HDAC3[1]
(E,E)-RGFP966 targets histone deacetylase 3 (HDAC3), a Class I HDAC involved in transcriptional regulation, neurodevelopment, and neurodegeneration. The compound exhibits high selectivity for HDAC3 with an IC50 of 0.08 μM and greater than 200-fold selectivity over other HDAC isoforms. By inhibiting HDAC3, the compound modulates histone acetylation and gene expression in the central nervous system.

An important epigenetic modification in Huntington's disease (HD) research is histone acetylation, which is regulated by histone acetyltransferase and histone deacetylase (HDAC) enzymes. HDAC inhibitors have proven effective in HD model systems, and recent work is now focused on functional dissection of the individual HDAC enzymes in these effects. Histone deacetylase 3 (HDAC3), a member of the class I subfamily of HDACs, has previously been implicated in neuronal toxicity and huntingtin-induced cell death. Hence, we tested the effects of RGFP966 ((E)-N-(2-amino-4-fluorophenyl)-3-(1-cinnamyl-1H-pyrazol-4-yl)acrylamide), a benzamide-type HDAC inhibitor that selectively targets HDAC3, in the N171-82Q transgenic mouse model of HD. We found that RGFP966 at doses of 10 and 25 mg/kg improves motor deficits on rotarod and in open field exploration, accompanied by neuroprotective effects on striatal volume. In light of previous studies implicating HDAC3 in immune function, we measured gene expression changes for 84 immune-related genes elicited by RGFP966 using quantitative PCR arrays. RGFP966 treatment did not cause widespread changes in cytokine/chemokine gene expression patterns, but did significantly alter the striatal expression of macrophage migration inhibitory factor (Mif), a hormone immune modulator associated with glial cell activation, in N171-82Q transgenic mice, but not WT mice. Accordingly, RGFP966-treated mice showed decreased glial fibrillary acidic protein (GFAP) immunoreactivity, a marker of astrocyte activation, in the striatum of N171-82Q transgenic mice compared to vehicle-treated mice. These findings suggest that the beneficial actions of HDAC3 inhibition could be related, in part, with lowered Mif levels and its associated downstream effects[1].

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In cell-free enzymatic assays, (E,E)-RGFP966 inhibits HDAC3 with an IC50 of 0.08 μM and exhibits greater than 200-fold selectivity over other HDAC isoforms. The compound's high selectivity for HDAC3 makes it a valuable tool for studying the specific role of HDAC3 in various biological processes. The HDAC inhibition assay typically uses fluorogenic substrates and purified recombinant HDAC enzymes.

In cell-based assays, (E,E)-RGFP966 selectively inhibits HDAC3 activity in cells. The compound is CNS-permeable, enabling activity in neuronal cells. The compound's effects on histone acetylation, gene expression, and cellular function are evaluated in cell culture models of Huntington's disease and other neurological disorders.

The cell-free HDAC3 inhibition assay involves incubation of purified recombinant HDAC3 enzyme with a fluorogenic substrate and the inhibitor at various concentrations. The reaction is incubated at 37°C for 30-60 minutes, and fluorescence is measured to determine the degree of inhibition. The IC50 of 0.08 μM is determined from dose-response curves. Selectivity is assessed by testing the compound against other HDAC isoforms.

Cell-based assays for (E,E)-RGFP966 involve culturing neuronal cell lines or primary neurons and treating them with the compound at concentrations ranging from 0.01 to 10 μM. Cells are incubated for 24-72 hours, and HDAC3 inhibition is assessed by measuring histone acetylation levels by Western blotting. The effect on gene expression is evaluated by qRT-PCR or RNA-seq. Neuroprotective effects are assessed in models of Huntington's disease.

In animal models, (E,E)-RGFP966 has been evaluated for its ability to inhibit HDAC3 in the CNS and improve outcomes in models of Huntington's disease. Typical studies involve administration of the compound to mice or rats via intraperitoneal or oral routes at doses ranging from 1-30 mg/kg. The compound's CNS permeability enables brain penetration. Pharmacodynamic endpoints include assessment of HDAC3 inhibition, histone acetylation, and behavioral outcomes in disease models.

(E,E)-RGFP966 exhibits favorable pharmacokinetic properties including CNS permeability. The compound is able to cross the blood-brain barrier to reach therapeutic concentrations in the brain. Detailed pharmacokinetic parameters including plasma half-life, brain penetration, and bioavailability have been reported. The compound's CNS permeability supports its use in neurological research.

Toxicity studies of (E,E)-RGFP966 have been conducted in animal models. As a selective HDAC3 inhibitor, the compound may have a favorable safety profile compared to pan-HDAC inhibitors. Standard toxicology studies include assessment of target organ toxicity, hematological effects, and determination of maximum tolerated dose. The compound is intended for research use only.

[1]. The Effects of Pharmacological Inhibition of Histone Deacetylase 3 (HDAC3) in Huntington's Disease Mice. PLoS One. 2016 Mar 31;11(3):e0152498.

N-(2-amino-4-fluorophenyl)-3-[1-(3-phenylprop-2-enyl)-4-pyrazolyl]-2-acrylamide is an aromatic amide and aromatic amine.

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(E,E)-RGFP966 is a research compound used to study HDAC3 inhibition and its role in neurological disorders. The compound is a selective and CNS-permeable HDAC3 inhibitor that can be used for research on Huntington's disease. It is not an approved pharmaceutical and has no clinical trial history as a standalone compound. This product is intended for research use only and is not for human therapeutic applications.

C21H19FN4O

362.40

362.154

C, 69.60; H, 5.28; F, 5.24; N, 15.46; O, 4.41

1396841-57-8

RGFP966;1357389-11-7

56650312

Typically exists as Off-white to light yellow solids at room temperature

4.623

2

4

6

27

532

0

FC1C([H])=C([H])C(=C(C=1[H])N([H])[H])N([H])C(/C(/[H])=C(\[H])/C1C([H])=NN(C=1[H])C([H])([H])/C(/[H])=C(\[H])/C1C([H])=C([H])C([H])=C([H])C=1[H])=O

BLVQHYHDYFTPDV-VCABWLAWSA-N

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+

(E)-N-(2-amino-4-fluorophenyl)-3-[1-[(E)-3-phenylprop-2-enyl]pyrazol-4-yl]prop-2-enamide

RGFP966; 1357389-11-7; 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;

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)