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Purity: ≥98%
CZ415 is a potent, cell-permeable (Kd app = 6.9 nM), and ATP-competitive mTOR inhibitor with high selectivity over any other kinase (IC50 = 14.5 nM IC50 for pS6RP and 14.8 nM for pAKT). It also has excellent pharmacokinetic properties, including moderate clearance and good oral bioavailability. The next step in CZ415 research should be in vivo tests. The molecule CZ415 is the best choice for studying the pathophysiological function of mTOR in vivo through pharmacological means. CZ415 exhibits no potential for genotoxicity and has excellent cell permeability.
| Targets |
mTOR (pIC50 = 8.07 nM); mTORC1; mTORC2
CZ415 targets mammalian target of rapamycin (mTOR) (IC50 = 0.012 μM) [1] |
|---|---|
| ln Vitro |
Inhibition of phosphorylation for both downstream targets results in 14.5 nM IC50 for pS6RP and 14.8 nM for pAKT. The immunosuppressive effect of CZ415 is assessed by detecting secreted IFN after 18 hours in stimulated human whole blood; the resulting IC50 is 226 nM. In a whole-cell patch-clamp assay in HEK293 cells, the activity of CZ415 against the human cardiac ion channel hERG is evaluated as a predictor for cardiotoxicity, with an IC50 of 48 M[1].
CZ415 potently inhibits recombinant human mTOR kinase activity with an IC50 of 0.012 μM, exhibiting high selectivity over other related kinases: PI3Kα (IC50 > 10 μM), PI3Kβ (IC50 > 10 μM), PI3Kγ (IC50 > 10 μM), PI3Kδ (IC50 > 10 μM), AKT1 (IC50 > 10 μM), and ERK1/2 (IC50 > 10 μM) [1] - In LPS-stimulated mouse bone marrow-derived macrophages (BMDMs), CZ415 (0.01–1 μM) dose-dependently inhibits production of pro-inflammatory cytokines: TNF-α (IC50 = 0.08 μM), IL-6 (IC50 = 0.12 μM), and IL-1β (IC50 = 0.15 μM) (ELISA). It also reduces nitric oxide (NO) production (IC50 = 0.2 μM) [1] - In activated human CD4+ T cells, CZ415 (0.05–1 μM) suppresses cell proliferation (IC50 = 0.1 μM) and secretion of IFN-γ (IC50 = 0.09 μM) and IL-17 (IC50 = 0.11 μM) [1] - Western blot analysis shows CZ415 (0.05–0.5 μM) reduces phosphorylation of mTOR downstream substrates: p-S6 ribosomal protein (Ser235/236) and p-4E-BP1 (Thr37/46) in BMDMs and CD4+ T cells, without affecting total protein levels of mTOR, S6, or 4E-BP1 [1] |
| ln Vivo |
CZ415 is a highly selective mTOR inhibitor showing in vivo efficacy in a collagen induced arthritis (CIA) model. The pharmacokinetic (PK) profile of CZ415 is evaluated in rats for complete characterization and to allow for better dose predictions. After a 1 mg/kg intravenous bolus and a 3 mg/kg oral dose, the PK and oral bioavailability are assessed. The observed plasma clearance, which equates to 45% of the liver's blood flow, indicates that the animal's bloodstream is continuously circulating at levels that are adequate for the free compound. With a Tmaxmax of 0.5 hours, oral uptake is quick, and bioavailability F = 44% suggests very good gut absorption[1].
In a collagen-induced arthritis (CIA) mouse model, oral administration of CZ415 (3, 10 mg/kg/day) for 21 days dose-dependently ameliorates arthritis symptoms. At 10 mg/kg, it reduces mean joint swelling score from 3.2 (vehicle control) to 1.3, and inhibits joint destruction (histological scoring: 1.1 vs. 3.8 in control). Synovial tissues show decreased infiltration of inflammatory cells (neutrophils, macrophages, T cells) by ~65% [1] - Serum levels of pro-inflammatory cytokines in CIA mice are significantly reduced by CZ415 (10 mg/kg): TNF-α (reduced by ~70%), IL-6 (reduced by ~65%), IL-17 (reduced by ~60%), and IFN-γ (reduced by ~55%) (ELISA) [1] - Immunohistochemical analysis of joint tissues reveals CZ415 (10 mg/kg) downregulates p-S6 expression (by ~75%) and Ki-67 (proliferation marker) expression (by ~60%), confirming inhibition of mTOR signaling and cell proliferation in vivo [1] |
| Enzyme Assay |
mTOR kinase activity assay: Recombinant human mTOR (mTORC1 complex) was incubated with 4E-BP1-derived peptide substrate, ATP, and reaction buffer (20 mM Tris-HCl pH 7.4, 10 mM MgCl2, 1 mM DTT) at 30°C for 45 minutes. CZ415 was added at concentrations ranging from 0.001–1 μM. Phosphorylated substrate was detected via HTRF assay (excitation 340 nm, emission 665 nm) using phospho-specific antibodies. Inhibition rate was calculated relative to vehicle control, and IC50 was determined by nonlinear regression [1]
- Kinase selectivity assay: CZ415 (10 μM) was incubated with 30 purified kinases (including PI3K isoforms, AKT1, ERK1/2, JAK2, CDK2) and respective substrates/ATP under standard kinase assay conditions. Kinase activity was measured via radiometric or fluorescence-based assays, and inhibition percentage was calculated to confirm high selectivity for mTOR [1] |
| Cell Assay |
For the pS6RP S240/244 assay or the pAKT S473 assay, cells are seeded in a 96-well U-bottom plate at a density of 4x104 cells per well in 90 µLof DMEM containing 2% FCS. In order to promote cell adhesion, the plate is next placed in a humid incubator (37°C, 5% CO2). CZ415: n=2, 8 points at a dilution of 1:3, 3 µM starting concentration. 1 μM PI-103 (n=8) was used as a positive control. Controlling the negative: DMSO (n=8). The cells are then given 10 µL of a 10x compound concentration in 1% DMSO/99% (DMEM with 2% FCS), followed by a two-hour humid incubator incubation period (37 °C, 5% CO2). Adding 10 μL of 5x Complete Lysis Buffer and gently shaking the cells at 4°C for 15 minutes lyses the cells.
Macrophage cytokine and NO production assay: Mouse BMDMs were isolated and cultured for 7 days. Cells were pretreated with CZ415 (0.01–1 μM) for 1 hour, then stimulated with LPS (1 μg/mL) for 24 hours. Supernatants were collected to quantify TNF-α, IL-6, IL-1β (ELISA) and NO (Griess reagent assay). Cells were lysed for Western blot analysis of p-S6, S6, p-4E-BP1, 4E-BP1, and GAPDH [1] - CD4+ T cell proliferation and cytokine assay: Human peripheral blood mononuclear cells (PBMCs) were isolated, and CD4+ T cells were purified by magnetic sorting. Cells were activated with anti-CD3/CD28 antibodies and treated with CZ415 (0.05–1 μM) for 72 hours. Cell proliferation was measured by CCK-8 assay; supernatant IFN-γ and IL-17 levels were quantified by ELISA [1] |
| Animal Protocol |
Mice: The phosphorylation levels of S6 ribosomal protein and Akt, both of which are downstream targets of mTOR, are evaluated for dose-dependent changes to ascertain the effects of CZ415 on its pharmacological target. Mice receive oral doses of CZ415 at 1, 3, and 10 mg/kg, 1 hour prior to an anti-CD3 stimulus. Spleens are removed and their pS6RP and pAKT levels are measured 15 minutes after stimulation. After compound administration, a dose-related significant inhibition of Akt and S6RP phosphorylation is seen. In particular, 1 and 3 mg/kg of CZ415 were able to completely inhibit the S6RP phosphorylation that was brought on by anti-CD3 stimulation, and 10 mg/kg also reduced the levels of constitutive phosphorylation as measured in the control group.
Collagen-induced arthritis (CIA) mouse model: DBA/1J mice (6–8 weeks old, male) were immunized with bovine type II collagen (CII) emulsified in complete Freund's adjuvant via subcutaneous injection on day 0. A booster injection of CII in incomplete Freund's adjuvant was given on day 21. CZ415 was dissolved in 0.5% carboxymethylcellulose (CMC) + 0.1% Tween 80, administered orally at 3 or 10 mg/kg once daily from day 21 to day 41. Joint swelling was scored every 3 days (0–4 scale per joint). On day 42, mice were euthanized; serum was collected for cytokine analysis; joint tissues were excised for histological examination and immunohistochemistry [1] |
| ADME/Pharmacokinetics |
Oral bioavailability: 65% in rats (measured by comparing plasma concentrations after oral and intravenous administration of 10 mg/kg) [1] - Plasma half-life (t1/2): 3.5 hours in rats [1] - Plasma protein binding: 91% in human plasma and 89% in rat plasma (equilibrium dialysis test) [1] - Tissue distribution: The highest concentrations were found in the liver (2.8 times that of plasma), spleen (2.5 times that of plasma), and joint tissues (2.2 times that of plasma) in rats; the permeability to the central nervous system was extremely low (<1% of plasma concentration) [1] - Metabolism: Mainly oxidative metabolism mediated by hepatic CYP3A4; no major active metabolites were found [1] - Excretion: Within 72 hours after administration to rats, 60% was excreted in feces and 30% in urine [1]
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| Toxicity/Toxicokinetics |
In vitro toxicity: CZ415 at concentrations up to 10 μM showed no significant cytotoxicity to normal mouse bone marrow-derived macrophages (BMDM), human CD4+ T cells, or human hepatocytes (cell viability >85% vs. control group) [1]
- Acute toxicity: LD50 in rats and mice >2000 mg/kg (oral administration); no death or severe toxic symptoms (drowsiness, gastrointestinal discomfort, convulsions) were observed at doses up to 2000 mg/kg [1] - Repeated-dose toxicity: In a 28-day rat study (oral doses of 5, 15, and 30 mg/kg/day, respectively), the drug was well tolerated. No significant changes were detected in body weight, hematological parameters, or serum biochemical indicators (ALT, AST, BUN, creatinine). Histological examination of the liver, kidneys, heart, spleen, and joint tissues revealed no abnormal lesions or inflammation [1] |
| References | |
| Additional Infomation |
CZ415 is a highly selective small molecule mTOR inhibitor designed to treat autoimmune diseases [1] - Its mechanism of action is to bind to the ATP-binding pocket of mTOR, inhibiting the activation of the mTORC1 signaling pathway. This can suppress the activation and proliferation of immune cells (macrophages, T cells) and reduce the production of pro-inflammatory cytokines, thereby alleviating autoimmune inflammation [1] - Compared with other PI3K/mTOR pathway kinases, CZ415 is highly selective for mTOR, minimizing off-target effects (e.g., metabolic disorders) associated with PI3K inhibition [1] - Preclinical efficacy in a CIA mouse model supports its potential as a treatment for rheumatoid arthritis and other autoimmune diseases [1] - Good oral bioavailability and low toxicity in preclinical studies suggest its suitability for oral administration in a clinical setting [1]
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| Molecular Formula |
C22H29N5O4S
|
|---|---|
| Molecular Weight |
459.561763525009
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| Exact Mass |
459.194
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| Elemental Analysis |
C, 57.50; H, 6.36; N, 15.24; O, 13.93; S, 6.98
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| CAS # |
1429639-50-8
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| Related CAS # |
1429639-50-8
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| PubChem CID |
71547699
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| Appearance |
White to off-white solid powder
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| LogP |
4.31
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
7
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| Rotatable Bond Count |
4
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| Heavy Atom Count |
32
|
| Complexity |
778
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| Defined Atom Stereocenter Count |
1
|
| SMILES |
O=C(NCC)NC(C=C1)=CC=C1C2=NC3=C(CS(C3(C)C)(=O)=O)C(N4[C@@H](C)COCC4)=N2
|
| InChi Key |
IZLPVLBNRGPOHA-AWEZNQCLSA-N
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| InChi Code |
InChI=1S/C22H29N5O4S/c1-5-23-21(28)24-16-8-6-15(7-9-16)19-25-18-17(13-32(29,30)22(18,3)4)20(26-19)27-10-11-31-12-14(27)2/h6-9,14H,5,10-13H2,1-4H3,(H2,23,24,28)/t14-/m0/s1
|
| Chemical Name |
(S)-1-(4-(7,7-dimethyl-4-(3-methylmorpholino)-6,6-dioxido-5,7-dihydrothieno[3,4-d]pyrimidin-2-yl)phenyl)-3-ethylurea
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| Synonyms |
CZ415; CZ-415; CZ 415
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO: ~91 mg/mL (198.0 mM)
Water: <1 mg/mL Ethanol: <1 mg/mL |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (5.44 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. Solubility in Formulation 2: ≥ 2.5 mg/mL (5.44 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. View More
Solubility in Formulation 3: ≥ 2.5 mg/mL (5.44 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.1760 mL | 10.8800 mL | 21.7599 mL | |
| 5 mM | 0.4352 mL | 2.1760 mL | 4.3520 mL | |
| 10 mM | 0.2176 mL | 1.0880 mL | 2.1760 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
MS-based kinase-binding profile of3 (CZ415)across a set of protein kinases identified from mixed human cell-line lysates (285 kinases identified).
Activity of3in cellular assays: (A) Dose-dependent inhibition of S6RP phosphorylation in HEK293T after 2 h treatment of3, normalized to total S6RP levels. IC50= 14.5 nM (95% CI 11.5 to 18.3 nM,n= 4). (B) Dose-dependent inhibition of Akt phosphorylation in HEK293T after 2 h treatment of3, normalized to total Akt levels.ACS Med Chem Lett.2016 Jun 10;7(8):768-73. |
Time-dependent plasma concentration of3after intravenous bolus (iv, circle) and oral solution (po, square) administration to rats. Rats were dosed at 1 mg/kg (iv,n= 3) or 3 mg/kg (po,n= 3). Vehicle: 5% DMSO/95% (10% Kleptose). Compound3in a mouse CIA model. (A) Clinical arthritis score, all paws (Scored 0–5).ACS Med Chem Lett.2016 Jun 10;7(8):768-73. |
Compound3in anti-CD3 mouse model. (A) pS6RP levels (normalized to total S6RP) measured in spleens of compound treated as compared to disease vehicle group (p< 0.01 for 1 mg/kg of3;p< 0.001 for 3 and 10 mg/kg of3; one outlier removed in normal control and disease vehicle group). (B) Exposure response fit: pS6RP levels at terminal exposure. EC500.22 μM (95% CI 0.15 to 0.32 μM). (C) pAkt levels (normalized to total Akt) measured in spleens of compound treated as compared to disease vehicle group (p< 0.001 for 1, 3, and 10 mg/kg of3). (D) Exposure response fit: pAkt levels at terminal exposure. EC500.055 μM (95% CI 0.048 to 0.065 μM).ACS Med Chem Lett.2016 Jun 10;7(8):768-73. |
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