Histone deacetylase 3 (HDAC3) ( IC50 = 80 nM )

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]

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Using a substrate-dependent biochemical assay, it was found that RGFP966 could inhibit HDAC3 with an IC50 of 80 nM, and had no significant inhibitory effect on other HDACs when the concentration was up to 15 μM. When tested on cutaneous T-cell lymphoma (CTCL) cell lines, RGFP966 could significantly inhibit cell growth by targeting HDAC3, and the mechanism was related to promoting cell apoptosis [1]

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]

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- In male C57BL/6J mice, after administering RGFP966, the mice were trained in object recognition (ORM) and location-dependent object recognition (OLM). The results showed that RGFP966 could significantly increase the novel object preference in a dose-dependent manner and enhance long-term OLM memory [1]
- RGFP966 can attenuate cardiac dysfunction induced by prolonged hypothermic preservation in rat hearts. It inhibits the hypothermic preservation-induced increase of the phosphorylated (p)-MST1/MST1 and p-YAP/YAP ratios, prevents a reduction in total YAP protein expression, and increases the nuclear YAP protein level. It also increases the expression of antioxidant enzymes such as superoxide dismutase, catalase, and glutathione peroxidase, inhibits the overexpression of pro-apoptotic proteins Bax and cleaved caspase-3, increases the expression of anti-apoptotic protein Bcl-2, and reduces cardiomyocyte apoptosis. The mechanism may involve inactivation of the YAP pathway [2]

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.

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A substrate-dependent biochemical assay was used to detect the inhibitory effect of RGFP966 on HDACs. Prepare the reaction system, add HDAC3 enzyme, substrate and different concentrations of RGFP966 into the reaction well, incubate at an appropriate temperature for a certain period of time, and then detect the reaction product by a specific detection method (the specific detection method is not described in the literature), so as to obtain the inhibitory curve of RGFP966 on HDAC3 and calculate the IC50 value [1]

For cutaneous T-cell lymphoma (CTCL) cell lines: Cells were cultured in appropriate medium and seeded into plates. After attachment, RGFP966 at different concentrations was added. Cell growth was monitored over a period of time, and cell viability was assessed using relevant methods. Additionally, the effect on cell apoptosis was evaluated through techniques such as detecting apoptotic markers [1]
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.

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.

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- In the memory-related experiment: Male C57BL/6J mice were used, and RGFP966 was administered to the mice. Then, the mice were trained in ORM and OLM tests. The specific training method is: first, let the mice familiarize with the experimental environment, then place a familiar object in the test area, let the mice explore, and then place a new object, and record the time that the mice spent exploring the new object and the familiar object to calculate the novel object preference. In the OLM test, change the position of the object, and record the exploration time of the mice to evaluate the long-term OLM memory. Different doses of RGFP966 were set, and the dose-response relationship was analyzed according to the test results [1]
- In the cardiac dysfunction experiment: Rat hearts were hypothermically preserved in Celsior solution with or without RGFP966 for 12 h, followed by 60 min of reperfusion. The hemodynamic parameters during reperfusion were evaluated. The expression and phosphorylation levels of MST1 and YAP were determined by western blotting, and cell apoptosis was measured by the TUNEL method [2]

[1]. Proc Natl Acad Sci U S A . 2013 Feb 12;110(7):2647-52.

[2]. PLoS One . 2013 Jul 22;8(7):e68915.

DAC3 is an isoform of the HDACs family, which plays an important role in DNA transcriptional regulation. HDACs are involved in many biological processes, such as DNA repair, replication, transcription and chromatin structure. RGFP966 is a potent and selective HDAC3 inhibitor, which has potential application value in the treatment of related diseases by regulating the function of HDAC3 [1]
N-(2-amino-4-fluorophenyl)-3-[1-(3-phenylprop-2-enyl)-4-pyrazolyl]-2-propenamide is an aromatic amide and an aromatic amine.

C21H19FN4O

362.4

362.154

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

1357389-11-7

56650312

White solid powder

1.2±0.1 g/cm3

630.4±55.0 °C at 760 mmHg

335.0±31.5 °C

0.0±1.8 mmHg at 25°C

1.607

3.95

2

4

6

27

532

0

O=C(NC1=CC=C(F)C=C1N)/C=C/C2=CN(C/C=C/C3=CC=CC=C3)N=C2.[E]

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; 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

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)

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.

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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.

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Solubility in Formulation 3: 1% DMSO+30% polyethylene glycol+1% Tween 80: 30mg/mL

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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.

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