RGFP966 (RGF-P966) is a novel, potent and highly selective inhibitor of HDAC3 (histone deacetylase 3) with potential anticancer activity. In a cell-free assay, it inhibits HDAC3 with an IC50 of 80 nM and shows >200-fold selectivity for HDAC3 over other HDACs.

Physicochemical Properties

Molecular Formula	C21H19FN4O
Molecular Weight	362.4
Exact Mass	362.154
Elemental Analysis	C, 69.60; H, 5.28; F, 5.24; N, 15.46; O, 4.41
CAS #	1357389-11-7
Related CAS #	1396841-57-8
PubChem CID	56650312
Appearance	White solid powder
Density	1.2±0.1 g/cm3
Boiling Point	630.4±55.0 °C at 760 mmHg
Flash Point	335.0±31.5 °C
Vapour Pressure	0.0±1.8 mmHg at 25°C
Index of Refraction	1.607
LogP	3.95
Hydrogen Bond Donor Count	2
Hydrogen Bond Acceptor Count	4
Rotatable Bond Count	6
Heavy Atom Count	27
Complexity	532
Defined Atom Stereocenter Count	0
SMILES	O=C(NC1=CC=C(F)C=C1N)/C=C/C2=CN(C/C=C/C3=CC=CC=C3)N=C2.[E]
InChi Key	BLVQHYHDYFTPDV-VCABWLAWSA-N
InChi Code	InChI=1S/C21H19FN4O/c22-18-9-10-20(19(23)13-18)25-21(27)11-8-17-14-24-26(15-17)12-4-7-16-5-2-1-3-6-16/h1-11,13-15H,12,23H2,(H,25,27)/b7-4+,11-8+
Chemical Name	(E)-N-(2-amino-4-fluorophenyl)-3-[1-[(E)-3-phenylprop-2-enyl]pyrazol-4-yl]prop-2-enamide
Synonyms	RGFP966; RGFP-966; 1396841-57-8; (E)-N-(2-amino-4-fluorophenyl)-3-(1-cinnamyl-1H-pyrazol-4-yl)acrylamide; RGFP 966; RGFP-966; (E,E)-RGFP966; CHEMBL4078477; RGFP 966
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	HDAC3 ( IC50 = 80 nM )
ln Vitro	In vitro activity: RGFP966 is a competitive tight-binding HDAC inhibitor that exhibits slow-on/slow-off behavior. Its IC50 for HDAC3 is 0.08μM, and at concentrations up to 15μM, it does not effectively inhibit any other HDAC.[1] Two CTCL cell lines treated with RGFP966 for 24 hours before western blot analysis showed increased acetylation at H3K9/K14, H3K27, and H4K5, but not at H3K56ac. Due to enhanced apoptosis linked to DNA damage and impeded S phase progression, RGFP966 reduces cell growth in CTCL (cutaneous T cell lymphoma) cell lines. Within the first hour of medication treatment, RGFP966 significantly lowers the DNA replication fork velocity.[2]
ln Vivo	RGFP966 treatment (10 mg/kg)improves object memory's long-term memory. RGFP966 (3 or 10 mg/kg, s.c.) lessens the chance of reinstating cocaine-conditioned place preference and promotes its extinction.[1]
Enzyme Assay	Assays for acetylation are derived from the homogenous fluorescence release method. Following pre-incubation periods ranging from 0 to 3 hours, purified recombinant enzymes are incubated in the standard HDAC buffer with serially diluted inhibitors at the concentrations shown in the figures. After pre-incubation, acetyl-Lys(Ac)-AMC substrate (at 10 μM, corresponding to the Km for both HDAC1 and HDAC3) is added. An hour is given for the reaction to continue. An Tecan M200 96-well plate reader is used to measure the fluorescence emission after an hour of adding the trypsin peptidase developer at a final concentration of 5 mg/ml.
Cell Assay	At 26105 cells/mL, cells are counted and divided into T25 (Corning) flasks. After that, cells are treated once at hour 0 with either DMSO or HDIs. At 0, 24, 48, and 72 hours following treatment, 100 ml aliquots are taken in triplicate from each flask, distributed into a flat bottom 96-well plate, and 10 ml of alamar blue is added to each well. Using the Biotek Synergy MX Microplate Reader, fluorescence is measured after a 4-hour incubation period.
Animal Protocol	Mice: The N171-82Q transgenic mice are kept in their housing and given free access to food and water, as well as a typical 12-hour light/dark cycle beginning at 6:00 a.m. Beginning at 8 weeks of age, mice are given 3 injections per week by subcutaneous injection of RGFP966 (10 or 25 mg/kg) for a duration of 10 weeks. A solution of 75% polyethylene glycol 200/25% sodium acetate (6.25 mM) is used to dissolve RGFP966; mice in the control group were given the same volume of drug vehicle. Weights are recorded twice a week for the body. At eighteen weeks of age, mice are killed by overdosing on isofluorane anesthesia, six hours after the last injection. Brains are taken out, and 4% paraformaldehyde is either intracardially infused, or the cortex and striata are separated out for gene expression experiments. Rats: The experiment uses thirty-three adult male Sprague Dawley rats weighing between 275 and 350 grams. A posttraining systemic injection of either RGPF966 (10 mg/kg, s.c.) or vehicle (at a volume similar to drug treatment) is administered to each subject right after the daily training session.
References	[1]. Proc Natl Acad Sci U S A . 2013 Feb 12;110(7):2647-52. [2]. PLoS One . 2013 Jul 22;8(7):e68915.
Additional Infomation	N-(2-amino-4-fluorophenyl)-3-[1-(3-phenylprop-2-enyl)-4-pyrazolyl]-2-propenamide is an aromatic amide and an aromatic amine.

Solubility Data

Solubility (In Vitro)

DMSO: 50~72 mg/mL (138~198.7 mM) Water: <1 mg/mL Ethanol: <1 mg/mL

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.90 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 (6.90 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. Solubility in Formulation 3: 1% DMSO+30% polyethylene glycol+1% Tween 80: 30mg/mL Solubility in Formulation 4: 7.69 mg/mL (21.22 mM) in 50% PEG300 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	2.7594 mL	13.7969 mL	27.5938 mL
	5 mM	0.5519 mL	2.7594 mL	5.5188 mL
	10 mM	0.2759 mL	1.3797 mL	2.7594 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.