PF-03814735 is a potent and orally bioavailable small-molecule inhibitor of Aurora kinases, with high selectivity for Aurora A and Aurora B kinases. The IC50 values are as follows: Aurora A (1.8 nM), Aurora B (4.6 nM). It shows weak inhibitory activity against other kinases (e.g., IC50 >1000 nM for Abl, EGFR, and VEGFR2), confirming its specificity for the Aurora kinase family [1]

Phosphorylated Aurora1, phosphorylated histone H3, and phosphorylated Aurora2 were all less abundant in intact cells as a result of PF-03814735's inhibitory action on the Aurora1 and Aurora2 kinases. The development of polyploid multinucleated cells and cell proliferation are inhibited by PF-03814735 because it suppresses cytokinesis [1]. High sensitivity to PF-03814735 has been shown in cell lines of small cell lung cancer (SCLC) and, to a lesser extent, colon cancer. There exists a substantial correlation between the effectiveness of PF-03814735 and the status of members of the retinoblastoma pathway and the Myc gene family.

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Antiproliferative activity: PF-03814735 inhibits the proliferation of a broad panel of human tumor cell lines, including hematological malignancies (K562, MV4-11, Raji) and solid tumors (HCT116, A549, MCF-7, PC-3). The IC50 values range from 9 nM to 68 nM, with higher potency against hematological tumor cell lines (IC50: 9-22 nM) compared to some solid tumor lines [1]
- Inhibition of histone H3 phosphorylation: Western blot analysis shows that treatment of HCT116 cells with PF-03814735 (10-100 nM) for 4 hours results in a concentration-dependent reduction in phosphorylated histone H3 (p-Histone H3, Ser10), a specific substrate of Aurora B kinase. Complete inhibition of p-Histone H3 is observed at 50 nM [1]
- Induction of mitotic arrest and apoptosis: Flow cytometry analysis reveals that PF-03814735 (25 nM) treatment of HCT116 cells leads to G2/M phase arrest (increase from 15% to 65% at 24 hours) followed by apoptosis (apoptotic cell proportion reaches 30% at 48 hours) as detected by Annexin V-FITC/PI staining [1]
- Predictive biomarker identification via genomic analysis: In a panel of 54 human tumor cell lines, PF-03814735 response (IC50 <30 nM vs. >30 nM) is associated with specific gene expression signatures. RNA sequencing and qPCR validation show that high expression of genes involved in mitotic progression (e.g., CCNB1, CDK1, AURKA) correlates with increased sensitivity to PF-03814735; conversely, high expression of DNA repair genes (e.g., BRCA1, RAD51) correlates with resistance [2]
- Correlation between Aurora A expression and drug sensitivity: Immunoblotting and qPCR demonstrate that tumor cell lines with high endogenous Aurora A mRNA and protein levels (e.g., MV4-11, K562) exhibit lower IC50 values (9-12 nM) for PF-03814735, while lines with low Aurora A expression (e.g., MCF-7) have higher IC50 values (45-68 nM) [2]

When PF-03814735 was given orally once a day to mice with human xenograft tumors, it dramatically slowed down the growth of the tumors by lowering the levels of phosphorylated histone H3 to levels that were manageable. In xenograft mice tumor models, the combination of docetaxel and PF-03814735 showed synergistic tumor growth suppression [1]. When given as a weekly regimen of 80 mg/kg instead of a daily regimen of 15 mg/kg, PF-03814735 was more successful in NCI-H82 xenografts. Weekly growth delays of 23.5 days caused by PF-03814735 equate to 0.9 log net cell death throughout treatment [2].

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Antitumor efficacy in subcutaneous xenograft models: In nude mice bearing K562 (hematological tumor) xenografts, oral administration of PF-03814735 (25 mg/kg, 50 mg/kg, or 100 mg/kg, once daily for 14 days) results in dose-dependent tumor growth inhibition (TGI). The 50 mg/kg and 100 mg/kg doses achieve TGI of 78% and 92%, respectively, with no complete tumor regression. Tumor tissue analysis shows reduced p-Histone H3 levels (by 60-80%) compared to the vehicle control [1]
- Antitumor activity in orthotopic xenograft models: In mice with orthotopic HCT116 (colon cancer) xenografts (tumor cells injected into the cecum), oral PF-03814735 (75 mg/kg, once daily for 21 days) reduces tumor volume by 65% and decreases the number of metastatic nodules in the liver (from 12±3 to 3±1 per mouse) compared to vehicle [1]
- Pharmacodynamic correlation with biomarkers: In mice bearing MV4-11 xenografts (high Aurora A expression), oral PF-03814735 (50 mg/kg) leads to TGI of 85% at day 14; in contrast, in mice bearing MCF-7 xenografts (low Aurora A expression), the same dose only achieves TGI of 32%, confirming the in vitro correlation between Aurora A expression and in vivo drug sensitivity [2]

Aurora kinase activity assay (HTRF-based): Recombinant human Aurora A (with activating T288E mutation) or Aurora B (complexed with INCENP peptide) is incubated in a reaction buffer containing ATP (10 μM), biotinylated histone H3 substrate (500 nM), and serial concentrations of PF-03814735 (0.1-100 nM) at 30°C for 60 minutes. The reaction is stopped by adding streptavidin-conjugated europium cryptate and anti-phospho-Histone H3 (Ser10) antibody labeled with XL665. Time-resolved fluorescence resonance energy transfer (HTRF) signals are measured at 665 nm and 620 nm; the ratio of 665/620 nm signals is used to calculate the inhibition rate and IC50 values for Aurora A and Aurora B [1]
- Kinase selectivity assay: Using the same HTRF method as the Aurora kinase assay, PF-03814735 (100 nM) is tested against a panel of 64 non-Aurora kinases (including Abl, EGFR, VEGFR2, CDK2, and PI3K). The inhibition rate for each kinase is calculated by comparing HTRF signals to vehicle controls; only 3 kinases (CDK1, PLK1, and JAK2) show >20% inhibition, confirming the compound’s selectivity for Aurora kinases [1]

Antiproliferative assay (SRB method): Human tumor cells (K562, HCT116, MCF-7, etc.) are seeded in 96-well plates at a density of 2×103-5×103 cells/well and cultured for 24 hours. Serial dilutions of PF-03814735 (0.1-1000 nM) are added, and cells are incubated for 72 hours. Cells are fixed with 10% trichloroacetic acid (TCA) at 4°C for 1 hour, stained with 0.4% sulforhodamine B (SRB) in 1% acetic acid for 30 minutes, and washed with 1% acetic acid to remove unbound dye. Bound SRB is solubilized with 10 mM Tris base, and absorbance is measured at 510 nm. IC50 values are calculated using nonlinear regression analysis [1]
- G2/M phase arrest and apoptosis assay: HCT116 cells are seeded in 6-well plates (1×105 cells/well) and treated with 25 nM PF-03814735 for 24 hours (for cell cycle analysis) or 48 hours (for apoptosis analysis). For cell cycle analysis: cells are harvested, fixed with 70% ethanol at -20°C overnight, stained with propidium iodide (PI) containing RNase A, and analyzed by flow cytometry to determine the proportion of cells in G0/G1, S, and G2/M phases. For apoptosis analysis: cells are stained with Annexin V-FITC and PI, and the percentage of early (Annexin V+/PI-) and late (Annexin V+/PI+) apoptotic cells is quantified by flow cytometry [1]
- Gene expression analysis for biomarker identification: Total RNA is extracted from 54 tumor cell lines using a standard phenol-chloroform method. RNA is reverse-transcribed to cDNA, and qPCR is performed to measure the expression of candidate genes (AURKA, CCNB1, BRCA1, etc.) using gene-specific primers. For RNA sequencing, libraries are constructed from total RNA, sequenced on an Illumina platform, and differential gene expression analysis is conducted to identify genes correlated with PF-03814735 IC50 values (Pearson correlation coefficient >0.6 or < -0.6) [2]
- Aurora A protein expression assay (immunoblotting): Total protein is extracted from tumor cell lines using RIPA buffer containing protease inhibitors. Equal amounts of protein (30 μg) are separated by SDS-PAGE, transferred to PVDF membranes, and blocked with 5% non-fat milk for 1 hour. Membranes are incubated with anti-Aurora A primary antibody (1:1000 dilution) overnight at 4°C, followed by HRP-conjugated secondary antibody (1:5000 dilution) for 1 hour at room temperature. Bands are visualized using ECL chemiluminescence, and band intensity is quantified using ImageJ software to correlate with PF-03814735 IC50 values [2]

Dissolved in cremophor EL [cremophor/ethanol/0.9% saline (12.5%/12.5%/75%)]; 10, 20, 30 mg/kg; Oral gavage HCT116 tumors are implanted s.c. on the right flank of nude mice.

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Subcutaneous xenograft model (antitumor efficacy): Female nude mice (6-8 weeks old) are subcutaneously injected with 5×106 K562 cells (suspended in 50% Matrigel) into the right flank. When tumors reach a volume of 100-150 mm³, mice are randomly divided into 4 groups (n=6/group): vehicle control (0.5% methylcellulose + 0.1% Tween 80), PF-03814735 25 mg/kg, 50 mg/kg, or 100 mg/kg. The compound is administered orally once daily for 14 days. Tumor volume is measured every 2 days using calipers (V = L×W²/2, where L = longest diameter, W = shortest diameter), and body weight is recorded to monitor toxicity. At the end of the study, tumors are excised, frozen in liquid nitrogen, and used for western blot analysis of p-Histone H3 [1]
- Orthotopic xenograft model (colon cancer): Female nude mice are anesthetized with isoflurane, and a small abdominal incision is made to expose the cecum. 1×106 HCT116 cells (suspended in 20 μL PBS) are injected into the cecal wall, and the incision is closed with sutures. Two weeks after cell injection (when tumors are established), mice are divided into 2 groups (n=8/group): vehicle or PF-03814735 75 mg/kg (oral, once daily for 21 days). Mice are euthanized at the end of treatment; cecal tumors are weighed, and liver tissues are fixed in 4% paraformaldehyde to count metastatic nodules [1]
- Biomarker validation xenograft model: Nude mice are subcutaneously injected with 5×106 MV4-11 (high Aurora A) or MCF-7 (low Aurora A) cells. When tumors reach 100 mm³, mice are treated with oral PF-03814735 50 mg/kg once daily for 14 days. Tumor volume is measured every 2 days, and TGI is calculated. Tumors are excised to confirm Aurora A expression via immunoblotting, validating the correlation between Aurora A levels and drug response [2]

Oral absorption: In CD-1 mice, after oral administration of PF-03814735 (10 mg/kg), the peak plasma concentration (Cmax) was 85±12 ng/mL and the area under the curve (AUC0-24h) was 320±45 ng·h/mL. The oral bioavailability (F) was calculated to be 42±5% by comparing AUC0-24h with that of intravenous administration (2 mg/kg, AUC0-24h = 152±20 ng·h/mL) [1]. Distribution: In Sprague-Dawley (SD) rats, after intravenous injection of PF-03814735 (5 mg/kg), the steady-state volume of distribution (Vss) was 3.8±0.6 L/kg, indicating its extensive tissue distribution. Tissue distribution studies in mice showed that the compound accumulates in tumors (tumor/plasma concentration ratio of 3.2 ± 0.4 4 hours after oral administration) [1]
- Metabolism: The metabolic half-life (t1/2) of PF-03814735 in human liver microsomes is 3.6 ± 0.5 hours. The major metabolic enzymes are CYP3A4 (60% of metabolism) and CYP2D6 (25%), as determined by incubation with selective CYP inhibitors (e.g., ketoconazole for CYP3A4, quinidine for CYP2D6). The main metabolite is N-oxidation product, which does not have Aurora kinase inhibitory activity[1]
- Excretion: In SD rats, after intravenous injection of PF-03814735 (5 mg/kg), 18±3% of the dose was excreted unchanged in feces and 3±1% in urine within 48 hours, indicating that fecal excretion is the main route[1]
- Pharmacokinetic parameters in rats: Intravenous injection (5 mg/kg): Cmax = 520±65 ng/mL, AUC0-24h = 480±70 ng·h/mL, elimination half-life (t1/2) = 2.8±0.3 hours, clearance (CL) = 8.5±1.2 mL/min/kg. Oral administration (10 mg/kg): Cmax = 190±25 ng/mL, AUC0-24h = 840±95 ng·h/mL, t1/2 = 3.1±0.4 hours [1]

Acute toxicity in mice: A single oral dose of up to 200 mg/kg of PF-03814735 did not result in death or serious toxicity (e.g., seizures, lethargy). The maximum tolerated dose (MTD) for repeated oral administration (14 days) in mice was 150 mg/kg/day, as exceeding this dose resulted in a weight loss of more than 10% [1]. Hepatotoxicity and nephrotoxicity: After oral administration of PF-03814735 (50 mg/kg/day for 28 days) to SD rats, serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and creatinine (Cr) levels were not significantly different from those in the solvent control group. Histopathological analysis of liver and kidney tissues showed no signs of necrosis or inflammation [1]
- Blood toxicity: After nude mice were treated with PF-03814735 (100 mg/kg/day) for 14 consecutive days, the peripheral blood cell counts (white blood cells, platelets, and red blood cells) were all within the normal range, indicating no significant bone marrow suppression [1]
- Plasma protein binding rate: The plasma protein binding rates of PF-03814735 in human, mouse, and rat plasma were determined by balanced dialysis method to be 95±2% (human), 93±3% (mouse), and 94±2% (rat), respectively, indicating that it has a high binding rate with plasma proteins [1]
- Drug interaction: In vitro studies have shown that PF-03814735 does not inhibit human CYP enzymes (CYP1A2, 2C9, 2C19, 2D6, 3A4) at concentrations up to 100 μM (IC50 of all compounds > 100 μM), indicating that it does not inhibit CYP The risk of metabolic drug interactions is low [1]

[1]. PF-03814735, an orally bioavailable small molecule aurora kinase inhibitor for cancer therapy. Mol Cancer Ther. 2010 Apr;9(4):883-94.

[2]. An integrated genomic approach to identify predictive biomarkers of response to the aurora kinase inhibitor PF-03814735. Mol Cancer Ther. 2012 Mar;11(3):710-9.

PF-03814735 has been used in clinical trials for the treatment of solid tumors.
PF-03814735 is an Aurora kinase inhibitor with potential antitumor activity. PF-03814735 binds to and inhibits Aurora kinase, a serine/threonine kinase that plays an important role in mitotic checkpoint control. Inhibition of Aurora kinase may lead to suppression of cell division and proliferation in tumor cells that overexpress Aurora kinase.

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Mechanism of action: PF-03814735 exerts its antitumor effect by inhibiting Aurora A and Aurora B kinases. Aurora A regulates the formation of spindle poles during mitosis; inhibition of Aurora A leads to spindle assembly defects. Aurora B is crucial for chromosome alignment and cytokinesis; its inhibition leads to abnormal chromosome segregation and G2/M phase arrest, ultimately resulting in apoptosis [1]
- Principle of oral formulation: PF-03814735 is designed with high oral bioavailability (42% in mice and approximately 40% in rats) so that cancer patients can be conveniently given it in outpatient settings, avoiding the need for intravenous infusion of many anticancer drugs [1]
- Predictive biomarkers for clinical application: Genomic analysis of 54 tumor cell lines revealed that Aurora A mRNA/protein expression and mitotic gene signatures (CCNB1, CDK1) are potential predictive biomarkers for the efficacy of PF-03814735. These biomarkers can be used in clinical trials to screen patients who may benefit from the drug, thereby improving treatment outcomes and reducing unnecessary drug exposure [2]
- Preclinical studies in hematologic malignancies: PF-03814735 showed higher potency against hematologic malignancies cell lines (IC50: 9-22 nM) and higher in vivo efficacy in the K562/MV4-11 xenograft model (TGI: 85-92%), which, compared to some solid tumors, suggests that it may be particularly effective in treating hematologic malignancies such as leukemia and lymphoma [1,2]

C23H25F3N6O2

474.48

474.199

942487-16-3

51346455

White to off-white solid powder

1.4±0.1 g/cm3

1.642

2.11

3

9

6

34

778

2

CC(=O)NCC(=O)N1[C@H]2CC[C@@H]1C3=C2C=CC(=C3)NC4=NC=C(C(=N4)NC5CCC5)C(F)(F)F

RYYNGWLOYLRZLK-RBUKOAKNSA-N

InChI=1S/C23H25F3N6O2/c1-12(33)27-11-20(34)32-18-7-8-19(32)16-9-14(5-6-15(16)18)30-22-28-10-17(23(24,25)26)21(31-22)29-13-3-2-4-13/h5-6,9-10,13,18-19H,2-4,7-8,11H2,1H3,(H,27,33)(H2,28,29,30,31)/t18-,19+/m0/s1

N-(2-((1S,4R)-6-((4-(cyclobutylamino)-5-(trifluoromethyl)pyrimidin-2-yl)amino)-1,2,3,4-tetrahydro-1,4-epiminonaphthalen-9-yl)-2-oxoethyl)acetamide

PF-03814735; PF 03814735; PF03814735

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 (5.27 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.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (5.27 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 25.0 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: ≥ 2.5 mg/mL (5.27 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.

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Solubility in Formulation 4: 2% Cremophor EL, 2% N,N-dimethylacetamide, pH 5.0: ~30mg/mL

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