- Cdc25B2 (in vitro IC50 = 206 ± 75 nM). [1]
- Cdc25A (Ki = 29 ± 7 nM). [1]
- Cdc25B (Ki = 95 ± 14 nM). [1]
- Cdc25C (Ki = 89 ± 18 nM). [1]
- VHR phosphatase (IC50 = 4.0 ± 0.1 µM; 20-fold selectivity over Cdc25B2). [1]
- PTP1B phosphatase (IC50 > 100 µM; 450-fold selectivity over Cdc25B2). [1]
- NSD2 (WT) (IC50 = 0.17 µM). [2]
- NSD2 (E1099K) (IC50 = 0.11 µM). [2]
- NSD2 (T1150A) (IC50 = 0.17 µM). [2]
- NSD1 (IC50 = 0.11 µM). [2]
- NSD3 (IC50 = 0.26 µM). [2]

The NCI 60-cell human tumor panel showed a mean IC50 of 1.5 ± 0.6 μM for NSC 663284 (3-100 μM; 48 hours), while human breast cancer MDA-MB-435 and MDA-N cells had an IC50 of 0.2 μM. 1.7 μM is the IC50 value in human breast MCF-7 cells that have been grown [1]. NSC 663284's relative IC50 value for Cdc25B2 (IC50=0.21 μM) is greater than that of PTP1B (IC50>4.0 μM) or VHR (IC50=4.0 μM) [1].

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- Enzymatic Inhibition: NSC-663284 inhibits Cdc25B2 with an IC50 of 206 ± 75 nM. It exhibits mixed competitive kinetics against Cdc25A, Cdc25B, and Cdc25C with Ki values of 29, 95, and 89 nM, respectively. It shows selectivity for Cdc25B2 over VHR (20-fold, IC50 = 4.0 µM) and PTP1B (450-fold, IC50 > 100 µM). [1]
- Methyltransferase Inhibition: NSC-663284 (referenced as DA3003-1) inhibits full-length WT NSD2 with an IC50 of 0.17 µM, NSD2 E1099K with an IC50 of 0.11 µM, and NSD2 T1150A with an IC50 of 0.17 µM using the HotSpot radiolabel assay. It also inhibits NSD1 (IC50 = 0.11 µM) and NSD3 (IC50 = 0.26 µM). A methyltransferase profiling panel showed it inhibits 27 out of 38 methyltransferases tested with submicromolar potencies. [2]
- Cell Proliferation Inhibition: NSC-663284 has a mean IC50 of 1.5 ± 0.6 µM in the NCI 60 Human Tumor Cell Panel after 48 hours of treatment. The most sensitive cells are human breast cancer MDA-MB-435 and MDA-N cells (IC50 = 0.2 µM). For human breast MCF-7 cells, the IC50 for growth inhibition after 48 hours is 1.7 µM. After only a 3-hour exposure, the IC50 for MCF-7 cells is approximately 35 µM. The regioisomer DA3003-2 is 3-fold less cytotoxic after a 3-hour exposure. [1]
- Inhibition of Cellular Cdc25A: In a chemical complementation assay using HeLa cells, treatment with 10 µM NSC-663284 for 1 hour reversed the depression of phospho-Erk (phosphorylated ERK1/2) levels caused by ectopic overexpression of Cdc25A, returning it to constitutive levels. The compound had no significant effect on phospho-Erk levels in mock-transfected cells. [1]
- GSH Depletion in Vitro: DA3003-1 (5 µM) incubated with mouse liver cytosol or with purified glutathione S-transferase (GST) and glutathione (GSH) resulted in rapid metabolism, with loss of the parent compound and formation of a dechlorinated glutathione conjugate. [3]

The growth of human floating HT29 xenografts obtained via SCID intravenous subcutaneous injection is inhibited by NSC 663284 (iv; 2, 3, and 5 mg/kg). After a single dosage of 5 mg/kg, NSC 663284 was not identifiable in tissues or veins for more than five minutes. Glutathione levels in HT29 tumors decreased more and persisted longer following NSC 663284 treatment in tumor-bearing SCID mice than in kidney and liver cancer [3].

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- Antitumor Efficacy in HT29 Xenografts: In C.B.-17 SCID mice bearing subcutaneous human colon HT29 xenografts, DA3003-1 administered intravenously every 4 days for 6 doses at 5 mg/kg resulted in significant tumor growth inhibition. The maximum mean % T/C (treated tumor volume/control tumor volume) was 50% on day 25. The mean time for tumors to double was significantly prolonged to 29.1 ± 3.9 days compared to 16.4 ± 3.4 days for vehicle control (p ≤ 0.05). The efficacy was slightly less than that of gemcitabine (50 mg/kg). Two mice in the 5 mg/kg group were moribund and euthanized on days 25 and 28. Doses of 2 and 3 mg/kg were less effective. No significant tumor growth inhibition was observed against MDA-MB-435 breast cancer xenografts. [3]
- GSH Depletion in Tissues: Following i.v. administration of 5 mg/kg DA3003-1 to HT29 tumor-bearing mice, reduced glutathione (GSH) concentrations decreased in liver, kidney, and HT29 tumor tissue. The decrease was most sustained in the tumor tissue, where GSH remained lower even 4 hours after treatment, while hepatic and renal levels had returned to normal. No decrease in GSH was detected in red blood cells. [3]

- In Vitro Cdc25 Phosphatase Assay: The activities of GST-fusion Cdc25A, Cdc25B2, Cdc25C, VHR, and human recombinant PTP1B were measured using o-methyl fluorescein phosphate as a substrate in a miniaturized 96-well microtiter plate assay. Fluorescence emission from the product (o-methyl fluorescein) was measured after a 60-minute incubation at ambient temperature using a multiwell plate reader with excitation at 485/20 nm and emission at 530/30 nm. [1]
- HotSpot Methyltransferase Assay (Radioisotope): The assay was performed using standard substrate concentrations (5 µM peptide/protein or 0.05 mg/ml for nucleosomes/core histones) and 1 µM SAM. Test compounds were diluted in DMSO and added to enzyme/substrate mixtures via acoustic technology with a 20-minute preincubation. The reaction was initiated with [³H] SAM and incubated at 30°C for 1 hour. The reaction was detected by a filter-binding method. Data analysis was performed using graphing software. [2]
- Methyltransferase-Glo (MTase-Glo) Assay: The assay was performed in a multi-step format in 1,536-well plates. 23 nl of compound (or DMSO) were pin-transferred into 3 µl of reaction buffer containing 666.7 nM nucleosomes and 10.7 nM full-length WT NSD2 enzyme (or no enzyme for low control). After a 30-minute incubation, 1 µl of 4 µM SAM was added to initiate the 15-minute reaction. Then, 1 µl of MTase-Glo reagent was added to convert SAH to ADP. After 30 minutes, 5 µl of MTase-Glo Detection Solution was added to convert ADP to ATP, and luminescence was measured after a 30-minute incubation. [2]
- EPIgenous HTRF Methyltransferase Assay: The assay was performed in white 1,536-well plates. 23 nl of compound were pin-transferred into 3 µl of reaction buffer with nucleosomes and NSD2 enzyme, followed by a 30-minute incubation. 1 µl of 4 µM SAM was added to start the 15-minute reaction. Then, 0.8 µl of Detection Buffer One was added, followed by a 10-minute incubation. Anti-SAH/Lumi4-Tb solution was added, and finally the SAH-d2 conjugate was added. The plate was incubated for 1 hour before HTRF signal detection. [2]
- Amplex Red Redox Activity Assay: 23 nl of compound were pin-transferred into 2.5 µl of HBSS in black 1,536-well plates. Compound fluorescence was measured immediately (READ 0). 2.5 µl of 2X Amplex Red solution (containing Amplex Red, DTT, and horseradish peroxidase) was added. Fluorescence was measured again after a 15-minute incubation (READ 1). Activity was calculated using corrected fluorescence values (READ 1 minus READ 0). [2]
- Surface Plasmon Resonance: Human recombinant NSD2-SET domain was immobilized on a sensorchip using classic amine coupling. Single-cycle kinetic measurements were performed in running buffer (50 mM Tris, pH 8.8, 50 mM NaCl, 0.5 mM TCEP, 5 mM MgCl2, 0.05% P20, 2% DMSO). Compounds were diluted in a 3-fold series and injected over the chip at 80 µl/min for 80 seconds, followed by a dissociation period of 200-600 seconds. Data was analyzed using a 1:1 kinetic binding model. For DA3003-1, the binding kinetics showed a KD of 370 nM. [2]

- Antiproliferative Assay (MCF-7 cells): Human MCF-7 breast cancer cells were seeded in microtiter plates and allowed to attach overnight. Cells were then treated with vehicle or NSC-663284 either continuously or for 3 hours. After a 48-hour incubation, the medium was replaced with serum-free medium containing MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). Plates were incubated for another 3 hours, and total cell number was determined spectrophotometrically at 540 nm. The 48-hour IC50 was 1.7 µM, and the 3-hour exposure IC50 was approximately 35 µM. [1]
- Chemical Complementation Assay (HeLa cells): HeLa cells were transiently transfected with a plasmid encoding full-length wild-type Cdc25A or a mock control. Transfected cells were treated with 10 µM NSC-663284 or vehicle for 1 hour. Whole-cell lysates were then analyzed by Western blot for phospho-Erk (pERK), total ERK, and β-tubulin (loading control). Cdc25A overexpression caused a >50% decrease in Erk phosphorylation, and treatment with NSC-663284 reversed this depression, returning pERK to constitutive levels. [1]
- Cellular H3K36me2 Reduction and Cytotoxicity (U-2 OS cells): U-2 OS human osteosarcoma cells were treated with DA3003-1 in a 10-point, 3-fold dilution series (0.0025 - 50 µM) for 96 hours. Whole-cell lysates were subjected to Western blotting with anti-H3K36me2 and anti-total H3 antibodies. Densitometry showed a dose-dependent reduction in H3K36me2 with an IC50 of 545 nM. However, higher drug concentrations resulted in cytotoxicity with a CC50 of 270 nM. [2]

- Efficacy Study in HT29 Xenografts: C.B.-17 SCID mice bearing subcutaneous human colon HT29 tumor fragments (approx. 300 mm³) were stratified into treatment groups (n=9-10). DA3003-1 was administered intravenously at doses of 2, 3, or 5 mg/kg in sterile 5% dextrose (0.01 ml/g body weight). The dosing schedule was every 4 days for a total of 6 doses. Body weights and tumor volumes were recorded twice weekly. The positive control was gemcitabine at 50 mg/kg i.v. on the same schedule. [3]
- Efficacy Study in MDA-MB-435 Xenografts: C.B.-17 SCID mice bearing MDA-MB-435 human breast cancer xenografts were treated similarly, with paclitaxel at 20 mg/kg i.v. every 7 days as the positive control. No significant tumor growth inhibition was observed for DA3003-1. [3]
- Pharmacokinetic Study in Tumor-Bearing Mice: C.B.-17 SCID mice bearing HT29 xenografts were fasted overnight and treated with DA3003-1 at a single i.v. dose of 5 mg/kg. Groups of 2 mice were euthanized at 2, 5, 10, 15, 30, 45, 60, 120, 240, 420, and 1440 minutes post-dose. Blood (cardiac puncture), liver, kidneys, spleen, lungs, and tumor were collected, weighed, and snap-frozen for analysis. [3]
- Toxicity Study for MTD Determination: Female Balb/C and C.B.-17 SCID mice received single intravenous doses of 5, 7, 10, or 20 mg/kg DA3003-1 in sterile 5% dextrose (0.01 ml/g body weight). The maximum tolerated dose (MTD) was determined to be 5 mg/kg. Mice receiving 7 mg/kg lost >10% body weight, and those receiving 10 or 20 mg/kg were moribund by day 4. [3]

- Plasma and Tissue Distribution: After a single i.v. dose of 5 mg/kg in HT29 tumor-bearing mice, DA3003-1 was detected in plasma (0.44, 0.49 µg/ml), red blood cells (0.14, 0.18 µg/ml), liver (0.12, 0.14 µg/g), kidney (0.23, 0.25 µg/g), and lung (1.70, 1.72 µg/g) at 2 minutes post-dose. The parent compound was not detectable in the tumor at this time point. At 5 minutes post-dose, plasma concentration was halved (0.21 µg/ml), and it was no longer detectable in the liver or kidney. It was not detectable in plasma or any normal tissues by 15 minutes. [3]
- In Vitro Metabolism: DA3003-1 was rapidly metabolized by mouse liver subcellular fractions. Cytosolic metabolism was much faster than microsomal metabolism, with a slight preference for NADPH over NADH. Incubation with cytosol resulted in the loss of parent compound and the appearance of a more polar metabolite. LC/MS analysis identified the major metabolite as a dechlorinated glutathione conjugate (m/z 595, double-charged positive ion [m+2H] of 297.5). [3]
- Role of GST in Metabolism: Incubation of DA3003-1 with purified glutathione S-transferase (GST) and glutathione (GSH) resulted in almost quantitative conversion to the same glutathione conjugate within 2-5 minutes, confirming the role of GST in this metabolic pathway. No significant conjugation occurred without GST. [3]

- Maximum Tolerated Dose (MTD): In both Balb/c and C.B.-17 SCID mice, the maximum tolerated intravenous dose of DA3003-1 was 5 mg/kg when administered as a single dose. Mice receiving 7 mg/kg lost more than 10% of their body weight. Single doses of 10 or 20 mg/kg were lethal, with mice becoming moribund by day 4 post-injection. [3]
- Treatment-Related Toxicity in Efficacy Study: In the HT29 xenograft efficacy study, two mice treated with 5 mg/kg DA3003-1 (every 4 days for 6 doses) were moribund and euthanized on days 25 and 28 of the study. No significant decrease in body weight was observed with any of the treatments (2, 3, or 5 mg/kg). [3]

[1]. Discovery and biological evaluation of a new family of potent inhibitors of the dual specificity protein phosphatase Cdc25. J Med Chem. 2001 Nov 22;44(24):4042-9.

[2]. High-throughput screening with nucleosome substrate identifies small-molecule inhibitors of the human histone lysine methyltransferase NSD2. J Biol Chem. 2018 Aug 31;293(35):13750-13765.

[3]. Pharmacology and antitumor activity of a quinolinedione Cdc25 phosphatase inhibitor DA3003-1 (NSC 663284). Anticancer Res. 2007 Sep-Oct;27(5A):3067-73.

6-Chloro-7-[2-(4-morpholino)ethylamino]quinoline-5,8-dione is a quinolone compound. NSC-663284 is a potent quinoline dione Cdc25 phosphatase inhibitor.
- Chemical Name and Synonyms: NSC-663284 is also known as DA3003-1, and its chemical name is 6-chloro-7-(2-morpholin-4-ylethylamino)quinoline-5,8-dione. Its regioisomer is 7-chloro-6-(2-morpholin-4-ylethylamino)quinoline-5,8-dione (DA3003-2). [1]
- Proposed Mechanism of Action: NSC-663284 inhibits Cdc25 phosphatases, leading to cell cycle arrest at G1 and G2/M phases by preventing the dephosphorylation and activation of cyclin-dependent kinases like Cdk1/Cdc2. It can also generate cellular reactive oxygen species and participate in Michael adduction in the catalytic domain of Cdc25A. [1][3]
- Pharmacophore and Structure-Activity Relationship: The quinoline-5,8-dione pharmacophore is critical for Cdc25 inhibitory activity. The presence of a 7-substituted amino group (such as 2-morpholin-4-ylethylamino) significantly enhances potency and selectivity. The regioisomer with the chlorine at the 7-position (DA3003-2) is 3-fold less active against Cdc25B2 in vitro and less potent as a growth inhibitor. Computational electrostatic potential mapping suggests the need for an electron-deficient 7-position for maximal inhibitor activity. [1]

C15H16CLN3O3

321.758842468262

321.088

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

383907-43-5

379077

Pink 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(C2=CC=CN=C2C(C=1NCCN1CCOCC1)=O)=O

BMKPVDQDJQWBPD-UHFFFAOYSA-N

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

6-Chloro-7-(2-morpholin-4-yl-ethylamino)quinoline-5,8-dione

SPS8I1; DA30031; NSC-663284; NSC-663,284; DA3003-1; CY8W2F69MW; 6-Chloro-7-((2-morpholinoethyl)amino)quinoline-5,8-dione; SPS-8I1; DA3003 1;NSC 663284; SPS 8I1; DA-3003-1; NSC663284;

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)

DMSO : ≥ 100 mg/mL (~310.79 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (7.77 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.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (7.77 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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (7.77 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.

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