive oxygen species and oxidative DNA damage, which in turn causes cell cycle arrests and an increase in DNA double strand breaks, ultimately resulting in cell death. [2]
ln Vivo
Enzyme Assay	The components of the BER reaction buffer are 0.5 mM DTT, 0.1 mM EDTA, 5 mM MgCl2, and 40 mM HEPES–KOH (pH 7.8). 0.75 ng of reduced AP site double-stranded oligonucleotide, purified protein (3.3 nM final concentration of APE1), and BER buffer mix were added to a 10 μl AP site cleavage reaction. The mixture is incubated for one hour at 37°C. After adding 1 μl of stop buffer (consisting of 50% glycerol, 10 mM Tris–HCl, 1 mM EDTA, 0.1% bromophenol blue, and 0.1% Xylene cyanol), the sample mixture is denatured for two minutes at 90–100°C. The sample is then electrophoresed at a constant current of 30 mA for 30 minutes on a 15% TBE CriterionTM Pre-Cast Gel. The radiolabeled substrate and reaction products are then visualized using a phosphorImager. From 0.1 to 100 μM drug concentrations, the inhibitory activity of putative APE1-targeting compounds is scrutinized. IC50 values are computed by finding the concentration of the inhibitor that decreased APE1 activity to 50% of the control values. The quantification of the resolved radiolabeled bands is done using ImageQuant software investigation.
Cell Assay	The culture medium used for HT1080 fibrosarcoma cells is 2% RPMI supplemented with 0.06 g/l penicillin, 0.1 g/l streptomycin (pH 7.0), 10% fetal bovine serum, and 4 mM glutamine. For clonogenic survival analysis, only cells with a plating efficiency of ≥60% are employed. 500 cells are seeded into 10-cm tissue culture dishes, and the cultures are kept in an environment of 95% air and 5% CO2 at 37°C in a humidified incubator. In order to assess the toxicity profile of potential APE1 inhibitors, the medium is supplemented with inhibitor at different concentrations (100–500 μM), and cultures are incubated for 7–10 days until cell colonies form. After fixing (75 percent methanol and 25 percent acetic acid) for 30 minutes, the colonies are stained for four hours at room temperature using crystal violet (1 mg/ml in distilled water). A colony counter is used to count colonies that are visible.
Animal Protocol
References	[1]. Nucleic Acids Res . 2005 Aug 19;33(15):4711-24. [2]. Cancer Res . 2009 Sep 15;69(18):7285-93.

Solubility Data

Solubility (In Vitro)

DMSO : 41~50 mg/mL (198.9~242.5 mM) Ethanol : ~1 mg/mL (~4.9 mM)

Solubility (In Vivo)

Solubility in Formulation 1: 2.5 mg/mL (12.13 mM) in 10% DMSO + 40% PEG300 +5% Tween-80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication. 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.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	4.8508 mL	24.2542 mL	48.5084 mL
	5 mM	0.9702 mL	4.8508 mL	9.7017 mL
	10 mM	0.4851 mL	2.4254 mL	4.8508 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.