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Purity: ≥98%
AT-9283 L-lactate is a novel, potent multikinase inhibitor with potential antineoplastic activity and has the potential for the treatment of multiple myeloma. It inhibits JAK2/3 with IC50 of 1.2 nM/1.1 nM in cell-free assays. AT9283 binds to and inhibits Aurora kinases A and B, JAK2 and the kinase BCR-ABL, which may result in the inhibition of cellular division and proliferation and the induction of apoptosis in tumor cells that overexpress these kinases.
| Targets |
In vitro activity: AT9283 leads to a clear polyploid phenotype by inhibiting the activity of Aurora B kinase in HCT116 cells with IC50 of 30 nM. Furthermore, AT9283 also produces the potent inhibition on HCT116 colony formation.
Kinase Assay: Assays for Aurora A and B are performed in a DELFIA format. Aurora A enzyme is incubated with AT9283 and 3 μM cross-tide substrate (biotin-CGPKGPGRRGRRRTSSFAEG) in 10 mM MOPS, pH 7, 0.1 mg/mL BSA, 0.001% Brij-35, 0.5% glycerol, 0.2 mM EDTA, 10 mM MgCl2, 0.01% β-mercaptoethanol, 15 μM ATP, and 2.5% DMSO. Aurora B enzyme is incubated with AT9283, 3 μM of the above substrate in 25 mM Tris, pH 8.5, 5 mM MgCl2, 0.1 mg/mL BSA, 0.025% Tween-20, 1 mM DTT, 15 μM ATP, and 2.5% DMSO. Reactions are allowed to proceed for 60 minutes and 45-90 minutes for Aurora A and Aurora B, respectively, before quenching with EDTA. The reaction mixtures are then transferred to a neutravidin-coated plate, and phosphorylated peptide is quantified by means of a phospho-specific antibody and a europium labeled secondary antibody using time-resolved fluorescence (excitation, 337 nm; emission, 620 nm). IC50 values for the control compounds are 92 nM (Aurora A assay) and 17 nM (Aurora B). Cell Assay: HCT 116 cells are cultured in DMEM + 10% FBS + GLUTAMAX I. Black 96-well flat-bottomed (clear) tissue culture treated plates are seeded in 200 μL of medium and incubated for approximately 16 hours at 37°C in a humidified atmosphere of 5% CO2 in air. Cells are treated with test compound at nine different concentrations (spanning 1 nM to 10 μM, plus DMSO vehicle control) and then incubated for 72 hours. Polyploidy morphological observations of the cells are then noted. The concentration of AT9283 required to produce a distinct polyploid phenotype is reported. Cells are seeded at a concentration of 75−100 cells/mL relevant culture media onto 6- or 24-well tissue culture plates and allowed to recover for 16 hours. Test compound (11 concentrations spanning 0.1 nM to 10 μM) or vehicle control (DMSO) is added to duplicate wells to give a final DMSO concentration of 0.1%. Following compound addition, colonies are allowed to grow between 10 and 14 days for optimum discrete colony counting. Colonies are fixed in 2 mL of Carnoys fixative (25% acetic acid, 75% MeOH) and stained in 2 mL of 0.4% w/v crystal violet. The numbers of colonies in each well is counted. IC50 values are calculated by sigmoidal dose-response (variable slope) IC50 curves using Prism Graphpad software. |
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| ln Vitro |
In vitro activity: AT9283 leads to a clear polyploid phenotype by inhibiting the activity of Aurora B kinase in HCT116 cells with IC50 of 30 nM. Furthermore, AT9283 also produces the potent inhibition on HCT116 colony formation.
Kinase Assay: Assays for Aurora A and B are performed in a DELFIA format. Aurora A enzyme is incubated with AT9283 and 3 μM cross-tide substrate (biotin-CGPKGPGRRGRRRTSSFAEG) in 10 mM MOPS, pH 7, 0.1 mg/mL BSA, 0.001% Brij-35, 0.5% glycerol, 0.2 mM EDTA, 10 mM MgCl2, 0.01% β-mercaptoethanol, 15 μM ATP, and 2.5% DMSO. Aurora B enzyme is incubated with AT9283, 3 μM of the above substrate in 25 mM Tris, pH 8.5, 5 mM MgCl2, 0.1 mg/mL BSA, 0.025% Tween-20, 1 mM DTT, 15 μM ATP, and 2.5% DMSO. Reactions are allowed to proceed for 60 minutes and 45-90 minutes for Aurora A and Aurora B, respectively, before quenching with EDTA. The reaction mixtures are then transferred to a neutravidin-coated plate, and phosphorylated peptide is quantified by means of a phospho-specific antibody and a europium labeled secondary antibody using time-resolved fluorescence (excitation, 337 nm; emission, 620 nm). IC50 values for the control compounds are 92 nM (Aurora A assay) and 17 nM (Aurora B). Cell Assay: HCT 116 cells are cultured in DMEM + 10% FBS + GLUTAMAX I. Black 96-well flat-bottomed (clear) tissue culture treated plates are seeded in 200 μL of medium and incubated for approximately 16 hours at 37°C in a humidified atmosphere of 5% CO2 in air. Cells are treated with test compound at nine different concentrations (spanning 1 nM to 10 μM, plus DMSO vehicle control) and then incubated for 72 hours. Polyploidy morphological observations of the cells are then noted. The concentration of AT9283 required to produce a distinct polyploid phenotype is reported. Cells are seeded at a concentration of 75−100 cells/mL relevant culture media onto 6- or 24-well tissue culture plates and allowed to recover for 16 hours. Test compound (11 concentrations spanning 0.1 nM to 10 μM) or vehicle control (DMSO) is added to duplicate wells to give a final DMSO concentration of 0.1%. Following compound addition, colonies are allowed to grow between 10 and 14 days for optimum discrete colony counting. Colonies are fixed in 2 mL of Carnoys fixative (25% acetic acid, 75% MeOH) and stained in 2 mL of 0.4% w/v crystal violet. The numbers of colonies in each well is counted. IC50 values are calculated by sigmoidal dose-response (variable slope) IC50 curves using Prism Graphpad software. AT-9283 (Compound 16) is a potent, dual Aurora A/Aurora B inhibitor with IC₅₀ values in the low nanomolar range (approximately 3 nM). It inhibits the growth and survival of HCT116 colon carcinoma cells, inducing a distinct polyploid cellular phenotype characteristic of Aurora B kinase inhibition at a concentration of 0.03 µM. In a secondary HCT116 colony formation assay, AT-9283 exhibits potent anti-proliferative activity with an IC₅₀ of 12 nM. The compound demonstrates a clean CYP450 inhibition profile, with IC₅₀ values >10 µM for CYP3A4, 2D6, 1A2, 2C9, and 2C19. [1] |
| ln Vivo |
In HCT116 human colon carcinoma xenograft bearing mice, AT9283 treatment (15 mg/kg and 20 mg/kg) for 16 days results in a significant tumor growth inhibition of 67% and 76%, respectively. In addition, AT9283 also exhibits a significantly longer half-life in tumors(2.5 hours) compared with plasma (0.5 hour) and modest oral bioavailability in mice (Fp.o. = 24%).
AT-9283 demonstrated significant in vivo antitumor efficacy in an early-stage HCT116 human colon carcinoma xenograft model in immunocompromised mice. When administered intraperitoneally (ip) on an intermittent schedule (15 and 20 mg/kg, twice daily for 2 days, followed by 2 days off, repeated for 5 cycles), it produced significant tumor growth inhibition of 67% (%T/C = 33%) and 76% (%T/C = 24%), respectively, on day 16 of treatment compared to vehicle control. The doses were well-tolerated, with mean body weight maintained above 90% of the starting weight. [1] |
| Enzyme Assay |
Assays for Aurora A and Aurora B kinase activity were performed in a DELFIA format.
For Aurora A, the enzyme was incubated with the test compound and a biotinylated peptide substrate in a buffer containing MOPS, BSA, MgCl₂, ATP, and DMSO. The reaction proceeded for 60 minutes before being quenched with EDTA. Phosphorylated peptide was quantified using a phospho-specific primary antibody and a europium-labeled secondary antibody via time-resolved fluorescence. For Aurora B, a similar procedure was followed using a Tris-based buffer, with the reaction allowed to proceed for 45-90 minutes. IC₅₀ values were determined from dose-response curves. [1] |
| Cell Assay |
Primary HCT116 Cellular Assay: HCT116 cells were seeded in 96-well plates and allowed to adhere overnight. Cells were treated with a range of concentrations of AT-9283 (spanning 1 nM to 10 µM) or DMSO vehicle control for 72 hours. Following incubation, cells were examined microscopically for morphological signs of polyploidy. The lowest concentration required to produce a distinct polyploid phenotype was reported.
HCT116 Colony Forming Assay (Secondary Assay): Cells were seeded at low density in multi-well plates. After recovery, cells were treated with AT-9283 at 11 concentrations (spanning 0.1 nM to 10 µM) or vehicle control. Colonies were allowed to grow for 10-14 days, then fixed, stained, and counted. IC₅₀ values for inhibition of colony formation were calculated using sigmoidal dose-response curve fitting. [1] |
| Animal Protocol |
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.
Efficacy Study: The hydrochloride salt of AT-9283 was formulated in 10% DMSO, 20% water, and 70% hydroxypropyl-β-cyclodextrin (25% w/v aqueous solution). Mice bearing HCT116 xenograft tumors (~100 mm³) were dosed intraperitoneally (ip) with 15 or 20 mg/kg of AT-9283, twice daily for 2 days, followed by 2 days without treatment. This cycle was repeated five times. Tumor dimensions were measured periodically, and volumes were calculated. Control animals received the vehicle only on the same schedule. [1] Pharmacokinetic/Tumor Distribution Study: For iv dosing, AT-9283 hydrochloride was administered in 100% saline at 5 or 10 mg/kg. For oral (po) dosing, it was given at 10 mg/kg in 10% saline and 90% water. For ip dosing, it was administered at 20 mg/kg in a vehicle of 10% DMSO, 20% water, and 70% hydroxypropyl-β-cyclodextrin (25% w/v aqueous). Plasma and tumor samples were collected at timed intervals for analysis. [1] |
| ADME/Pharmacokinetics |
In mice, after intravenous (iv) administration of 5 mg/kg, AT-9283 exhibited a plasma clearance of 114 mL/min/kg, a steady-state volume of distribution (Vss) of 3.9 L/kg, and a plasma half-life (T₁/₂) of 0.5 h. In tumors, intravenous administration of 10 mg/kg significantly prolonged the half-life to 2.5 h. After oral (po) administration of 10 mg/kg, the maximum concentration (Cmax) was 0.45 µM, the AUC₀–t was 0.91 µM·h, and the oral bioavailability (F) was 24%.
After intraperitoneal injection (ip) at a dose of 20 mg/kg, absorption was nearly complete (F ≈ 100%), with a Cmax of 8.4 µM and an AUC₀–t of 7.7 µM·h. The plasma protein binding rate in mice was 81.5%. The compound exhibited good thermodynamic solubility (2.0 mg/mL at pH 7.0 and 13 mg/mL at pH 5.5). [1] |
| References |
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| Additional Infomation |
AT-9283 (compound 16) is a multi-target kinase inhibitor discovered through fragment optimization of the pyrazole-benzimidazole core structure. It effectively inhibits Aurora A and Aurora B kinases, leading to cell cycle defects (polyploidy) and apoptosis. Its kinase inhibition spectrum also extends to other cancer-related targets, such as JAK2 and Abl (T315I), which may provide an advantage in treating malignancies driven by multiple kinases. Based on its balanced in vitro activity, physicochemical properties, and in vivo efficacy, AT-9283 was selected for preclinical development and has entered a Phase I clinical trial for cancer treatment. [1]
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| Molecular Formula |
C22H29N7O5
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| Molecular Weight |
471.52
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| Exact Mass |
381.191
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| CAS # |
896466-76-5
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| Related CAS # |
AT9283;896466-04-9
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| PubChem CID |
135566103
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| Appearance |
Typically exists as solid at room temperature
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| Density |
1.5±0.1 g/cm3
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| Index of Refraction |
1.715
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| LogP |
0.92
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| Hydrogen Bond Donor Count |
6
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| Hydrogen Bond Acceptor Count |
8
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| Rotatable Bond Count |
6
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| Heavy Atom Count |
34
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| Complexity |
614
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| Defined Atom Stereocenter Count |
1
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| SMILES |
O=C(NC1CC1)NC2=CNN=C2C=3NC4=C(N3)C=C(C=C4)CN5CCOCC5.C[C@H](O)C(O)=O
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| InChi Key |
RWYNKTMXFIMBFE-WNQIDUERSA-N
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| InChi Code |
InChI=1S/C19H23N7O2.C3H6O3/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;1-2(4)3(5)6/h1,4,9-10,13H,2-3,5-8,11H2,(H,20,25)(H,22,23)(H2,21,24,27);2,4H,1H3,(H,5,6)/t;2-/m.0/s1
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| Chemical Name |
1-cyclopropyl-3-[5-[6-(morpholin-4-ylmethyl)-1H-benzimidazol-2-yl]-1H-pyrazol-4-yl]urea;(2S)-2-hydroxypropanoic acid
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| Synonyms |
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
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| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
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| Solubility (In Vivo) |
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| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.1208 mL | 10.6040 mL | 21.2080 mL | |
| 5 mM | 0.4242 mL | 2.1208 mL | 4.2416 mL | |
| 10 mM | 0.2121 mL | 1.0604 mL | 2.1208 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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