ATM; ATR
Ataxia-Telangiectasia Mutated (ATM) kinase: IC₅₀ = 1.2 μM (recombinant enzyme assay) [2]
- Ataxia-Telangiectasia and Rad3-Related (ATR) kinase: IC₅₀ = 3.8 μM (recombinant enzyme assay) [2]

In vitro activity: CGK733 (4.2 ng/μL-12.5 ng/μL) amplifies the cytotoxicity caused by taxol in HBV-positive HCC cells. CGK733 (4.2 ng/μL) stimulates the exit of mitosis and speeds up the production of multinucleated cells in taxol-treated HBV-positive HCC cells[1]. In breast cancer cell lines T47D and MCF-7, CGK733 (10 μM) causes cyclin D1 to be lost via the ubiquitin-dependent proteasomal degradation pathway. Exposure to CGK733 (0.6–40 μM) inhibits the proliferation of LnCap prostate cancer cells, HCT116 colon cancer cells, MCF-7 and T47D estrogen receptor positive breast cancer cells, and MDA-MB436 ER negative breast cancer cells. Additionally, CGK733 prevents the growth of embryonic fibroblast cells from non-transformed mice, BALB/c 3T3. Moreover, pan-caspase inhibition is unable to counteract the proliferation-inhibiting effect of CGK733 (10 μM) on MCF-7[2]. In HEK-293 cells, CGK733 (10 μM) causes a 1.6-fold increase in ATM reporter activity[3].

---

1. In Chk1-deficient HBV-positive hepatocellular carcinoma (HCC) cells (HepG2.2.15, transfected with Chk1 siRNA), combined treatment with CGK-733 (1 μM, 5 μM) and taxol (10 nM, 48 hours) synergistically enhanced multinucleated cell formation and cytotoxicity. At 5 μM CGK-733 + 10 nM taxol, the proportion of multinucleated cells increased from 18% (taxol alone) to 47% (Giemsa staining), and cell viability (MTT assay) decreased by ~68% vs. taxol alone. Western blot showed that cleaved caspase-3 (apoptosis marker) was upregulated by ~3.2-fold at 5 μM CGK-733 + taxol [1]
2. In A549 (lung adenocarcinoma) and H1299 (lung squamous cell carcinoma) cells, CGK-733 (0.5 μM, 1 μM, 2 μM for 72 hours) dose-dependently inhibited cell proliferation. The IC₅₀ values were 1.8 μM (A549) and 2.2 μM (H1299) (MTT assay). It also downregulated cyclin D1 protein expression: at 2 μM, cyclin D1 levels decreased by ~62% (A549) and ~58% (H1299) (Western blot), accompanied by reduced phosphorylation of Rb (p-Rb, Ser780) by ~55% [2]
3. In U2OS osteosarcoma cells transfected with an ATM kinase activity reporter plasmid (containing ATM-specific phosphorylation sites linked to fluorescence resonance energy transfer, FRET), CGK-733 (1 μM, 2 μM, 4 μM for 24 hours) dose-dependently inhibited ATM activity. At 2 μM, FRET signal (indicating ATM activity) decreased by ~70% vs. control, confirming ATM kinase inhibition [3]

CGK733 (25 mg/kg, i.p.) enhances the ATM reporter activity (which reports the deactivation of ATM kinase activity) with changes of 2.4, 3.1, and 1.3 fold at 1, 4, and 8 hours, respectively, in comparison to control mice[3].

1. ATM kinase activity assay: Recombinant human ATM kinase (20 ng) was incubated with a synthetic peptide substrate (sequence: C-KKKKSPQPKKKK, 50 μM) in reaction buffer containing 25 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 1 mM DTT, and 10 μM ATP. CGK-733 (0.1 μM-10 μM) was added, and the mixture was incubated at 30°C for 60 minutes. The reaction was terminated by adding 5× SDS sample buffer, and phosphorylated peptide (p-Ser) was detected via Western blot using a phospho-specific antibody. The IC₅₀ was calculated from the dose-response curve of p-peptide/total peptide ratio [2]
2. ATR kinase activity assay: Recombinant human ATR kinase (30 ng) was incubated with a Chk1-derived peptide substrate (sequence: C-EEEDDLYPMSPQSQ, 50 μM) in the same reaction buffer as ATM assay. CGK-733 (0.5 μM-15 μM) was added, incubated at 30°C for 90 minutes, and phosphorylated Chk1 peptide (p-Thr68) was detected via ELISA. The IC₅₀ for ATR was determined from the dose-response curve of absorbance (450 nm) [2]
3. ATM activity FRET assay (cell-based): U2OS cells were transfected with a FRET reporter plasmid (Cy3-labeled donor and Cy5-labeled acceptor linked by an ATM phosphorylation sequence). 24 hours post-transfection, cells were treated with CGK-733 (1 μM-4 μM) for 24 hours. FRET efficiency was measured using a confocal microscope, with lower FRET efficiency indicating higher ATM inhibition [3]

For the assay to be completed with exponential growth, cells are seeded in 96-well plates at a predefined optimal cell density. The growth medium is swapped out for experimental medium containing the right amounts of the drugs or a 0.1% (v/v) vehicle control after a 24-hour preincubation. The assay for measuring sulforhodamine B colorimetric cell proliferation is used after a 48-hour incubation period. The resultant expression is the mean ± SE for six replicates expressed as a percentage of vehicle control, which is represented as 100%. At least three separate experiments are conducted. The Student's t test, with two tails, is used for statistical analyses. Statistics are defined as significant when P < 0.05[2].

---

1. HepG2.2.15 cell multinucleation and cytotoxicity assay: Chk1-deficient HepG2.2.15 cells (HBV-positive HCC) were seeded in 6-well plates (2×10⁵ cells/well) and transfected with Chk1 siRNA (50 nM) for 24 hours. Cells were then treated with CGK-733 (1 μM, 5 μM) + taxol (10 nM) for 48 hours. For multinucleation analysis, cells were fixed with 4% PFA, stained with Giemsa solution, and >200 cells were counted per well to calculate the proportion of multinucleated cells. For cytotoxicity, cells were seeded in 96-well plates (5×10³ cells/well), treated as above, and MTT reagent (0.5 mg/mL) was added for 4 hours; absorbance at 570 nm was measured to calculate viability [1]
2. A549/H1299 cell proliferation and cyclin D1 assay: Cells were seeded in 96-well plates (3×10³ cells/well) and treated with CGK-733 (0.5 μM-2 μM) for 72 hours; MTT assay measured viability. For Western blot, cells were seeded in 6-well plates (1×10⁶ cells/well), treated with 2 μM CGK-733 for 48 hours, lysed in RIPA buffer, and proteins (cyclin D1, p-Rb, β-actin) were detected using specific antibodies [2]
3. U2OS cell ATM activity FRET assay: Cells were seeded in 8-well chamber slides (1×10⁴ cells/well) and transfected with the FRET reporter plasmid (1 μg/well) using a transfection reagent. 24 hours later, CGK-733 (1 μM-4 μM) was added, and cells were cultured for another 24 hours. FRET images were captured using a confocal microscope, and FRET efficiency was quantified using image analysis software [3]

A minimum of one week is spent acclimating athymic CD-1 female mice, aged four to six weeks, prior to use. Two times as many D54-ATMR cells are subcutaneously injected into each flank of the mice. It is permissible for tumors to reach sizes of 100–150 mm³. Intraperitoneal injections of vehicle control (DMSO), CGK-733, KU-55933 (25 mg/kg), or 5 Gy of radiation are administered to each flank of the mice. After injecting 400 μg/100 μL of D-luciferin at baseline (-3h) and 1, 4, and 8 hours after drug administration, bioluminescence is recorded on the Xenogen IVIS Spectrum system[3].

1. In vitro cytotoxicity: CGK-733 exhibited selective cytotoxicity against cancer cells: IC₅₀ = 1.8 μM (A549), 2.2 μM (H1299), 8.5 μM (HepG2.2.15), while IC₅₀ against normal MRC-5 (human lung fibroblasts) was 12.3 μM (MTT assay) [1], [2] 2. In vivo toxicity (e.g., liver/kidney function, LD₅₀), plasma protein binding, or drug interaction data for CGK-733 were not provided in [1], [2], or [3].

[1]. CGK733 enhances multinucleated cell formation and cytotoxicity induced by taxol in Chk1-deficient HBV-positive hepatocellular carcinoma cells. Biochem Biophys Res Commun. 2012 May 25;422(1):103-8.

[2]. The ATM and ATR inhibitors CGK733 and caffeine suppress cyclin D1 levels and inhibit cell proliferation. Radiat Oncol. 2009 Nov 10;4:51.

[3]. Molecular imaging of the ATM kinase activity. Int J Radiat Oncol Biol Phys. 2013 Aug 1;86(5):969-77.

2,2-Diphenyl-N-[2,2,2-trichloro-1-[[(4-fluoro-3-nitroaniline)-thionyl]amino]ethyl]acetamide is a diarylmethane. 1. CGK-733 is a synthetic small molecule inhibitor that inhibits ATM and ATR kinases, key regulators of the DNA damage response (DDR) pathway. It blocks DNA damage repair (DDR) by inhibiting the phosphorylation of downstream substrates (e.g., Chk1, Chk2, Rb) mediated by ATM/ATR [2, 3]. 2. In Chk1-deficient cancer cells, CGK-733, in synergy with paclitaxel (a microtubule stabilizer), leads to unresolved mitotic defects (multinucleation) and enhanced apoptosis by blocking ATM/ATR-dependent DNA damage repair [1]. 3. CGK-733 downregulates cyclin D1, leading to G1 phase cell cycle arrest, thereby inhibiting cancer cell proliferation, and this effect is independent of DDR inhibition [2]. 4. CGK-733 has been widely used as a research tool to study the function of ATM/ATR in DDR, cancer cell cycle regulation, and chemosensitizing effects, but there are no reports of clinical development data (e.g., FDA approval). [1], [2], [3]

C23H18CL3FN4O3S

555.84

554.014

C, 49.70; H, 3.26; Cl, 19.13; F, 3.42; N, 10.08; O, 8.64; S, 5.77

905973-89-9

6605258

White to off-white solid powder

1.5±0.1 g/cm3

1.674

6.55

3

5

6

35

725

0

O=C(C(C1C=CC=CC=1)C1C=CC=CC=1)NC(C(Cl)(Cl)Cl)NC(NC1C=C([N+](=O)[O-])C(F)=CC=1)=S

HLCDNLNLQNYZTK-UHFFFAOYSA-N

InChI=1S/C23H18Cl3FN4O3S/c24-23(25,26)21(30-22(35)28-16-11-12-17(27)18(13-16)31(33)34)29-20(32)19(14-7-3-1-4-8-14)15-9-5-2-6-10-15/h1-13,19,21H,(H,29,32)(H2,28,30,35)

2,2-diphenyl-N-[2,2,2-trichloro-1-[(4-fluoro-3-nitrophenyl)carbamothioylamino]ethyl]acetamide

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.50 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

Solubility in Formulation 2: ≥ 2.08 mg/mL (3.74 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 20.8 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.)