HDAC6-IN-4 (C10) is a potent, orally bioactive, selective HDAC6 inhibitor (antagonist) with IC50 of 23 nM. HDAC6-IN-4 causes apoptosis in tumor cells and has significant anti-tumor effects without obvious toxicity.

Physicochemical Properties

Molecular Formula	C30H38N2O5
Molecular Weight	506.63
Exact Mass	506.278
CAS #	2709103-20-6
PubChem CID	163322236
Appearance	Typically exists as solid at room temperature
LogP	5.1
Hydrogen Bond Donor Count	3
Hydrogen Bond Acceptor Count	6
Rotatable Bond Count	15
Heavy Atom Count	37
Complexity	615
Defined Atom Stereocenter Count	0
InChi Key	NWHRMDIXMQBLHY-UHFFFAOYSA-N
InChi Code	InChI=1S/C30H38N2O5/c1-22-24(14-11-15-26(22)23-12-7-6-8-13-23)21-37-25-18-28(35-2)27(29(19-25)36-3)20-31-17-10-5-4-9-16-30(33)32-34/h6-8,11-15,18-19,31,34H,4-5,9-10,16-17,20-21H2,1-3H3,(H,32,33)
Chemical Name	7-[[2,6-dimethoxy-4-[(2-methyl-3-phenylphenyl)methoxy]phenyl]methylamino]-N-hydroxyheptanamide
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

ln Vitro	HDAC6-IN-4 (C10) (0-50 µM, 72 h) has low cytotoxicity but substantial anti-proliferative effect against several cancer cells [1]. HDAC6-IN-4 (0–6 µM, 24 hours) exhibits greater HDAC6 selectivity compared to HDAC1[1]. B16 and CT26 cells' migratory activity is time- and dose-dependently inhibited by HDAC6-IN-4 [1]. B16 cells undergo dose-dependent apoptosis when exposed to HDAC6-IN-4 (0-8 µM) for 24 hours [1]. HDAC6-IN-4 shows notable action in human (half-life 101.91 min) and mouse liver (half-life 67.94 min) microsomes, as well as significant plasma stability in humans (97% retention after 6 hours). exceptional stability in metabolism[1].
ln Vivo	In a dose-dependent manner and without causing significant toxicity, HDAC6-IN-4 (C10) (0-100 mg/kg; ig; once daily for 21 days) significantly promotes T cell responses and shows excellent anti-tumor activity[1].
Cell Assay	Cell Proliferation Assay[1] Cell Types: B16, HepG2, A549 and CT26 Cell Tested Concentrations: 0-50 µM Incubation Duration: 72 hrs (hours) Experimental Results: demonstrated anti-proliferative activity with IC50 values of 1.52, 2.36, 5.77 and 2.09 µM against B16 , HepG2, A549 and CT26 cells respectively. Western Blot Analysis[1] Cell Types: B16 and CT26 Cancer Cell Tested Concentrations: 2, 4 and 6 µM Incubation Duration: 2, 4, 8, 12 and 24 hrs (hours) Experimental Results: Ac-Tub (Acetyl-α-Tubulin) in a dose-dependent and time-dependent manner. It has almost no effect on the content of Ac-H3 (acetyl-H3). Apoptosis analysis[1] Cell Types: B16 Cells Tested Concentrations: 4, 6 and 8 µM Incubation Duration: 24 hrs (hours) Experimental Results: Moderate to strong induction of apoptosis in B16 cell line in a dose-dependent manner. Upregulates the expression of PARP, which cleaves apoptotic proteins.
Animal Protocol	Animal/Disease Models: Fiveweeks old C57BL/6 mice (immune-related CT26 xenograft model) [1]. Doses: 50 and 100 mg/kg Route of Administration: po (oral gavage) one time/day for 21 days Experimental Results: Significant inhibition of tumor growth and tumor tissue size in a dose-dependent manner. Displayed Dramatically high antitumor activity (TGI = 75%) at 100 mg/kg. Increases plasma IFN-g levels and the number of CD+ and CD3+CD+ (activated cytotoxic T) cells. CD4+CD25+CD127low/- T regulatory cells diminished. No obvious toxicity was seen.
References	[1]. Novel biphenyl-based scaffold as potent and selective histone deacetylase 6 (HDAC6) inhibitors: Identification, development and pharmacological evaluation. Eur J Med Chem. 2022 Apr 5;233:114228.

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	1.9738 mL	9.8691 mL	19.7383 mL
	5 mM	0.3948 mL	1.9738 mL	3.9477 mL
	10 mM	0.1974 mL	0.9869 mL	1.9738 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.