Aurora A (IC50 = 13 nM); Aurora B (IC50 = 79 nM); Aurora C (IC50 = 61 nM)

The viability of C13 and A2780cp cells is severely reduced by denusertib (0.01 to 50 μM). After 24 and 48 hours of treatment, the IC50s for C13 cells are 10.40 and 1.83 μM, and for A2780cp cells, they are 19.89 and 3.88 μM. In C13 and A2780cp cells, dunusertib promotes a cell arrest cycle in the G2/M phase. After being treated with denusertib, the proportion of cells arrested in the G2/M phase rises noticeably, and polyploidy accumulates in C13 and A2780cp cells. Danusertib increases the expression of p21 Waf1/Cip1, p27 Kip1, and p53 while depressing CDK1/CDC2 and cyclin B1 expression. Danusertib activates the PI3K/Akt/mTOR signaling pathway in C13 and A2780cp cells to cause autophagy[1]. All examined leukemic cell lines are significantly inhibited from proliferating by PHA-739358, with IC50 values ranging from 0.05 μM to 3.06 μM. IM-resistant M351T, E255K, and T315I mutants are among the BaF3-p210 cells in which PHA-739358 exerts antiproliferative effects. BaF3 -p210 wt cells and IM-resistant mutants exhibit decreased phosphorylation of CrkL in response to PHA-739358 (5 μM)[2]. In vitro, dacartib totally prevents GEP-NET cell proliferation and induces cell-cycle arrest[3].

PHA-739358 (15 mg/kg twice day, ip) and IM are well tolerated; over the course of the 10-day treatment period, they virtually suppressed tumor growth and significantly inhibited K562 cell proliferation[2]. When compared to controls or mice given streptozotocine/5-fluorouracil, danusertibsertib (2×15 mg/kg/d, ip) dramatically slows down the growth of tumors in vivo in a subcutaneous murine xenograft model[3].

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Danusertib treatment inhibits growth of subcutaneous GEP-NET xenografts and lowers serum chromogranin levels [3]
Treatment of tumor-bearing mice with danusertib at a dose of 2 × 15 mg/kg/d decreased growth of BON1 and QGP xenografts (Fig. 3A and B). BON1 tumor growth was significantly inhibited from day 4 (P < 0.001) until the end of the experiment (P < 0.001). The mean absolute tumor volume was reduced by 88.2%, and the mean percental tumor growth was also reduced by 88.2%, compared with vehicle-treated controls (Fig. 3A, Table 2). In mice with QGP xenografts, treatment with danusertib led to a virtual shrinkage of QGP tumors from day 4 (P < 0.001), until the end of the experiment (P < 0.001). The final tumor volume was only 6.3 ± 8.2% of the original tumor volume (Fig. 3B, Table 2). Mean absolute and percental tumor growth in danusertib-treated mice was reduced by 98.4% and 98.6%, respectively, compared with vehicle-treated controls. When compared with treatment with streptozotocine/5-fluoruracil (STZ/5-FU), which is a frequently used cytostatic therapy for GEP-NETs, the antiproliferative effect of danusertib was significantly higher from day 12 (P < 0.001), in both BON1 and QGP tumors (Fig. 3A and B, Table 2).

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Treatment of GEP-NET liver metastases with danusertib [3]
In transplanted mice, treatment with danusertib or vehicle was commenced when liver metastases became clearly detectable and were eligible for volumetric analysis, which was after 2 to 3 weeks following cell transplantation. Average size of BON1 metastases (single nodules) before treatment was 2.9 ± 1.3 mm3 (n = 20). Although liver metastases of BON1 tumors in vehicle-treated controls continued to grow until large areas of the liver were replaced by tumor tissue, treatment with danusertib significantly inhibited growth of liver metastases (day 12 after start of treatment 55.9 ± 39.7 mm3, n = 10 vs. 5.2 ± 3.8 mm3; n = 10; P < 0.01; Fig. 6A and C, Table 3). This corresponds to a relative tumor volume compared with day 0 (100%) of 1,495.3 ± 1,277.9% in controls versus 164.5 ± 50.2% in danusertib-treated mice (P < 0.01). Mean absolute and percental tumor growth in danusertib-treated mice was reduced by 90.7% and 89.0%, respectively, compared with vehicle-treated controls. Average size of QGP metastases in the liver before treatment was 6.8 ± 3.7 mm3 (n = 9). Danusertib entirely inhibited growth of QGP metastases, although no shrinkage was observed, as in subcutaneous tumors (control: day 12, 51.7 ± 35.0 mm3; n = 4 vs. danusertib 6.1 ± 4.3 mm3; n = 5, P < 0.05; Fig. 6B and C, Table 3). This corresponds to a relative tumor volume of 810.7 ± 355.4% versus 108.7 ± 58.9%, P < 0.01. The mean absolute tumor volume was reduced by 88.2%, and the mean percental tumor growth was reduced by 86.6%, compared with vehicle-treated controls.[3]

The emergence of resistance to imatinib (IM) mediated by mutations in the BCR-ABL domain has become a major challenge in the treatment of chronic myeloid leukemia (CML). Here, we report on studies performed with a novel small molecule inhibitor, PHA-739358, which selectively targets Bcr-Abl and Aurora kinases A to C. PHA-739358 exhibits strong antiproliferative and proapoptotic activity against a broad panel of human BCR-ABL–positive and –negative cell lines and against murine BaF3 cells ectopically expressing wild-type (wt) or IM-resistant BCR-ABL mutants, including T315I. Pharmacologic synergism of IM and PHA-739358 was observed in leukemia cell lines with subtotal resistance to IM. Treatment with PHA-739358 significantly decreased phosphorylation of histone H3, a marker of Aurora B activity and of CrkL, a downstream target of Bcr-Abl, suggesting that PHA-739358 acts via combined inhibition of Bcr-Abl and Aurora kinases. Moreover, strong antiproliferative effects of PHA-739358 were observed in CD34+ cells derived from untreated CML patients and from IM-resistant individuals in chronic phase or blast crisis, including those harboring the T315I mutation. Thus, PHA-739358 represents a promising new strategy for treatment of IM-resistant BCR-ABL-positive leukemias, including those harboring the T315I mutation. Clinical trials investigating this compound in IM-resistant CML have recently been initiated[2].

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PHA-739358 shares activity of both Aurora and Abl kinase inhibitors [2]
The influence of PHA-739358 on Aurora kinase activity was assessed by the phosphorylation status of histone H3 at Ser10 (Figures 4,5C). K562 cells exposed to PHA-739358 showed strong reduction of phosphorylation with only 0.1% phospho-H3 (Figure 4C) compared with 3.5% in untreated (Figure 4A) and 3.7% in IM-treated cells (Figure 4B). Inhibitory activity of PHA-739358 on Bcr-Abl kinase was evaluated by determining phosphorylation of CrkL and Stat5, 2 well-known downstream targets of Bcr-Abl (Figure 5). PHA-739358 treatment produced distinct inhibition of c-Abl autophosphorylation on Tyr393 (Figure 5C). In addition, pronounced inhibition of phosphorylation of CrkL (28% residual phosphorylation) and Stat5 (37% residual phosphorylation) was observed to a similar degree as for IM (19% and 22% residual phosphorylation, respectively; Figure 5A,B).

Ovarian carcinoma (OC) is one of the most common gynecological malignancies, with a poor prognosis for patients at advanced stage. Danusertib (Danu) is a pan-inhibitor of the Aurora kinases with unclear anticancer effect and underlying mechanisms in OC treatment. This study aimed to examine the cancer cell killing effect and explore the possible mechanisms with a focus on proliferation, cell cycle progression, apoptosis, autophagy, and epithelial to mesenchymal transition (EMT) in human OC cell lines C13 and A2780cp. The results showed that Danu remarkably inhibited cell proliferation, induced apoptosis and autophagy, and suppressed EMT in both cell lines. Danu arrested cells in G₂/M phase and led to an accumulation of polyploidy through the regulation of the expression key cell cycle modulators. Danu induced mitochondria-dependent apoptosis and autophagy in dose and time-dependent manners. Danu suppressed PI3K/Akt/mTOR signaling pathway, evident from the marked reduction in the phosphorylation of PI3K/Akt/mTOR, contributing to the autophagy inducing effect of Danu in both cell lines. In addition, Danu inhibited EMT. In aggregate, Danu exerts potent inducing effect on cell cycle arrest, apoptosis, and autophagy, but exhibits a marked inhibitory effect on EMT. PI3K/Akt/mTOR signaling pathway contributes, partially, to the cancer cell killing effect of Danu in C13 and A2780cp cells[1].

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Cell analysis, immunostaining, and flow cytometry [3]
Cells were seeded in DMEM, fixed and immunostained for aurora-A, aurora-B, and histone H3 phosphorylation, as described. For analysis of cell proliferation, increasing concentrations of danusertib (dissolvent control; 5 nmol/L–5 μmol/L) were added after 24 hours of cell culture. Forty-eight to 120 hours later, the number of viable cells was determined. Analysis of DNA content and H3 phosphorylation were carried out by flow cytometry as described.

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Short-term expansion of CD34+ cells [2]
For short-term expansion assays, 103 CD34+ cells were plated in triplicates in 96-well plates containing 100 μL of serum-free medium per well supplemented with human stem-cell factor (100 ng/mL), human Flt-3 Ligand (100 ng/mL), human thrombopoietin (50 ng/mL), human interleukin-3 and -6 (IL-3 and IL-6, respectively, both 20 ng/mL), and granulocyte colony-stimulating factor (20 ng/mL) plus Danusertib (PHA739358)or IM at the indicated concentrations. After 5 days, an additional 100 μL of cytokine and PHA-739358 or IM containing medium were added. Cell numbers within each individual well were evaluated at days 3, 6, and 9 or at days 3, 6, and 12 for healthy donor samples.

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MTT assay [2]
Cells were seeded in triplicates in 96-well flat-bottomed microtiter plates at a density of 1.5 × 104 cells/well in 150 μL of their respective media. After 24 hours increasing concentrations of Danusertib (PHA739358) (0-5.0 μM) or/and IM (0-20 μM) were added. After 48 hours of treatment, the viable cells were assayed for their ability to transform 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) into purple formazan, as described previously.23 The compound concentration that inhibits response by 50% (IC50) is defined as the concentration resulting in 50% growth inhibition that corresponds to the fraction affected (Fa value) of 0.5. All tested cells were exposed to 10 different concentrations of each compound. Fraction affected (Fa) and dose-effect relationship at the point of IC50 were analyzed by CalcuSyn Software.

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Analysis of DNA content and apoptosis by flow cytometry [2]
Cells were plated in triplicates in 6-well plates and cultured in 2 mL of their respective media. After 24 hours, cells were exposed to increasing concentrations of Danusertib (PHA739358) for 48 hours, then washed with phosphate-buffered saline (PBS), and fixed in cold 70% ethanol overnight at −20°C. For flow cytometric analysis, cells were washed twice with PBS, resuspended in PBS containing RNAse A (100 μg/mL) and propidium iodide (10 μg/mL), and incubated for 30 minutes on ice; 10 000 cells were analyzed from each sample.

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Assessment of phosphorylation status by intracellular flow cytometry [2]
Cells exposed to 5 μM Danusertib (PHA739358) or 5 μM IM for 2 hours or 24 hours were collected, fixed in 2% formaldehyde for 10 minutes at 37°C, chilled on ice for 1 minute, and then permeabilized with ice-cold 90% methanol for 30 minutes on ice. From each sample, 5 × 105 cells were washed with 2 mL incubation buffer (PBS/0.5% bovine serum albumin), centrifuged at 50g for 5 minutes, and resuspended in 100 μL of incubation buffer with 2.0 μL of Phospho-CrkL, Phospho-Stat5, or Phospho-Histone H3-(Ser10) specific antibody. After 45 minutes of incubation at RT, cells were washed twice, resuspended in 100 μL incubation buffer with 0.5 μL of the secondary antibody, and incubated at RT for 30 minutes in the dark. Again, cells were washed twice with washing buffer. Samples stained with specific Phospho-Histone H3-(Ser10) antibody were also stained with propidium iodide. All samples were analyzed by flow cytometry, and isotype controls were included for cytometer setup. The amount of phosphorylated proteins (P-CrkL and P-Stat5) was determined as the geometric mean fluorescence intensity, and the changes of the phosphorylation status were expressed as a percentage of the no-drug control. CML CD34+ cells were incubated with either 5 μM Danusertib (PHA739358) for 72 hours and 96 hours or with 5 μM IM for 96 hours. After collection, cells were prepared as described above.

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Western blotting [2]
K562 cells cultured under standard conditions were exposed for 1 hour to 2 μM and 5 μM of Danusertib (PHA739358) or IM. Collected cells were lysed in sodium dodecyl sulfate (SDS) buffer (125 mM Tris-HCl, pH 6.8, 2% SDS) and after sonication and boiling, 20 μg total extract of the indicated samples was loaded on SDS-polyacrylamide gel electrophoresis precast gels and immunoblotted. The following antibodies were used: c-Abl, Phospho-H3-(Ser10; Upstate), Histone-H3, Stat5. Anti-PathScan Bcr-Abl activity assay was used for the detection of Phospho-Bcr-Abl, Phospho-Stat5, and Phospho-CrkL.

In a subcutaneous murine xenograft model, danusertib significantly reduced tumor growth in vivo compared with controls or mice treated with streptozotocine/5-fluorouracil. As a consequence, decreased levels of tumor marker chromogranin A were found in mouse serum samples. In a newly developed orthotopic model for GEP-NET liver metastases by intrasplenic tumor cell transplantation, dynamic MRI proved significant growth inhibition of BON1- and QGP-derived liver metastases.[3]

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Experimental protocol [3]
Mice bearing BON1 or QGP tumors were divided into a treatment group receiving Danusertib (PHA739358) intraperitoneally at a dose of 2 × 15 mg/kg/d or a control group receiving the same volume of vehicle solution (5% dextrose). Streptozotocine and 5-fluorouracil were applied intraperitoneally at a dose of 1 × 15 mg/kg/d.

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Evaluation of Danusertib (PHA739358) and IM in vivo [2]
To evaluate the efficacy and toxicity of Danusertib (PHA739358) in vivo, a subcutaneous animal model for CML was used; 5 × 107 K562 cells were injected into the flanks of female SCID mice and tumor growth was monitored daily by palpation. On day 7, when tumors reached an estimated weight of 100 to 150 mg, animals were assigned to 3 experimental groups by random selection and received the following treatment for a period of 10 days: group 1, control, vehicle solution (7 mice); group 2, PHA-739358 twice a day intraperitoneally at a dose of 15 mg/kg (7 mice); and group 3, IM twice a day per os at 100 mg/kg. Tumor growth was assessed by caliper, and tumor weight was calculated according to the following formula: Tumor weight = [length (mm) × width2 (mm)]/2. Toxicity was monitored by changes in body weight and vitality of the animals.

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DMSO; 15 mg/kg; i.p.

Female SCID mice

[1]. Danusertib Induces Apoptosis, Cell Cycle Arrest, and Autophagy but Inhibits Epithelial to Mesenchymal Transition Involving PI3K/Akt/mTOR Signaling Pathway in Human Ovarian Cancer Cells. Int J Mol Sci. 2015 Nov 13;16(11):27228-51.

[2]. Simultaneous targeting of Aurora kinases and Bcr-Abl kinase by the small molecule inhibitor PHA-739358 is effective against imatinib-resistant BCR-ABL mutations including T315I. Blood. 2008 Apr 15;111(8):4355-64.

[3]. Targeting Aurora Kinases with Danusertib (PHA-739358) Inhibits Growth of Liver Metastases from Gastroenteropancreatic Neuroendocrine Tumors in an Orthotopic Xenograft Model. Clin Cancer Res. 2012 Sep 1;18(17):4621-32. Epub 2012 Jul 2.

N-[5-[(2R)-2-methoxy-1-oxo-2-phenylethyl]-4,6-dihydro-1H-pyrrolo[3,4-c]pyrazol-3-yl]-4-(4-methyl-1-piperazinyl)benzamide is a member of piperazines.
Danusertib has been used in trials studying the treatment of Leukemia.
Danusertib is a small-molecule 3-aminopyrazole derivative with potential antineoplastic activity. Danusertib binds to and inhibits the Aurora kinases, which may result in cell growth arrest and apoptosis in tumor cells in which Aurora kinases are overexpressed. This agent may preferentially bind to and inhibit Aurora B kinase. Aurora kinases, a family of serine-threonine kinases, are important regulators of cellular proliferation and division.

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The emergence of resistance to imatinib (IM) mediated by mutations in the BCR-ABL domain has become a major challenge in the treatment of chronic myeloid leukemia (CML). Here, we report on studies performed with a novel small molecule inhibitor, PHA-739358, which selectively targets Bcr-Abl and Aurora kinases A to C. PHA-739358 exhibits strong antiproliferative and proapoptotic activity against a broad panel of human BCR-ABL-positive and -negative cell lines and against murine BaF3 cells ectopically expressing wild-type (wt) or IM-resistant BCR-ABL mutants, including T315I. Pharmacologic synergism of IM and PHA-739358 was observed in leukemia cell lines with subtotal resistance to IM. Treatment with PHA-739358 significantly decreased phosphorylation of histone H3, a marker of Aurora B activity and of CrkL, a downstream target of Bcr-Abl, suggesting that PHA-739358 acts via combined inhibition of Bcr-Abl and Aurora kinases. Moreover, strong antiproliferative effects of PHA-739358 were observed in CD34(+) cells derived from untreated CML patients and from IM-resistant individuals in chronic phase or blast crisis, including those harboring the T315I mutation. Thus, PHA-739358 represents a promising new strategy for treatment of IM-resistant BCR-ABL-positive leukemias, including those harboring the T315I mutation. Clinical trials investigating this compound in IM-resistant CML have recently been initiated.[2]

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Purpose: Aurora kinases play a crucial role in cell-cycle control. Uncontrolled expression of aurora kinases causes aneuploidy and tumor growth. As conservative treatment options for advanced gastroenteropancreatic neuroendocrine tumors (GEP-NET) are disappointing, aurora kinases may be an interesting target for novel therapeutic strategies. Experimental design: Human GEP-NETs were tested for aurora kinase expression. The efficacy of the new aurora kinase inhibitor danusertib was evaluated in two human GEP-NET cell lines (BON1 and QGP) in vitro and in vivo. Results: The majority of ten insulinomas and all 33 nonfunctional pancreatic or midgut GEP-NETs expressed aurora A despite a mostly high degree of cell differentiation. Both human GEP-NET cell lines expressed aurora kinase A and B, and high Ser10 phosphorylation of histone H3 revealed increased aurora B activity. Remarkably, danusertib led to cell-cycle arrest and completely inhibited cell proliferation of the GEP-NET cells in vitro. Decreased phosphorylation of histone H3 indicated effective aurora B inhibition. In a subcutaneous murine xenograft model, danusertib significantly reduced tumor growth in vivo compared with controls or mice treated with streptozotocine/5-fluorouracil. As a consequence, decreased levels of tumor marker chromogranin A were found in mouse serum samples. In a newly developed orthotopic model for GEP-NET liver metastases by intrasplenic tumor cell transplantation, dynamic MRI proved significant growth inhibition of BON1- and QGP-derived liver metastases. Conclusions: These results show that danusertib may impose a new therapeutic strategy for aurora kinase expressing metastasized GEP-NETs.[3]

C26H30N6O3

474.55

474.237

C, 65.80; H, 6.37; N, 17.71; O, 10.11

827318-97-8

11442891

Typically exists as Off-white to yellow solids at room temperature

1.3±0.1 g/cm3

664.1±55.0 °C at 760 mmHg

355.5±31.5 °C

0.0±2.0 mmHg at 25°C

1.663

2.03

2

6

6

35

731

1

N(C1=NNC2CN(CC1=2)C(=O)[C@@H](C1C=CC=CC=1)OC)C(C1C=CC(N2CCN(C)CC2)=CC=1)=O

XKFTZKGMDDZMJI-HSZRJFAPSA-N

InChI=1S/C26H30N6O3/c1-30-12-14-31(15-13-30)20-10-8-19(9-11-20)25(33)27-24-21-16-32(17-22(21)28-29-24)26(34)23(35-2)18-6-4-3-5-7-18/h3-11,23H,12-17H2,1-2H3,(H2,27,28,29,33)/t23-/m1/s1

N-[5-[(2R)-2-methoxy-2-phenylacetyl]-4,6-dihydro-1H-pyrrolo[3,4-c]pyrazol-3-yl]-4-(4-methylpiperazin-1-yl)benzamide

Danusertib; 827318-97-8; PHA-739358; Danusertib (PHA-739358); (R)-N-(5-(2-methoxy-2-phenylacetyl)-1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazol-3-yl)-4-(4-methylpiperazin-1-yl)benzamide; PHA 739358; Danusertib [INN]; CHEMBL402548;

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: ≥ 2.08 mg/mL (4.38 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 20.8 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: ≥ 2.08 mg/mL (4.38 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 20.8 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: ≥ 2.08 mg/mL (4.38 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 1% DMSO +30% polyethylene glycol+1% Tween 80 : 30mg/mL

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