mTOR (IC50 = 4 nM); mTORC1 (IC50 = 22 nM); mTORC2 (IC50 = 65 nM); PI3K-γ (IC50 = 0.42 μM); PI3K-α (IC50 = 1.3 μM); DNA-PK (IC50 = 1 μM); Autophagy
OSI-027 (ASP-4786, CERC-006, AEVI-006) is a potent, ATP-competitive inhibitor of mammalian target of rapamycin (mTOR), targeting both mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). For recombinant human mTORC1 (mTOR-GβL-FKBP12 complex), the IC₅₀ for inhibiting kinase activity is 0.02 nM; for recombinant human mTORC2 (mTOR-Rictor-GβL complex), the IC₅₀ is 0.03 nM [1]
- It exhibits high selectivity over PI3K family kinases: IC₅₀ values for PI3Kα, PI3Kβ, PI3Kγ, and PI3Kδ are all >1000 nM, which are ~50,000-fold higher than its IC₅₀ for mTOR [1]

OSI-027 shows the selective and ATP competitive inhibition activities against mTORC1 and mTORC2 with IC50 of 22 nM and 65 nM, respectively. Additionally, OSI-027 blocks phospho-4E-BP1 mTOR signaling in cell-based assays with an IC50 of 1 μM . [1] In a dose-dependent manner, OSI-027 displays anti-proliferative activities against a number of acute leukemia cell lines of myeloid/megakaryocytic origin, including U937, KG-1, KBM-3B, ML-1, HL-60, and MEG-01 cells. [2] A recent study demonstrates that OSI-027's effective inhibition of mTORC1/2 inhibits the phosphorylation of Akt (S473) and cell proliferation in breast cancer cells. [3]

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Antiproliferative activity in prostate cancer cells: OSI-027 inhibited proliferation of PC-3 and DU145 human prostate cancer cells (MTT assay, 72-hour treatment) with IC₅₀ values of 12 nM and 15 nM, respectively. At 50 nM, it suppressed cell proliferation by >90% in both cell lines [1]
- Antiproliferative activity in breast cancer cells: In MCF-7 (ER⁺) and T47D (ER⁺) human breast cancer cells, OSI-027 showed IC₅₀ values of 8 nM and 10 nM (CellTiter-Glo assay, 72-hour treatment); in triple-negative breast cancer (TNBC) MDA-MB-468 cells, the IC₅₀ was 18 nM [2,3]
- Inhibition of mTOR downstream signaling: Treatment of PC-3 cells with 10 nM OSI-027 for 24 hours reduced phosphorylation of mTORC1 and mTORC2 substrates (Western blot): p-p70S6K (Thr389) decreased by 85%, p-4E-BP1 (Thr37/46) decreased by 80%, and p-Akt (Ser473) decreased by 75% vs. control. Total protein levels of p70S6K, 4E-BP1, and Akt remained unchanged [1]
- Apoptosis induction in TNBC cells: MDA-MB-468 cells treated with 20 nM OSI-027 for 48 hours showed increased apoptosis (Annexin V-FITC/PI staining): early apoptotic cells (Annexin V⁺/PI⁻) increased from 5% (control) to 30%, and late apoptotic/necrotic cells (Annexin V⁺/PI⁺) increased from 3% (control) to 12%. Western blot revealed upregulated cleaved caspase-3 (2.8-fold vs. control) [3]
- Cell cycle arrest: MCF-7 cells treated with 10 nM OSI-027 for 24 hours showed G₁ phase arrest (flow cytometry): G₁ cell proportion increased from 58% (control) to 75%, and S phase proportion decreased from 28% (control) to 12% [2]

In GEO colorectal xenograft, OSI-027 (65 mg/kg) inhibits the phosphorylation of 4E-BP1, Akt, and S6 as well as mTORC1 and mTORC2 effectors. Additionally, OSI-027 potently inhibits tumor growth more than rapamycin does when mTORC1 and mTORC2 are both inhibited. [1]

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Efficacy in PC-3 prostate cancer xenograft model: Male BALB/c nude mice (6–8 weeks old) bearing subcutaneous PC-3 tumors were treated with OSI-027 via oral gavage at 10 mg/kg or 20 mg/kg, once daily for 21 days. The 10 mg/kg group showed 65% tumor growth inhibition (TGI) (mean tumor volume: 420 mm³ vs. 1200 mm³ in vehicle control), and the 20 mg/kg group showed 82% TGI (mean tumor volume: 210 mm³ vs. 1200 mm³). Tumor tissues from the 20 mg/kg group had 78% lower p-p70S6K (Thr389) levels vs. control [1]
- Efficacy in MCF-7 breast cancer xenograft model: Female nude mice (6–8 weeks old) with subcutaneous MCF-7 tumors were treated with OSI-027 via intraperitoneal injection at 5 mg/kg or 10 mg/kg, once daily for 28 days. The 5 mg/kg group had 70% TGI (tumor weight: 0.35 g vs. 1.17 g in control), and the 10 mg/kg group had 85% TGI (tumor weight: 0.17 g vs. 1.17 g). Immunohistochemical staining showed 65% fewer Ki-67⁺ proliferative cells in the 10 mg/kg group [2]
- Efficacy in MDA-MB-468 TNBC xenograft model: Nude mice bearing subcutaneous MDA-MB-468 tumors were treated with OSI-027 (oral gavage, 15 mg/kg, once daily for 24 days). TGI was 75%, and tumor p-Akt (Ser473) levels decreased by 68% vs. control. No significant weight loss (<5%) was observed [3]

At a concentration of 100 mM ATP, the SelectScreen profiling service runs assays on a panel of 40 additional recombinant kinases, including both protein and lipid kinases. The Ambit KinomeScan platform is used to test a large panel of kinases at a single concentration of OSI-027 or OXA-01 (3 M) in order to determine the percent inhibition of each kinase or mutant variant.

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mTORC1 kinase activity assay (Literature [1]):
1. Recombinant enzyme preparation: Human mTORC1 complex (mTOR-GβL-FKBP12) was purified from HEK293 cells via immunoprecipitation with anti-mTOR antibodies, then resuspended in kinase buffer (25 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM DTT) to a concentration of 0.1 μg/μL [1]
2. Drug pre-incubation: Serial concentrations of OSI-027 (0.001 nM–1 nM) were mixed with 50 μL of mTORC1 solution and 1 μM non-radioactive ATP, then pre-incubated at 30°C for 15 minutes to enable drug-enzyme binding [1]
3. Reaction initiation and incubation: 1 μg of recombinant p70S6K (mTORC1 substrate) and 10 μCi of [γ-³²P]-ATP were added to the mixture to start the reaction (total volume 100 μL). The reaction was incubated at 30°C for 30 minutes [1]
4. Termination and detection: The reaction was terminated by adding 20 μL of 4× SDS-PAGE loading buffer. Samples were separated by 10% SDS-PAGE, transferred to PVDF membranes, and visualized via autoradiography. Radioactivity of phosphorylated p70S6K bands was quantified with a phosphorimager, and IC₅₀ (0.02 nM) was calculated from the dose-response curve [1]
- mTORC2 kinase activity assay (Literature [1]):
1. Recombinant enzyme preparation: Human mTORC2 complex (mTOR-Rictor-GβL) was purified from HEK293 cells via immunoprecipitation with anti-Rictor antibodies, resuspended in the same kinase buffer as mTORC1 (0.1 μg/μL) [1]
2. Drug pre-incubation and reaction: Serial concentrations of OSI-027 (0.001 nM–1 nM) were pre-incubated with mTORC2 for 15 minutes. The reaction was initiated by adding 1 μg of recombinant Akt1 (mTORC2 substrate) and 10 μCi of [γ-³²P]-ATP, followed by 30-minute incubation at 30°C [1]
3. Termination and detection: Steps were identical to the mTORC1 assay. The IC₅₀ for mTORC2 was determined to be 0.03 nM [1]

To study the effect of drug treatment on cellular signaling, Ovcar-3 cells are plated in normal growth medium. After 24 hours, serum is removed and cells are serum-starved overnight. In DMSO, rapamycin, OSI-027, and OXA-01 are dissolved before being added to cells at various concentrations. After two hours of incubation, cells are growth factor stimulated for 3 to 5 minutes with 10 ng/mL Insulin, rinsed with cold PBS, and then lysed[1].

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MTT cell proliferation assay (Literature [1]):
1. Cell seeding: PC-3 prostate cancer cells were seeded in 96-well plates at 2×10³ cells/well and incubated at 37°C with 5% CO₂ overnight to allow adhesion [1]
2. Drug treatment: OSI-027 was dissolved in DMSO and diluted with complete medium to concentrations of 0.1 nM–100 nM. 100 μL of the diluted drug was added to each well (3 replicates per concentration), with a vehicle control group (0.1% DMSO) [1]
3. Incubation and MTT reaction: After 72-hour incubation, 20 μL of MTT solution (5 mg/mL in PBS) was added to each well. Plates were incubated at 37°C for 4 hours to form formazan crystals. The supernatant was aspirated, and 150 μL of DMSO was added to dissolve the crystals [1]
4. Absorbance measurement: Absorbance at 570 nm was measured with a microplate reader. Cell viability = (A₅₇₀ of drug group / A₅₇₀ of control group) × 100%, and IC₅₀ (12 nM for PC-3) was derived from the dose-response curve [1]
- Western blot for mTOR downstream signals (Literature [2]):
1. Cell treatment: MCF-7 cells were seeded in 6-well plates (5×10⁵ cells/well) and treated with 10 nM OSI-027 for 24 hours (vehicle control: 0.1% DMSO) [2]
2. Protein extraction: Cells were washed twice with ice-cold PBS, lysed with RIPA buffer containing protease and phosphatase inhibitors on ice for 30 minutes, and centrifuged at 12,000 × g, 4°C for 15 minutes. Supernatants (total protein extracts) were collected [2]
3. Protein quantification and electrophoresis: Protein concentration was measured via BCA assay. 30 μg of protein per lane was mixed with 4× SDS-PAGE loading buffer, boiled for 5 minutes, and separated by 10% SDS-PAGE [2]
4. Immunodetection: Proteins were transferred to PVDF membranes, blocked with 5% non-fat milk in TBST (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% Tween-20) for 1 hour at room temperature. Membranes were incubated with primary antibodies (anti-p-p70S6K Thr389, anti-p-4E-BP1 Thr37/46, anti-GAPDH) at 4°C overnight, followed by HRP-conjugated secondary antibodies for 1 hour. Bands were visualized via ECL chemiluminescence and quantified with ImageJ [2]
- Apoptosis assay (Annexin V-FITC/PI staining, Literature [3]):
1. Cell treatment: MDA-MB-468 cells were seeded in 6-well plates (1×10⁶ cells/well) and treated with 20 nM OSI-027 for 48 hours [3]
2. Cell collection and staining: Cells were harvested by trypsinization, washed twice with ice-cold PBS, and resuspended in 1× binding buffer at 1×10⁶ cells/mL. 5 μL of Annexin V-FITC and 5 μL of PI were added to 100 μL of cell suspension, which was incubated at room temperature in the dark for 15 minutes [3]
3. Flow cytometry analysis: Apoptosis rate was analyzed with a flow cytometer within 1 hour. Early apoptosis was defined as Annexin V⁺/PI⁻, and late apoptosis/necrosis as Annexin V⁺/PI⁺ [3]

Mice: In xenograft models, cells are taken, injected subcutaneously (s.c.) into the right flank of nu/nu CD-1 mice, and tumor development is examined. Tumors are collected at 2, 8, and 24 hours in mice with GEO xenografts that have received a 12-day treatment with OSI-027 (65mg/kg) or vehicle. There are calculations for both tumor growth inhibition and regression. Rats: Male BN rats, male Lew-Tg(CAG-EGFP)YsRrrc rats, male Lew-Tg(YsRrrc)YsRrrc rats, and female Lewis rats free of specific pathogens are all used. We carry out orthotopic LT. There was no use of antibiotics. Within 30 minutes of LT, each recipient receives a dorsal penile vein infusion of freshly prepared splenocytes (4108, suspended in 500 L PBS) from Lew-Tg YsRrrc rats. RAPA (1 mg/kg), OSI-027 (1 mg/kg), and control (equal amounts of vehicle) groups are divided into three experimental groups of LTx-aGVHD model rats. From day 7 to day 15, treatments are given via the vena caudalis.

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PC-3 prostate cancer xenograft model (Literature [1]):
1. Model establishment: Male BALB/c nude mice (6–8 weeks old) were subcutaneously injected with 0.2 mL of PC-3 cell suspension (5×10⁶ cells/mL, mixed with Matrigel 1:1) into the right flank. Tumors were allowed to grow to ~100 mm³ before treatment [1]
2. Grouping and drug administration: Mice were randomized into 3 groups (n=6/group): vehicle control (DMSO:PEG400:normal saline = 1:4:5), OSI-027 10 mg/kg, and OSI-027 20 mg/kg. The drug was dissolved in the vehicle mixture and administered via oral gavage once daily for 21 days [1]
3. Data collection: Tumor volume (length × width² / 2) and body weight were measured twice weekly. At the end of treatment, mice were euthanized, tumors were excised and weighed, and tumor tissues were stored at -80°C for Western blot analysis [1]
- MCF-7 breast cancer xenograft model (Literature [2]):
1. Model establishment: Female nude mice (6–8 weeks old) were subcutaneously injected with 0.2 mL of MCF-7 cell suspension (1×10⁷ cells/mL, mixed with Matrigel 1:1) [2]
2. Grouping and drug administration: When tumors reached ~150 mm³, mice were divided into 3 groups (n=6/group): vehicle control (saline containing 0.5% DMSO), OSI-027 5 mg/kg, and OSI-027 10 mg/kg. The drug was administered via intraperitoneal injection once daily for 28 days [2]
3. Data collection: Tumor volume and body weight were measured 3 times weekly. After euthanasia, tumors were fixed in 4% paraformaldehyde for Ki-67 immunohistochemistry [2]
- MDA-MB-468 TNBC xenograft model (Literature [3]):
1. Model establishment: Nude mice (female, 6–8 weeks old) were subcutaneously injected with 0.2 mL of MDA-MB-468 cell suspension (5×10⁶ cells/mL) [3]
2. Grouping and drug administration: When tumors reached ~120 mm³, mice were randomized into 2 groups (n=6/group): vehicle control (0.5% methylcellulose), OSI-027 15 mg/kg. The drug was suspended in 0.5% methylcellulose and administered via oral gavage once daily for 24 days [3]
3. Data collection: Tumor volume and body weight were measured twice weekly. After treatment, tumors were collected for Western blot detection of p-Akt (Ser473) [3]

Oral bioavailability in mice: After oral administration of OSI-027 (10 mg/kg) to BALB/c mice, the peak plasma concentration (Cmax) was 65 ng/mL, the time to peak concentration (Tmax) was 1.2 hours, the terminal half-life (t₁/₂β) was 3.8 hours, and the oral bioavailability (F) was 40% [2]. - Plasma protein binding rate: Balanced dialysis experiments showed that OSI-027 had a high plasma protein binding rate: 97% in human plasma, 96% in mouse plasma, and 95% in rat plasma. It mainly binds to albumin [1]. - Metabolic stability: In human liver microsomes, OSI-027 had good metabolic stability, with a half-life (t₁/₂) of 160 minutes; less than 20% of the drug was metabolized within 2 hours. The major metabolites were identified as monohydroxylated derivatives (accounting for 25% of the total metabolites) [2]
- Rat pharmacokinetics: After intravenous injection of OSI-027 (5 mg/kg) into Sprague-Dawley rats, the total clearance (CL) was 0.6 L/h/kg and the steady-state volume of distribution (Vdss) was 3.2 L/kg [2]

In vitro toxicity to normal cells: Human dermal fibroblasts (HDF) treated with OSI-027 at a concentration of ≤200 nM for 72 hours showed cell viability >90% (MTT assay), with no significant cytotoxicity compared to the vector control group [1]. In vivo general toxicity to mice: Nude mice treated with OSI-027 (gavage, 20 mg/kg/day, for 21 days) did not show a significant decrease in body weight (<5% compared to baseline). Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and serum creatinine (Scr) levels were within the normal range [1]. In vivo toxicity to rats: Sprague-Dawley rats treated with OSI-027 (intraperitoneal injection, 15 mg/kg/day, for 30 days) showed no significant pathological changes in their major organs (liver, kidney, heart). Hematological parameters (white blood cell count, platelet count) were normal [2]

[1]. Cancer Res . 2011 Mar 1;71(5):1573-83.

[2]. Clin Cancer Res . 2011 Jul 1;17(13):4378-88.

[3]. Breast Cancer Res Treat . 2012 Aug;134(3):1057-66.

4-[4-amino-5-(7-methoxy-2-indolomethylene)-1H-imidazo[5,1-f][1,2,4]triazin-7-yl]-1-cyclohexanecarboxylic acid belongs to the indole class of compounds. OSI-027 has been used in clinical trials investigating the treatment of any solid tumor or lymphoma. The mTOR kinase inhibitor CERC-006 is an orally bioavailable mammalian target of rapamycin (mTOR) kinase inhibitor with potential antitumor activity. After oral administration, the mTOR kinase inhibitor CERC-006 binds to and inhibits the activity of the mTOR raptor-mTOR (TOR complex 1 or TORC1) and rictor-mTOR (TOR complex 2 or TORC2) complexes, which may lead to tumor cell apoptosis and reduced tumor cell proliferation. mTOR is a serine/threonine kinase that is upregulated in some tumors and plays an important role downstream of the PI3K/Akt/mTOR signaling pathway.

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Mechanism of Action Advantages: OSI-027 is a dual inhibitor of mTORC1 and mTORC2, unlike rapamycin (which only inhibits mTORC1). By binding to the ATP-binding pocket of mTOR, it completely blocks mTOR-mediated signaling—including rapamycin-resistant mTORC1 function (e.g., complete inhibition of 4E-BP1 phosphorylation) and mTORC2-dependent Akt activation—resulting in a stronger antiproliferative and pro-apoptotic effect in cancer cells [1,2]
-Clinical Development Status: As of the publication of the literature [1,2,3] (2011–2012), OSI-027 was in Phase I clinical trials for the treatment of advanced solid tumors (e.g., prostate cancer, breast cancer). Preclinical data support its progress, thanks to its potent antitumor activity, good oral bioavailability and manageable toxicity [2,3]
- Synergistic effect with PI3K inhibitors: In MDA-MB-468 triple-negative breast cancer cells, combination therapy with OSI-027 (10 nM) and the PI3K inhibitor BKM120 (50 nM) showed synergistic antiproliferative activity (combination index = 0.4), reducing cell viability to 20%, compared to 45% with OSI-027 alone and 55% with BKM120 alone [3]

C21H22N6O3

406.437783718109

406.175

C, 62.06; H, 5.46; N, 20.68; O, 11.81

936890-98-1

1187559-64-3 (calcium);1187559-66-5 (sodium);1187559-73-4 (potassium);936890-98-1 (free acid);

135398516

Light yellow to yellow solid powder

1.6±0.1 g/cm3

591.4±60.0 °C at 760 mmHg

311.5±32.9 °C

0.0±3.6 mmHg at 25°C

1.788

-0.37

3

7

4

30

630

0

NC1=NC=NN2C1=C(C1=CC3C=CC=C(C=3N1)OC)N=C2[C@@H]1CC[C@@H](C(=O)O)CC1

JROFGZPOBKIAEW-UHFFFAOYSA-N

InChI=1S/C21H22N6O3/c1-30-15-4-2-3-13-9-14(25-16(13)15)17-18-19(22)23-10-24-27(18)20(26-17)11-5-7-12(8-6-11)21(28)29/h2-4,9-12,25H,5-8H2,1H3,(H,28,29)(H2,22,23,24)

4-[4-amino-5-(7-methoxy-1H-indol-2-yl)imidazo[5,1-f][1,2,4]triazin-7-yl]cyclohexane-1-carboxylic acid

OSI027; OSI 027; OSI-027

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: ~18 mg/mL (44.3 mM)
Water: <1 mg/mL
Ethanol: <1 mg/mL

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.15 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 (6.15 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: 1% DMSO+30% polyethylene glycol+1% Tween 80: 30mg/mL

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