- Cdc25 phosphatases (based on the compound's class as a Cdc25 inhibitor, though specific IC50 values for this compound are not provided in this study). [1]

- Antiproliferative Activity: In PC-3 human prostate cancer cells, DA 3003-2 exhibited an IC50 of approximately 5 µM after 48 hours of continuous exposure. The relative IC50 of DA 3003-2 was 2-fold lower (more potent) compared to its congener NSC 672121 (which had an IC50 of approximately 10 µM). [1]
- Cell Cycle Effects: Treatment of asynchronous PC-3 cells with 10 µM DA 3003-2 (an IC70 concentration) for 24 hours induced accumulation of cells in the G2/M phase. The cell cycle distribution at this concentration was: G1: 19.6±1.8%, S: 7.3±0.6%, G2/M: 70.3±1.4%. In contrast, untreated or DMSO-treated control cells had approximately 52-53% of cells in G2/M. A lower concentration (5 µM) did not cause a substantial change in G2/M accumulation. [1]
- Mechanism of Action - Cdc2 Hyperphosphorylation: DA 3003-2 caused hyperphosphorylation of Cdc2 on tyrosine 15 (Tyr¹⁵) in both cyclin B₁ and cyclin A complexes. Treatment with 10 µM DA 3003-2 for 1 hour resulted in increased phospho-Cdc2 (Tyr¹⁵) levels in these immunoprecipitated complexes. [1]
- Effect on Protein Expression Levels: DA 3003-2 did not downregulate the protein expression levels of Cdc25A, Cdc25B, Cdc25C, G2/M cyclins (cyclin A, cyclin B₁), or Cdks (Cdc2, Cdk2) after 24 hours of treatment. [1]

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In PC-3 cells, DA 3003-2 (0.3-30 µM; 48 h) exhibits anti-proliferative action with an IC50 value of 5 µM[1]. In PC-3 cells, DA 3003-2 (5, 10 µM; 24, 1 h) enhances the expression of P-tyr15 Cdc2 and promotes cell cycle arrest in the G2/M phase [1].

- MTT Cytotoxicity Assay: To determine the sensitivity of PC-3 cells to DA 3003-2 and NSC 672121, a microtiter assay was performed. Cells (4 x 10³) were plated in 96-well microtiter plates, cultured for 24 hours, and then continuously exposed to 0.3 - 30 µM of the test compounds for 48 hours. Cell viability was assayed by measuring the color development from the reduction of MTT, read spectrophotometrically at 540 nm. The IC50 for DA 3003-2 was found to be approximately 5 µM. [1]
- Flow Cytometry for Cell Cycle Analysis: PC-3 cells (5 x 10⁵ cells/dish) were plated and maintained for 24 hours, then treated with DMSO, NSC 672121, or DA 3003-2. After 24 hours, cells were trypsinized, washed with PBS, and stained with a solution containing propidium iodide and RNase A. Flow cytometric analysis was conducted to determine the cell cycle distribution. The concentration of DA 3003-2 used to induce G2/M accumulation was 10 µM (IC70 concentration). [1]
- Western Blotting and Immunoprecipitation: Vehicle- or compound-treated cells were harvested and lysed in a buffer containing HEPES, Triton X-100, NaCl, glycerol, MgCl₂, NaF, EDTA, Na₃VO₄, and protease inhibitors. After centrifugation, protein concentration was determined. For immunoprecipitation of cyclin B₁, cyclin A, Cdc2, and Cdk2 proteins, 2 µg of the respective antibodies and Protein G Sepharose 4B were incubated with 1 mg of lysate for 16 hours. Beads were washed, and equal amounts of protein were resolved by SDS-PAGE and transferred to nitrocellulose membranes. A chemiluminescence detection system was used for immunocomplex detection. To study Cdc2 phosphorylation, cells were treated with 5 or 10 µM DA 3003-2 or 20 µM NSC 672121 for 1 hour before lysis and immunoprecipitation with anti-cyclin B₁ or anti-cyclin A, followed by immunoblotting with anti-phospho-Cdc2 (Tyr¹⁵) and anti-Cdc2 antibodies. To study protein expression levels, cells were treated for 24 hours, and whole cell lysates were immunoblotted with antibodies against Cdc25A, Cdc25B, Cdc25C, cyclin A, cyclin B₁, Cdc2, Cdk2, and actin. [1]

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Cell Cytotoxicity Assay[1]
Cell Types: PC-3 cells
Tested Concentrations: 0.3-30 µM
Incubation Duration: 48 h
Experimental Results: demonstrated antiproliferative efficacy in a dose-dependent manner with an IC50 value of 5 µM.

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Cell Cycle Analysis[1]
Cell Types: PC-3 cells
Tested Concentrations: 5, 10 µM
Incubation Duration: 24 h
Experimental Results: Induced cell cycle arrest at G2/M phase.

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Western Blot Analysis[1]
Cell Types: PC-3 cells
Tested Concentrations: 5, 10 µM
Incubation Duration: 1 h
Experimental Results: Increased the expression of P-tyr15 Cdc2.

[1]. G2/M accumulation in prostate cancer cell line PC-3 is induced by Cdc25 inhibitor 7-chloro-6-(2-morpholin-4-ylethylamino) quinoline-5, 8-dione (DA 3003-2). Exp Ther Med. 2010 Jul;1(4):647-650.

- Chemical Name and Structure: DA 3003-2 is chemically named 7-chloro-6-(2-morpholin-4-ylethylamino)quinoline-5,8-dione. It is a regioisomer of NSC 663284 (DA3003-1), which is 6-chloro-7-(2-morpholin-4-ylethylamino)quinoline-5,8-dione. [1]
- Proposed Mechanism of Action: DA 3003-2 induces G2/M cell cycle accumulation in asynchronous PC-3 prostate cancer cells by causing hyperphosphorylation of Cdc2 on Tyr¹⁵ within the cyclin B₁-Cdc2 and cyclin A-Cdc2 complexes. This hyperphosphorylation inhibits the activation of the cyclin-Cdk complex, leading to cell cycle arrest. The compound does not appear to work by downregulating the expression of Cdc25s, cyclins, or Cdks. [1]
- Potential Therapeutic Relevance: The study suggests that Cdc25 inhibitors like DA 3003-2 may be important small molecular targets for the treatment of androgen-refractory prostate cancer, given the increased expression of G2/M transition activators (including Cdc25 and cyclin B₁) in advanced prostate cancer. [1]

C15H16CLN3O3

321.758842468262

321.088

C, 55.99; H, 5.01; Cl, 11.02; N, 13.06; O, 14.92

383907-47-9

379078

Brown to red solid powder

1.4±0.1 g/cm3

478.8±45.0 °C at 760 mmHg

243.4±28.7 °C

0.0±1.2 mmHg at 25°C

1.626

0.33

1

6

4

22

488

0

ClC1C(C2C(=CC=CN=2)C(C=1NCCN1CCOCC1)=O)=O

LGTBDMBBPVNDSZ-UHFFFAOYSA-N

InChI=1S/C15H16ClN3O3/c16-11-13(18-4-5-19-6-8-22-9-7-19)14(20)10-2-1-3-17-12(10)15(11)21/h1-3,18H,4-9H2

7-chloro-6-(2-morpholin-4-ylethylamino)quinoline-5,8-dione

DA3003-2; NSC 663285; 383907-47-9; DA-3003-2; NSC-663285; 7-chloro-6-(2-morpholin-4-ylethylamino)quinoline-5,8-dione; DA 3003-2; NSC663285;

2934.99.9001

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.

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

DMSO: 150 mg/mL (466.19 mM)

Solubility in Formulation 1: ≥ 3.75 mg/mL (11.65 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 37.5 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.

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