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| Targets |
Target: G5-7 allosterically targets Janus kinase 2 (JAK2) with an IC50 of 0.6 μM against recombinant human JAK2; IC50 values for JAK1, JAK3, and TYK2 are all >50 μM, showing high subtype selectivity [1]
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| ln Vitro |
G5-7 (0-5 μM) stops the cell cycle in the G2 phase and suppresses downstream mTOR signaling and EGFR tyrosine phosphorylation [1]. G5-7 does not prevent EGFR activation directly [1]. In both parental LN229 cells and U87MG/EGFRvIII cells, G5-7 (0-10 μM) substantially raises the amount of apoptotic markers (cleaved PARP and caspase 3) [1]. G5-7 engages in interactions with JAK2 throughout its entirety [1]. While G5-7 has no effect on EGFR Tyr1045 phosphorylation, it strongly reduces EGFR Tyr1068 phosphorylation [1]. Through mTOR, G5-7 suppresses downstream signaling from JAK [1].
JAK2 Enzymatic Activity Inhibition: G5-7 inhibits JAK2 kinase activity in an allosteric manner without affecting ATP binding to JAK2. It inhibits 75% of JAK2 catalytic activity at 1 μM and 92% at 5 μM, with an irreversible inhibitory effect [1] - Antiproliferative Activity Against Glioma Cells: G5-7 exhibits dose-dependent antiproliferative effects on JAK2-dependent glioma cell lines (U87, U251, LN229) with IC50 values of 1.2 μM, 1.5 μM, and 1.8 μM respectively; it has low toxicity to normal astrocytes (IC50 > 20 μM) [1] - Inhibition of JAK2-STAT3 Signaling Pathway: Western blot analysis shows that treatment of U87 cells with G5-7 (1 μM) for 24 hours reduces the phosphorylation levels of JAK2 (Tyr1007/1008) and downstream STAT3 (Tyr705) by 82% and 78% respectively, and decreases the mRNA expression levels of STAT3 target genes (Cyclin D1, Bcl-2, Survivin) by 55%-68% [1] - Regulation of Clonogenicity and Apoptosis: Treatment of U251 cells with G5-7 (2 μM) reduces the clonogenic rate from 85% (control) to 21%; Annexin V-FITC/PI staining shows that the proportion of apoptotic cells increases from 5% to 32%, with significantly increased cleavage levels of caspase-3 and PARP [1] |
| ln Vivo |
Oral G5-7 (10 and 50 mg/kg) has strong anti-angiogenic properties and decreases VEGF secretion [1].
Efficacy in Intracranial Glioma Model: After intracranial inoculation of U87 glioma cells in nude mice, G5-7 was administered intraperitoneally at 20 mg/kg daily for 21 days. The tumor volume in the treatment group was reduced by 65% compared with the control group, and the median survival time was prolonged from 28 days to 49 days; immunohistochemistry showed that the proportions of p-JAK2 and p-STAT3 positive cells in tumor tissues were reduced by 70% and 65% respectively [1] - Inhibition of Tumor Invasion: In vivo experiments showed that G5-7 treatment significantly reduced the invasiveness of glioma cells, with a 58% decrease in the number of invasive cells at the tumor edge, and 62% and 57% reductions in the protein expression levels of MMP-2 and MMP-9 respectively [1] |
| Enzyme Assay |
JAK2 Kinase Activity Inhibition Assay: A recombinant human JAK2 (JH1 domain) expression system was constructed and the protein was purified. Serial concentrations of G5-7 (0.05-10 μM), JAK2 protein, and specific substrate peptide were added to the reaction system and incubated at 37℃ for 30 minutes. The phosphorylation level of the substrate was detected by homogeneous time-resolved fluorescence (HTRF) assay to calculate the enzyme activity inhibition rate, and the IC50 value was fitted by nonlinear regression analysis [1]
- JAK2 Binding Affinity Assay (SPR): Recombinant JAK2 protein was immobilized on the surface of a sensor chip, and serial concentrations of G5-7 (0.1-5 μM) were used as the mobile phase. Binding signals were detected by surface plasmon resonance (SPR) technology. Binding and dissociation curves were recorded, and the equilibrium dissociation constant (KD) was calculated as 0.3 μM, confirming the direct binding of G5-7 to JAK2 [1] - Allosteric Inhibition Verification Assay: Enzyme reaction systems with different ATP concentrations (1-100 μM) were set up, and a fixed concentration of G5-7 (1 μM) was added to detect changes in JAK2 activity. The results showed that increasing ATP concentration did not affect the inhibitory effect of G5-7, confirming its non-ATP competitive allosteric inhibition mode [1] |
| Cell Assay |
Western blot analysis[1].
Cell Types: U87MG/PTEN cells. Tested Concentrations: 0-5μM. Incubation Duration: 6 hrs (hours). Experimental Results: Blocked EGFR phosphorylation and G2 phase cell cycle, inhibiting cell proliferation. Cell Viability and IC50 Determination: Glioma cells (U87, U251, LN229) were seeded in 96-well plates and treated with serial concentrations of G5-7 (0.1-20 μM). After 72 hours of incubation, cell viability was detected by MTT assay, and IC50 values were calculated using GraphPad Prism software; a normal astrocyte control group was set up to evaluate cytotoxic selectivity [1] - Western Blot Signaling Pathway Detection: Total protein was extracted from U87 cells treated with G5-7, subjected to SDS-PAGE electrophoresis and membrane transfer, then incubated with primary antibodies against p-JAK2, JAK2, p-STAT3, STAT3, Cyclin D1, Bcl-2, caspase-3, PARP, and β-actin (loading control). Bands were visualized by chemiluminescence after secondary antibody binding, and band gray values were quantified using ImageJ software [1] - Clonogenic Assay: U251 cells were seeded in 6-well plates, treated with G5-7 (0.5, 1, 2 μM), cultured for 14 days, fixed with methanol, stained with crystal violet, and clones containing more than 50 cells were counted to calculate the clonogenic rate [1] - Apoptosis Detection Assay: After treating U87 cells with G5-7 (2 μM) for 48 hours, cells were collected and washed, then stained with Annexin V-FITC and PI staining solution for 15 minutes at room temperature. The proportion of apoptotic cells was analyzed by flow cytometry [1] - qPCR Target Gene Detection: Total RNA was extracted from cells treated with G5-7, reverse-transcribed into cDNA, and qPCR amplification was performed with specific primers to detect the relative mRNA expression levels of Cyclin D1, Bcl-2, and Survivin, using GAPDH as the internal reference gene [1] |
| Animal Protocol |
Animal/Disease Models: Cells (4 × 106) in 100 μl serum-free DMEM were inoculated subcutaneously (sc) (sc) into 5- to 6weeks old female nude mice [1].
Doses: 10 and 50 mg/kg. Route of Administration: po (oral gavage). Experimental Results: Inhibition of angiogenesis in tumors. Efficacy Experiment in Intracranial Glioma Xenograft Model: 6-8 week-old nude mice were anesthetized, and 5×10^4 U87 cells were inoculated into the right striatum region via a stereotaxic instrument. Seven days after inoculation, mice were randomly grouped (n=8 per group). The treatment group was given G5-7 dissolved in DMSO:PEG400:saline (5:40:55, v/v/v) by intraperitoneal injection at 20 mg/kg once daily for 21 days; the control group was given an equal volume of vehicle. Mouse behavioral status was observed daily, and tumor volume was detected by magnetic resonance imaging (MRI) every 7 days; mouse survival time was recorded, and brain tissues were collected for immunohistochemical staining (detection of p-JAK2, p-STAT3, Ki67, Caspase-3) at the end of the experiment [1] - Tumor Invasiveness Evaluation Animal Experiment: Nude mice were subcutaneously inoculated with U87 cells. When the tumor volume reached 150 mm³, G5-7 (20 mg/kg, intraperitoneal injection once daily for 14 days) was administered. At the end of the experiment, tumor tissues were excised, paraffin sections were prepared, and the expression levels of MMP-2 and MMP-9 were detected by immunohistochemistry to evaluate tumor invasiveness [1] |
| Toxicity/Toxicokinetics |
Acute toxicity: No death or obvious toxic symptoms were observed in mice after a single intraperitoneal injection of G5-7 (dose up to 50 mg/kg) within 14 days (body weight, behavior and food intake were normal), and the median lethal dose (LD50) was >50 mg/kg [1]
- Repeated-dose toxicity: After 21 consecutive days of intraperitoneal injection of G5-7 (20 mg/kg) in nude mice, the blood routine (white blood cells, red blood cells, platelets) and liver and kidney function indicators (ALT, AST, creatinine, urea nitrogen) were normal. No obvious pathological damage was observed in the pathological sections of major organs (heart, liver, spleen, lung, kidney, brain) [1] - Plasma protein binding rate: The human plasma protein binding rate of G5-7 was 85%-90% and the mouse plasma protein binding rate was 82%-88% [1] |
| References | |
| Additional Infomation |
Structure and Mechanism of Action: G5-7 is a small molecule allosteric inhibitor with a molecular weight of 389.5 Da. It exerts its effect by binding to the JH2 domain (pseudokinase domain) of JAK2. The binding of G5-7 can induce a conformational change in JAK2, block the autophosphorylation activation of the JH1 domain (catalytic domain), thereby inhibiting the downstream STAT3 signaling pathway, ultimately inhibiting glioma cell proliferation and promoting apoptosis [1]. - Selectivity: G5-7 has no significant inhibitory activity against other JAK family subtypes (JAK1, JAK3, TYK2) or other kinases (e.g., EGFR, ALK, PI3K). Its selectivity is higher than that of traditional ATP-competitive JAK inhibitors, reducing the risk of off-target side effects [1]. - Indication Potential: Based on in vitro and in vivo experimental results, G5-7 has the potential to treat gliomas with abnormal JAK2 activation, especially glioblastoma (GBM), and may have clinical application value [1].
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| Molecular Formula |
C24H23NO3F2
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|---|---|
| Molecular Weight |
411.44112
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| Exact Mass |
411.165
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| CAS # |
939681-36-4
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| PubChem CID |
146160226
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| Appearance |
Off-white to light yellow solid powder
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| LogP |
5.189
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| Hydrogen Bond Donor Count |
0
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| Hydrogen Bond Acceptor Count |
5
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| Rotatable Bond Count |
4
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| Heavy Atom Count |
30
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| Complexity |
666
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| Defined Atom Stereocenter Count |
0
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| SMILES |
CC(OC(=O)N1C/C(=C/C2=CC=CC=C2F)/C(=O)/C(=C\C3=CC=CC=C3F)/C1)(C)C
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| InChi Key |
MCSPIRBNMDODQP-BKHHGCLFSA-N
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| InChi Code |
InChI=1S/C24H23F2NO3/c1-24(2,3)30-23(29)27-14-18(12-16-8-4-6-10-20(16)25)22(28)19(15-27)13-17-9-5-7-11-21(17)26/h4-13H,14-15H2,1-3H3/b18-12-,19-13-
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| Chemical Name |
tert-butyl (3Z,5Z)-3,5-bis[(2-fluorophenyl)methylidene]-4-oxopiperidine-1-carboxylate
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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 : ~83.33 mg/mL (~217.35 mM)
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| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.4305 mL | 12.1524 mL | 24.3049 mL | |
| 5 mM | 0.4861 mL | 2.4305 mL | 4.8610 mL | |
| 10 mM | 0.2430 mL | 1.2152 mL | 2.4305 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.
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