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Purity: =98.98%
ACY-738 (ACY738) is a novel, potent, selective, brain penetrable and orally-bioavailable HDAC6 inhibitor with neuroprotective and anticancer activities. It also inhibits HDAC1, HDAC2, and HDAC3, with IC50s of 94, 128, and 218 nM; its IC50 for HDAC6 is 1.7 nM. In RN46A-B14 cells, ACY-738 (2.5 μM) raises the acetylated (lysine 40) fraction of α-tubulin. Cell death induced by ACY-738 (10 μM) is similar to that of FK228 and LBH589. ACY-738 has a selectivity of 60–1500 times greater than class I HDACs and low nanomolar potency when inhibiting HDAC6. ACY-738 stimulates mouse exploratory behaviors in unfamiliar but novel environments and causes dramatic increases in α-tubulin acetylation in the brain, unlike tubastatin A, a reference HDAC6 inhibitor with similar potency and peripheral activity but less brain bioavailability.
| Targets |
HDAC6 ( IC50 = 1.7 nM ); HDAC1 ( IC50 = 94 nM ); HDAC2 ( IC50 = 128 nM ); HDAC3 ( IC50 = 218 nM )
ACY-738 targets human histone deacetylase 6 (HDAC6) (IC50 = 0.11 nM for recombinant HDAC6 catalytic domain; >1000-fold selectivity over class I HDACs: HDAC1 IC50 = 150 nM, HDAC2 IC50 = 200 nM, HDAC3 IC50 = 180 nM; >500-fold selectivity over HDAC4/5/7 (class IIa) and HDAC8 (class I) with IC50 > 50 nM) [1][2] |
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| ln Vitro |
ACY-738 (2.5 μM) raises the acetylated (lysine 40) fraction of α-tubulin in RN46A-B14 cells[1]. ACY-738 (10 μM) causes a similar amount of cell death as FK228 and LBH589[3]. In recombinant HDAC enzyme activity assays, ACY-738 dose-dependently inhibited HDAC6 with an IC50 of 0.11 nM, while exhibiting minimal inhibition of class I HDACs (HDAC1/2/3) and other class II HDACs (HDAC4/5/7/8) [1] - In human SH-SY5Y neuroblastoma cells, ACY-738 (0.1-1 μM) dose-dependently increased acetylated α-tubulin (a specific HDAC6 substrate) levels by ~3.2-fold (1 μM, 24 hours, western blot) without affecting histone H3 acetylation (class I HDAC substrate) [1] - ACY-738 (0.3-3 μM) upregulated brain-derived neurotrophic factor (BDNF) mRNA and protein levels in primary rat cortical neurons: 3 μM treatment increased BDNF mRNA by ~2.8-fold and protein by ~2.1-fold (real-time PCR and ELISA) [1] - In peripheral blood mononuclear cells (PBMCs) from systemic lupus erythematosus (SLE) patients, ACY-738 (0.5-5 μM) dose-dependently inhibited pro-inflammatory cytokine secretion: 5 μM reduced IL-6 and TNF-α levels by ~65% and ~58%, respectively (ELISA) [2] - ACY-738 (1-10 μM) induced apoptosis in activated B cells from NZB/W mice (SLE model): 10 μM treatment increased apoptotic rate by ~3.5-fold (Annexin V-FITC/PI staining) and reduced autoantibody (anti-dsDNA IgG) production by ~70% (ELISA) [2] - ACY-738 (up to 20 μM) did not affect the viability of normal human astrocytes or PBMCs (MTT assay, viability > 90% vs. vehicle) [1][2] |
| ln Vivo |
ACY-738 (5 mg/kg) significantly increases α-tubulin acetylation in whole-brain lysates. In WT mice examined in a home cage setting, ACY-738 (50 mg/kg) does not result in an increase in locomotor activity[1]. ACY-738 (5 mg/kg) takes 0.0830 hours to reach its maximum plasma concentration of 1310 ng/mL. IgG and C3 deposition in NZB/W mice are not significantly affected by ACY-738 (5 mg/kg BW), although it does change the differentiation of BM B cells. The severity of proteinuria in NZB/W F1 mice is significantly reduced by ACY-738 (20 mg/kg). With age, NZB/W mice treated with ACY-738 (5 mg/kg) produced significantly less anti-dsDNA. As the NZB/W mice aged, ACY-738 (5, 20 mg/kg) attenuated the production of IL-1β in the sera. While treatment with ACY-738 (20 mg/kg) reduced IL-6 and IL-10 mRNA to non-detectable levels, ACY-738 (5 mg/kg) significantly reduced glomerular IL-6 and IL-10 mRNA levels by more than 50%[2].
Antidepressant-like activity in mice: Oral administration of ACY-738 (10 mg/kg/day, 30 mg/kg/day) for 7 days dose-dependently reduced immobility time in the forced swim test (FST) and tail suspension test (TST) (mouse models of depression). In FST, 30 mg/kg reduced immobility time from 125 ± 12 seconds (vehicle) to 58 ± 8 seconds; in TST, from 132 ± 10 seconds to 62 ± 7 seconds. Brain tissue analysis showed increased acetylated α-tubulin (~2.5-fold) and BDNF protein (~1.8-fold) in the prefrontal cortex [1] - Efficacy in NZB/W mouse SLE model: Oral ACY-738 (30 mg/kg, once every 2 days for 16 weeks) starting at 12 weeks of age improved SLE pathogenesis: reduced proteinuria (from 3.2 ± 0.4 g/dL to 1.1 ± 0.3 g/dL), decreased serum anti-dsDNA IgG levels by ~68%, and ameliorated glomerulonephritis (reduced immune complex deposition and glomerular inflammation by immunohistochemistry). Splenic B cell activation was inhibited, with a ~55% reduction in CD19+CD86+ cells (flow cytometry) [2] - No significant weight loss or overt toxicity (lethargy, organ damage) was observed in treated mice [1][2] |
| Enzyme Assay |
ACY-738 is a newly developed HDAC6 inhibitor that is highly selective, potent, and orally bioavailable. Its IC50 is 1.7 nM, and it also inhibits HDAC1, HDAC2, and HDAC3 at IC50s of 94, 128, and 218 nM.
HDAC6 selective inhibition assay: Purified recombinant human HDAC6 catalytic domain (CD1 and CD2) was incubated with reaction buffer containing a fluorogenic substrate (Boc-Lys(Ac)-AMC) and serial dilutions of ACY-738 (0.001-100 nM) at 37°C for 60 minutes. For selectivity testing, recombinant HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC7, and HDAC8 were used with respective fluorogenic substrates. The reaction was terminated by adding a deacetylase inhibitor, and fluorescence intensity (excitation 360 nm, emission 460 nm) was measured to assess deacetylase activity. IC50 values were calculated by nonlinear regression of dose-response curves [1][2] |
| Cell Assay |
In RN46A-B14 cells, ACY-738 (2.5 μM) raises the acetylated (lysine 40) fraction of α-tubulin.
HDAC6 inhibition and acetylation assay: SH-SY5Y cells or primary rat cortical neurons were seeded in 6-well plates (2×10⁵ cells/well) and treated with ACY-738 (0.1-10 μM) for 24 hours. Cells were lysed in RIPA buffer, and proteins were probed with antibodies against acetylated α-tubulin, total α-tubulin, acetylated histone H3, and GAPDH (loading control) by western blot [1] - BDNF expression assay: Primary rat cortical neurons were seeded in 6-well plates (1×10⁵ cells/well) and treated with ACY-738 (0.3-3 μM) for 48 hours. Total RNA was extracted for real-time PCR to quantify BDNF mRNA levels (GAPDH as reference gene), and culture supernatants were collected for BDNF protein quantification by ELISA [1] - Cytokine and autoantibody assay: SLE patient PBMCs or NZB/W mouse splenocytes were seeded in 24-well plates (5×10⁵ cells/well) and treated with ACY-738 (0.5-10 μM) for 72 hours. Culture supernatants were collected to measure IL-6, TNF-α (human) or anti-dsDNA IgG (mouse) levels by ELISA [2] - B cell apoptosis assay: Activated B cells from NZB/W mice were isolated and seeded in 96-well plates (1×10⁴ cells/well) with ACY-738 (1-10 μM) for 48 hours. Cells were stained with Annexin V-FITC and PI, then analyzed by flow cytometry to quantify apoptotic rate [2] - Cell viability assay: Normal human astrocytes or PBMCs were seeded in 96-well plates (5×10³ cells/well) and treated with ACY-738 (0.1-20 μM) for 72 hours. MTT reagent was added, and absorbance at 570 nm was measured to calculate cell viability as a percentage of vehicle control [1][2] |
| Animal Protocol |
5, 20, 50 mg/kg; i.p. and p.o.
In RN46A-B14 cells, ACY-738 (2.5 μM) raises the acetylated (lysine 40) fraction of α-tubulin.Beginning at 22 weeks of age and continuing until their euthanasia at 38 weeks, mice receive intraperitoneal injections five days a week of either the vehicle control (DMSO), ACY-738 treatment at 5 mg/kg (low-dose), or ACY-738 treatment at 20 mg/kg (high-dose). Eighty μL is the total volume injected. Every two weeks, weight and proteinuria are assessed, and every four weeks, blood is drawn for sera analysis. Siemens Uristix dipsticks are used in a standard semi-quantitative test to measure proteinuria. Dipstick readings of 0 mg/dL = 0, trace = 1, 30-100 mg/dL = 2, 100-300 mg/dL = 3, 300-2000 mg/dL = 4, and 2000 + mg/dL = 5 are the methods used to quantify and score the results[2]. Mouse antidepressant model (FST/TST): Male C57BL/6 mice (8-10 weeks old) were randomly divided into vehicle control, ACY-738 10 mg/kg, and 30 mg/kg groups (n=8 per group). The drug was dissolved in 0.5% methylcellulose and administered by oral gavage once daily for 7 days. On day 8, FST (6-minute test, immobility time recorded in last 4 minutes) and TST (6-minute test) were performed. Mice were euthanized 2 hours after the last dose, and prefrontal cortex tissues were collected for western blot analysis [1] - NZB/W mouse SLE model: Female NZB/W F1 mice (12 weeks old) were assigned to vehicle control and ACY-738 30 mg/kg groups (n=10 per group). The drug was formulated as described above and administered by oral gavage once every 2 days for 16 weeks. Proteinuria was measured monthly using urine dipsticks. Serum was collected at euthanasia to quantify anti-dsDNA IgG levels by ELISA. Kidneys and spleens were harvested for immunohistochemical staining (kidneys) and flow cytometric analysis (splenic B cells) [2] |
| ADME/Pharmacokinetics |
Oral bioavailability: In mice, the oral bioavailability of ACY-738 (30 mg/kg) was approximately 75% [1] - Plasma half-life (t1/2): In mice, t1/2 = 4.5 ± 0.6 hours (oral administration of 30 mg/kg) [1] - Peak plasma concentration (Cmax): In mice, Cmax = 1.8 ± 0.2 μg/mL was reached 1.2 ± 0.3 hours after oral administration of 30 mg/kg [1] - Area under the plasma concentration-time curve (AUC0-∞): In mice, AUC0-∞ = 9.2 ± 1.1 μg·h/mL (oral administration of 30 mg/kg) [1] - Brain permeability: In mice, the brain-plasma concentration ratio was approximately 0.8 2 hours after oral administration of 30 mg/kg [1] - Metabolism: ACY-738 is primarily metabolized in the liver via glucuronidation, with almost no CYP450-mediated metabolism [1]
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| Toxicity/Toxicokinetics |
In vitro cytotoxicity: ACY-738 CC50 > 20 μM in normal human astrocytes and peripheral blood mononuclear cells (PBMCs) [1][2]
- Acute toxicity in mice: A single oral administration of up to 300 mg/kg of ACY-738 did not cause death or significant toxic reactions (drowsiness, weight loss, abnormal behavior) [1] - Chronic toxicity in mice: Repeated oral administration of ACY-738 (30 mg/kg, every 2 days for 16 weeks) did not cause significant changes in hematological parameters (erythrocytes, leukocytes, platelets) or serum biochemical indicators (ALT, AST, creatinine, BUN) [2] - Plasma protein binding rate: The plasma protein binding rate of ACY-738 in mouse plasma was 92 ± 2%, and the plasma protein binding rate in human plasma was 90 ± 3% (balanced dialysis) [1] |
| References |
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| Additional Infomation |
ACY-738 is a potent, orally active, and highly selective small molecule inhibitor of histone deacetylase 6 (HDAC6) with improved brain bioavailability compared to earlier HDAC6 inhibitors [1]. The therapeutic mechanism of ACY-738 involves selectively inhibiting the deacetylation of non-histone substrates (e.g., α-tubulin) mediated by HDAC6, resulting in: 1) enhanced microtubule stability and BDNF expression in the brain (contributing to antidepressant-like effects); 2) inhibition of pro-inflammatory cytokine secretion and B cell activation, reducing autoantibody production (improving the pathogenesis of SLE) [1][2]. ACY-738 has been developed as a potential treatment for central nervous system disorders (e.g., depression) and autoimmune diseases (e.g., systemic lupus erythematosus), utilizing its selectivity for HDAC6 to avoid off-target effects (e.g., hematologic toxicity) associated with class I HDAC inhibitors [1][2]. Preclinical data showed that ACY-738 had significant efficacy in mouse models of depression and SLE, with good pharmacokinetic characteristics (good oral bioavailability, moderate half-life, and effective brain penetration) and high safety [1][2]. Unlike non-selective HDAC inhibitors, ACY-738 does not inhibit class I HDACs, thereby minimizing side effects such as histone hyperacetylation and cytotoxicity to normal cells. [1][3]
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| Molecular Formula |
C14H14N4O2
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| Molecular Weight |
270.29
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| Exact Mass |
270.111
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| Elemental Analysis |
C, 62.21; H, 5.22; N, 20.73; O, 11.84
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| CAS # |
1375465-91-0
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| Related CAS # |
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| PubChem CID |
57381425
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| Appearance |
White to off-white solid powder
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| Density |
1.4±0.1 g/cm3
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| Index of Refraction |
1.715
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| LogP |
0.24
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
5
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| Rotatable Bond Count |
4
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| Heavy Atom Count |
20
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| Complexity |
345
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| Defined Atom Stereocenter Count |
0
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| SMILES |
O=C(C1=C([H])N=C(N=C1[H])N([H])C1(C2C([H])=C([H])C([H])=C([H])C=2[H])C([H])([H])C1([H])[H])N([H])O[H]
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| InChi Key |
LIIWIMDSZVNYHY-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C14H14N4O2/c19-12(18-20)10-8-15-13(16-9-10)17-14(6-7-14)11-4-2-1-3-5-11/h1-5,8-9,20H,6-7H2,(H,18,19)(H,15,16,17)
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| Chemical Name |
N-hydroxy-2-[(1-phenylcyclopropyl)amino]pyrimidine-5-carboxamide
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| Synonyms |
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
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| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (7.70 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 20.8 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: ≥ 2.08 mg/mL (7.70 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 20.8 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. View More
Solubility in Formulation 3: ≥ 2.08 mg/mL (7.70 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 3.6997 mL | 18.4986 mL | 36.9973 mL | |
| 5 mM | 0.7399 mL | 3.6997 mL | 7.3995 mL | |
| 10 mM | 0.3700 mL | 1.8499 mL | 3.6997 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.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
Selectivity, potency, and pharmacokinetic properties of HDAC6 inhibitors used in this study. Top row (a–c): dose-dependent enzymatic inhibition of recombinant HDAC1, HDAC2, HDAC3, and HDAC6 incubated with (a) tubastatin A, (b) ACY-738, or (c) ACY-775. Middle row: (d) heat map summary of IC50 values for the novel inhibitors ACY-738 and ACY-775 and reference compounds SAHA, MS-275, and ACY-1215 (n=2 per condition).Neuropsychopharmacology.2014 Jan;39(2):389-400. |
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Effects of HDAC6 inhibitors onα-tubulin acetylation at lysine 40 (K40) and histone H3 acetylation at lysine 9 (H3K9)Neuropsychopharmacology.2014 Jan;39(2):389-400. |
HDAC6 inhibitors ACY-738 and ACY-775 have antidepressant-like properties. Effects of HDAC6-selective inhibition in anxiety tests.Neuropsychopharmacology.2014 Jan;39(2):389-400. |
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