Aurora B (IC50 = 0.37 nM)

In newly isolated leukemia cells, barasertib-HQPA (3 μM, 3 hours) dramatically lowers the expression of phosphorylated variants of histone H3 [1]. In plasma, barasertib-HQPA is quickly transformed into active barasertib-HQPA [2]. In the LNCaP cell line, barasertib-HQPA therapy causes suboptimal cell survival, polyploidy, and cell death [3]. Significant antiproliferative effects caused by barasertib-HQPA are accompanied by polyploid population emergence, which typically results in apoptosis [4].

The development and weight of tumors treated with AZD1152 were considerably decreased by barasertib-HQPA (AZD1152, 25 mg/kg) [1]. In human MOLM13 leukemia xenografts, barasertib-HQPA (AZD1152, 5 mg/kg) amplifies the suppression of proliferation caused by vincristine or daunorubicin [1]. Effectively inhibiting human colon, lung, and hematological tumor xenografts in immunodeficient mice, barasertib-HQPA (AZD1152, 10–150 mg/kg/d) (mean tumor growth inhibition range, 55% to z100%; 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.

---

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.

---

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.

---

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.

---

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]

---

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]. AZD1152-HQPA induces growth arrest and apoptosis in androgen-dependent prostate cancer cell line (LNCaP) via producing aneugenic micronuclei and polyploidy. Tumour Biol. 2015 Feb;36(2):623-32.

[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 member of the of quinazolines that is 4-aminoquinazolin-7-ol in which the amino group at position 4 has been substituted by a 5-[2-(3-fluoroanilino)-2-oxoethyl]-1H-pyrazol-3-yl group, while the hydroxy group at position 7 has been converted into the corresponding 3-[ethyl(2-hydroxyethyl)aminopropyl ether. It has a role as an antineoplastic agent and an Aurora kinase inhibitor. It is a member of quinazolines, a secondary carboxamide, a tertiary amino compound, a secondary amino compound, a member of pyrazoles, a primary alcohol, a member of monofluorobenzenes and an anilide.
Defosbarasertib is a small-molecule inhibitor of the serine-threonine kinase Aurora B, with potential antineoplastic activity. Upon administration, defosbarasertib specifically binds to and inhibits Aurora kinase B, which disrupts spindle checkpoint functions and chromosome alignment, and results in the disruption of chromosome segregation and cytokinesis. This inhibits cell division and cell proliferation and induces apoptosis in Aurora kinase B-overexpressing tumor cells. Aurora kinase B, a serine/threonine protein kinase that functions in the attachment of the mitotic spindle to the centromere, is overexpressed in a wide variety of cancer cell types.

---

Aurora kinases play an important role in chromosome alignment, segregation, and cytokinesis during mitosis. We have recently shown that hematopoietic malignant cells including those from acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) aberrantly expressed Aurora A and B kinases, and ZM447439, a potent inhibitor of Aurora kinases, effectively induced growth arrest and apoptosis of a variety of leukemia cells. The present study explored the effect of AZD1152, a highly selective inhibitor of Aurora B kinase, on various types of human leukemia cells. AZD1152 inhibited the proliferation of 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 with an IC50 ranging from 3 nM to 40 nM, as measured by thymidine uptake on day 2 of culture. These cells had 4N/8N DNA content followed by apoptosis, as measured by cell-cycle analysis and annexin V staining, respectively. Of note, AZD1152 synergistically enhanced the antiproliferative activity of vincristine, a tubulin depolymerizing agent, and daunorubicin, a topoisomerase II inhibitor, against the MOLM13 and PALL-2 cells in vitro. Furthermore, AZD1152 potentiated the action of vincristine and daunorubicin in a MOLM13 murine xenograft model. Taken together, AZD1152 is a promising new agent for treatment of individuals with leukemia. The combined administration of AZD1152 and conventional chemotherapeutic agent to patients with leukemia warrants further investigation. [1]

---

Purpose: In the current study, we examined the in vivo effects of AZD1152, a novel and specific inhibitor of Aurora kinase activity (with selectivity for Aurora B). Experimental design: The pharmacodynamic effects and efficacy of AZD1152 were determined in a panel of human tumor xenograft models. AZD1152 was dosed via several parenteral (s.c. osmotic mini-pump, i.p., and i.v.) routes. Results: AZD1152 potently inhibited the growth of human colon, lung, and hematologic tumor xenografts (mean tumor growth inhibition range, 55% to > or =100%; P < 0.05) in immunodeficient mice. Detailed pharmacodynamic analysis in colorectal SW620 tumor-bearing athymic rats treated i.v. with AZD1152 revealed a temporal sequence of phenotypic events in tumors: transient suppression of histone H3 phosphorylation followed by accumulation of 4N DNA in cells (2.4-fold higher compared with controls) and then an increased proportion of polyploid cells (>4N DNA, 2.3-fold higher compared with controls). Histologic analysis showed aberrant cell division that was concurrent with an increase in apoptosis in AZD1152-treated tumors. Bone marrow analyses revealed transient myelosuppression with the drug that was fully reversible following cessation of AZD1152 treatment. Conclusions: These data suggest that selective targeting of Aurora B kinase may be a promising therapeutic approach for the treatment of a range of malignancies. In addition to the suppression of histone H3 phosphorylation, determination of tumor cell polyploidy and apoptosis may be useful biomarkers for this class of therapeutic agent. AZD1152 is currently in phase I trials. [2]

---

Prostate cancer is the frequent non-cutaneous tumor with high mortality in men. Prostate tumors contain cells with different status of androgen receptor. Androgen receptor plays important roles in progression and treatment of prostate cancer. Aurora B kinase, with oncogenic potential, is involved in chromosome segregation and cytokinesis, and its inhibition is a promising anti-cancer therapy. In the present study, we aimed to investigate the effects of Aurora B inhibitor, AZD1152-HQPA, on survival and proliferation of androgen receptor (AR)-positive prostate cancer cells. LNCaP was used as androgen-dependent prostate cancer cell line. We explored the effects of AZD1152-HQPA on cell viability, DNA content, micronuclei formation, and expression of genes involved in apoptosis and cell cycle. Moreover, the expression of Aurora B and AR were investigated in 23 benign prostatic hyperplasia and 38 prostate cancer specimens. AZD1152-HQPA treatment induced defective cell survival, polyploidy, and cell death in LNCaP cell line. Centromeric labeling with fluorescence in situ hybridization (FISH) showed that the loss of whole chromosomes is the origin of micronuclei, indicating on aneugenic action of AZD1152-HQPA. Treatment of AZD1152-HQPA decreased expression of AR. Moreover, we found weak positive correlations between the expression of Aurora B and AR in both benign prostatic hyperplasia and prostate cancer specimens (r = 0.25, r = 0.41). This is the first time to show that AZD1152-HQPA can be a useful therapeutic strategy for the treatment of androgen-dependent prostate cancer cell line. AZD1152-HQPA induces aneugenic mechanism of micronuclei production. Taken together, this study provides new insight into the direction to overcome the therapeutic impediments against prostate cancer. [3]

---

Aurora kinases play a critical role in regulating mitosis and cell division, and their overexpression has been implicated in the survival and proliferation of human cancer. In this study, we report the in vitro and in vivo activities of AZD1152, a compound that has selectivity for aurora B kinase, in acute myeloid leukemia (AML) cell lines, primary AML samples, and cord blood cells. AZD1152 exerted antiproliferative or cytotoxic effects in all cell lines studied, inhibited the phosphorylation of histone H3 (pHis H3) on Ser10 in a dose-dependent manner, and resulted in cells with >4N DNA content. THP-1 cells treated with AZD1152 accumulated in a state of polyploidy and showed a senescent response to the drug, in contrast to the apoptotic response seen in other cell lines. Accordingly, AZD1152 profoundly affected the growth of AML cell lines and primary AML in an in vivo xenotransplantation model. However, concentration-dependent effects on cell growth, apoptosis, and cell cycle progression were also observed when human cord blood and primary lineage-negative stem and progenitor cells were analyzed in vitro and in vivo. These data suggest that the inhibition of aurora B kinase may be a useful therapeutic strategy in the treatment of AML and that further exploration of dosing and treatment schedules is warranted in clinical trials.[4]

C26H30FN7O3

507.56

507.239

C, 61.53; H, 5.96; F, 3.74; N, 19.32; O, 9.46

722544-51-6

Barasertib;722543-31-9 (free acid); 722543-50-2 (2HCl); 957104-91-5; 722544-51-6

16007391

Typically exists as white to yellow solids at room temperature

1.4±0.1 g/cm3

796.7±60.0 °C at 760 mmHg

435.6±32.9 °C

0.0±2.9 mmHg at 25°C

1.677

2.82

4

9

13

37

693

0

FC1=C([H])C([H])=C([H])C(=C1[H])N([H])C(C([H])([H])C1=C([H])C(=NN1[H])N([H])C1C2C([H])=C([H])C(=C([H])C=2N=C([H])N=1)OC([H])([H])C([H])([H])C([H])([H])N(C([H])([H])C([H])([H])[H])C([H])([H])C([H])([H])O[H])=O

QYZOGCMHVIGURT-UHFFFAOYSA-N

InChI=1S/C26H30FN7O3/c1-2-34(10-11-35)9-4-12-37-21-7-8-22-23(16-21)28-17-29-26(22)31-24-14-20(32-33-24)15-25(36)30-19-6-3-5-18(27)13-19/h3,5-8,13-14,16-17,35H,2,4,9-12,15H2,1H3,(H,30,36)(H2,28,29,31,32,33)

2-(3-((7-(3-(ethyl(2-hydroxyethyl)amino)propoxy)quinazolin-4-yl)amino)-1H-pyrazol-5-yl)-N-(3-fluorophenyl)acetamide

INH-34; INH34; AZD 2811; AZD2811; INH 34; AZD1152-HQPA; AZD1152; AZD-1152; AZD 1152 HQPA; AZD-2811; AZD-1152HQPA; AZD 1152HQPA; AZD1152HQPA; AZD1152 HQPA; AZD1152-HQPA; AZD1152HQPA.AZD1152-HQPA; AZD-1152HQPA; barasertib-hQPA; Barasertib (AZD1152-HQPA); defosbarasertib; INH 34; 2-(3-((7-(3-(ethyl(2-hydroxyethyl)amino)propoxy)quinazolin-4-yl)amino)-1H-pyrazol-5-yl)-N-(3-fluorophenyl)acetamide;

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 (4.93 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.

---

Solubility in Formulation 2: ≥ 2.5 mg/mL (4.93 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.

---

View More

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

---

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