ACY-775, a novel pyrimidine hydroxyl amide small molecule, is a brain bioavailable, highly potent and isoform-selective inhibitor of HDAC6 with IC50 of 7.5 nM. ACY-738 and ACY-775 exhibit a selectivity of 60–1500 times greater than class I HDACs while inhibiting HDAC6 with low nanomolar potency. When it comes to stimulating mouse exploratory behaviors in unfamiliar but novel environments, ACY-738 and ACY-775 cause dramatic increases in α-tubulin acetylation in the brain, unlike tubastatin A, a reference HDAC6 inhibitor with similar potency and peripheral activity but more limited brain bioavailability. Remarkably, ACY-738 and ACY-775 exhibit antidepressant-like effects in the tail suspension test and social defeat paradigm, similar to other HDAC inhibitors like SAHA and MS-275, despite having no discernible effect on histone acetylation. The central inhibition of HDAC6 is directly responsible for the effects of ACY-738 and ACY-775, as these effects are completely abolished in mice that have a loss of function of HDAC6 that is specific to the neurons. Moreover, a behaviorally inactive dosage of ACY-738 combined with a subactive dose of the selective serotonin reuptake inhibitor citalopram significantly enhances the anti-immobility effect. These findings support the viability of this HDAC isoform as a possible target for the development of antidepressants and validate novel isoform-selective probes for in vivo pharmacological investigations of HDAC6 in the CNS.

Physicochemical Properties

Molecular Formula	C17H19FN4O2
Molecular Weight	330.36
Exact Mass	330.15
Elemental Analysis	C, 61.81; H, 5.80; F, 5.75; N, 16.96; O, 9.69
CAS #	1375466-18-4
Related CAS #	1375466-18-4
PubChem CID	57382288
Appearance	White to off-white solid powder
Density	1.1±0.1 g/cm3
Boiling Point	589.7±45.0 °C at 760 mmHg
Flash Point	310.4±28.7 °C
Vapour Pressure	0.0±1.7 mmHg at 25°C
Index of Refraction	1.566
LogP	1.54
Hydrogen Bond Donor Count	3
Hydrogen Bond Acceptor Count	6
Rotatable Bond Count	4
Heavy Atom Count	24
Complexity	423
Defined Atom Stereocenter Count	0
SMILES	FC1=CC=CC(=C1)C1(CCCCC1)NC1N=CC(C(NO)=O)=CN=1
InChi Key	IYBURCQQEUNLDL-UHFFFAOYSA-N
InChi Code	InChI=1S/C17H19FN4O2/c18-14-6-4-5-13(9-14)17(7-2-1-3-8-17)21-16-19-10-12(11-20-16)15(23)22-24/h4-6,9-11,24H,1-3,7-8H2,(H,22,23)(H,19,20,21)
Chemical Name	2-[[1-(3-fluorophenyl)cyclohexyl]amino]-N-hydroxypyrimidine-5-carboxamide
Synonyms	ACY 775; ACY775; ACY-775
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 = 7.5 nM); HDAC1 (IC50 = 2123 nM); HDAC2 (IC50 = 2570 nM); HDAC3 (IC50 = 11223 nM)
ln Vitro	In cells treated with the vehicle, histone 3 is obviously acetylated, but α-tubulin is primarily seen in its deacetylated state. ACY-775 treatment results in a definite increase in α-tubulin acetylation, but not in histone acetylation. Through the use of immunofluorescence, α-tubulin acetylation can be seen, and the intensity of this signal in the neuronal neurites can be measured and compared to its length. The DRG neurons treated with vehicles already have acetylated α-tubulin. Acetylated α-tubulin significantly increases in signal intensity after treatment with ACY-775. When comparing the neurites to DRG neurons treated with a vehicle, a notable increase in both the motility and total number of mitochondria was noted. Compared to cells treated with a vehicle, DRG neurons treated with ACY-775 exhibit a noticeably higher number of retrogradely transport mitochondria[1].
ln Vivo	ACY-738, ACY-775, and tubastatin A biodistribution profiles are investigated following acute dosing at 5 or 50 mg/kg over a 2-hour period. The plasma levels of ACY-738 and ACY-775 at t=30 min following an acute 50 mg/kg injection are 515 ng/mL (1.9 μM) and 1359 ng/mL (4.1 μM), respectively. It is rapidly eliminated from plasma; after two hours, the concentration falls below 10 ng/mL, with a plasmatic half-life of 12 minutes. However, ratios greater than one result from areas under concentration time curves for brain and plasm computed over a 2-hour period for both ACY-738 and ACY-775. In wild-type mice, significant increases in α-tubulin acetylation are observed in all tested brain regions when ACY-738 (5 mg/kg) or ACY-775 (50 mg/kg) are administered repeatedly at 24 h, 4 h, and 30 min before killing[2].
Cell Assay	The line of immortalized rat raphe neuronal precursors, known as undifferentiated RN46A-B14 cells, is cultured. For four hours, they are given either 2.5 μM ACY-738, ACY-775, tubastatin A, 0.6 μM TSA, or 0.1% DMSO as a vehicle. Samples are prepared with a histone extraction kit, and protein assay is used to quantify the results.
Animal Protocol	5, 50 mg/kg; i.p. In the TST, mice are tested for immobility. A combination of the previous, or vehicle, mice are attached to the test rig at 30 minutes or 2 hours after intraperitoneal injection of ACY-738 (5, 50 mg/kg), ACY-775 (5, 50 mg/kg), and citalopram (0.5, 2, 20 mg/kg). The amount of time the mice remain immobile for 6 minutes is recorded. Mice are injected with ACY-738 or ACY-775 at 5, 10, or 50 mg/kg or a vehicle, and then given free rein to explore in an open-field environment. Recorded activity exists [2].
References	[1]. Development of Improved HDAC6 Inhibitors as Pharmacological Therapy for Axonal Charcot-Marie-Tooth Disease. Neurotherapeutics. 2017 Apr; 14(2): 417-428. [2]. Antidepressant-Like Properties of Novel HDAC6-Selective Inhibitors with Improved Brain Bioavailability. Neuropsychopharmacology. 2014 Jan; 39(2): 389-400. [3]. Target deconvolution of HDAC pharmacopoeia reveals MBLAC2 as common off-target. Nat Chem Biol. 2022 Apr 28.

Solubility Data

Solubility (In Vitro)

DMSO: ~100 mg/mL Water: N/A Ethanol: N/A

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.5 mg/mL (7.57 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 (7.57 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. Solubility in Formulation 3: ≥ 2.5 mg/mL (7.57 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.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	3.0270 mL	15.1350 mL	30.2700 mL
	5 mM	0.6054 mL	3.0270 mL	6.0540 mL
	10 mM	0.3027 mL	1.5135 mL	3.0270 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.