IC50: 0.37 nM (Aurora B)

Barasertib-HQPA (3 μM, 3 hours) significantly reduces the expression of phosphorylated forms of histone H3 in freshly isolated leukemic cells[1]. Barasertib-hydroxyquinazoline pyrazole aniline (HQPA)] is rapidly converted to active Barasertib-HQPA in plasma[2]. Barasertib-HQPA induces a significant antiproliferative effect accompanied by the appearance of polyploid populations, which in most cases leads to apoptosis[3].

Barasertib (AZD1152, 25 mg/kg) significantly inhibited the growth and weight of AZD1152 dihydrochloride-treated tumors[1]. Barasertib (AZD1152, 5 mg/kg) enhanced the ability of vincristine or daunorubicin to inhibit the proliferation of human MOLM13 leukemia xenografts[1]. Barasertib (AZD1152, 10-150 mg/kg/d) effectively inhibited the growth of human colon, lung, and hematologic tumor xenografts in immunodeficient mice (mean tumor growth inhibition range, 55% to 100%; P < 0.05)[2].

In vitro studies. [2] Phospho-histone H3 (PhH3) suppression was determined by high-content image analysis screening. SW620 cells, seeded in 96-well plates, were incubated with AZD1152-HQPA for 24 h before being fixed in 3.7% formaldehyde for 30 min. Cells were then washed with PBS, permeabilized with 0.5% Triton X-100, and stained with rabbit anti-PhH3 (Ser10) antibodies (1:100) for 1 h at room temperature. After washing with PBS, cells were incubated with Alexa Fluor 488 goat anti-rabbit antibodies (1:200) and Hoechst stain (1:10,000) for 1 h at room temperature. Cellular levels of PhH3 were analyzed on the Array Scan II using the Target Activation algorithm to calculate the percentage of PhH3-positive cells. Individual IC50 values were calculated in Origin (version 7.5) and the data were summarized using the geometric mean (i.e., the average of the logarithmic values converted back to a base 10 number).

Cell Proliferation Assay[1].
Cell Types: AML lines (HL-60, NB4, MOLM13), ALL line (PALL-2), biphenotypic leukemia (MV4-11), acute eosinophilic leukemia (EOL-1), and the blast crisis of chronic myeloid leukemia K562 cells.
Tested Concentrations: 0-100 nM. (Barasertib -HQPA)
Incubation Duration: 48 h.
Experimental Results: IC50 values ranged from 3 nM to 40 nM.

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Colony-forming assay[1]
The effects of AZD1152 on clonogenic growth of leukemia cells as well as normal bone marrow mononuclear cells were assessed by colony-forming assay using methylcellulose medium H4534, as previously described.

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Cell-cycle analysis by flow cytometry[1]
Cell-cycle analysis was performed on leukemia cells incubated with AZD1152-HQPA (1-10 nM) for 2 days at 5 × 105 cells/mL in 12-well plates.

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Apoptosis assays[1]
The ability of AZD1152-HQPA to induce apoptosis of leukemia cells was measured by annexin V–FITC apoptosis detection kit according to the manufacturer's instructions.

Mice[1]
Female immune-deficient BALB/c nude mice at 4 weeks of age were were maintained in pathogen-free conditions with irradiated chow. Animals were bilaterally, subcutaneously injected with 2 × 106 MOLM13 cells/tumor in 0.1 mL Matrigel or every another day, respectively. Daunorubicin (1 mg/kg) was given to mice by intraperitoneal injection 6 times during 2 weeks of treatment either alone or in combination with AZD1152 (5 mg/kg). The dose of these agents was determined by our preliminary studies (data not shown). Control diluent was given to the untreated control mice. Body weight and tumors were measured twice a week. Tumor sizes were calculated by the formula: a × b × c, where “a” is the length, “b” is the width, and “c” is the height in millimeters.

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In vivo studies. Male Swiss nude (nu/nu genotype), SCID-bg mice (CB17/Icr.Cg.PrkdcSCIDLystbg/Crl), or nude rats (Nude:Hsd Han:RNU-rnu; AstraZeneca) were housed in negative pressure isolators or in an individually ventilated cage system. Experiments were conducted on 8- to 12-week-old animals. Human tumor xenografts were established by s.c. injecting 100 to 200 μL tumor cells (between 1 × 106 and 1 × 107 cells mixed 50:50 with Matrigel; Becton Dickinson) on the flank. Animals were randomized into treatment groups (n = 8-11 per group) when tumors reached a defined palpable size (0.2-0.3 cm3 and 0.5-1 cm3 for mice and rats, respectively). AZD1152 was prepared in Tris buffer (pH 9) and administered either as a bolus injection (i.v. or i.p.) or as a continuous 48-h infusion via s.c. implanted osmotic mini-pumps (two 24-h pumps implanted sequentially.) in accordance with the manufacturer's instructions. Tumors were measured up to three times weekly with calipers, tumor volumes were calculated, and the data were plotted using the geometric mean for each group versus time. Tumor volume and tumor growth inhibition were calculated as described previously. Statistical analysis of any change in tumor volume was carried out using a Student's one-tailed t test (P value of <0.05 was considered to be statistically significant).[2]
For pharmacodynamic time course studies, nude rats bearing established SW620 tumor xenografts received vehicle or AZD1152 (25 mg/kg/d) as a daily i.v. bolus dose for 4 consecutive days (days 1-4). At multiple time points after dosing (days 0, 5, 9, 12, 16, and 19), two subgroups (n = 3 per group) of either vehicle- or AZD1152-treated animals were humanely killed and tumor and normal proliferating tissues (including bone marrow) were excised and assessed for pharmacodynamic effects using flow cytometric, histologic, or immunohistochemical analysis.[2]

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Dissolved in 3M Tris, pH 9.0, at a concentration of 2.5 mg/mL; 5, 25 mg/kg; i.p. injection

Female immune-deficient BALB/c nude mice subcutaneously injected with MOLM13 cells

[1]. AZD1152, a novel and selective aurora B kinase inhibitor, induces growth arrest, apoptosis, and sensitization for tubulin depolymerizing agent or topoisomerase II inhibitor in human acute leukemia cells in vitro and in vivo.Blood. 2007 Sep 15;110(6):2034-40.

[2]. AZD1152, a selective inhibitor of Aurora B kinase, inhibits human tumor xenograft growth by inducing apoptosis. Clin Cancer Res. 2007 Jun 15;13(12):3682-8.

[3]. The selective Aurora B kinase inhibitor AZD1152 is a potential new treatment for multiple myeloma. Br J Haematol. 2008 Feb;140(3):295-302.

[4]. AZD1152 rapidly and negatively affects the growth and survival of human acute myeloid leukemia cells in vitro and in vivo. Cancer Res. 2009 May 15;69(10):4150-8.

AZD-1152 is a quinazoline compound with the chemical name 4-aminoquinazoline-7-ol, in which the amino group at position 4 is replaced by 5-[2-(3-fluoroaniline)-2-oxoethyl]-1H-pyrazol-3-yl, and the hydroxyl group at position 7 is replaced by the corresponding 3-[ethyl(2-hydroxyethyl)aminopropyl] ether. It is an antitumor drug and an Aurora kinase inhibitor. AZD-1152 belongs to the quinazoline, secondary amide, tertiary amine, secondary amine, pyrazole, primary alcohol, monofluorobenzene, and aniline classes of compounds. Defosbarasertib is a small molecule inhibitor of the serine/threonine kinase Aurora B with potential antitumor activity. After administration, defosbarasertib specifically binds to and inhibits Aurora kinase B, thereby disrupting spindle checkpoint function and chromosome alignment, leading to chromosome segregation and cytokinesis arrest. This inhibits cell division and proliferation and induces apoptosis in tumor cells overexpressing Aurora kinase B. Aurora kinase B is a serine/threonine protein kinase that functions to attach the mitotic spindle to the centromere and is overexpressed in various cancer cell types. Aurora kinase plays a crucial role in chromosome alignment, segregation, and cytokinesis during mitosis. We recently discovered that hematopoietic malignancies, including acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL), abnormally express Aurora A and B kinases, and that the potent Aurora kinase inhibitor ZM447439 effectively induces growth arrest and apoptosis in various leukemia cell lines. This study investigated the effects of the highly selective Aurora B kinase inhibitor AZD1152 on various human leukemia cell lines. AZD1152 inhibited the proliferation of acute myeloid leukemia (AML) cell lines (HL-60, NB4, MOLM13), acute lymphoblastic leukemia (ALL) cell line (PALL-2), biphenotypic leukemia (MV4-11), acute eosinophilic leukemia (EOL-1), and chronic myeloid leukemia (CML) K562 cells during the blast crisis, with IC50 values ranging from 3 nM to 40 nM, determined by thymidine uptake on day 2 of culture. These cells had a DNA content of 4N/8N and subsequently underwent apoptosis, as detected by cell cycle analysis and annexin V staining. Notably, AZD1152 synergistically enhanced the antiproliferative activity against MOLM13 and PALL-2 cells in vitro with vincristine (a microtubule depolymerizer) and daunorubicin (a topoisomerase II inhibitor). Furthermore, AZD1152 enhanced the effects of vincristine and daunorubicin in a MOLM13 mouse xenograft model. In summary, AZD1152 is a promising new drug for the treatment of leukemia. Further research is warranted on the combination of AZD1152 with traditional chemotherapy drugs for the treatment of leukemia patients. [1]

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Objective: This study aimed to investigate the in vivo effects of AZD1152 (a novel specific Aurora kinase inhibitor selective for Aurora B). Experimental design: The pharmacodynamic effects and efficacy of AZD1152 were determined in various human tumor xenograft models. AZD1152 was administered via various parenteral routes (subcutaneous osmotic pump, intraperitoneal injection and intravenous injection). Results: AZD1152 effectively inhibited the growth of human colon cancer, lung cancer and hematological malignancies xenografts in immunodeficient mice (mean tumor growth inhibition rate ranged from 55% to ≥100%; P<0.05). After intravenous treatment with AZD1152 in athymic rats carrying SW620 colorectal cancer tumors, detailed pharmacodynamic analysis revealed the time sequence of tumor phenotypic events: first, transient inhibition of histone H3 phosphorylation, followed by accumulation of intracellular 4N DNA (2.4 times higher than the control group), and then an increase in the proportion of polyploid cells (>4N DNA, 2.3 times higher than the control group). Histological analysis showed abnormal cell division and increased apoptosis in the tumors treated with AZD1152. Bone marrow analysis showed that the drug caused transient myelosuppression, which was completely reversible after discontinuation of AZD1152 treatment. Conclusion: These data suggest that selective targeting of Aurora B kinase may be a promising approach for the treatment of a variety of malignancies. In addition to inhibition of histone H3 phosphorylation, the detection of tumor cell polyploidy and apoptosis may also be useful biomarkers for such therapeutics. AZD1152 is currently undergoing a phase I clinical trial. [2]

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Prostate cancer is the most common non-skin cancer in men and has a high mortality rate. Prostate tumor cells exhibit different androgen receptor states. Androgen receptors play a crucial role in the progression and treatment of prostate cancer. Aurora B kinase possesses oncogenic potential and participates in chromosome segregation and cytokinesis; inhibition of Aurora B kinase is a promising anticancer therapy. This study aimed to investigate the effects of the Aurora B inhibitor AZD1152-HQPA on the survival and proliferation of androgen receptor (AR)-positive prostate cancer cells. We used LNCaP as the androgen-dependent prostate cancer cell line. We investigated the effects of AZD1152-HQPA on cell viability, DNA content, micronucleus formation, and the expression of apoptosis and cell cycle-related genes. Furthermore, we examined the expression of Aurora B and AR in 23 benign prostatic hyperplasia (BPH) specimens and 38 prostate cancer specimens. AZD1152-HQPA treatment induced LNCaP cell survival defects, polyploidization, and cell death. Fluorescence in situ hybridization (FISH) centromere labeling showed that the deletion of the entire chromosome was the source of micronuclei, indicating that AZD1152-HQPA had an aneuploidy-inducing effect. AZD1152-HQPA treatment reduced AR expression. In addition, we found that the expression of Aurora B and AR in benign prostatic hyperplasia and prostate cancer specimens was weakly positively correlated (r = 0.25, r = 0.41). This is the first time that AZD1152-HQPA can be used as an effective treatment strategy for androgen-dependent prostate cancer cell lines. AZD1152-HQPA can induce aneuploidy in micronuclei. In summary, this study provides a new idea for overcoming the obstacles in prostate cancer treatment. [3]

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Aurora kinase plays a key role in regulating mitosis and cell division, and its overexpression is associated with the survival and proliferation of human cancer cells. This study reports the in vitro and in vivo activities of compound AZD1152 (a compound selective for Aurora B kinase) in acute myeloid leukemia (AML) cell lines, primary AML samples, and cord blood cells. AZD1152 exhibited antiproliferative or cytotoxic effects in all studied cell lines, inhibiting phosphorylation of histone H3 Ser10 site (pHis H3) in a dose-dependent manner and resulting in cellular DNA content >4N. In contrast to the apoptotic response observed in other cell lines, AZD1152-treated THP-1 cells accumulated in a polyploid state and exhibited a senescent response. Accordingly, AZD1152 significantly affected the growth of AML cell lines and primary AML in an in vivo xenograft model. However, concentration-dependent effects on cell growth, apoptosis, and cell cycle progression were also observed when analyzing human cord blood and primary lineage-negative stem cells and progenitor cells in vitro and in vivo. These data suggest that inhibition of Aurora B kinase may be an effective therapeutic strategy for AML, and further exploration of dosage and treatment regimens is necessary in clinical trials. [4]

C26H33CL2FN7O6P

660.46

659.159

C, 47.28; H, 5.04; Cl, 10.73; F, 2.88; N, 14.85; O, 14.53; P, 4.69

722543-50-2

722543-31-9 (free acid); 722543-50-2 (2HCl); 957104-91-5

16221572

Typically exists as solid at room temperature

5.292

7

12

15

43

859

0

PEVRMFUIHQMEHQ-UHFFFAOYSA-N

InChI=1S/C26H31FN7O6P.2ClH/c1-2-34(10-12-40-41(36,37)38)9-4-11-39-21-7-8-22-23(16-21)28-17-29-26(22)31-24-14-20(32-33-24)15-25(35)30-19-6-3-5-18(27)13-19;;/h3,5-8,13-14,16-17H,2,4,9-12,15H2,1H3,(H,30,35)(H2,36,37,38)(H2,28,29,31,32,33);2*1H

2-[ethyl-[3-[4-[[5-[2-(3-fluoroanilino)-2-oxoethyl]-1H-pyrazol-3-yl]amino]quinazolin-7-yl]oxypropyl]amino]ethyl dihydrogen phosphate;dihydrochloride

AZD1152 dihydrochloride; Barasertib dihydrochloride; 722543-50-2; 5-[[7-[3-[ethyl[2-(phosphonooxy)ethyl]amino]propoxy]-4-quinazolinyl]amino]-n-(3-fluorophenyl)-1h-pyrazole-3-acetamide dihydrochloride; H3T2NXF7ZK; Barasertib (dihydrochloride); AZD 1152 (hydrochloride); 2-[ethyl-[3-[4-[[5-[2-(3-fluoroanilino)-2-oxoethyl]-1H-pyrazol-3-yl]amino]quinazolin-7-yl]oxypropyl]amino]ethyl dihydrogen phosphate;dihydrochloride; 1H-Pyrazole-3-acetamide, 5-((7-(3-(ethyl(2-(phosphonooxy)ethyl)amino)propoxy)-4-quinazolinyl)amino)-N-(3-fluorophenyl)-, hydrochloride (1:2);

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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