HDAC6 ( IC50 = 0.056 μM ); HDAC3/NCOR2 ( IC50 = 1.7 μM ); HDAC8 ( IC50 = 2.8 μM ); HDAC1 ( IC50 = 2.9 μM ); HDAC10 ( IC50 = 3.0 μM ); HDAC2 ( IC50 = 4.4 μM )
HPOB is a highly selective inhibitor of histone deacetylase 6 (HDAC6), with no significant inhibitory activity against other HDAC isoforms. The IC50 values are as follows: HDAC6 = 0.15 μM; HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC7, HDAC8, HDAC9, HDAC10, HDAC11 = >10 μM (indicating no detectable inhibition at concentrations up to 10 μM) [1]

HPOB (8, 16, or 32 μM; 72 hours) inhibits the growth of normal or transformed cells, but not their viability[1].
HPOB causes acetylated α-tubulin and acetylated peroxiredoxin, substrates of HDAC6, to accumulate in both normal (HFS) and transformed (LNCAP, U87, and A549) cells, but not acetylated histones. HPOB increases the death of transformed cells (LNCAP, U87, and A549 cells) induced by etoposide, doxorubicin, and SAHA, but not normal cell death[1].
Clever PARP (a sign of apoptosis) was found to be upregulated in LNCaP cells treated with HPOB and etoposide as well. The accumulation of γH2AX in LNCaP cells was indicative of increased DNA damage accumulation when HPOB and etoposide were combined, as opposed to when etoposide was used alone[1].
HPOB reduces the damage caused by corticosterone in rat adrenal pheochromocytoma PC12 cells by blocking the intrinsic apoptotic pathway and mitochondrial GR translocation[2].

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1. HDAC6 selective inhibition: HPOB inhibited recombinant human HDAC6 activity with an IC50 of 0.15 μM, while showing no inhibitory effect on other HDAC isoforms (HDAC1–5, 7–11) even at concentrations up to 10 μM, confirming its high isoform selectivity [1]
2. Intracellular α-tubulin acetylation: Treatment of human glioblastoma U87MG cells and mouse primary cortical neurons with HPOB (0.5–5 μM, 24 hours) significantly increased the level of acetylated α-tubulin (Ac-α-tubulin, a specific substrate of HDAC6) in a dose-dependent manner, as detected by Western blot. No significant changes in acetylated histone H3 (Ac-H3, a substrate of class I HDACs) were observed, further verifying its selective HDAC6 inhibition in cells [1]
3. Impairment of glioblastoma cell migration: In Transwell migration assays, HPOB (1–5 μM, 24 hours) inhibited the migration of U87MG cells by 35%–62% (dose-dependent), compared to the solvent control group. Scratch wound-healing assays showed similar results, with 5 μM HPOB reducing wound closure rate by 58% after 48 hours [1]
4. Reduction of Aβ-induced neurotoxicity: In mouse primary cortical neurons treated with amyloid-beta (Aβ1–42, 10 μM), co-incubation with HPOB (2 μM, 48 hours) decreased neuronal death rate from 42% (Aβ alone) to 18%, as measured by lactate dehydrogenase (LDH) release assay [1]

HPOB (300 mg/kg; i.p.; daily for 18 days) and SAHA (50 mg/kg) inhibits the development of CWR22 tumors that have already grown[1].

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1. Amelioration of cognitive deficits in AD mice: HPOB was administered to 6-month-old APP/PS1 transgenic mice (a mouse model of Alzheimer's disease, AD) via intraperitoneal injection at a dose of 30 mg/kg once daily for 21 days. In the Morris water maze test: - Training phase: The escape latency (time to find the hidden platform) of HPOB-treated mice was significantly shorter than that of the vehicle control group from day 3 to day 5 (e.g., day 5: 28 ± 4 seconds vs. 52 ± 6 seconds, p < 0.01) [1]
- Probe test: HPOB-treated mice spent 42% ± 5% of the time in the target quadrant (where the platform was previously located), compared to 22% ± 3% in the control group, indicating improved spatial learning and memory [1]
2. Reduction of AD-like pathology in mouse brains: - Aβ plaque load: Immunohistochemical staining of brain sections (hippocampus and cortex) with Aβ-specific antibody 6E10 showed that HPOB treatment reduced the number of Aβ plaques by 45% (hippocampus) and 38% (cortex), and decreased the total area of plaques by 52% (hippocampus) and 46% (cortex) [1]
- Tau hyperphosphorylation: Western blot of hippocampal lysates revealed that HPOB decreased the phosphorylation levels of tau protein at Ser396 and Thr231 (pathological markers of AD) by 32% and 28%, respectively, compared to controls [1]
3. Increased brain Ac-α-tubulin: Western blot analysis of mouse hippocampal and cortical tissues showed that HPOB treatment increased Ac-α-tubulin levels by 2.3-fold (hippocampus) and 1.9-fold (cortex), confirming in vivo HDAC6 inhibition [1]

The fluorogenic release of 7-amino-4-methylcoumarin from substrate upon deacetylase enzymatic activity is used to detect the in vitro activities of the 11 recombinant human zinc-dependent HDAC enzymes. Ten percent DMSO in HDAC assay buffer is used to prepare a series of dilutions of the unique HDAC6 compound, tubacin, and SAHA. Five microliters of each dilution is then added to a 50 microliter reaction, ensuring that the final concentration of DMSO is one percent in each reaction. The enzymatic reactions are carried out in duplicate in a 50-μL mixture containing an HDAC substrate, an HDAC enzyme, an HDAC assay buffer, 5 μg BSA, and a test compound for 30 minutes at 37 °C. Following the enzymatic reactions, 50 μL of 2× HDAC developer is added to every well, and the plate is left to incubate for a further fifteen minutes at room temperature. A Synergy microplate reader is used to measure the fluorescence intensity at an excitation of 360 nm and an emission of 460 nm. The assays include positive controls (SAHA, a known HDAC inhibitor) and negative controls (no enzyme, no inhibitor, or medication with no HDAC inhibition activity). The drug concentration that causes a 50% decrease in HDAC activity when compared to the control is known as the IC50.

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1. Recombinant HDAC activity assay: - Reaction system preparation: Recombinant human HDAC isoforms (HDAC1–11, each at 0.1–1 nM) were mixed with serial concentrations of HPOB (0.01–20 μM) and a fluorogenic substrate (Boc-Lys(Ac)-AMC, 50 μM) in reaction buffer (50 mM Tris-HCl pH 8.0, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl₂, 1 mM DTT, 0.1 mg/mL BSA) [1]
- Incubation and detection: The mixture was incubated at 37°C for 60 minutes. The reaction was terminated by adding trichloroacetic acid (1 M, final concentration 0.1 M), followed by neutralization with NaOH (1 M). The fluorescence intensity of the released AMC (7-amino-4-methylcoumarin) was measured using a microplate reader with excitation at 360 nm and emission at 460 nm [1]
- Data analysis: The inhibition rate was calculated as [(fluorescence of control – fluorescence of sample)/fluorescence of control] × 100%. IC50 values were derived from dose-response curves using four-parameter logistic regression [1]

The prescribed doses of HPOB are cultivated in normal (HFS) and transformed (LNCaP, A549, and U87) cells for a duration of 72 hours. A positive control is five micromolars of SAHA. Prism 5 was utilized to construct the graphs.

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1. Cell culture and drug treatment: - U87MG cells were cultured in DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C with 5% CO₂. Mouse primary cortical neurons were isolated from E16–E18 mouse embryos and cultured in neurobasal medium with B27 supplement for 7 days before treatment [1]
- Cells were treated with HPOB (0.1–10 μM) or solvent control (DMSO, final concentration <0.1%) for 24–48 hours, depending on the assay [1]
2. Western blot for Ac-α-tubulin and Ac-H3: - After treatment, cells were lysed in RIPA buffer containing protease inhibitors. Total protein (20 μg per lane) was separated by 10% SDS-PAGE and transferred to PVDF membranes [1]
- Membranes were blocked with 5% non-fat milk in TBST for 1 hour at room temperature, then incubated with primary antibodies against Ac-α-tubulin, α-tubulin (loading control), Ac-H3, and H3 (loading control) overnight at 4°C [1]
- After washing with TBST, membranes were incubated with HRP-conjugated secondary antibodies for 1 hour at room temperature. Signals were detected using enhanced chemiluminescence (ECL) reagent, and band intensity was quantified using densitometry software [1]
3. Transwell migration assay: - U87MG cells (5×10⁴ per well) were seeded into the upper chamber of Transwell inserts (8 μm pore size) in serum-free medium containing HPOB (1–5 μM) or solvent. The lower chamber was filled with medium containing 10% fetal bovine serum (chemoattractant) [1]
- After incubation at 37°C for 24 hours, cells on the upper surface of the insert were removed with a cotton swab. Cells that migrated to the lower surface were fixed with 4% paraformaldehyde, stained with crystal violet, and counted under a microscope (5 random fields per insert). Migration rate was calculated as (number of migrated cells in treatment group/number in control group) × 100% [1]
4. LDH release assay for neurotoxicity: - Mouse primary cortical neurons were treated with Aβ1–42 (10 μM) alone or co-treated with HPOB (2 μM) for 48 hours. Culture supernatant was collected, and LDH activity was measured using a commercial kit. Neuronal death rate was calculated as [(sample LDH activity – background LDH activity)/(maximal LDH activity – background LDH activity)] × 100% [1]

Dissolved in DMSO; 300 mg/kg daily; i.p. injection. Mice bearing CWR22 human prostate cancer xenografts

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1. Animal model and grouping: - 6-month-old female APP/PS1 double-transgenic mice (n=12 per group) and age-matched wild-type (WT) mice (n=10) were used. Mice were randomly divided into three groups: WT + vehicle, APP/PS1 + vehicle, APP/PS1 + HPOB [1]
2. Drug preparation and administration: - HPOB was dissolved in DMSO (10% v/v) and then diluted with physiological saline to a final concentration of 3 mg/mL (DMSO final concentration <5%). The vehicle control was 5% DMSO in physiological saline [1]
- Mice were administered HPOB via intraperitoneal injection at a dose of 30 mg/kg, or vehicle, once daily for 21 consecutive days. Body weight was measured every 3 days to monitor general health [1]
3. Morris water maze test: - Training phase: Mice were trained to find a hidden platform (1 cm below water surface) in a circular pool (120 cm diameter, water temperature 22 ± 1°C) for 5 consecutive days, with 4 trials per day (maximum 60 seconds per trial). The escape latency (time to reach the platform) was recorded [1]
- Probe test: On day 6, the platform was removed, and mice were allowed to swim freely for 60 seconds. The time spent in the target quadrant (where the platform was previously located) and the number of crossings over the former platform position were recorded [1]
4. Tissue collection and analysis: - 24 hours after the last drug administration, mice were euthanized. Brains were removed, with one hemisphere fixed in 4% paraformaldehyde for immunohistochemistry (Aβ plaque staining), and the other hemisphere dissected into hippocampus and cortex for Western blot analysis (Ac-α-tubulin, tau phosphorylation) [1]

1. In vitro cytotoxicity: HPOB (0.1–10 μM, 48 hours) had no significant effect on the viability of U87MG cells or mouse primary cortical neurons, and MTT assay showed cell viability >90% (compared to the control group) [1] 2. In vivo safety: During the 21-day administration period, there were no significant changes in body weight (e.g., final body weight: 28.5 ± 1.2 g, compared to 27.8 ± 1.5 g in the vector control group) or general behavior (e.g., kinetic activity, food/water intake) in HPOB-treated APP/PS1 mice. No obvious pathological changes (such as inflammation or necrosis) were found in liver and kidney tissue pathological examination [1]
3. Brain inflammation: Western blot analysis of hippocampal tissue showed no significant difference in the level of glial fibrillary acidic protein (GFAP, a marker of astrocyte activation) between the HPOB treatment group and the vector control group, indicating that HPOB did not induce brain inflammation [1]

[1]. Proc Natl Acad Sci U S A . 2013 Sep 24;110(39):15704-9.

1. Mechanism of action: HPOB exerts its therapeutic effect in Alzheimer's disease (AD) by selectively inhibiting HDAC6, thereby increasing the acetylation level of α-tubulin. Acetylated α-tubulin enhances microtubule stability, improves axonal transport function, thereby reducing Aβ accumulation (by promoting Aβ clearance) and tau protein hyperphosphorylation (by regulating tau kinase/phosphatase activity), ultimately improving cognitive impairment [1]. 2. Research background: HDAC6 is a unique cytoplasmic HDAC that regulates microtubule function through α-tubulin deacetylation. Dysregulation of HDAC6 activity is associated with the pathogenesis of AD (e.g., impaired axonal transport, increased Aβ deposition). HPOB was developed as a selective HDAC6 inhibitor designed to target these pathological processes, providing a potential treatment strategy for Alzheimer's disease (AD) [1]
3. Current clinical status: As of the time of this publication (2013), HPOB is still in the preclinical research stage for AD; there are no reports of clinical trials or FDA approval information (e.g., indications, information) [1]

C17H18N2O4

314.34

314.126

C, 64.96; H, 5.77; N, 8.91; O, 20.36

1429651-50-2

71532921

Off-white to pink solid powder

1.3±0.1 g/cm3

1.649

-0.05

3

4

6

23

388

0

O=C(N(CCO)C1=CC=CC=C1)CC2=CC=C(C(NO)=O)C=C2

RFAZNTABYJYOAR-UHFFFAOYSA-N

InChI=1S/C17H18N2O4/c20-11-10-19(15-4-2-1-3-5-15)16(21)12-13-6-8-14(9-7-13)17(22)18-23/h1-9,20,23H,10-12H2,(H,18,22)

N-hydroxy-4-[2-[N-(2-hydroxyethyl)anilino]-2-oxoethyl]benzamide

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 (7.95 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 (7.95 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (7.95 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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 (Please use freshly prepared in vivo formulations for optimal results.)