Nexturastat A (AG-CR1-3901), an aryl urea analog, is a novel, potent and selective inhibitor of histone deacetylase 6 (HDAC6) with potential anticancer activity. It shows >190-fold selectivity for HDAC6 over other HDACs and inhibits HDAC6 with an IC50 of 5 nM. Based on an aryl urea HDACI, structural modifications were used to develop it.

Physicochemical Properties

Molecular Formula	C19H23N3O3
Molecular Weight	341.4
Exact Mass	341.173
Elemental Analysis	C, 66.84; H, 6.79; N, 12.31; O, 14.06
CAS #	1403783-31-2
Related CAS #	1403783-31-2
PubChem CID	71462653
Appearance	White solid powder
Density	1.228±0.06 g/cm3
Index of Refraction	1.622
LogP	2.37
Hydrogen Bond Donor Count	3
Hydrogen Bond Acceptor Count	3
Rotatable Bond Count	7
Heavy Atom Count	25
Complexity	416
Defined Atom Stereocenter Count	0
SMILES	O=C(N([H])C1C([H])=C([H])C([H])=C([H])C=1[H])N(C([H])([H])C1C([H])=C([H])C(C(N([H])O[H])=O)=C([H])C=1[H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H]
InChi Key	JZWXMCPARMXZQV-UHFFFAOYSA-N
InChi Code	InChI=1S/C19H23N3O3/c1-2-3-13-22(19(24)20-17-7-5-4-6-8-17)14-15-9-11-16(12-10-15)18(23)21-25/h4-12,25H,2-3,13-14H2,1H3,(H,20,24)(H,21,23)
Chemical Name	4-[[butyl(phenylcarbamoyl)amino]methyl]-N-hydroxybenzamide
Synonyms	Nexturastat A; AG-CR13901
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 = 5.02 nM ); HDAC8 ( IC50 = 0.954 μM ); HDAC1 ( IC50 = 3.02 μM ); HDAC7 ( IC50 = 4.46 μM ); HDAC11 ( IC50 = 5.14 μM ); HDAC3 ( IC50 = 6.68 μM ); HDAC9 ( IC50 = 6.72 μM ); HDAC2 ( IC50 = 6.92 μM ); HDAC10 ( IC50 = 7.57 μM ); HDAC4 ( IC50 = 9.39 μM ); HDAC5 ( IC50 = 11.7 μM )
ln Vitro	In vitro activity: Nexturastat A causes a dose-dependent rise in acetyl α-tubulin levels in B16 murine melanoma cells, but not in histone H3 acetylation. Furthermore, Nexturastat A has a GI50 of 14.3 μM and effectively suppresses the growth of B16 melanoma cells. [1]
ln Vivo	N/A
Enzyme Assay	Human recombinant full-length HDAC1 and -6 are isolated and used in baculovirus expression systems in Sf9 cells by Reaction Biology Corp. to conduct HDAC inhibition assays. A substrate in the form of RHKKAc, an acetylated fluorogenic peptide derived from p53 residues 379–382, is employed. 127 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, 1 mg/mL BSA, 50 mM Tris-HCl pH 8.0, and a final concentration of 1% DMSO make up the reaction buffer. Following a 5-to 10-minute preincubation period and a 2-hour incubation period at 30°C, compounds are added to the enzyme mixture in DMSO. To quench the reaction and produce fluorescence, respectively, trichostatin A and developer are added. In order to create a 10-dose plot, dose-response curves are created using three-fold serial dilutions beginning at 30 μM compound. The resulting plots are then used to calculate IC50 values, which are expressed as the average of the duplicate trials ± standard deviation.
Cell Assay	B16 murine melanoma cells are plated in 96-well flat-bottom plates at 5000 cells per well. The medium is swapped out the next day for one that contains different HDACi concentrations or matched DMSO vehicle concentrations diluted in full medium, all done in triplicate. 48 hours are spent incubating the cells at 37°C with 5% CO2. According to the manufacturer's instructions, the density of metabolically active, viable cells is measured using a standard MTS assay. In summary, each well receives 20μL of reagent, which is then incubated for three hours at 37°C. Spectrophotometric measurements of absorbances at 490 nM are made after subtracting the background at 690 nM. After normalization, each value is given as a percentage of the medium control (100%).
Animal Protocol	NA NA
References	[1]. Selective histone deacetylase 6 inhibitors bearing substituted urea linkers inhibit melanoma cell growth. J Med Chem. 2012 Nov 26;55(22):9891-9. [2]. The selective HDAC6 inhibitor Nexturastat A induces apoptosis, overcomes drug resistance and inhibits tumor growth in multiple myeloma. Biosci Rep. 2019 Mar 22;39(3):BSR20181916.

Solubility Data

Solubility (In Vitro)

DMSO: ~68 mg/mL (~199.2 mM) Water: <1 mg/mL Ethanol: ~2 mg/mL (~5.9 mM)

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.5 mg/mL (7.32 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 (7.32 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 (7.32 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	2.9291 mL	14.6456 mL	29.2912 mL
	5 mM	0.5858 mL	2.9291 mL	5.8582 mL
	10 mM	0.2929 mL	1.4646 mL	2.9291 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.