Tubacin (known also as tubulin acetylation inducer) is a specific HDAC6 (histone deacetylase 6) inhibitor with potential anticancer activity. In a cell-free assay, it inhibits HDAC6 with an IC50 of 4 nM and shows approximately ~350-fold higher selectivity for HDAC6 than HDAC1.

Physicochemical Properties

Molecular Formula	C41H43N3O7S
Molecular Weight	721.86
Exact Mass	721.282
Elemental Analysis	C, 68.22; H, 6.00; N, 5.82; O, 15.51; S, 4.44
CAS #	537049-40-4
Related CAS #	537049-40-4
PubChem CID	6675804
Appearance	White to off-white solid powder
Density	1.3±0.1 g/cm3
Index of Refraction	1.668
LogP	5.82
Hydrogen Bond Donor Count	4
Hydrogen Bond Acceptor Count	9
Rotatable Bond Count	16
Heavy Atom Count	52
Complexity	1060
Defined Atom Stereocenter Count	3
SMILES	OCC(C=C1)=CC=C1[C@@H]2C[C@H](CSC3=NC(C4=CC=CC=C4)=C(C5=CC=CC=C5)O3)O[C@H](C6=CC=C(NC(CCCCCCC(NO)=O)=O)C=C6)O2
InChi Key	BHUZLJOUHMBZQY-YXQOSMAKSA-N
InChi Code	InChI=1S/C41H43N3O7S/c45-26-28-17-19-29(20-18-28)35-25-34(27-52-41-43-38(30-11-5-3-6-12-30)39(51-41)31-13-7-4-8-14-31)49-40(50-35)32-21-23-33(24-22-32)42-36(46)15-9-1-2-10-16-37(47)44-48/h3-8,11-14,17-24,34-35,40,45,48H,1-2,9-10,15-16,25-27H2,(H,42,46)(H,44,47)/t34-,35+,40+/m1/s1
Chemical Name	N-[4-[(2R,4R,6S)-4-[(4,5-diphenyl-1,3-oxazol-2-yl)sulfanylmethyl]-6-[4-(hydroxymethyl)phenyl]-1,3-dioxan-2-yl]phenyl]-N'-hydroxyoctanediamide
Synonyms	Tubacin; tubulin acetylation inducer
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	HDAC6 ( IC50 = 4 nM ); HDAC3 ( IC50 = 1.27 μM ); HDAC8 ( IC50 = 1.27 μM ); HDAC1 ( IC50 = 1.40 μM ); HDAC5 ( IC50 = 3.35 μM ); HDAC10 ( IC50 = 3.71 μM ); HDAC11 ( IC50 = 3.79 μM ); HDAC9 ( IC50 = 4.31 μM ); HDAC2 ( IC50 = 6.27 μM ); HDAC7 ( IC50 = 9.70 μM ); HDAC4 ( IC50 = 17.30 μM )
ln Vitro	Tubacin causes an increase in α-tubulin acetylation in A549 cells with an EC50 of 2.5 μM without directly stabilizing microtubules. Tubacin suppresses the migration of both HDAC6-overexpressing and wild-type cells by blocking HDAC6-mediated α-tubulin deacetylation.[2] Paclitaxel and tubulin acetylation are enhanced synergistically by tubbacin.[3] Tubacin activates caspases to cause cell apoptosis and dramatically suppresses the growth of both drug-sensitive and drug-resistant MM cells, with an IC50 of 5–20 μM.[4]
ln Vivo	In chick embryos, inhibition of HDAC6 activity by Tubacin reduces the formation of new blood vessels in matrigel/nylon mesh. In angioreactors implanted in mice, Tubacin also impairs the formation of new blood vessels.
Enzyme Assay	The Reaction Biology HDAC Spectrum platform is utilized for the execution of enzyme inhibition experiments. Isolated recombinant human protein was utilized in the HDAC1, 2, 4, 5, 6, 7, 8, 9, 10, and 11 assays; the HDAC3/NcoR2 complex is utilized in the HDAC3 test. Fluorogenic peptide derived from p53 residues 379–382 (RHKKAc) serves as the substrate for HDAC1, 2, 3, 6, 10, and 11 assays; fluorogenic diacyl peptide derived from p53 residues 379–382 (RHKAcKAc) serves as the substrate for HDAC8. For HDAC4, 5, 7, and 9 assays, acetyl-Lys(trifluoroacetyl)-AMC substrate is utilized. After dissolving compounds in DMSO, they are tested in 10-dose IC50 mode using a 3-fold serial dilution protocol that begins at 30 μM. Trichostatin A (TSA), the control compound, is tested in a 10-dose IC50 using a 3-fold serial dilution that begins at 5 μM. Curve-fitting the dose/response slopes yields IC50 values.
Cell Assay	The assay uses tubacin, TBSA, VPA, and TSA as HDAC inhibitors. TE671 and BHK-21 cells are used to test the cytotoxicity of HDACi using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. In 96-well plates, 5 × 104 cells are seeded per well, and each HDACi is then added at the indicated concentration. Following a 48-hour treatment period, each well receives 25 μL of MTT solution (5 mg/mL), which is then incubated for three hours at 37 °C with 5% CO2. To dissolve formazan crystals, 100 μL of DMSO is added to each well following three washes with phosphate buffer saline (PBS). The micro-ELISA reader measures OD570−630, and the survival rate is computed to show the inhibitory effects of each HDACi on the viability of BHK-21 and TE671 cells. ((Acontrol − Aexperiment)/Acontrol) × 100% is the survival rate (%). The values of the 50% cytotoxic concentration (CC50) are determined by computer programs.
Animal Protocol	Athymic nude mice implanted with angioreactors Tubacin is filled in semiclosed angioreactors, and then implanted into the mice.
References	[1]. J Am Chem Soc . 2010 Aug 11;132(31):10842-6. [2]. Proc Natl Acad Sci U S A . 2003 Apr 15;100(8):4389-94. [3]. Cancer Res . 2005 May 1;65(9):3883-93. [4]. Proc Natl Acad Sci U S A . 2005 Jun 14;102(24):8567-72. [5]. Protein Cell . 2011 Feb;2(2):150-60.
Additional Infomation	N-[4-[(2R,4R,6S)-4-[[(4,5-diphenyl-2-oxazolyl)thio]methyl]-6-[4-(hydroxymethyl)phenyl]-1,3-dioxan-2-yl]phenyl]-N'-hydroxyoctanediamide is a member of 1,3-oxazoles.

Solubility Data

Solubility (In Vitro)

DMSO: ~100 mg/mL (~138.5 mM) Water: <1 mg/mL Ethanol: <1 mg/mL

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.5 mg/mL (3.46 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 (3.46 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: 2% DMSO+30% PEG 300+5% Tween 80+ddH2O: 10 mg/mL  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	1.3853 mL	6.9266 mL	13.8531 mL
	5 mM	0.2771 mL	1.3853 mL	2.7706 mL
	10 mM	0.1385 mL	0.6927 mL	1.3853 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.