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 )
Histone Deacetylase 6 (HDAC6): In recombinant human HDAC6 enzyme assay, the IC50 of Tubacin (tubulin acetylation inducer) for HDAC6 inhibition was 4 nM [2]
- Histone Deacetylase 6 (HDAC6): In human breast cancer MDA-MB-231 cells, the EC50 of Tubacin (tubulin acetylation inducer) for increasing acetylated α-tubulin (a downstream marker of HDAC6 inhibition) was 12 nM [3]
- Histone Deacetylase 6 (HDAC6) (vs. other HDAC subtypes): Tubacin (tubulin acetylation inducer) showed high selectivity for HDAC6; IC50 for HDAC1/2/3 (class I HDACs) was >10 μM, IC50 for HDAC7/9 (class II HDACs) was >5 μM, and IC50 for HDAC11 (class IV HDAC) was >20 μM [4]

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]

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In recombinant HDAC6 enzyme system: Tubacin (tubulin acetylation inducer) inhibited HDAC6 activity in a dose-dependent manner (IC50=4 nM). At 50 nM, it increased the acetylation level of recombinant tubulin (substrate of HDAC6) by 3.8-fold compared to the control, as detected by Western blot with anti-acetylated α-tubulin antibody [2]
- In human breast cancer cell lines (MDA-MB-231, MCF-7): Tubacin (tubulin acetylation inducer) inhibited cell proliferation with IC50 values of 18 nM (MDA-MB-231) and 25 nM (MCF-7) at 72 h (MTT assay). Flow cytometry (Annexin V/PI staining) showed that 20 nM treatment for 48 h increased apoptotic rates from 2.1% (control) to 28.3% (MDA-MB-231) and 24.5% (MCF-7). Western blot revealed upregulated acetylated α-tubulin (4.2-fold in MDA-MB-231), cleaved caspase-3 (3.5-fold), and cleaved PARP (2.8-fold) [3]
- In human pancreatic cancer PANC-1 cells: Tubacin (tubulin acetylation inducer) at 15 nM suppressed cell migration (Transwell assay: migrated cells reduced by 62% vs. control) and invasion (Matrigel assay: invasive cells reduced by 58% vs. control) at 24 h. PCR results showed downregulated mRNA levels of MMP-2 (55%) and MMP-9 (60%), and upregulated mRNA of E-cadherin (2.3-fold, an epithelial marker) [4]
- In mouse embryonic fibroblast (MEF) cells (wild-type vs. HDAC6 knockout): Tubacin (tubulin acetylation inducer) (10 nM) increased acetylated α-tubulin by 3.9-fold in wild-type MEFs, but no significant change was observed in HDAC6 knockout MEFs, confirming its HDAC6-specific action. Additionally, 20 nM treatment induced G2/M cell cycle arrest in wild-type MEFs (G2/M phase ratio increased from 18% to 42%), but not in HDAC6 knockout MEFs [5]

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.

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In nude mice bearing MDA-MB-231 breast cancer xenografts: Mice were randomly divided into control (DMSO/saline) and Tubacin (tubulin acetylation inducer) groups (2 mg/kg, intraperitoneal injection, once every 2 days for 21 days). The treatment group showed a 68% reduction in tumor volume (from 1050 mm³ to 336 mm³) and a 62% decrease in tumor weight (from 1.2 g to 0.46 g) vs. control. Median survival was prolonged by 22 days (control: 45 days; treatment: 67 days). Immunohistochemistry of tumor tissues showed increased acetylated α-tubulin (4.5-fold) and cleaved caspase-3 (3.2-fold), and decreased Ki-67 (proliferation marker, 48% reduction) [3]
- In SCID mice with PANC-1 pancreatic cancer liver metastasis (via intrasplenic injection): Tubacin (tubulin acetylation inducer) was administered at 1.5 mg/kg via tail vein injection once every 3 days for 28 days. The treatment group had 72% fewer liver metastatic nodules (control: 28 ± 4 nodules; treatment: 8 ± 2 nodules) and reduced metastatic lesion area (from 35% of liver tissue to 12%). Western blot of liver metastatic tissues showed upregulated acetylated α-tubulin (3.8-fold) and downregulated MMP-9 (65%) [4]

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.

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Recombinant HDAC6 Activity Assay: Prepare reaction mixtures containing 50 nM recombinant human HDAC6, 100 μM fluorogenic substrate (peptide conjugated to 7-amino-4-methylcoumarin, specific for HDAC6), and Tubacin (tubulin acetylation inducer) (0.1–100 nM) in assay buffer (50 mM Tris-HCl, pH 8.0, 137 mM NaCl, 2.7 mM KCl). Incubate the mixture at 37°C for 45 minutes. Add a stop solution (100 mM Tris-HCl, pH 4.5, containing trypsin) to terminate the reaction and release fluorescent product. Measure fluorescence intensity at excitation 360 nm and emission 460 nm using a microplate reader. Calculate HDAC6 inhibition rate as [(control fluorescence – sample fluorescence)/control fluorescence] × 100%. Plot dose-response curves to determine IC50 [2]
- HDAC Subtype Selectivity Assay: Set up parallel reactions for recombinant HDAC1, HDAC2, HDAC3, HDAC7, HDAC9, and HDAC11 (each at 50 nM) using their respective fluorogenic substrates. Treat each reaction with Tubacin (tubulin acetylation inducer) (0.1 nM–50 μM) and incubate at 37°C for 60 minutes. Detect fluorescence as described above and calculate IC50 for each HDAC subtype to assess selectivity [4]

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.

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Breast Cancer Cell Proliferation Assay: Seed MDA-MB-231/MCF-7 cells in 96-well plates at 3×10³ cells/well. After 24 h attachment, treat with Tubacin (tubulin acetylation inducer) (1, 5, 10, 20, 50 nM; control: DMSO). Incubate for 24, 48, 72 h. Add MTT reagent (5 mg/mL) and incubate for 4 h. Remove supernatant, add DMSO to dissolve formazan crystals. Measure absorbance at 570 nm. Calculate proliferation inhibition rate = [1 – (absorbance of treatment group/absorbance of control group)] × 100%. Determine IC50 using GraphPad Prism software [3]
- MEF Cell Acetylated α-Tubulin Detection (Western Blot): Seed wild-type and HDAC6 knockout MEFs in 6-well plates at 2×10⁵ cells/well. Incubate for 24 h, then treat with Tubacin (tubulin acetylation inducer) (1, 5, 10, 20 nM) for 16 h. Lyse cells with RIPA buffer containing protease inhibitors. Separate proteins by SDS-PAGE, transfer to PVDF membranes, and block with 5% non-fat milk for 1 h. Incubate with primary antibodies against acetylated α-tubulin and α-tubulin (loading control) at 4°C overnight. Incubate with HRP-conjugated secondary antibody for 1 h at room temperature. Detect chemiluminescence signals and quantify band intensity using ImageJ software [5]
- PANC-1 Cell Invasion Assay: Coat Transwell inserts (8 μm pores) with Matrigel (1:3 dilution in serum-free medium) and incubate at 37°C for 2 h to form a gel. Seed PANC-1 cells (5×10⁴ cells/insert) in the upper chamber with Tubacin (tubulin acetylation inducer) (5, 10, 15 nM); add complete medium to the lower chamber. Incubate for 24 h, fix cells on the lower surface with 4% paraformaldehyde, stain with crystal violet. Count stained cells under a microscope (5 fields/insert) and calculate invasion inhibition rate vs. control [4]

Athymic nude mice implanted with angioreactors
Tubacin is filled in semiclosed angioreactors, and then implanted into the mice.

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MDA-MB-231 Breast Cancer Xenograft Model: Female nude mice (6–8 weeks old) were injected subcutaneously with 4×10⁶ MDA-MB-231 cells into the right flank. When tumors reached 100–150 mm³, mice were randomly divided into 2 groups (n=6/group): control group (intraperitoneal injection of 10% DMSO in 0.9% saline, once every 2 days) and Tubacin (tubulin acetylation inducer) group (intraperitoneal injection of 2 mg/kg Tubacin (tubulin acetylation inducer) dissolved in 10% DMSO/0.9% saline, once every 2 days). Treatments continued for 21 days. Every 3 days, measure tumor volume (formula: volume = length × width² / 2) and mouse body weight. Monitor mouse survival for 80 days to calculate median survival. At the end of treatment, sacrifice mice, excise tumors for immunohistochemistry (acetylated α-tubulin, cleaved caspase-3, Ki-67) [3]
- PANC-1 Pancreatic Cancer Liver Metastasis Model: Male SCID mice (7–9 weeks old) were anesthetized, and 2×10⁶ PANC-1 cells were injected into the spleen. After 7 days (to establish initial metastasis), mice were divided into control (tail vein injection of 5% DMSO in 0.9% saline, once every 3 days) and Tubacin (tubulin acetylation inducer) group (tail vein injection of 1.5 mg/kg Tubacin (tubulin acetylation inducer) dissolved in 5% DMSO/0.9% saline, once every 3 days). Treatments continued for 28 days. At the endpoint, sacrifice mice, harvest livers, count metastatic nodules under a dissecting microscope, and measure metastatic lesion area via H&E staining. Collect metastatic tissues for Western blot (acetylated α-tubulin, MMP-9) [4]

In male SD rats (250–300 g), a single intravenous injection of 2 mg/kg Tubacin (a microtubule acetylation inducer) was administered. Plasma concentration-time curves were determined using ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). The maximum plasma concentration (Cmax) was reached 85.6 ng/mL 5 minutes after administration. The area under the plasma concentration-time curve (AUC₀₋∞) was 128.4 ng·h/mL. The elimination half-life (t₁/₂) was 1.8 h. Tissue distribution analysis showed that the highest drug concentrations were found in the liver (12.3 μg/g at 1 hour) and kidney (8.5 μg/g at 1 hour), while the lowest drug concentrations were found in brain tissue (0.3 μg/g at 1 hour) [5]
- In male C57BL/6 mice (20–25 g), a single oral dose of 5 mg/kg Tubacin (a microtubule acetylation inducer) resulted in an oral bioavailability of 12.5% (calculated by comparing the AUC₀₋∞ of oral and intravenous administration). Urinary excretion within 24 hours was 18.3% (primarily as metabolites), and fecal excretion was 65.2% (of which 30% was the original drug) [5]

In nude mice treated with 2 mg/kg Tubacin (a microtubule acetylation inducer) via intraperitoneal injection (once every 2 days for 21 days): no significant weight loss (weight change: -2.8% vs. control group: +3.1%, P > 0.05) or significant toxic symptoms (drowsiness, diarrhea, hair loss) were observed. Serum biochemical parameters: ALT (27.3 U/L vs. control group 25.1 U/L), AST (43.2 U/L vs. control group 41.5 U/L), BUN (14.8 mg/dL vs. control group 14.2 mg/dL) and creatinine (0.78 mg/dL vs. control group 0.75 mg/dL) were not significantly different from the control group [3] - In SD rats treated with 2 mg/kg Tubacin (a microtubule acetylation inducer) (intravenous injection, single dose): plasma protein binding (measured by ultrafiltration) was 78.5%. No significant necrosis or inflammation was observed in liver and kidney tissue pathology 24 hours after administration [5]
- In SCID mice treated with 1.5 mg/kg Tubacin (a microtubule acetylation inducer) (tail vein injection, every 3 days for 28 days): no significant changes were observed in food intake (treatment group: 4.2 g/day vs. control group: 4.5 g/day) or hematological parameters (erythrocytes, leukocytes, platelets) [4]

[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.

N-[4-[(2R,4R,6S)-4-[[(4,5-diphenyl-2-oxazolyl)thio]methyl]-6-[4-(hydroxymethyl)phenyl]-1,3-dioxane-2-yl]phenyl]-N'-hydroxyoctadiamide is a member of the 1,3-oxazolyl class of compounds. Tubacin (a microtubule acetylation inducer) is a first-in-class selective HDAC6 inhibitor. Unlike non-selective HDAC inhibitors, it specifically targets HDAC6 (a cytoplasmic deacetylase). Its mechanism of action involves inhibiting HDAC6-mediated α-tubulin deacetylation, leading to an increase in acetylated α-tubulin, thereby disrupting microtubule dynamics, cytoskeleton organization, and intracellular transport [2]
- In breast cancer, Tubacin (tubulin acetylation inducer) exerts its antitumor effect by inducing G2/M phase cell cycle arrest and apoptosis, which is related to the accumulation of acetylated α-tubulin induced by HDAC6 inhibition and subsequent activation of the caspase-dependent apoptosis pathway [3]
- For pancreatic cancer metastasis, Tubacin (tubulin acetylation inducer) inhibits cell migration and invasion by downregulating matrix metalloproteinases (MMP-2, MMP-9) and upregulating E-cadherin, which is mediated by actin cytoskeleton remodeling induced by HDAC6 inhibition [4]
- In preclinical studies, Tubacin (tubulin acetylation inducer) has shown antitumor activity due to its inhibition of HDAC6. The selectivity of these inhibitors has shown potential in solid tumors (breast cancer, pancreatic cancer) and hematologic malignancies (not reported in the literature for these), and this selectivity reduces off-target toxicity compared to pan-HDAC inhibitors [5].

C41H43N3O7S

721.86

721.282

C, 68.22; H, 6.00; N, 5.82; O, 15.51; S, 4.44

537049-40-4

6675804

White to off-white solid powder

1.3±0.1 g/cm3

1.668

5.82

4

9

16

52

1060

3

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

BHUZLJOUHMBZQY-YXQOSMAKSA-N

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

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

Tubacin; tubulin acetylation inducer

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

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.

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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.

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Solubility in Formulation 3: 2% DMSO+30% PEG 300+5% Tween 80+ddH2O: 10 mg/mL

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 (Please use freshly prepared in vivo formulations for optimal results.)