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.

Physicochemical Properties

Molecular Formula	C16H21N2
Molecular Weight	276.81
Exact Mass	240.162
CAS #	749886-87-1
Related CAS #	545380-34-5
PubChem CID	16760588
Appearance	Pale purple to purple solid powder
Density	1.1±0.1 g/cm3
Boiling Point	418.7±40.0 °C at 760 mmHg
Melting Point	104.4-105.0℃
Flash Point	245.0±30.9 °C
Vapour Pressure	0.0±1.0 mmHg at 25°C
Index of Refraction	1.630
LogP	3.66
Hydrogen Bond Donor Count	2
Hydrogen Bond Acceptor Count	2
Rotatable Bond Count	5
Heavy Atom Count	18
Complexity	223
Defined Atom Stereocenter Count	0
Synonyms	JSH-23 HCL
HS Tariff Code	2934.99.9001
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)

Biological Activity

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].
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].
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	JSH-23 at concentrations up to 100 μM did not show significant cytotoxic effects on RAW 264.7 macrophages over a 24-hour period, as assessed by the WST-1 assay. Cytotoxicity was observed at 300 μM.[1] JSH-23 alone (30 μM) did not induce apoptotic cell death in RAW 264.7 macrophages.[1]
References	[1]. Inhibitory action of novel aromatic diamine compound on lipopolysaccharide-induced nuclear translocation of NF-kappaB without affecting IkappaB degradation. FEBS Lett. 2004 Jul 30;571(1-3):50-4. [2]. JSH-23 targets nuclear factor-kappa B and reverses various deficits in experimental diabetic neuropathy: effect on neuroinflammation and antioxidant defence. Diabetes Obes Metab. 2011 Aug;13(8):750-8.
Additional Infomation	JSH-23 is a diamine that is 1,2-phenylenediamine carrying a methyl substituent at position 4 and a 3-phenylpropyl substituent at position N1. It has a role as a NF-kappaB inhibitor. It is a diamine and a substituted aniline. It is functionally related to a 1,2-phenylenediamine. JSH-23 (4-Methyl-N¹-(3-phenyl-propyl)-benzene-1,2-diamine) is a novel synthetic aromatic diamine compound with a purity of ≥98%.[1] Its mechanism of action is considered rare, as it inhibits NF-κB nuclear translocation without blocking IκBα degradation, potentially by interfering with the nuclear import machinery of NF-κB, similar to the synthetic peptide SN50 which targets the nuclear localization signal (NLS).[1] The compound's inhibitory effects on pro-inflammatory cytokine and enzyme expression suggest potential utility in inflammation-related disorders such as arthritis, cancer, and septic shock.[1] The study was conducted entirely in vitro using the murine macrophage cell line RAW 264.7.[1]

Solubility Data

Solubility (In Vitro)

DMSO: >50 mg/mL Water: N/A Ethanol: N/A

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. Solubility in Formulation 3: 0.5% hydroxyethyl cellulose: 30 mg/mL  (Please use freshly prepared in vivo formulations for optimal results.)

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.