KDM5B-IN-4 (compound 11ad) is a novel lysine demethylase 5B (KDM5B) inhibitor (antagonist) with IC50 of 0.025 μM for KDM5B. KDM5B-IN-4 increases the concentration of substrate H3K4me1/2/3 by inhibiting KDM5B in PC-3 cells. KDM5B-IN-4 can arrest PC-3 cells in the G2/M phase. KDM5B-IN-4 can downregulate PI3K/AKT pathway proteins. KDM5B-IN-4 reduces tumor volume in mice and is less toxic to organs.

Physicochemical Properties

Molecular Formula	C30H30N6O
Molecular Weight	490.60
Appearance	White to off-white solid powder
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	IC 50: 0.025 μM (Lysine demethylase 5B, KDM5B)[1].
ln Vitro	KDM5B-IN-4 (20 μM, 72 h) stimulates the synthesis of H3K4me1/2/3 in PC-3 cells and has a specific inhibitory effect on KDM5B [1]. In addition to targeting KDM5B in cells, KDM5B-IN-4 (0–20 μM; 72 h) causes PC-3 cells to accumulate H3K4me1/2/3 [1]. KDM5B-IN-4 (0-10 μM, 0-24 h) suppresses prostate cancer cell migration and proliferation, interrupts the PC-3 cycle in the G2/M phase, and to some extent can cause PC-3 cell apoptosis. Demise [1].
ln Vivo	KDM5B-IN-4 (50 mg/kg, ig, 50 mg/kg/d, 13 days) has a somewhat greater tumor-inhibiting activity than DOX [1]. Mice treated with KDM5B-IN-4 (50 mg/kg, ig, 50 mg/kg/d, 25 days) did not exhibit any overt toxicity or adverse effects, as reported in [1].
Cell Assay	Western Blot Analysis[1] Cell Types: PC-3 Tested Concentrations: 0, 2.5, 5, 10, 20 μM Incubation Duration: 72 h Experimental Results: Induced the concentrations of H3K4me1/2/3 Dramatically different from those of the control, and the results obtained were similar to those obtained using the CPI-455 positive control. Had no significant effect on P110α, P85, and pAKT at low concentration levels (0-10μM), and P110α, P85, and pAKT were Dramatically diminished when 11ad was at high concentration(20 μM). Cell Migration Assay [1] Cell Types: PC-3 Tested Concentrations: 0, 5, 10 μM Incubation Duration: 0, 6, 12, 24 h Experimental Results: Inhibited colony formation in a dosedependent manner, especially at high doses(10 μM). Cell Cycle Analysis[1] Cell Types: PC-3 Tested Concentrations: 0, 2.5, 5, 10 μM Incubation Duration: 24 h Experimental Results: Blocked the cell cycle at G2/M phase in 10 μM. Apoptosis Analysis[1] Cell Types: PC-3 Tested Concentrations: 0, 2.5, 5, 10 μM Incubation Duration: 24 h Experimental Results: Induced PC-3 cell apoptosis in a dose-dependent manner(7.58%, 26.14%, 28.20%, 4
Animal Protocol	Animal/Disease Models: PC-3 xenograft model in male Spraguee-Dawley rats[1]. Doses: 25, 50 mg/kg Route of Administration: intragastric (po) administration to mice (ig) for 25 days, administered one time/day. Experimental Results: Compared with NaCl treatment, treatment with 25 mg/kg and 50 mg/kg Dramatically diminished tumor volume. Animal/Disease Models: PC-3 xenograft model in male Spraguee-Dawley rats[1]. Doses: 2 g/kg Route of Administration: intragastric (po) administration to mice (ig) for 14 days, administered one time/day. Experimental Results: No significant loss of major organs in the high-dose and low-dose groups.
References	[1]. Discovery of a novel 1H-pyrazole- [3,4-b] pyridine-based lysine demethylase 5B inhibitor with potential anti-prostate cancer activity that perturbs the phosphoinositide 3-kinase/AKT pathway. Eur J Med Chem. 2023 May 5;251:115250.

Solubility Data

Solubility (In Vitro)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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 (e.g. IP/IV/IM/SC) Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline) *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). 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)] *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. Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline) Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline) Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline) Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil) Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL 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). Oral Formulation 3: Dissolved in PEG400 Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose Oral Formulation 6: Mixing with food powders Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	2.0383 mL	10.1916 mL	20.3832 mL
	5 mM	0.4077 mL	2.0383 mL	4.0766 mL
	10 mM	0.2038 mL	1.0192 mL	2.0383 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.