Tubulin/HDAC-IN-4 (compound 9n) is a dual inhibitor of microtubules (Tubulin) and HDACs, with IC50 values of 0.73, 0.43, 0.62, and 2.34 µM for HDAC1, HDAC2, HDAC6, and HDAC7, respectively. Tubulin/HDAC-IN-4 inhibits tubulin polymerization by targeting the colchicine binding site. Tubulin/HDAC-IN-4 induces apoptosis and cell cycle arrest at the G2/M phase. Tubulin/HDAC-IN-4 induces a significant increase in intracellular ROS levels. Tubulin/HDAC-IN-4 exhibits anti-angiogenic and anti-cancer activities.

Physicochemical Properties

Molecular Formula	C24H26N2O6
Molecular Weight	438.47
Appearance	Typically exists as solid at room temperature
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	HDAC1 0.73 μM (IC50) HDAC2 0.43 μM (IC50) HDAC6 0.62 μM (IC50) HDAC7 2.34 μM (IC50)
ln Vitro	Tubulin/HDAC-IN-4 (compound 9n) (0-10 µM; 72 h) exhibited cytotoxicity against MDA-MB-231 and A549 cells with IC50s of 0.34, 0.29, 0.016, 0.15, and 0.16 µM for PC-3, U251, and MCF-7 cells, respectively[1]. Tubulin/HDAC-IN-4 (2.5, 5, 10, 20, and 40 nM; 24 h) inhibited PC-3 cell colony formation in a dose-dependent manner[1]. Tubulin/HDAC-IN-4 (0.2, 1, 5, and 25 µM) inhibited tubulin polymerization with an IC50 value of 4.82 µM[1]. Tubulin/HDAC-IN-4 (0.08, 0.16, 0.32; 24 h) increased the expression of Ac-α-tubulin and Ac-Histone H3 in PC-3 cells[1]. Tubulin/HDAC-IN-4 (0.08, 0.16, 0.32 µM; 24 h) induced apoptosis and G2/M phase cell cycle arrest[1]. Tubulin/HDAC-IN-4 (0.08, 0.16, 0.32 µM; 24 h) induced a significant increase in intracellular ROS levels[1].
ln Vivo	Tubulin/HDAC-IN-4 (10, 20 mg/kg; iv; every two days for 21 days) showed anticancer activity[1].
Cell Assay	Cell Cytotoxicity Assay[1] Cell Types: MDA-MB-231, A549, PC-3, U251, MCF-7 Tested Concentrations: 0-10 µM Incubation Duration: 72 h Experimental Results: Showed cytotoxicity with IC50s of 0.34, 0.29, 0.016, 0.15, 0.16 µM for MDA-MB-231, A549, PC-3, U251, MCF-7 cells, respectively. Western Blot Analysis[1] Cell Types: PC-3 Tested Concentrations: 0.08, 0.16, 0.32 µM Incubation Duration: 24 h Experimental Results: Increased in both the expression of HDAC6 substrate Ac-α-tubulin and HDAC1/2/3 substrate Ac-Histone H3 in a dose-dependent manner. Cell Cycle Analysis[1] Cell Types: PC-3 Tested Concentrations: 0.08, 0.16, 0.32 µM Incubation Duration: 24 h Experimental Results: Dose-dependently arrested PC-3 cells at G2/M phase, decreased in expression level of p-Cdc25cSer216, p-Cdc2Thr216 and p-CdcTyr15, increased the expression of Cyclin B1. Apoptosis Analysis[1] Cell Types: PC-3 Tested Concentrations: 0.08, 0.16, 0.32 µM Incubation Duration: 24 h Experimental Results: Induced apoptosis and increased the expression of cleaved PARP and cleaved Caspase 3, decreased the expression of Bim and Bcl-2.
Animal Protocol	Animal/Disease Models:5-week-old male BALB/c nude mouse (PC-3 cells)[1] Doses: 10, 20 mg/kg Route of Administration: I.v.; every two days for 21 days Experimental Results: Inhibited the growth of tumor with the tumor growth inhibition (TGI) reached 90.07% at 20 mg/kg.
References	[1]. Design and biological evaluation of dual tubulin/HDAC inhibitors based on millepachine for treatment of prostate cancer. European Journal of Medicinal Chemistry. 2024, 268(15): 116301.

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	2.2807 mL	11.4033 mL	22.8066 mL
	5 mM	0.4561 mL	2.2807 mL	4.5613 mL
	10 mM	0.2281 mL	1.1403 mL	2.2807 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.