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Purity: ≥98%
JSH-23 HCl is designed as an inhibitor of NF-κB transcriptional activity with IC50 of 7.1 μM in RAW 264.7 cell line. In LPS-stimulated macrophages RAW 264.7, JSH-23 inhibits NF-κB transcriptional activity in a dose-dependent fashion. Its cytotoxicity is not the cause of this outcome. JSH-23 is discovered to significantly reduce the LPS-induced DNA binding activity of NF-κB while reducing nuclear NF-κB p65 amounts in the same condition. JSH-23 performs these functions without affecting IκB degradation. Additionally, JSH-23 exhibits inhibition effects on the expression of pro-inflammatory transcripts and enzymes, such as IL-6, IL-1β, COX-2, and TNF-α. JSH-23 also prevents the chromatin condensation brought on by apoptosis that is induced by LPS.
| Targets |
NF-κB (IC50 = 7.1 μM)
JSH-2 (1-300 μM; 24 hours) at less than <100 μM has no discernible cytotoxic effects on RAW 264.7 cells[1]. After being exposed to LPS for 1 h, NF-B p65 nuclear amount is significantly increased. JSH-23 (30 μM; 1 hour) treatment reduces nuclear NF-κB p65 content in RAW 264.7 cells stimulated by LPS in a dose-dependent manner[1]. |
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| ln Vitro |
JSH-2 (1-300 μM; 24 hours) at less than <100 μM has no discernible cytotoxic effects on RAW 264.7 cells[1].
After being exposed to LPS for 1 h, NF-B p65 nuclear amount is significantly increased. JSH-23 (30 μM; 1 hour) treatment reduces nuclear NF-κB p65 content in RAW 264.7 cells stimulated by LPS in a dose-dependent manner[1]. JSH-23 inhibited LPS-induced NF-κB transcriptional activity in a dose-dependent manner in RAW 264.7 macrophages stably transfected with a pNF-κB-SEAP reporter construct, showing 23%, 68%, and 103% inhibition at 3, 10, and 30 μM, respectively.[1] JSH-23 decreased LPS-induced DNA binding activity of NF-κB in a dose-dependent manner, as demonstrated by electrophoretic mobility shift assay (EMSA).[1] Western immunoblot analysis showed that JSH-23 dose-dependently inhibited LPS-induced nuclear translocation of the NF-κB p65 subunit (49%, 75%, and 95% inhibition at 3, 10, and 30 μM, respectively), without affecting LPS-induced IκBα degradation or its cytoplasmic recovery.[1] Semi-quantitative RT-PCR analysis revealed that JSH-23 differentially inhibited LPS-induced mRNA expression of pro-inflammatory mediators: it inhibited IL-6 and inducible nitric oxide synthase (iNOS) at concentrations ≥3 μM, IL-1β and cyclooxygenase-2 (COX-2) at ≥10 μM, and tumor necrosis factor-alpha (TNF-α) at ≥30 μM.[1] JSH-23 inhibited LPS-induced apoptosis (chromatin condensation) in RAW 264.7 cells in a dose-dependent manner, showing 44%, 63%, and 93% inhibition at 3, 10, and 30 μM, respectively.[1] |
| ln Vivo |
JSH-23 (orally given at doses of 1 mg/kg or 3 mg/kg twice daily for two weeks) significantly improves nerve conduction and blood flow deficits in diabetic rats[2].
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| Cell Assay |
To measure NF-κB transcriptional activity, RAW 264.7 macrophages stably harboring a pNF-κB-SEAP-NPT reporter plasmid were treated with LPS (1 μg/mL) and/or JSH-23 for 16 hours. Secreted alkaline phosphatase (SEAP) activity in the cell-free culture medium was then measured as a readout for NF-κB-driven gene expression.[1]
For electrophoretic mobility shift assay (EMSA), RAW 264.7 cells were treated with LPS (1 μg/mL) plus JSH-23 for 1 hour. Nuclear extracts were prepared and incubated with a ³²P-labeled oligonucleotide containing a κB binding site. The DNA-protein complexes were resolved on a non-denaturing 6% polyacrylamide gel, followed by autoradiography.[1] For Western immunoblot analysis of NF-κB p65 nuclear translocation, cells were treated with LPS (1 μg/mL) and/or JSH-23 for 1 hour. Nuclear extracts were prepared and subjected to immunoblotting using an antibody against NF-κB p65.[1] For Western immunoblot analysis of IκBα degradation, cells were treated with LPS (1 μg/mL) and/or JSH-23 (30 μM) for various times (10 minutes to 8 hours). Cytoplasmic extracts were prepared and subjected to immunoblotting using an antibody against IκBα.[1] For semi-quantitative RT-PCR analysis of cytokine and enzyme expression, cells were treated with LPS (1 μg/mL) and/or JSH-23 for 6 hours. Total RNA was extracted, reverse transcribed, and amplified by PCR using specific primers for TNF-α, IL-1β, IL-6, iNOS, COX-2, and β-actin (as an internal control). PCR products were separated by agarose gel electrophoresis and visualized with ethidium bromide staining.[1] To assess apoptosis, cells were treated with LPS (1 μg/mL) and/or JSH-23 for 24 hours, then stained with 4',6-diamidino-2-phenylindole (DAPI). Cells were examined under fluorescence microscopy, and nuclei with condensed or fragmented chromatin were counted as apoptotic.[1] To assess cytotoxicity, cells were treated with various concentrations of JSH-23 for 24 hours. Cell viability/proliferation was measured using a WST-1 assay, and absorbance was read at 450 nm.[1] |
| Animal Protocol |
Male Sprague Dawley diabetic rats (250-270 g)
1 mg/kg, 3 mg/kg Orally administered; daily; for 2 weeks |
| Toxicity/Toxicokinetics |
At concentrations up to 100 μM, JSH-23 did not show significant cytotoxicity against RAW 264.7 macrophages within 24 hours (WST-1 assay). Cytotoxicity was observed at a concentration of 300 μM. [1] JSH-23 alone (30 μM) did not induce apoptosis in RAW 264.7 macrophages. [1]
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| References |
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| Additional Infomation |
JSH-23 is a diamine with the structure 1,2-phenylenediamine, a methyl substituent at the 4-position, and a 3-phenylpropyl substituent at the N1-position. It is an NF-κB inhibitor. It is a diamine and a substituted aniline. It is functionally related to 1,2-phenylenediamine. JSH-23 (4-methyl-N¹-(3-phenylpropyl)-phenyl-1,2-diamine) is a novel synthetic aromatic diamine compound with a purity ≥98%. [1] Its mechanism of action is relatively rare, as it inhibits the nuclear translocation of NF-κB without blocking IκBα degradation, which may be achieved by interfering with the nuclear input mechanism of NF-κB, similar to the synthetic peptide SN50 targeting nuclear localization signals (NLS). [1] The inhibitory effect of this compound on the expression of pro-inflammatory cytokines and enzymes suggests its potential application value in inflammation-related diseases such as arthritis, cancer, and septic shock. [1]
This study was conducted entirely in vitro using the mouse macrophage cell line RAW 264.7. [1] |
| Molecular Formula |
C16H21N2
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| Molecular Weight |
276.81
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| Exact Mass |
240.162
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| CAS # |
749886-87-1
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| Related CAS # |
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| PubChem CID |
16760588
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| Appearance |
Pale purple to purple solid powder
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| Density |
1.1±0.1 g/cm3
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| Boiling Point |
418.7±40.0 °C at 760 mmHg
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| Melting Point |
104.4-105.0℃
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| Flash Point |
245.0±30.9 °C
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| Vapour Pressure |
0.0±1.0 mmHg at 25°C
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| Index of Refraction |
1.630
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| LogP |
3.66
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
2
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| Rotatable Bond Count |
5
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| Heavy Atom Count |
18
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| Complexity |
223
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| Defined Atom Stereocenter Count |
0
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| Synonyms |
JSH-23 HCL
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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 |
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| 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) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (10.40 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 (10.40 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. View More
Solubility in Formulation 3: 0.5% hydroxyethyl cellulose: 30 mg/mL |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 3.6126 mL | 18.0629 mL | 36.1259 mL | |
| 5 mM | 0.7225 mL | 3.6126 mL | 7.2252 mL | |
| 10 mM | 0.3613 mL | 1.8063 mL | 3.6126 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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