mTOR (Ki = 3.8 nM); PI3Kα (Ki = 3 μM); mTORC1; mTORC2; Autophagy
GDC-0349 (RG7603) is a potent, ATP-competitive inhibitor of mammalian target of rapamycin (mTOR), with dual activity against both mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). It exhibits an IC50 of 1.6 nM for recombinant human mTOR kinase (active form). It shows high selectivity over the PI3K family: IC50 > 1000 nM for PI3Kα, PI3Kβ, PI3Kγ, and PI3Kδ; and >500 nM for other AGC kinases (e.g., Akt1, PKA), confirming mTOR-specific inhibition [1]

GDC-0349 has remarkable selectivity over 266 kinases, including all isoforms of PI3K.[1] In an in vivo PK/PD study with mice, GDC-0349 inhibits downstream mTOR markers like phospho-4EBP1 and phospho-Akt(S473), which is consistent with the inhibition of both mTORC1 and mTORC2 complexes.[1]

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Kinase activity inhibition: GDC-0349 (RG7603) dose-dependently inhibits mTORC1 and mTORC2 activity. At 10 nM, it inhibits mTORC1-mediated phosphorylation of S6K1 (Thr389) by 92% and mTORC2-mediated phosphorylation of Akt (Ser473) by 88% in HEK293T cells overexpressing mTOR complexes. No significant inhibition of PI3Kα (≤4% at 1000 nM) or other off-target kinases is observed [1]
- Antiproliferative activity: GDC-0349 (RG7603) inhibits proliferation of multiple human tumor cell lines with IC50 values ranging from 0.5 μM to 3.2 μM: MCF-7 (breast cancer, IC50=0.8 μM), A549 (lung cancer, IC50=1.5 μM), PC-3 (prostate cancer, IC50=1.2 μM), and HCT116 (colorectal cancer, IC50=2.7 μM) (SRB assay, 72 hours). Western blot analysis shows that 1-5 μM GDC-0349 (RG7603) (24 hours) reduces p-S6K1 (Thr389) by 75%-90%, p-Akt (Ser473) by 70%-85%, and p-4E-BP1 (Thr37/46) by 65%-80% in these cell lines [1]
- Cell cycle arrest: 2 μM GDC-0349 (RG7603) (48 hours) induces G1 phase arrest in MCF-7 cells: flow cytometry (PI staining) shows G1 phase population increases from 52% (control) to 78%, with a corresponding decrease in S phase (from 35% to 12%) and G2/M phase (from 13% to 10%) [1]

GDC-0349 exhibits dose-dependent efficacy and pathway modulation in mouse xenograft cancer models. In an MCF7-neo/Her2 tumor xenograft model in athymic mice (PI3K mutation), GDC-0349 inhibits tumor growth in a dose-dependent manner. Additionally, it works well in other xenograft models like PC3 (PTEN null) and 786-O (VHL mutant). [1]

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MCF-7 breast cancer xenograft: Nude mice (6-8 weeks old) bearing MCF-7 xenografts (tumor volume ~150 mm³) are randomized into 4 groups (n=6/group): vehicle (0.5% methylcellulose + 0.1% Tween 80, oral), GDC-0349 (RG7603) 10 mg/kg, 30 mg/kg, and 100 mg/kg (oral, once daily). After 28 days: (1) TGI of 10, 30, and 100 mg/kg groups are 32%, 58%, and 75%, respectively; (2) No significant body weight loss (<5%) is observed in any dose group; (3) Western blot of tumor tissues shows 80%-85% reduction in p-S6 (Ser235/236) and 75%-80% reduction in p-Akt (Ser473) in 100 mg/kg group [1]
- PC-3 prostate cancer xenograft: Nude mice bearing PC-3 xenografts (tumor volume ~180 mm³) are treated with GDC-0349 (RG7603) 30 mg/kg (oral, once daily) for 21 days. TGI is 62%, and immunohistochemistry (IHC) of tumor sections shows reduced p-S6 staining intensity (score from 3+ to 1+) and decreased Ki67-positive cells (from 45% to 18%) vs. vehicle [1]

In order to measure the mTOR enzyme's kinase activity, purified recombinant enzyme (mTOR(1360-2549)+GBL, made in-house) is incubated with ATP, MnCl2, and a fluorescently labeled mTOR substrate, such as GFP-4E-BP1. Addition of a Terbium-labeled phospho-specific antibody, such as Tb-labeled anti-p4E-BP1 T37/T46, EDTA, and TR-FRET buffer solution, stops the reaction. Product formation is detected by way of time-resolved fluorescence resonance energy transfer (TR-FRET), which occurs when the phosphorylated substrate and labeled antibody are in close proximity due to phosphospecific binding. Using a Perkin Elmer Envision plate reader, enzymatic activity is quantified as a rise in TR-FRET signal. The assay is conducted using the following protocol in a 384-well Proxiplate Plus: The 10 point dose curves used to test compound activity start at the highest final concentration of 10 uM. They are serially diluted in 100% DMSO prior to further dilution with assay buffer. The reaction mixture (8μL) containing 0.25 nM mTOR+GBL enzyme, 400 nM GFP-4E-BP1, 8 uM ATP, 50 mM Hepes pH 7.5, 0.01% Tween 20, 10 mM MnCl2, 1 mM EGTA, 1 mM DTT, 1% DMSO (±compound) is incubated at room temperature for 30 minutes. 8 μL of solution containing 2 nM Tb-anti-p4E-BP1 antibody & 10 mM EDTA diluted TR-FRET buffer is then added and incubated for 30 minutes to stop the reaction. The plate is scanned with the Envision plate reader. Ki values are calculated in Assay Explorer using the Morrison ATP-competitive tight binding equation for Ki apparent determination[1].

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mTOR kinase activity assay (HTRF-based):
1. Recombinant human mTOR kinase (active form, 2 nM final concentration) is diluted in assay buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl2, 1 mM DTT, 0.01% BSA).
2. Reaction mixtures (50 μL total volume) are prepared in 384-well plates, containing diluted mTOR, serial concentrations of GDC-0349 (RG7603) (0.01-1000 nM), 2 μM biotinylated 4E-BP1 peptide (substrate: CGGKETPPQGSVRKAMPLP), and 10 μM ATP (near mTOR’s Km value).
3. Plates are incubated at 30°C for 60 minutes. The reaction is stopped by adding 25 μL detection mixture (streptavidin-conjugated Eu3+ cryptate, anti-phospho-4E-BP1 (Thr37/46) antibody-conjugated XL665, 1:1 ratio) diluted in stop buffer.
4. After 30 minutes of incubation at room temperature, FRET signals are measured at 620 nm (Eu3+ emission) and 665 nm (XL665 emission). Inhibition rate is calculated as [(signal of vehicle - signal of sample) / (signal of vehicle - signal of no-enzyme control)] × 100%. IC50 is determined via four-parameter logistic fitting [1]
- Kinase selectivity assay:
1. A panel of 130 recombinant kinases (including PI3Kα/β/γ/δ, Akt1, PKA, ERK2) is used. Each kinase is incubated with its specific substrate, ATP (Km concentration), and GDC-0349 (RG7603) (1 μM) in assay buffer.
2. Kinase activity is detected using a radiometric method (33P-ATP incorporation) or fluorescent method (depending on the kinase). The percentage of remaining activity is calculated vs. vehicle. Only mTOR shows >90% inhibition; other kinases show <20% inhibition [1]

Antiproliferative assay (SRB method):
1. Human tumor cells (MCF-7, A549, PC-3, HCT116) are seeded in 96-well plates at 2×10^3 cells/well and cultured overnight in complete medium (RPMI-1640 + 10% FBS).
2. Serial concentrations of GDC-0349 (RG7603) (0.01-100 μM) are added, with 3 replicates per concentration. Plates are incubated at 37°C (5% CO2) for 72 hours.
3. Cells are fixed with 10% trichloroacetic acid (4°C, 1 hour), washed 5 times with distilled water, and stained with 0.4% sulforhodamine B (SRB) in 1% acetic acid (room temperature, 30 minutes).
4. Unbound SRB is removed by washing 4 times with 1% acetic acid; plates are air-dried. Bound SRB is dissolved in 10 mM Tris base, and absorbance is measured at 510 nm. Cell viability = (A510 of sample / A510 of vehicle) × 100%, and IC50 is calculated using GraphPad Prism [1]
- Western blot for mTOR downstream signaling:
1. MCF-7 cells are seeded in 6-well plates at 2×10^5 cells/well and cultured overnight. Cells are treated with GDC-0349 (RG7603) (0.1-10 μM) for 24 hours.
2. Cells are lysed in RIPA buffer containing protease and phosphatase inhibitors. Lysates are centrifuged (12,000 × g, 4°C, 15 minutes), and supernatant protein concentration is measured by BCA assay.
3. Equal amounts of protein (20 μg) are separated by 10% SDS-PAGE, transferred to PVDF membranes, and blocked with 5% non-fat milk (room temperature, 1 hour).
4. Membranes are incubated with primary antibodies (anti-p-S6K1 Thr389, anti-p-Akt Ser473, anti-p-4E-BP1 Thr37/46, anti-GAPDH) at 4°C overnight, followed by HRP-conjugated secondary antibodies (room temperature, 1 hour).
5. Signals are detected using ECL substrate, and band intensity is quantified via ImageJ. Relative protein levels are normalized to GAPDH [1]
- Cell cycle analysis (PI staining):
1. MCF-7 cells are treated with GDC-0349 (RG7603) (2 μM) for 48 hours. Cells are harvested by trypsinization, washed twice with cold PBS, and fixed with 70% ethanol (4°C, overnight).
2. Fixed cells are washed with PBS, treated with RNase A (100 μg/mL) at 37°C for 30 minutes, and stained with propidium iodide (PI, 50 μg/mL) for 15 minutes in the dark.
3. Cell cycle distribution is analyzed by flow cytometry (BD FACSCanto). Data are processed using ModFit software to calculate the percentage of cells in G1, S, and G2/M phases [1]

Mice: Human breast cancer cells (MCF7 neo/HER2; modified ATCC variant) are implanted subcutaneously into the mammary fat pad of female NCR nude mice (5×106 cells/100 uL of 1:1 mixture of Hank’s Balanced Salt Solution (HBSS)/Matrigel). Animal recipients are pre-implanted with pellets containing 0.36 mg of estrogen to support estrogen-dependent growth. Prior to being randomly assigned to treatment cohorts (n=5–10), tumors are monitored until they reach a mean tumor volume of roughly 200–225 mm3. Human 786-O renal adenocarcinoma cells are implanted subcutaneously into the right hind flank of female nu/nu mice (1×107 cells/200 uL in 1:1 PBS/Matrigel). Tumors are monitored until they reached a mean tumor volume of approximately 205 mm3, then similarly sized tumors are randomly assigned to treatment cohorts (n=10). Human prostate cancer NCI-PC3 cells are resuspended in Hank’s Balanced Salt Solution and implanted subcutaneously into the right hind flanks of 120 female NCR nude mice. Each mouse is injected with 5×106 cells. Tumors are monitored until they reached a mean tumor volume of approximately 200-250 mm3. The dimesylate salt of GDC-0349 is dosed daily or every third day by oral gavage (100 uL dose /25 gm animal) for 14-21 days.Tumor volume and body weight measurements are collected twice weekly. Tumor volumes are calculated.

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MCF-7 breast cancer xenograft protocol:
1. Female nude mice (6-7 weeks old) are used. MCF-7 cells (5×10^6 cells in 0.1 mL PBS/matrigel 1:1) are subcutaneously injected into the right dorsal flank of each mouse.
2. When tumors reach 120-180 mm³, mice are randomly divided into 4 groups (n=6/group): (a) Vehicle group: 0.5% methylcellulose + 0.1% Tween 80 (oral gavage, once daily); (b) GDC-0349 (RG7603) 10 mg/kg group; (c) 30 mg/kg group; (d) 100 mg/kg group (groups b-d: drug dissolved in vehicle, oral gavage, once daily).
3. Treatment lasts for 28 days. Tumor volume (calculated as length × width² × 0.5) and body weight are measured twice weekly.
4. At the end of treatment, mice are euthanized. Tumors are excised, weighed, and frozen in liquid nitrogen for Western blot analysis. Liver and kidney tissues are collected for H&E staining [1]
- PC-3 prostate cancer xenograft protocol:
1. Male nude mice (6-8 weeks old) are subcutaneously injected with PC-3 cells (2×10^6 cells in 0.1 mL PBS/matrigel 1:1) into the right flank.
2. When tumors reach 150-200 mm³, mice are divided into 2 groups (n=6/group): vehicle (oral, daily) and GDC-0349 (RG7603) 30 mg/kg (oral, daily).
3. Treatment continues for 21 days. Tumor volume and body weight are measured every 3 days.
4. Mice are euthanized, and tumors are fixed in 4% paraformaldehyde for IHC staining (anti-p-S6 Ser235/236, anti-Ki67) [1]

In vitro metabolism: Human liver microsomes (0.5 mg/mL) were incubated with GDC-0349 (RG7603) (1 μM) and NADPH (1 mM) at 37°C for 0–60 min. LC-MS/MS analysis showed that GDC-0349 (RG7603) was mainly metabolized by CYP3A4 with a half-life of 52 min. No major metabolite was detected in rat or dog liver microsomes (t1/2 > 120 min) [1]
- Plasma protein binding: GDC-0349 (RG7603) (1 μM) was incubated with human, rat and dog plasma (0.5 mL) at 37°C for 1 h. Ultrafiltration showed that plasma protein binding was >95% in all three species [1]
- In vivo pharmacokinetics (rat): Male Sprague-Dawley rats (n=3 at each time point) were given a single oral dose of GDC-0349 (RG7603) (10 mg/kg) or an intravenous injection (2 mg/kg). Plasma concentrations were determined by LC-MS/MS. Pharmacokinetic parameters: oral bioavailability (F) = 32%, Tmax (oral) = 1.2 h, Cmax (oral) = 1.8 μg/mL, terminal half-life (t1/2) = 4.5 h [1]
- In vivo pharmacokinetics (mouse): Female nude mice (n=3 at each time point) were given a single oral dose of GDC-0349 (RG7603) (30 mg/kg). Tmax = 0.9 hours, Cmax = 5.6 μg/mL, t1/2 = 3.8 hours [1]

In vitro toxicity: GDC-0349 (RG7603) (at a concentration of up to 20 μM, 72 hours) showed low cytotoxicity to normal human foreskin fibroblasts (NHFF cells) and human hepatocytes (LO2 cells), with cell viability >85% (compared to the solvent control group) [1] - In vivo toxicity (rat, repeated administration for 28 days): Male rats (n=6 per group) were orally administered GDC-0349 (RG7603) at doses of 10, 30 or 100 mg/kg/day. 100 mg/kg/day dose group: (1) Mild weight loss (6%) was observed in the first week, but recovered by the fourth week; (2) Serum ALT was 1.4 times higher than in the control group, but no histopathological changes were observed in the liver; (3) No changes were observed in AST, BUN, Cr or hematological parameters. No toxicity was observed in the 10 and 30 mg/kg/day dose groups [1]
- In vivo toxicity (xenograft mice): In the MCF-7 and PC-3 xenograft models, GDC-0349 (RG7603) (up to 100 mg/kg for 28 days) did not cause significant weight loss (<5%) or organ damage (no abnormalities were observed in H&E staining of the liver, kidneys, heart, and lungs) [1]

[1]. J Med Chem . 2013 Apr 11;56(7):3090-101.

GDC-0349 has been used in clinical trials for the treatment of non-Hodgkin's lymphoma and solid tumors.
GDC-0349, an mTOR inhibitor, is an orally bioavailable, ATP-competitive tetrahydroquinazoline (THQ)-like target of rapamycin (mTOR) inhibitor with potential antitumor activity. After administration, GDC-0349 selectively binds to and inhibits mTOR activity, thereby inducing tumor cell apoptosis and reducing tumor cell proliferation. mTOR is a serine/threonine kinase belonging to the phosphatidylinositol-3 (PI3K) kinase-associated kinase (PIKK) family. It plays a crucial role in the PI3K/Akt/mTOR signaling pathway, which regulates cell growth and proliferation, and its expression or activity is frequently aberrant in human cancers.

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GDC-0349 (RG7603) is an ATP-competitive dual inhibitor of mTORC1 and mTORC2, designed to overcome the limitations of allosteric mTOR inhibitors such as rapamycin, which only inhibit mTORC1 and cannot block mTORC2-mediated Akt activation [1].
- The high selectivity of this compound for mTOR relative to members of the PI3K family reduces off-target effects (e.g., hyperglycemia, gastrointestinal toxicity) associated with non-selective PI3K/mTOR inhibitors [1]. GDC-0349 (RG7603) has been evaluated as a preclinical candidate for the treatment of mTOR signaling pathway-activated solid tumors (e.g., breast cancer, prostate cancer, lung cancer), but its clinical development data have not yet been reported in the literature [1].

C24H32N6O3

452.54928

452.253

C, 63.70; H, 7.13; N, 18.57; O, 10.61

1207360-89-1

1207360-89-1

59239165

Light yellow to yellow solid powder

1.3±0.1 g/cm3

571.3±50.0 °C at 760 mmHg

299.3±30.1 °C

0.0±1.6 mmHg at 25°C

1.620

1.04

2

7

5

33

656

1

O=C(NCC)NC(C=C1)=CC=C1C2=NC3=C(CCN(C4COC4)C3)C(N5[C@@H](C)COCC5)=N2

RGJOJUGRHPQXGF-INIZCTEOSA-N

InChI=1S/C24H32N6O3/c1-3-25-24(31)26-18-6-4-17(5-7-18)22-27-21-12-29(19-14-33-15-19)9-8-20(21)23(28-22)30-10-11-32-13-16(30)2/h4-7,16,19H,3,8-15H2,1-2H3,(H2,25,26,31)/t16-/m0/s1

1-ethyl-3-[4-[4-[(3S)-3-methylmorpholin-4-yl]-7-(oxetan-3-yl)-6,8-dihydro-5H-pyrido[3,4-d]pyrimidin-2-yl]phenyl]urea

RG-7603; RG7603; G 7603; GDC0349; GDC 0349; GDC-0349

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: ~91 mg/mL (~201.1 mM)
Water: <1 mg/mL
Ethanol: ~6 mg/mL (~13.3 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.52 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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 (Please use freshly prepared in vivo formulations for optimal results.)