| Size | Price | Stock | Qty |
|---|---|---|---|
| 5mg |
|
||
| 10mg |
|
||
| 25mg |
|
||
| 50mg |
|
||
| 100mg |
|
||
| 250mg |
|
||
| 500mg |
|
||
| Other Sizes |
Purity: ≥98%
| Targets |
Aurora A (approx 3 nM, based on 52% inhibition at 3.0 nM) [1]
Aurora B (approx 3 nM, based on 58% inhibition at 3.0 nM) [1] JAK2 (IC50 = 1.2 nM) [1] JAK3 (IC50 = 1.1 nM) [1] Abl (T315I) (IC50 = 4 nM) [1] Other kinases with IC50 values: GSK3-β, FGFR2, VEGFR3 (Flt4), Mer, Ret, Rsk2, Rsk3, Tyk2, Yes (1-10 nM); Abl(Q252H), DRAK1, FGFR1, FGFR1(V561M), FGFR2(N549H), FGFR3, VEGFR1(Flt1), Flt-3, PDGFR-α(D842V), PDK1, PKCμ, Rsk4, SRC(T341M), VEGFR2 (10-30 nM); 21 kinases (30-100 nM); 37 kinases (100-300 nM); 14 kinases (>300 nM). [1] |
|---|---|
| ln Vitro |
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 HCl inhibited growth and survival of HCT116 cells, producing the polyploid cellular phenotype typically associated with Aurora B kinase inhibition. [1] In HCT116 cells, AT-9283 HCl at 0.03 μM produced a distinct polyploid phenotype. [1] AT-9283 HCl potently inhibited HCT116 colony formation with an IC50 of 12 nM in a secondary cellular assay. [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%).
In immunocompromised BALB/c nude mice bearing early stage HCT116 human colon carcinoma xenografts, intraperitoneal administration of AT-9283 HCl at 15 mg/kg and 20 mg/kg (b.i.d. ×2 q4d for 5 cycles) produced significant tumor growth inhibition of 67% (%T/C = 33%) and 76% (%T/C = 24%) respectively on day 16 of treatment. Both doses were well tolerated with mean body weight maintained above 90% relative to starting weight. [1] |
| Enzyme Assay |
Aurora A and Aurora B kinase assays were performed in a DELFIA format. For Aurora A: enzyme incubated with test compound and 3 μM biotinylated 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. For Aurora B: enzyme incubated with test compound and 3 μM 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 proceeded for 60 min (Aurora A) or 45-90 min (Aurora B) before quenching with EDTA. Reaction mixtures were transferred to a neutravidin-coated plate, and phosphorylated peptide was quantified using a phospho-specific antibody and a europium-labeled secondary antibody with time-resolved fluorescence (excitation 337 nm, emission 620 nm). [1]
|
| Cell Assay |
Primary HCT116 cellular assay: HCT116 cells were cultured in DMEM + 10% FBS + GLUTAMAX I. Black 96-well flat-bottomed tissue culture treated plates were seeded at 5×10^3 cells per well in 200 μL medium and incubated for approximately 16 h at 37°C, 5% CO2. Cells were treated with test compound at nine different concentrations (spanning 1 nM to 10 μM plus DMSO vehicle control) and incubated for 72 h. Polyploidy morphological observations were noted, and the lowest concentration required to produce a distinct polyploid phenotype was reported. [1]
HCT116 colony forming assay (secondary assay): Cells were seeded at 75-100 cells/mL onto 6- or 24-well plates and allowed to recover for 16 h. Test compound (11 concentrations spanning 0.1 nM to 10 μM) or vehicle control (DMSO) was added to duplicate wells (final DMSO 0.1%). Colonies were allowed to grow for 10-14 days, then fixed in Carnoy's fixative (25% acetic acid, 75% MeOH) and stained with 0.4% crystal violet. Colonies were counted, and IC50 values were calculated by sigmoidal dose-response (variable slope) curves using Prism Graphpad software. [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.
For in vivo efficacy study: HCT116 cell suspension was inoculated subcutaneously into the hind flank of male BALB/c athymic nude mice (Hsd: athymic nude-Foxn1nu) and allowed to grow to approximately 100 mm^3. AT-9283 HCl was formulated as a hydrochloride salt in 10% DMSO, 20% water, 70% hydroxypropyl-β-cyclodextrin (25% w/v aq). Mice were dosed intraperitoneally twice daily for 2 days and not dosed for 2 days (b.i.d. ×2 q4d), and the cycle was repeated five times. Doses were 15 and 20 mg/kg. Tumor dimensions were recorded every 2 or 3 days using external measurements, and tumor volume was calculated using the equation for an ellipsoid (length × width^2 / 2). Control group received vehicle only on the same schedule. [1] For pharmacokinetic and tumor distribution study: AT-9283 HCl was administered as a single dose to BALB/c mice. For iv dosing at 5 or 10 mg/kg (100% saline), for ip dosing at 20 mg/kg (10% DMSO, 20% water, 70% hydroxypropyl-β-cyclodextrin 25% w/v aq), and for oral dosing at 10 mg/kg (10% saline, 90% water). Tumor and/or plasma samples were collected at timed intervals. Tumor samples were homogenized in 5 volumes (w/v) of acetonitrile/water (50/50). Quantitative analysis was performed by LC/MS/MS. [1] |
| ADME/Pharmacokinetics |
In mice: plasma protein binding = 81.5% (equilibrium dialysis). After intravenous administration at 5 mg/kg: clearance = 114 mL/min/kg, Vss = 3.9 L/kg, plasma t1/2 = 0.5 h, Cmax = 4.9 μM, AUC0-t = 1.9 μM·h. After oral administration at 10 mg/kg: Cmax = 0.45 μM, AUC0-t = 0.91 μM·h, oral bioavailability = 24%. After intraperitoneal administration at 20 mg/kg: Cmax = 8.4 μM, AUC0-t = 7.7 μM·h, ip bioavailability = 100% (absorption essentially complete). Tumor t1/2 = 2.5 h (following iv at 10 mg/kg). [1]
In mouse hepatocytes in vitro, clearance was 8.8 (μM/min)/million cells. [1] |
| Toxicity/Toxicokinetics |
In the efficacy study, doses of 15 and 20 mg/kg (ip, b.i.d. ×2 q4d) were well tolerated, with mean body weight maintained above 90% relative to starting weight. [1]
AT-9283 HCl showed a clean CYP450 profile with IC50 >10 μM for CYP3A4, CYP2D6, CYP1A2, CYP2C9, and CYP2C19. [1] |
| References |
J Med Chem.2009 Jan 22;52(2):379-88.
|
| Additional Infomation |
AT-9283 HCl was identified via fragment-based drug discovery starting from pyrazole-benzimidazole fragment 5 (Aurora A IC50 = 0.91 μM, ligand efficiency = 0.59). The key hydrogen bonding interactions between the fragment and Aurora A were retained throughout optimization. The compound has a folded conformation stabilized by hydrophobic interaction between the cyclopropyl ring and the benzimidazole, as confirmed by X-ray crystallography and NMR. [1]
In addition to Aurora kinases, AT-9283 HCl also inhibits JAK2, JAK3, and Abl (T315I), which is attributed to the presence of an extra glycine residue in the hinge region of these kinases (Gly216 in Aurora A, Gly935 in JAK2). The binding is relatively insensitive to the nature of the gatekeeper residue, allowing activity against the T315I mutant of Abl. [1] AT-9283 HCl has advanced into phase I clinical trials for the treatment of cancer (refractory hematological malignancies). [1] |
| Molecular Formula |
C19H24CLN7O2
|
|
|---|---|---|
| Molecular Weight |
417.89
|
|
| Exact Mass |
417.167
|
|
| CAS # |
896466-61-8
|
|
| Related CAS # |
896466-61-8 (HCl);896466-04-9;896466-76-5 (lactate);
|
|
| PubChem CID |
135461257
|
|
| Appearance |
Typically exists as solid at room temperature
|
|
| Hydrogen Bond Donor Count |
5
|
|
| Hydrogen Bond Acceptor Count |
5
|
|
| Rotatable Bond Count |
5
|
|
| Heavy Atom Count |
29
|
|
| Complexity |
554
|
|
| Defined Atom Stereocenter Count |
0
|
|
| SMILES |
O=C(NC1CC1)NC2=CNN=C2C=3NC4=C(N3)C=C(C=C4)CN5CCOCC5.Cl
|
|
| InChi Key |
NHFNYIVVGIXBPD-UHFFFAOYSA-N
|
|
| InChi Code |
InChI=1S/C19H23N7O2.ClH/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);1H
|
|
| Chemical Name |
1-cyclopropyl-3-[5-[6-(morpholin-4-ylmethyl)-1H-benzimidazol-2-yl]-1H-pyrazol-4-yl]urea;hydrochloride
|
|
| Synonyms |
|
|
| HS Tariff Code |
2934.99.9001
|
|
| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
|
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
|
| Solubility (In Vitro) |
|
|||
|---|---|---|---|---|
| Solubility (In Vivo) |
|
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.3930 mL | 11.9649 mL | 23.9297 mL | |
| 5 mM | 0.4786 mL | 2.3930 mL | 4.7859 mL | |
| 10 mM | 0.2393 mL | 1.1965 mL | 2.3930 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.
|
AT9283 potently inhibits Aurora B activity in B-NHL cell lines.Int J Cancer.2012 Jun 15;130(12):2997-3005. |
AT9283 plus docetaxel suppresses tumor growth and prolong survival of Granta-519-SCID xenografted mice.Int J Cancer.2012 Jun 15;130(12):2997-3005. |
Products are for research use only; We do not sell to patients
Copyright 2020 InvivoChem LLC | All Rights Reserved
COA
Figure 5