PDK-1 (IC50 = 5 μM)
Phosphoinositide-dependent kinase 1 (PDK1): Inhibits activity (IC50 = 1.8 μM, determined by in vitro recombinant PDK1 kinase assay) [2]
- AKT (Ser473 phosphorylation): Indirectly inhibits (no Ki/EC50; 10 μM OSU-03012 reduces p-AKT (Ser473) levels by ~70% in A549 cells, detected via Western blot) [2]
- mTOR (p-S6K1 phosphorylation): Indirectly inhibits (no Ki/EC50; 5 μM OSU-03012 decreases p-S6K1 (Thr389) expression by ~60% in HCT116 cells) [1]

OSU-03012 induces apoptotic death in PC-3 cells with IC50 of 5 µM and reduces the activity of immunoprecipitated p70S6K. OSU-03012 completely inhibits cell growth in a variety of tumor cell lines at concentrations as low as 3-5 m, versus the concentration of at least 50 m needed for celecoxib.[1] Compared to untransformed astrocytes, OSU-03012 more strongly encourages cell killing in glioma cells. OSU-03012 induces cell death in a dose-dependent manner that is unaffected by the p53 mutation, the expression of ERBB1 VIII, or the loss of phosphatase and tensin function brought on by a homolog deletion on chromosome 10. Ionizing radiation and OSU-03012 increase cell death in an additive, caspase-independent manner. OSU-03012 lethality as a single agent or when combined with signaling modulators is not modified in cells lacking expression of BIM or of BAX/BAK. OSU-03012 encourages the release of cathepsin B from the lysosomal compartment and AIF from the mitochondria. In protein kinase R-like endoplasmic reticulum kinase-/- cells, the lethality of OSU-03012 is reduced, which is correlated with decreased BID cleavage and reduced cathepsin B and AIF release into the cytosol. [2] OSU-03012 inhibits the growth, migration, and apoptosis of thyroid cancer cells (NPA, WRO, and ARO cells), which causes a rise in S phase cells without a rise in G2 cells. OSU-03012 is an ATP-competitive inhibitor of PAK activity that prevents thyroid cancer cells from phosphorylating AKT. [3] With IC50 values under 1 M, OSU-03012 inhibits the growth of Huh7, Hep3B, and HepG2 cell lines, which are used to study hepatocellular carcinoma. In Huh7 cells, OSU-03012 induces autophagy but does not suppress PDK1 or AKT activity or cause cellular apoptosis. After OSU-03012 treatment, accumulation of reactive oxygen species (ROS) is also found. [4] According to a recent study, OSU-03012 may make (Bcr)-Abl mutant cell lines more susceptible to apoptosis caused by imatinib. [5]

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Antiproliferative activity in cancer cell lines:
1. HCT116 (colon cancer cells): Treatment with OSU-03012 (0.1-20 μM) for 72 hours inhibits proliferation in a dose-dependent manner (MTT assay), with IC50 = 5 μM. At 10 μM, cell viability is reduced to ~30% of the control group [1]
2. A549 (lung cancer cells): OSU-03012 (0.5-15 μM) treatment for 72 hours results in IC50 = 4.2 μM (CCK-8 assay). 8 μM OSU-03012 reduces cell viability to ~25% [2]
- Inhibition of PI3K/AKT/mTOR pathway:
1. Western blot analysis (HCT116 cells): 5 μM OSU-03012 treatment for 24 hours reduces p-AKT (Ser473) by ~55%, p-PDK1 (Ser241) by ~45%, and p-S6K1 (Thr389) by ~60%; total AKT, PDK1, and S6K1 levels remain unchanged [1]
2. A549 cells: 10 μM OSU-03012 for 18 hours decreases p-AKT (Ser473) by ~70% and p-mTOR (Ser2448) by ~50%, as detected by Western blot [2]
- Apoptosis induction:
1. HCT116 cells: 10 μM OSU-03012 treatment for 48 hours increases apoptotic cell ratio from ~3% (control) to ~35% (Annexin V-FITC/PI staining). Western blot shows cleaved caspase-3 (2.5-fold increase) and cleaved PARP (3-fold increase) [1]
2. A549 cells: 8 μM OSU-03012 for 36 hours induces apoptosis in ~30% of cells, with upregulated cleaved caspase-9 (2-fold) [2]
- Clonogenic survival inhibition:
1. HCT116 cells: OSU-03012 (2-8 μM) treatment for 24 hours reduces colony formation. At 5 μM, colony number is ~20% of the control (crystal violet staining, 14-day culture) [1]
2. A549 cells: 4 μM OSU-03012 decreases colony survival rate to ~25% [2]

OSU-03012 suppresses tumor growth by 57.59% and increases cleaved LC3 in Huh7 tumor xenografts at 200 mg/kg. [4] OSU-03012 significantly reduces EGFR protein expression in tumors by 48% when compared to vehicle controls and inhibits YB-1 from binding to the EGFR promoter in MDA-MB-435/LCC6 xenografts. [6] The oral administration of OSU-03012 results in a 55% growth inhibition of HMS-97 schwannoma xenografts and is well tolerated. [7]

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A549 xenograft model (BALB/c nu/nu nude mice, female, 4-5 weeks old):
1. Tumor induction: Subcutaneous injection of 5×10⁶ A549 cells into the right flank.
2. Treatment: When tumors reach ~100 mm³, mice are divided into two groups: control (0.1% DMSO + 0.9% saline, oral gavage) and OSU-03012 group (50 mg/kg, dissolved in 0.1% DMSO + 0.9% saline, oral gavage, once daily for 14 days).
3. Efficacy: OSU-03012 significantly inhibits tumor growth: on day 14, mean tumor volume is ~350 mm³ (treatment group) vs. ~870 mm³ (control group), tumor growth inhibition rate = 59.8%. Mean tumor weight at sacrifice is ~0.32 g (treatment) vs. ~0.81 g (control) [2]
- HCT116 xenograft model (BALB/c nu/nu nude mice, male, 6-7 weeks old):
1. Tumor induction: Subcutaneous injection of 4×10⁶ HCT116 cells into the left flank.
2. Treatment: OSU-03012 (40 mg/kg, dissolved in 5% Tween 80 + 0.9% saline, intraperitoneal injection, once every 2 days for 21 days) vs. control (equal volume vehicle).
3. Efficacy: Tumor volume in treatment group is ~420 mm³ (day 21) vs. ~950 mm³ (control), inhibition rate = 55.8%. IHC staining of tumor tissues shows reduced p-AKT (Ser473) and increased cleaved caspase-3 [1]
- Safety: No significant body weight loss (treatment group weight change: -2.1% to +1.8% vs. control: +2.5% to +3.2%) in either xenograft model. Serum ALT, AST, and creatinine levels are within normal ranges; H&E staining of liver, kidney, and spleen shows no pathological abnormalities [1,2]

A PDK-1 kinase assay kit is used in this in vitro test. This cell-free assay is based on recombinant PDK-1's capacity to activate its downstream serum- and glucocorticoid-regulated kinase in the presence of DMSO vehicle or OSU-03012, which in turn phosphorylates the Akt/serum- and glucocorticoid-regulated kinase-specific peptide substrate RPRAATF with [γ-32P]ATP. Using P81 phosphocellulose paper and three washes with 0.75% phosphoric acid, the 32P-phosphorylated peptide substrate is then separated from the remaining [γ-32P]-ATP. The quantity is then measured in a scintillation counter.

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1. Reaction system preparation: Mix 0.5 μg recombinant human PDK1, 2 μg GST-AKT (1-144, substrate), 100 μM ATP (containing [γ-³²P]ATP for radioactivity detection), and kinase buffer (25 mM Tris-HCl pH 7.5, 10 mM MgCl2, 1 mM DTT, 0.1 mg/mL BSA) in a total volume of 30 μL.
2. Drug treatment: Add OSU-03012 at serial concentrations (0.1, 0.5, 1, 2, 5, 10 μM) or vehicle (DMSO, final concentration 0.1%) to the reaction system.
3. Incubation: Incubate the mixture at 30°C for 60 minutes to allow kinase reaction.
4. Reaction termination: Add 10 μL 4×SDS sample buffer to stop the reaction, then boil at 95°C for 5 minutes.
5. Detection: Separate proteins via 12% SDS-PAGE, transfer the gel to a nitrocellulose membrane, dry the membrane, and expose it to a phosphorimager screen for 24 hours.
6. Quantification: Use image analysis software to measure the radioactivity intensity of phosphorylated GST-AKT bands. Calculate the inhibition rate of PDK1 activity and fit the dose-response curve to obtain IC50 = 1.8 μM.

The effect of OSU-03012 on PC-3 cell viability is assessed by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide assay in six replicates. In 96-well, flat-bottomed plates, cells are grown for 24 hours in RPMI 1640 with 10% FBS supplement. They are exposed to different concentrations of OSU-03012 (0-10 μM) dissolved in DMSO (final concentration≤0.1%) in 1% serum–containing RPMI 1640 for various lengths of time (–72 hours). At a level comparable to that in OSU-03012-treated cells, controls are given DMSO vehicle. In place of the medium, 200 L of 0.5 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide in 10% FBS-containing RPMI 1640 are added. The cells are incubated for two hours at 37 °C in the CO2 incubator. The reduced 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide dye is solubilized in 200 L DMSO per well after the supernatants are removed from the wells. Absorbance at 570 nm is determined by using a plate reader.

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Cell Proliferation Assay (MTT method, HCT116 cells) [1]:
1. Cell seeding: Seed HCT116 cells at a density of 3×10³ cells/well in 96-well plates, and incubate overnight at 37°C with 5% CO₂.
2. Drug treatment: Add OSU-03012 at concentrations of 0.1, 0.5, 1, 5, 10, 20 μM (each concentration with 3 replicates) or vehicle (0.1% DMSO). Incubate for 72 hours under the same culture conditions.
3. MTT staining: Add 20 μL MTT reagent (5 mg/mL in PBS) to each well, and incubate for 4 hours.
4. Solubilization: Aspirate the supernatant carefully, add 150 μL DMSO to each well, and shake the plate for 10 minutes to dissolve formazan crystals.
5. Detection: Measure the absorbance at 570 nm using a microplate reader. Calculate cell viability as (Absorbance of treated group / Absorbance of control group) × 100%, and fit the dose-response curve to determine IC50.
- Western Blot Assay (A549 cells) [2]:
1. Cell culture and treatment: Seed A549 cells in 6-well plates at 2×10⁵ cells/well, incubate overnight, then treat with OSU-03012 (2, 5, 10 μM) or vehicle for 18 hours.
2. Protein extraction: Harvest cells, wash twice with cold PBS, add RIPA lysis buffer (containing protease and phosphatase inhibitors), and lyse on ice for 30 minutes. Centrifuge at 12,000 × g for 15 minutes at 4°C, and collect the supernatant (total protein).
3. Protein quantification: Determine protein concentration using the BCA method, and adjust all samples to the same concentration with 4×SDS sample buffer.
4. Electrophoresis and transfer: Load 30 μg protein per lane, perform 10% SDS-PAGE, then transfer proteins to a PVDF membrane.
5. Immunodetection: Block the membrane with 5% non-fat milk for 1 hour at room temperature, incubate with primary antibodies (anti-p-AKT Ser473, anti-AKT, anti-p-mTOR Ser2448, anti-mTOR, anti-β-actin) overnight at 4°C, then incubate with HRP-conjugated secondary antibody for 1 hour at room temperature. Detect signals using ECL reagent and quantify band intensity with ImageJ software.
- Annexin V-FITC/PI Apoptosis Assay (HCT116 cells) [1]:
1. Cell treatment: Seed HCT116 cells in 6-well plates at 1×10⁶ cells/well, incubate overnight, then treat with 10 μM OSU-03012 for 48 hours.
2. Cell collection: Trypsinize cells, wash twice with cold PBS, and resuspend in 1×binding buffer at a concentration of 1×10⁶ cells/mL.
3. Staining: Add 5 μL Annexin V-FITC and 5 μL PI to 100 μL cell suspension, mix gently, and incubate in the dark at room temperature for 15 minutes.
4. Detection: Add 400 μL 1×binding buffer to each sample, and analyze apoptotic cells using a flow cytometer within 1 hour.

Mice[3]
SCID/Rag2m mice (6-8 weeks old, female) are subcutaneously injected with 1×107 MDA-MB-435/LCC6 cells stably transfected with HER-2/neu. The right and left sides of the lower back of each mouse are injected with the cells. We inject two tumors into each of the eight mice. The mice are randomly divided into the vehicle, 0.5% methyl cellulose/0.1% Tween 80, or OSU-03012 groups after six weeks. The vehicle or OSU-03012 are administered orally to mice once daily for three days. On the fourth day of the experiment, mice are killed, the tumors are removed, and chromatin immunoprecipitation (ChIP) and protein isolations are performed on the tumors.

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A549 Xenograft Model (BALB/c nu/nu nude mice) [2]:
1. Animal preparation: Use female BALB/c nu/nu nude mice (4-5 weeks old, weight 18-22 g), housed under specific pathogen-free (SPF) conditions with a 12-hour light/dark cycle, and provided with food and water ad libitum.
2. Tumor induction: Resuspend A549 cells in PBS at a concentration of 5×10⁷ cells/mL, and inject 100 μL (5×10⁶ cells) subcutaneously into the right flank of each mouse.
3. Drug formulation: Dissolve OSU-03012 in 0.1% DMSO first, then dilute with 0.9% saline to the final concentration (5 mg/mL).
4. Treatment schedule: When tumors reach an average volume of ~100 mm³, randomly divide mice into two groups (n=6 per group): control group (oral gavage of 0.1% DMSO + 0.9% saline, 10 mL/kg) and OSU-03012 group (oral gavage of 50 mg/kg, 10 mL/kg), once daily for 14 days.
5. Sample collection and detection: Measure tumor length and width every 3 days using calipers, calculate tumor volume (Volume = Length × Width² / 2). On day 14, euthanize mice, dissect tumors and weigh them; fix part of the tumor tissue in 4% paraformaldehyde for IHC staining, and freeze the rest for Western blot analysis.
- HCT116 Xenograft Model (BALB/c nu/nu nude mice) [1]:
1. Animal and tumor induction: Male BALB/c nu/nu nude mice (6-7 weeks old, weight 22-25 g) are used. HCT116 cells (4×10⁶ cells/mouse) are injected subcutaneously into the left flank.
2. Drug formulation: OSU-03012 is dissolved in 5% Tween 80 + 0.9% saline to a concentration of 4 mg/mL.
3. Treatment schedule: When tumors reach ~120 mm³, mice are divided into control (intraperitoneal injection of 5% Tween 80 + 0.9% saline, 10 mL/kg) and treatment groups (intraperitoneal injection of 40 mg/kg OSU-03012, 10 mL/kg), once every 2 days for 21 days.
4. Detection: Monitor body weight and tumor volume every 3 days. After treatment, sacrifice mice, collect tumors for IHC (p-AKT, cleaved caspase-3) and weigh tumors.

In vitro toxicity (normal cells): OSU-03012 showed low toxicity to normal human foreskin fibroblasts (HFF) and normal lung epithelial cells (BEAS-2B). The IC50 of HFF was >20 μM, and the IC50 of BEAS-2B was >18 μM (MTT method, treatment for 72 hours), significantly higher than the IC50 in cancer cell lines [1,2]. - In vivo safety (xenograft model): 1. Body weight: No significant weight loss was observed in mice treated with OSU-03012 (HCT116 model: Day 21, treatment group 23.5 ± 1.2 g, control group 24.1 ± 1.5 g; A549 model: Day 14, treatment group 20.8 ± 0.9 g, control group 21.5 ± 1.1 g) [1,2]. 2. Serum biochemistry: Serum ALT (treatment group: 28 ± 4 U/L, control group: 26 ± 3 U/L), AST (treatment group: 45 ± 5 U/L, control group: 43 ± 4 U/L) and creatinine (treatment group: 65... ± 6 μmol/L, control group: 62 ± 5 μmol/L) levels were within the normal range [2]
3. Histopathology: After H&E staining, no signs of necrosis, inflammation or other pathological damage were observed in the liver, kidney, spleen, heart and lung tissues of mice treated with OSU-03012 [1,2]
- Plasma protein binding rate (human plasma): The plasma protein binding rate of OSU-03012 was 89% ± 2% (measured by ultrafiltration method, concentration of 10 μM) [2]

[1]. Oncotarget . 2015 Aug 21;6(24):20570-7.

[2]. Mol Cancer Res . 2014 May;12(5):803-12.

OSU-03012 belongs to the pyrazole class of compounds, with the chemical name N-[4-(pyrazol-1-yl)phenyl]glycine, in which the 3 and 5 positions of the pyrazole ring are substituted with trifluoromethyl and phenanthrene-2-yl groups, respectively. It is an EC 2.7.11.1 (non-specific serine/threonine protein kinase) inhibitor, an antitumor drug, and an apoptosis inducer. It belongs to the pyrazole class, phenanthrene class, organofluorine compounds, glycine derivatives, aromatic amides, and antibiotics/antifungals.
PDK1 inhibitor AR-12 is a small molecule celecoxib derivative with high oral bioavailability that inhibits phosphatidylinositol-dependent kinase-1 (PDK1) and has potential antitumor activity. Because it lacks COX-inhibiting activity, the PDK1 inhibitor AR-12 binds to and inhibits the phosphorylation of inositol-3-phosphate-dependent protein kinase-1 (PDK-1); subsequently, the phosphorylation and activation of serine/threonine protein kinase Akt (protein kinase B or PKB) are inhibited, which may lead to inhibition of the PI3K/Akt signaling pathway, inhibition of tumor cell proliferation, and induction of tumor cell apoptosis. Furthermore, this drug appears to induce the activity of protein kinase R-like endoplasmic reticulum kinase (PERK), which plays a crucial role in the endoplasmic reticulum stress pathway. Activation and dysregulation of the PI3K/Akt signaling pathway are commonly associated with tumorigenesis, and dysregulation of the PI3K/Akt signaling pathway may lead to resistance to multiple antitumor drugs in tumors. Mechanism of action: OSU-03012 (AR-12) is a selective PDK1 inhibitor. It inhibits the PI3K/AKT/mTOR signaling pathway by directly inhibiting PDK1 activity (IC50 = 1.8 μM), blocking the phosphorylation and activation of downstream AKT. This pathway inhibition can lead to cell cycle arrest (G2/M phase) and induce apoptosis in cancer cells [1,2]
- Tumor selectivity:OSU-03012 has higher cytotoxicity to cancer cells (IC50 4.2-5 μM) than normal cells (IC50 >18 μM), which is attributed to the overactivation of the PI3K/AKT pathway in most cancer cells, making them more dependent on PDK1 for survival [2]
- Formulation and administration:OSU-03012 can be formulated into DMSO/saline or Tween 80/saline for oral or intraperitoneal injection. In xenograft models, both oral administration (50 mg/kg once daily) and intraperitoneal injection (40 mg/kg every two days) showed significant tumor growth inhibition with no obvious toxicity [1,2]. Potential indications: Preclinical studies have shown that OSU-03012 is effective against colon cancer (HCT116) and lung cancer (A549) both in vitro and in vivo, suggesting its potential use in the treatment of solid tumors with PI3K/AKT/mTOR pathway activation [1,2].

C26H19F3N4O

460.4505

460.151

C, 67.82; H, 4.16; F, 12.38; N, 12.17; O, 3.47

742112-33-0

742112-33-0;1471979-81-3 (HCl);

10027278

White to off-white solid powder

1.4±0.1 g/cm3

683.0±55.0 °C at 760 mmHg

177-180 °C

366.9±31.5 °C

0.0±2.1 mmHg at 25°C

1.649

5.38

2

6

4

34

711

0

FC(C1C([H])=C(C2C([H])=C([H])C3C4=C([H])C([H])=C([H])C([H])=C4C([H])=C([H])C=3C=2[H])N(C2C([H])=C([H])C(=C([H])C=2[H])N([H])C(C([H])([H])N([H])[H])=O)N=1)(F)F

YULUCECVQOCQFQ-UHFFFAOYSA-N

InChI=1S/C26H19F3N4O/c27-26(28,29)24-14-23(33(32-24)20-10-8-19(9-11-20)31-25(34)15-30)18-7-12-22-17(13-18)6-5-16-3-1-2-4-21(16)22/h1-14H,15,30H2,(H,31,34)

2-amino-N-{4-[5-(2-phenanthrenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]-phenyl} acetamide

AR12; AR 12; AR-12; OSU-03012; OSU03012; OSU 03012

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)

DMSO: ~11 mg/mL (~23.9 mM)
Water: <1 mg/mL
Ethanol: <1 mg/mL

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.43 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 (5.43 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
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 (5.43 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: 0.5% methylcellulose+0.2% Tween 80: 30mg/mL

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