ART558 is a nanomolar, potent, selective, low molecular weight, allosteric DNA polymerase active Polθ inhibitor (IC50=7.9 nM). ART558 may be used in cancer-related research.

Physicochemical Properties

Molecular Formula	C21H21F3N4O2
Molecular Weight	418.412255048752
Exact Mass	418.161
CAS #	2603528-97-6
PubChem CID	156068882
Appearance	White to off-white solid powder
LogP	3.9
Hydrogen Bond Donor Count	1
Hydrogen Bond Acceptor Count	8
Rotatable Bond Count	3
Heavy Atom Count	30
Complexity	679
Defined Atom Stereocenter Count	2
SMILES	FC(C1C=C(C)N=C(C=1C#N)N1CC[C@H]([C@H]1C(N(C)C1C=CC=C(C)C=1)=O)O)(F)F
InChi Key	YHMDHAMZFMNMTF-MSOLQXFVSA-N
InChi Code	InChI=1S/C21H21F3N4O2/c1-12-5-4-6-14(9-12)27(3)20(30)18-17(29)7-8-28(18)19-15(11-25)16(21(22,23)24)10-13(2)26-19/h4-6,9-10,17-18,29H,7-8H2,1-3H3/t17-,18+/m1/s1
Chemical Name	(2S,3R)-1-[3-cyano-6-methyl-4-(trifluoromethyl)pyridin-2-yl]-3-hydroxy-N-methyl-N-(3-methylphenyl)pyrrolidine-2-carboxamide
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 Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.
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

ln Vitro	In an isogenic model of BRCA1 deficiency that has been previously described, ART558 (0~10 μM; 7 days) demonstrated synthetic lethality when combined with PARPi olaparib [1]. γH2AX accumulates in cells treated with ART558 (5μM; 0~72 hours; BRCA2 wild-type or BRCA2-/- cells) [1]. Without affecting nonhomologous end joining, ART558 suppresses the primary Polβ-mediated DNA repair mechanism, Theta-mediated end joining. In tumor cells with BRCA1 or BRCA2 mutations, ART558 causes damage to DNA, promotes synthetic lethality, and amplifies the effects of PARP inhibitors. In 53BP1-deficient cells, ART558 raises biomarkers and synthetic lethality of single-stranded DNA; however, these effects are reversed when DNA nucleases that induce end resection are inhibited, indicating that these effects are connected to the process of synthetic lethality. YFP-labeled full-length Polθ has a longer residency duration at the site of laser-induced DNA damage when treated with ART558 [1].
Cell Assay	Cell Viability Assay[1] Cell Types: BRCA2 wild type or BRCA2−/− Cell Tested Concentrations: 0~10 μM Incubation Duration: 7 days Experimental Results: Demonstrated synthetic lethality in previously described BRCA1-isogenic model and with PARPi olaparib The combined effect is insufficient. Western Blot Analysis [1] Cell Types: BRCA2 wild type or BRCA2−/− Cell Tested Concentrations: 5μM Incubation Duration: 0~72 hrs (hours) Experimental Results: Shows intracellular γH2AX accumulation.
References	[1]. Polθ inhibitors elicit BRCA-gene synthetic lethality and target PARP inhibitor resistance. Nat Commun. 2021 Jun 17;12(1):3636.

Solubility Data

Solubility (In Vitro)

DMSO : ~170 mg/mL (~406.30 mM)

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 5 mg/mL (11.95 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 50.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. Solubility in Formulation 2: 4.25 mg/mL (10.16 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 42.5 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	2.3900 mL	11.9500 mL	23.9000 mL
	5 mM	0.4780 mL	2.3900 mL	4.7800 mL
	10 mM	0.2390 mL	1.1950 mL	2.3900 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.