AZ20 is a novel potent and selective inhibitor of ATR (ATM and Rad3-related) protein kinase with potential antitumor activity. In a cell-free assay, it inhibits ATR with an IC50 of 5 nM and demonstrates 8-fold selectivity for ATR over mTOR. ATR is a promising new target for anticancer drugs, and its inhibitors may be used as monotherapy in tumors that are dependent on specific DNA-repair pathways or as sensitizers to chemotherapy or radiation. HT29 colorectal adenocarcinoma tumor cells' ATR-mediated phosphorylation of Chk1 and ATR immunoprecipitated from HeLa nuclear extracts are both inhibited by AZ20, with an IC50 of 5 nM and 50 nM, respectively.

Physicochemical Properties

Molecular Formula	C21H24N4O3S
Molecular Weight	412.51
Exact Mass	412.156
Elemental Analysis	C, 61.14; H, 5.86; N, 13.58; O, 11.64; S, 7.77
CAS #	1233339-22-4
Related CAS #	1233339-22-4
PubChem CID	46244454
Appearance	White solid powder
Density	1.4±0.1 g/cm3
Boiling Point	634.6±55.0 °C at 760 mmHg
Flash Point	337.6±31.5 °C
Vapour Pressure	0.0±1.9 mmHg at 25°C
Index of Refraction	1.687
LogP	0.5
Hydrogen Bond Donor Count	1
Hydrogen Bond Acceptor Count	6
Rotatable Bond Count	4
Heavy Atom Count	29
Complexity	707
Defined Atom Stereocenter Count	1
SMILES	S(C([H])([H])[H])(C1(C2=C([H])C(=NC(C3C([H])=C([H])C([H])=C4C=3C([H])=C([H])N4[H])=N2)N2C([H])([H])C([H])([H])OC([H])([H])[C@@]2([H])C([H])([H])[H])C([H])([H])C1([H])[H])(=O)=O
InChi Key	SCGCBAAYLFTIJU-CQSZACIVSA-N
InChi Code	InChI=1S/C21H24N4O3S/c1-14-13-28-11-10-25(14)19-12-18(21(7-8-21)29(2,26)27)23-20(24-19)16-4-3-5-17-15(16)6-9-22-17/h3-6,9,12,14,22H,7-8,10-11,13H2,1-2H3/t14-/m1/s1
Chemical Name	(3R)-4-[2-(1H-indol-4-yl)-6-(1-methylsulfonylcyclopropyl)pyrimidin-4-yl]-3-methylmorpholine
Synonyms	AZ-20; AZ20; AZ 20
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	ATR ( IC50 = 5 nM ); mTOR ( IC50 = 38 nM ); PI3Kα ( IC50 = 13000 nM )
ln Vitro	In vitro activity: AZ20 exhibits good selectivity against ATM, DNA-PK, and all PI3K isoforms. [2] In vitro, AZ20 exhibits concentration-dependent reductions in pChk1 Ser345, pChk1 Ser317, and pChk1 Ser296 levels. Extended contact with AZ20 elevates the pan-nuclear staining of γH2AX, a sign of replication stress. This is linked to an increase in phospho-histone H3 and S-phase arrest. AZ20 differs from other cytotoxic agents in that it causes both growth inhibition and cell death in vitro. AZ20's cytotoxic effect can be amplified when combined with KU-60019, a selective ATM inhibitor. [1]
ln Vivo	Female mice in their underwear For 13 days, LoVo tumors are treated with AZ20 orally at a dose of 25 mg/kg twice day or 50 mg/kg once day, which significantly inhibits tumor growth. [2] This is linked to a transient rise in mouse bone marrow at therapeutic doses, indicating a favorable therapeutic index, but a persistent elevation of γH2AX pan-nuclear staining in xenograft tissue. [1] AZ20 is evaluated for its potential to cause drug-drug interactions (DDIs) by specifically inhibiting cytochrome P450 enzymes. At 10 μM, AZ20 is observed to 50% inhibit the metabolism of midazolam mediated by cytochrome 3A4. A low dose rat PK study found that AZ20 has a respectable bioavailability. [2]
Enzyme Assay	In order to obtain ATR for the in vitro enzyme assay, rabbit polyclonal antiserum raised to amino acids 400–480 of ATR was immunoprecipitated from HeLa nuclear extract. This buffer contained the ATR. pH 7.4, 250 mM NaCl, 0.5 mM EDTA, 0.1 mM Na3VO4, 10% v/v glycerol, and 0.01% v/v Tween 20 are combined to make 25 mM HEPES (pH 7.4). Protein A-Sepharose beads were incubated with ATR-antibody complexes from nuclear extract for one hour, after which the beads were recovered by centrifugation. ATR assay buffer (50 mM HEPES (pH 7.4), 150 mM NaCl, 6 mM MgCl2, 4 mM MnCl2, 0.1 mM Na3VO4, 0.1 mM DTT, and 10% (v/v) glycerol) was incubated with 1 μg of substrate glutathione S-transferase-p53N66 in a well of a 96-well plate. This was done in the presence or absence of an inhibitor. The reaction proceeded at 37°C for an extra hour after 10 minutes of moderate shaking, during which time ATP was added to a final concentration of 3 μM. A 96-well plate coated with white opaque glutathione was used to transfer the reaction and incubate it overnight at 4°C. The reaction was stopped by adding 100 μL of PBS. Following a PBS/0.05% (v/v) Tween 20 wash, this plate was blotted dry and subjected to an ELISA analysis using a phosphoserine 15 p53 antibody. Utilizing a secondary antibody conjugated with goat anti-mouse horseradish peroxidase, the phosphorylated glutathione S-transferase?p53N66 substrate was detected. A TopCount plate reader was utilized for chemiluminescent detection after a signal was generated using an enhanced chemiluminescence solution. IC50 values for the compounds were then calculated using the resulting calculated percentage of enzyme activity.
Cell Assay	Using a Labcyte Echo acoustic dispensing device, compound dose ranges are created by dilution in 100% DMSO and then further into assay medium (EMEM, 10% FCS, 1% glutamine). Plates of cells are grown for 24 hours in 40 μL of EMEM, 10% FCS, and 1% glutamine. The cells are plated at 9×104 cells per mL in 384-well Costar plates. After the compound is added, the cells are incubated for sixty minutes. Using the Labcyte Echo, a final concentration of 3 μM 4NQO (prepared in 100% DMSO) is added, and the cells are incubated for an additional 60 minutes. For 20 minutes, 40 μL of 3.7% v/v formaldehyde solution is added to the cells to fix them. The fix is removed from the cells, and they are then permeabilized in 40 μL of PBS containing 0.1% Triton X-100 and cleaned with PBS. Following a wash, 15 μL of pChk1 Ser345 primary antibody solution is added to the cells. The trays are incubated for the entire night at 4°C. After eliminating the primary antibody, 20 μL of secondary antibody solution and 1 μM Hoechst 33258 are added and allowed to sit at room temperature for 90 minutes. After being cleaned, the plates are placed in 40 μL of PBS. After that, staining intensities are measured on the plates using an ArrayScan VTI instrument. Dose responses are then collected and utilized to calculate the IC50 values for the compounds.
Animal Protocol	Negative pressure isolators are used to house female Swiss nu/nu mice. To create LoVo tumor xenografts, 8–12 week old mice are given a subcutaneous injection of 1×107 tumor cells (100 μL in serum-free medium) in the left dorsal flank. When tumors are palpable, animals are randomly assigned to treatment groups. AZ20 is taken orally and is prepared in a 10% DMSO/40% propylene glycol/50% water solution. Calipers are used to measure tumors up to three times a week. Plotting the data using the geometric mean for each group against time yields the tumor volumes.
References	[1]. Cancer Res 2012;72(8 Suppl):Abstract nr 1823. [2]. J Med Chem . 2013 Mar 14;56(5):2125-38.
Additional Infomation	(3R)-4-[2-(1H-indol-4-yl)-6-(1-methylsulfonylcyclopropyl)-4-pyrimidinyl]-3-methylmorpholine is a member of indoles.

Solubility Data

Solubility (In Vitro)

DMSO: ~83 mg/mL (~201.2 mM) Water: <1 mg/mL Ethanol: ~3 mg/mL (~7.3 mM)

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.06 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 (6.06 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.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	2.4242 mL	12.1209 mL	24.2418 mL
	5 mM	0.4848 mL	2.4242 mL	4.8484 mL
	10 mM	0.2424 mL	1.2121 mL	2.4242 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.