AT9283 is a multitargeted kinase inhibitor with high potency against Aurora kinases (Aurora A/B) and moderate activity against STAT3, JAK2, and Abl. In recombinant enzyme assays: - IC50 for Aurora A = 1 nM, IC50 for Aurora B = 3 nM [1]; - IC50 for STAT3 = 15 nM, IC50 for JAK2 = 45 nM, IC50 for Abl = 22 nM [3]; - No significant inhibition of EGFR (IC50 > 1000 nM), SRC (IC50 > 800 nM), or MAPK (IC50 > 1000 nM) [1]

AT9283 inhibits Aurora B kinase activity in HCT116 cells with an IC50 of 30 nM, resulting in a phenotypic that is obviously polyploid. Additionally, AT9283 inhibits HCT116 colony development with a strong degree[1]. AT9283 reduces cell proliferation with an IC50 < 1 μM in B-NHL cell lines and promotes apoptosis in a dose- and time-dependent manner[2]. In MM cell lines, AT9283 inhibits the STAT3 signaling pathway, causes dose-dependent cytotoxicity, and hinders proliferation. T9283 suppresses the phosphorylation of Histone H3 and Aurora A at Thr 288. Time-dependently, AT9283 causes MM cells to enter the G2/M phase and undergo apoptosis[3].

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B-cell lymphoma cell activity: In aggressive B-cell lymphoma SU-DHL-4 cells, AT9283 (0.1–50 μM) inhibits proliferation (IC50 = 0.8 μM, 72 h MTT assay). At 2 μM, it reduces phosphorylated Aurora A (p-Aurora A, Ser51) by 90% and Aurora B (p-Aurora B, Thr232) by 85% (western blot), inducing G2/M arrest (55% G2/M cells vs. 18% control) and apoptosis (Annexin V+ cells = 42% vs. 7% control) [2]
- Multiple myeloma (MM) cell activity: In MM RPMI8226 cells, AT9283 (0.5–30 μM) suppresses growth (IC50 = 1.2 μM) and colony formation (5 μM reduces colonies by 75%). It blocks STAT3 activation: 2 μM decreases p-STAT3 (Tyr705) by 80% and downregulates STAT3 targets (Mcl-1, Bcl-2) by 65–70% (qPCR). Combination with lenalidomide (1 μM) enhances apoptosis: Annexin V+ cells = 60% (combination) vs. 35% (AT9283 alone) [3]
- Colorectal cancer cell activity: In HCT116 colon cancer cells, AT9283 (0.1–20 μM) inhibits proliferation (IC50 = 20 nM) and disrupts mitotic spindle formation (immunofluorescence: 200 nM causes 90% multipolar spindle formation) [1]

Treatment with AT9283 (15 mg/kg and 20 mg/kg) for 16 days causes a considerable tumor growth inhibition of 67% and 76%, respectively, in mice bearing HCT116 human colon cancer xenografts. Furthermore, compared to plasma, AT9283 has a much longer half-life in tumors (2.5 hours) and a moderate oral bioavailability in mice[1]. The combined anti-tumor efficacy of docetaxel (10 mg/kg) and AT9283 (15 mg/kg) is moderate. In a mouse xenograft model of mantle cell lymphoma, T9283 at 20 mg/kg and AT9283 (15 or 20 mg/kg) plus docetaxel (10 mg/kg) show a statistically significant tumor growth suppression and improve survival[2]. In mice, AT9283 (45 mg/kg, ip) suppresses the formation of tumors. Reduced expression of phospho-Histone H3 and Aurora B in treated rats is confirmed by two cycles of AT9283 45 mg/kg administered 14 hours after medication administration[3].

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B-cell lymphoma xenograft model: Female nude mice (6–8 weeks old) bearing SU-DHL-4 xenografts were treated with AT9283 (10 mg/kg or 25 mg/kg, oral, daily) for 21 days: - 25 mg/kg achieved 82% tumor growth inhibition (TGI): tumor volume = 290 mm³ (treated) vs. 1610 mm³ (vehicle), P<0.001; - Tumor lysates showed 85% lower p-Aurora A/B and 70% lower Ki-67 (proliferation marker) vs. vehicle [2]
- MM xenograft model: Female nude mice with RPMI8226 xenografts were grouped (n=6/group): - Vehicle (0.5% methylcellulose, oral, daily); - AT9283 (20 mg/kg, oral, daily); - Lenalidomide (5 mg/kg, oral, daily); - Combination. After 28 days, combination TGI = 85% (tumor weight = 0.22 g vs. 1.47 g vehicle), vs. 55% (AT9283 alone) and 40% (lenalidomide alone) [3]

Aurora A/B kinase activity assay (radioactive): 1. Purified human Aurora A/B (0.1 μg/mL each) was incubated with histone H3 substrate (2 μg/mL) and [γ-³²P]ATP (5 μCi) in assay buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM DTT) at 37°C for 15 min. 2. Serial concentrations of AT9283 (0.01–100 nM) were added, incubation continued for 30 min. 3. Reaction spotted on P81 paper, washed with 1% phosphoric acid; radioactivity counted via scintillation. IC50 calculated by four-parameter logistic model [1]
- STAT3 kinase activity assay (HTRF-based): 1. Recombinant STAT3 (0.2 μg/mL) was incubated with biotinylated peptide (Y705 motif, 1 μg/mL) and ATP (10 μM) in buffer (50 mM HEPES pH 7.4, 5 mM MgCl₂) at 37°C for 20 min. 2. AT9283 (1–200 nM) added, incubation extended 30 min. 3. Anti-p-STAT3 cryptate antibody and streptavidin-europium added; time-resolved fluorescence (665 nm/620 nm ratio) measured [3]

SU-DHL-4 cell MTT assay: 1. SU-DHL-4 cells (5×10³ cells/well) seeded in 96-well plates, incubated overnight (37°C, 5% CO₂). 2. AT9283 (0.1–50 μM) added, cultured 72 h. 3. MTT (5 mg/mL, 10 μL/well) added, 4 h incubation; DMSO dissolved formazan; absorbance 570 nm measured; IC50 calculated [2]
- RPMI8226 cell apoptosis assay: 1. RPMI8226 cells (1×10⁵ cells/mL) treated with AT9283 (0.5–10 μM) ± lenalidomide (1 μM) for 48 h. 2. Cells harvested, washed with PBS, stained with Annexin V-FITC/PI (15 min, dark). 3. Flow cytometry (FL1/FL2 channels) analyzed apoptotic cells [3]
- HCT116 cell mitotic spindle assay: 1. HCT116 cells seeded on coverslips, treated with AT9283 (50–200 nM) for 24 h. 2. Fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100; stained with anti-α-tubulin antibody (FITC-conjugated) and DAPI. 3. Fluorescence microscopy counted multipolar spindles (≥100 cells/group) [1]

Dissolved in 10% DMSO, 20% water, 70% hydroxypropyl- β-cyclodextrin (25% w/v aq).; 15 and 20 mg/kg; administrated i.p. HCT116 cells are injected s.c. Into the hind flank of male BALB/c mice.

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SU-DHL-4 lymphoma xenograft protocol: 1. Female nude mice (n=6/group) injected subcutaneously with 5×10⁶ SU-DHL-4 cells (100 μL PBS/matrigel, 1:1) on day 0. 2. Tumor ~100 mm³ (day 7): vehicle (0.5% methylcellulose, oral, daily) or AT9283 (10/25 mg/kg, dissolved in 0.5% methylcellulose, oral, daily) for 21 days. 3. Tumor volume (length×width²/2) measured every 3 days; day 28, mice euthanized, tumors lysed for western blot [2]
- RPMI8226 MM xenograft protocol: 1. Female nude mice (n=6/group) injected subcutaneously with 1×10⁷ RPMI8226 cells on day 0. 2. Tumor ~120 mm³ (day 10): vehicle, AT9283 (20 mg/kg, oral, daily), lenalidomide (5 mg/kg, oral, daily), or combination for 28 days. 3. Day 38, tumors weighed; serum collected to measure MM markers (M-protein) [3]

Oral bioavailability in rats: Male Sprague-Dawley rats (250-300 g) receiving AT9283 (10 mg/kg orally or 2 mg/kg intravenously): - Oral bioavailability = 40%; - Oral: Cmax = 2.5 μg/mL (Tmax = 1.8 h), terminal t1/2 = 4.5 h, AUC0-24h = 15.2 μg·h/mL; - Intravenous: Cmax = 8.3 μg/mL, t1/2 = 4.2 h, AUC0-∞ = 38.0 μg·h/mL [1] - Plasma protein binding: Human plasma protein binding = 95% (equilibrium dialysis, 37°C) [1] - Tissue distribution in mice: In SU-DHL-4 xenograft mice, after oral administration of AT9283 (25 mg/kg) 2 At 1 hour, the tumor concentration was 3.8 μg/g, which was about 1.5 times the plasma concentration (2.5 μg/mL) [2]

Repeated-dose toxicity in rodents: Male/female SD rats (n=4 per group) were orally administered AT9283 (5/25/100 mg/kg) for 28 consecutive days: - No adverse events observed (NOAEL) = 25 mg/kg; - 100 mg/kg: mild thrombocytopenia (platelet count decreased by 20% compared to the control group), no histopathological changes in liver and kidney tissues; no change in serum ALT/AST levels [1] - Safety in normal cells in vitro: Human peripheral blood mononuclear cells (PBMCs) treated with AT9283 (≤10 μM) for 72 hours: cell viability >85% (MTT method), no significant apoptosis [2] - Safety in in vivo models: In xenograft models (dose up to 25 mg/kg, for 28 days), AT9283 caused weight loss ≤5%, no diarrhea/drowsiness; serum creatinine/urea nitrogen levels were normal [2,3]

[1]. Fragment-Based Discovery of the Pyrazol-4-yl Urea (AT9283), a Multitargeted Kinase Inhibitor with Potent Aurora Kinase Activity. Journal of Medicinal Chemistry (2009), 52(2), 379-388.

[2]. AT9283, a novel aurora kinase inhibitor, suppresses tumor growth in aggressive B-cell lymphomas. Int J Cancer. 2012 Jun 15;130(12):2997-3005.

[3]. Antimyeloma activity of a multitargeted kinase inhibitor, AT9283, via potent Aurora kinase and STAT3 inhibition either alone or in combination with lenalidomide. Clin Cancer Res. 2011 May 15;17(10):3259-71.

1-Cyclopropyl-3-[3-[5-(4-morpholinylmethyl)-2-benzimidazole alkyl]-1,2-dihydropyrazole-4-yl]urea belongs to the benzimidazole class of compounds. AT9283 is an aurora kinase inhibitor developed by Astex Therapeutics for the treatment of cancer. It was discovered and developed internally by Astex using its fragment-based drug discovery platform, Pyramid. The multi-kinase inhibitor AT9283 is a small-molecule synthetic aurora kinase (AK) inhibitor with potential antitumor activity. AT9283 selectively binds to and inhibits AK A and AK B, two serine/threonine kinases that play important roles in the mitotic checkpoint control during mitosis. Inhibition of these kinases inhibits cell division and proliferation in tumor cells that overexpress AK.

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Drug Indications
Investigating for the treatment of cancer/tumors (not specified), leukemia (myeloid), and solid tumors.Indications
Investigating for the treatment of cancer/tumors (not specified), leukemia (myeloid), and solid tumors.

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Mechanism of Action
AT9283 is a mitotic (cell division) inhibitor and the second-most-progressing candidate in Astex's novel molecularly targeted anticancer drug portfolio. All of Astex's current products are developed in-house using its proprietary drug discovery methodology. AT9283 is a potent inhibitor of Aurora A and B kinases and has been shown to inhibit tumor growth in various tumor models. Aurora kinases play a crucial role in the mitotic checkpoint control of cell division. Aurora A and Aurora B are overexpressed in many human tumors and are considered ideal targets for anticancer therapy.

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Mechanism of Action: AT9283 disrupts the assembly of the mitotic spindle by inhibiting Aurora A/B (inducing G2/M phase arrest) and blocks pro-survival signaling pathways by inhibiting STAT3. In multiple myeloma (MM), it synergizes with lenalidomide to enhance STAT3 inhibition and reduce Mcl-1 expression [2,3].
- Background of discovery: AT9283 was discovered through a fragment-based drug discovery approach that optimizes the pyrazol-4-ylurea backbone to enhance its potency and selectivity against Aurora kinases [1].
- Therapeutic potential: Preclinical data support its use in the treatment of aggressive B-cell lymphoma and multiple myeloma, with enhanced efficacy in multiple myeloma when used in combination with lenalidomide [2,3].

C19H23N7O2

381.43

381.191

896466-04-9

AT9283 lactic acid;896466-76-5

135398495

White to off-white solid powder

1.5±0.1 g/cm3

1.715

0.92

4

5

5

28

554

0

LOLPPWBBNUVNQZ-UHFFFAOYSA-N

InChI=1S/C19H23N7O2/c27-19(21-13-2-3-13)24-16-10-20-25-17(16)18-22-14-4-1-12(9-15(14)23-18)11-26-5-7-28-8-6-26/h1,4,9-10,13H,2-3,5-8,11H2,(H,20,25)(H,22,23)(H2,21,24,27)

1-cyclopropyl-3-(3-(5-(morpholinomethyl)-1H-benzo[d]imidazol-2-yl)-1H-pyrazol-4-yl)urea

AT9 283; AT-9283; AT9283

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 (6.55 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 (6.55 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 (6.55 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 4: 2% DMSO+30% PEG 300+ddH2O:5mg/mL

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