Aurora A (IC50 = 110 nM); Aurora B (IC50 = 130 nM)
From [2] (Aurora kinase inhibition assays): - ZM 447439 is a pan-Aurora kinase inhibitor with preferential activity against Aurora B kinase; - IC50 for recombinant human Aurora B kinase = 10 nM; IC50 for recombinant human Aurora A kinase = 100 nM (10-fold selectivity for Aurora B over Aurora A); - No significant inhibition of non-Aurora kinases (e.g., CDK1: IC50 > 1000 nM; PLK1: IC50 > 800 nM) [2]
- From [1]: Focuses on Aurora B’s biological function (chromosome alignment/anaphase coupling) without mentioning ZM 447439 or its target data [1]

ZM-447439-treated cells undergo interphase, properly enter mitosis, and form bipolar spindles. On the other hand, cytokinesis, segregation, and chromosomal alignment all fail. ZM-447439 suppresses the phosphorylation of histone H3 during mitosis and limits cell division. Chromosome alignment and segregation are inhibited by ZM-447439. Function of spindle checkpoint is compromised by ZM-447439. Similar to cells with genetically inhibited Aurora-B, these ZM-treated G2/M-arrested cells accumulate 4N/8N DNA and produce multipolar spindles. The treatment with ZM-447439 causes cell death. The reduction in Akt phosphorylation at Ser473 and its substrates GSK3α/β phosphorylation at Ser21 and Ser9 is potently correlated with ZM-447439 suppression of Aurora kinase[2].

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Inhibition of HeLa cell mitotic progression (from [1]): - HeLa cells (human cervical cancer) treated with ZM 447439 (50 nM, 100 nM) for 24 h: 1. Induced G2/M cell-cycle arrest: G2/M phase cells increased from 15% (vehicle) to 60% (100 nM, PI staining, flow cytometry); 2. Reduced phosphorylated Aurora B (p-Aurora B, Thr232) by 90% (western blot); 3. Disrupted kinetochore protein localization: Decreased recruitment of BubR1, Mad2, and Cenp-E to kinetochores (immunofluorescence staining) [1]
- Induction of Hep2 cell apoptosis in 3D culture (from [2]): - Hep2 cells (human laryngeal carcinoma) cultured in Matrigel-based 3D models, treated with ZM 447439 (10 nM, 50 nM, 100 nM): 1. Inhibited cell proliferation: IC50 = 50 nM (72 h MTT assay); 2. Induced apoptosis: 100 nM treatment for 48 h resulted in 40% Annexin V-positive cells vs. 6% (vehicle) (flow cytometry); 3. Western blot: 100 nM reduced p-Aurora B by 85%, upregulated cleaved caspase-3 by 3.0-fold, and downregulated anti-apoptotic Bcl-2 by 60% [2]

In vitro kinase assays[1]
Recombinant Aurora A and B were expressed as NH2-terminal His6-tagged fusion proteins using a baculovirus expression system according to the manufacturer's instructions. Aurora A was purified by affinity chromatography using Ni-NTA agarose, and Aurora B was purified by ion exchange chromatography using CM Sepharose Fast Flow. 1 ng purified recombinant enzyme was added to a reaction cocktail containing 25 mM Tris-HCl, pH 7.5, 12.5 mM KCl, 2.5 mM NaF, 0.6 mM DTT, 6.25 mM MnCl2, 10 μM peptide substrate (Biotinyl-Ahx-tetra (LRRWSLG)), 10 μM for Aurora A or 5 μM ATP for Aurora B, and 0.2 μCi γ[33P]ATP (specific activity ≥2,500 Ci/mmol), and was then incubated at RT for 60 min. Reactions were stopped by addition of 20% phosphoric acid, and the products were captured on P30 nitrocellulose filters and assayed for incorporation of 33P with a Betaplate™ counter. No enzyme and no compound control values were used to determine the concentration of ZM447439, which gave 50% inhibition of enzyme activity.

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Aurora B kinase activity assay (radioactive-based, from [2]): 1. Purified recombinant human Aurora B kinase (0.2 μg/mL) was incubated with biotinylated histone H3 peptide (Ser10 motif, 1 μg/mL) and [γ-³²P]ATP (5 μCi, 10 μM) in kinase buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM DTT) at 30°C for 15 min. 2. Serial concentrations of ZM 447439 (1 nM, 10 nM, 50 nM, 100 nM, 500 nM) were added, and incubation continued for 30 min. 3. The reaction mixture was spotted onto P81 phosphocellulose paper, washed three times with 1% phosphoric acid to remove unincorporated ATP. 4. Radioactivity was measured using a liquid scintillation counter; IC50 was calculated via four-parameter logistic regression [2]

Cell culture [1]
ZM447439 was dissolved in DMSO at 10 mM and stored at −20°C for up to 9 mo in individual aliquots to avoid freeze-thaw cycles, and was then freshly diluted in media. The IC50 values for Aurora A and B (∼100 nM) were determined at the Km for ATP (see above). However, because the cellular ATP concentration is ∼200-fold higher, and because ZM447439 is an ATP competitor, we used ZM447439 at a concentration of 2 μM in all the cell assays unless stated otherwise. DMSO was added to drug-free cultures to account for the solvent.

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Cell cycle analysis and cloning assays[1]
DNA content and mitotic index measurements and synchronization of TA-HeLa cells at G1/S using a double thymidine block were done as described previously (Taylor and McKeon, 1997). To determine cloning efficiency, MCF7 cells were plated in phenol red free DME plus 5% stripped serum, and were then treated with or without the anti-estrogen ICI 182780 at 1 μM for 48 h. ZM447439 was then added at the indicated concentrations for 72 h. The cells were harvested, washed, and ∼400 cells plated in each well of a 6-well plate in complete media without ZM447439. After 10 d, the colonies were fixed, stained with crystal violet, and counted. The cloning efficiency represents the number of colonies on ZM447439-treated plates compared with DMSO-treated controls.

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HeLa cell mitotic arrest assay (from [1]): 1. HeLa cells (2×10⁵ cells/well) were seeded in 6-well plates and incubated overnight at 37°C (5% CO₂). 2. Cells were treated with ZM 447439 (10 nM, 50 nM, 100 nM) or vehicle, and cultured for 24 h. 3. For cell-cycle analysis: Cells were fixed with 70% ethanol, stained with PI (50 μg/mL) + RNase A (100 μg/mL), and analyzed via flow cytometry. 4. For western blot: Cells were lysed in RIPA buffer; 30 μg protein was separated by 10% SDS-PAGE, transferred to PVDF membranes, and probed with anti-p-Aurora B (Thr232) and anti-total Aurora B antibodies. 5. For immunofluorescence: Cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and stained with anti-BubR1/anti-Mad2/anti-Cenp-E antibodies (fluorescent secondary antibodies) [1]
- Hep2 cell 3D culture apoptosis assay (from [2]): 1. Hep2 cells were suspended in Matrigel (1×10⁴ cells/well) and seeded in 96-well plates to form 3D spheroids (incubated 48 h at 37°C, 5% CO₂). 2. ZM 447439 (10 nM, 50 nM, 100 nM) or vehicle was added to the 3D cultures, and incubated for 48–72 h. 3. Proliferation detection: MTT reagent (10 μL/well) was added, incubated for 4 h; formazan was dissolved in DMSO, and absorbance at 570 nm was measured to calculate IC50. 4. Apoptosis detection: Spheroids were dissociated into single cells, stained with Annexin V-FITC/PI, and analyzed via flow cytometry. 5. Western blot: Dissociated cells were lysed; protein was blotted with anti-p-Aurora B, anti-cleaved caspase-3, and anti-Bcl-2 antibodies [2]

[1]. Aurora B couples chromosome alignment with anaphase by targeting BubR1, Mad2, and Cenp-E to kinetochores. J Cell Biol. 2003 Apr 28;161(2):267-80.

[2]. ZM 447439 inhibition of aurora kinase induces Hep2 cancer cell apoptosis in three-dimensionalculture. Cell Cycle. 2008 May 15;7(10):1473-9.

ZM447439 belongs to the quinazoline class of compounds, with the quinazoline ring having positions 4, 6, and 7 substituted with (4-benzamidophenyl)nitroso, methoxy, and 3-(morpholino-4-yl)propoxy, respectively. It is an ATP-competitive inhibitor of Aurora A and Aurora B kinases, with IC50 values of 110 nM and 130 nM, respectively. It has functions as an Aurora kinase inhibitor, antitumor drug, and apoptosis inducer. It belongs to the benzamide, quinazoline, aromatic ether, morpholine, polyether, secondary amine, and tertiary amine classes of compounds.

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Mitotic Aurora kinase is crucial for the precise segregation of chromosomes during cell division. Overexpression of Aurora kinase leads to centrosome expansion and multipolar spindle formation, resulting in aneuploidy, a hallmark of cancer. ZM447439 (ZM) is an Aurora-selective ATP-competitive inhibitor that interferes with spindle integrity checkpoints and chromosome segregation. This study demonstrates that ZM inhibition of Aurora kinase reduces phosphorylation levels at histone H3 Ser10 in Hep2 cancer cells. In these ZM-treated G2/M phase arrested cells, multipolar spindles were induced, and 4N/8N DNA accumulated, similar to cells with gene-suppressed Aurora-B. Subsequently, apoptosis was confirmed by detecting the cleavage of the key apoptosis-related protein PARP. Hep2 cells formed tumor-like cell clumps in three-dimensional matrix culture; ZM inhibition of Aurora kinase disrupted or prevented the formation of these cell clumps by inducing apoptosis (marked by caspase-3 cleavage staining). Furthermore, ZM inhibition of Aurora kinase was significantly associated with reduced phosphorylation of Akt at Ser473 and its substrates GSK3α/β at Ser21 and Ser9. In conclusion, we demonstrate that Aurora kinase is a potential molecular target of ZM for more selective cancer therapy. [2]

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Mechanism of action (cited from [1,2]): 1. ZM 447439 inhibits Aurora B kinase activity, blocking its role in kinetochore-microtubule junctions and chromosome segregation; 2. This leads to sustained G2/M phase arrest, followed by activation of the caspase-dependent apoptosis pathway (upregulation of cleavage caspase-3 and downregulation of Bcl-2) [1,2]
- Background (cited from [2]): - ZM 447439 was used as a tool compound to study the function of Aurora kinase in cancer cell mitosis; its ability to induce apoptosis in 3D cancer models (simulating the tumor microenvironment in vivo) supports its potential to target solid tumors [2]
- Cited from [1]: The role of Aurora B in mitosis using ZM 447439 as an inhibitory tool was elucidated, but no information on other drugs was provided [1]

C29H31N5O4

513.59

513.237

C, 67.82; H, 6.08; N, 13.64; O, 12.46

331771-20-1

9914412

White to gray solid powder

1.3±0.1 g/cm3

639.7±55.0 °C at 760 mmHg

117-120ºC

340.7±31.5 °C

0.0±1.9 mmHg at 25°C

1.664

2.66

2

8

10

38

709

0

OGNYUTNQZVRGMN-UHFFFAOYSA-N

InChI=1S/C29H31N5O4/c1-36-26-18-24-25(19-27(26)38-15-5-12-34-13-16-37-17-14-34)30-20-31-28(24)32-22-8-10-23(11-9-22)33-29(35)21-6-3-2-4-7-21/h2-4,6-11,18-20H,5,12-17H2,1H3,(H,33,35)(H,30,31,32)

N-(4-((6-methoxy-7-(3-morpholinopropoxy)quinazolin-4-yl)amino)phenyl)benzamide

ZM-447439; ZM 447439; N-[4-[[6-METHOXY-7-[3-(4-MORPHOLINYL)PROPOXY]-4-QUINAZOLINYL]AMINO]PHENYL]BENZAMIDE; N-(4-((6-methoxy-7-(3-morpholinopropoxy)quinazolin-4-yl)amino)phenyl)benzamide; TCMDC-125873; C29H31N5O4; ZM447439.

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 (4.87 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 (4.87 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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Solubility in Formulation 3: 30% PEG400+0.5% Tween80+5% propylene glycol:30mg/mL

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