Nuclear factor of activated T-cells c1 (NFATc1). No direct binding IC50, Ki, EC50 values are provided. The mechanism is inferred from immunofluorescence showing inhibition of NFATc1 nuclear translocation. [1]

- Cytotoxicity in RAW264.7 cells (MTT assay): A04 showed no significant cytotoxic effects on RAW264.7 cell viability at concentrations up to 2.5 μM (survival rate >87%). The CC50 (concentration reducing viability by 50%) was 7.4 μM. [1]
- Inhibition of RANKL-induced osteoclast formation: RAW264.7 cells were treated with RANKL (100 ng/mL) and A04 at 2.5 μM. The number of TRAP-positive multinucleated cells (TRAP+ MNCs) was reduced to 0.1 ± 0.1% compared to RANKL-only control. The IC50 for inhibition of osteoclast formation was 1.57 ± 0.14 μM. [1]
- Dose-dependent inhibition of osteoclast formation: A04 at concentrations of 1.5, 2.0, and 2.5 μM significantly reduced the number and size of TRAP-positive multinucleated osteoclasts in a dose-dependent manner, with no cytotoxic effects at these concentrations. [1]
- Inhibition of bone resorption (pit formation assay): RAW264.7 cells were cultured on dentine slices with RANKL (100 ng/mL) and A04 at 1.5 or 2.0 μM for 4 days. A04 at 2.0 μM reduced bone resorption area to approximately 23.22% of the RANKL-treated group; at 1.5 μM, resorption area was reduced to approximately 63.98%. Total bone resorption pits were visualized under light microscopy and quantified using ImageJ software. [1]
- Inhibition of RANKL-induced NFATc1 nuclear translocation: RAW264.7 cells were treated with RANKL (100 ng/mL) for 24 h in the presence or absence of A04 (1.5 or 2.0 μM). Immunofluorescence staining showed that A04 blocked NFATc1 nuclear translocation and decreased NFATc1 protein levels. In unstimulated cells, NFATc1 was cytoplasmic. Upon RANKL stimulation, NFATc1 translocated to the nucleus (yellow in merged images). A04 treatment prevented this translocation. [1]

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NFATc1-IN-1 (Compound A04) (0-2.5 μM, 4 days) demonstrates strong inhibition of osteoclast formation and function, which leads to decreased bone resorption [1]. NFATc1-IN-1 (1.5-2.5 μM, 24 h) lowers NFATc1 levels and prevents NFATc1 nuclear translocation [1].

- Cell culture: Murine monocyte/macrophage RAW264.7 cells (osteoclast precursor cells) were cultured in DMEM supplemented with 10% heat-inactivated FBS at 37°C in 5% CO2. [1]
- MTT cytotoxicity assay: RAW264.7 cells were seeded in 96-well plates (2 × 10⁴ cells/well) and treated with various concentrations of compounds for 48 h. MTT solution (final concentration 0.5 mg/mL) was added and incubated for 2 h at 37°C. Formazan crystals were dissolved in DMSO, and absorbance was measured at 570 nm using an ELISA reader. Cell viability was expressed as a percentage of the control (vehicle-treated) cells. CC50 values (concentration reducing viability by 50%) were calculated. [1]
- Osteoclast differentiation assay (TRAP staining): RAW264.7 cells were seeded in 96-well plates (8 × 10³ cells/well) and cultured in α-MEM containing 10% FBS with RANKL (100 ng/mL) and test compounds (2.5 μM) for 4 days. Medium was replaced every 2 days with fresh medium containing RANKL and compounds. Cells were fixed with 4% paraformaldehyde for 20 min and stained for tartrate-resistant acid phosphatase (TRAP) using a commercial kit (Sigma-Aldrich 387A-1KT). TRAP-positive multinucleated cells (TRAP+ MNCs) with five or more nuclei were counted under a microscope. IC50 values were calculated as the concentration required to inhibit TRAP+ MNC formation by 50% compared to RANKL-treated controls. [1]
- Immunofluorescence for NFATc1 translocation: RAW264.7 cells were seeded in 8-well chamber slides (5 × 10³ cells/well) and incubated in α-MEM with 10% FBS in the presence or absence of RANKL (100 ng/mL) and test compounds for 24 h. Cells were washed with PBS, fixed in 4% paraformaldehyde for 20 min, washed with wash buffer (PBS containing 0.1% BSA), and permeabilized with blocking buffer (10% PBS, 10% BSA, 0.3% Triton X-100) for 45 min at room temperature. Cells were incubated overnight at room temperature with anti-NFATc1 monoclonal antibody (1:100) in dilution buffer (1% BSA, 1% FBS, 0.3% Triton X-100 in PBS), then washed and incubated with FITC-conjugated anti-mouse IgG antibody (1:50) for 1 h at room temperature. Nuclei were counterstained with DAPI (1 μg/mL) in PBS for 3 min. Fluorescence was visualized using a fluorescence microscope. [1]
- Pit formation assay (bone resorption): RAW264.7 cells (10⁴ cells/well) were seeded onto dentine slices in 24-well plates and incubated in α-MEM with 10% FBS in the presence or absence of RANKL (100 ng/mL) and test compounds for 4 days. Medium was replaced every 2 days. To observe resorption pits, wells were washed with PBS and 1 M ammonium hydroxide to completely remove attached cells. Total bone resorption pits were visualized under light microscopy. Bone resorbed percentage of compound-treated groups relative to RANKL-treated groups was quantified in the optical field of each slice using ImageJ software. [1]

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Cell viability assay [1]
Cell Types: Osteoclast precursor RAW 264.7 Cell
Tested Concentrations: 0, 0.5, 1.0, 1.5, 2.0 and 2.5 μM
Incubation Duration: 4 days
Experimental Results: At a certain concentration, red TRAP-positive multinucleated osteoclasts The number and size were Dramatically diminished at 1.5, 2.0 and 2.5 μM. Dramatically inhibits osteoclast formation in a dose-dependent manner (1.5, 2.0 and 2.5 μM) without having a cytotoxic effect on osteoclast precursor cells at concentrations up to 2.5 μM.

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Immunofluorescence[1]
Cell Types: RAW264.7 Cell
Tested Concentrations: 1.5 or 2.5 μM
Incubation Duration: 24 hrs (hours)
Experimental Results: Blocks NFATc1 nuclear translocation and reduces NFATc1 levels.

- In vitro cytotoxicity: A04 showed no significant cytotoxicity to RAW264.7 cells at concentrations up to 2.5 μM (cell viability >87%). The CC50 was 7.4 μM. [1]

[1]. Design, synthesis and SARs of novel salicylanilides as potent inhibitors of RANKL-induced osteoclastogenesis and bone resorption. Eur J Med Chem. 2016 Jul 19;117:70-84.

- Chemical name: 5-Fluoro-N-(2-fluoro-4-iodophenyl)-2-hydroxybenzamide. [1]
- Synthesis: A04 was synthesized by reacting 5-fluorosalicylic acid with thionyl chloride to form the acyl chloride, followed by reaction with 2-fluoro-4-iodoaniline in THF. Yield: 45%; white powder; melting point: 236-237°C. Characterization: 1H NMR, 13C NMR, and HRMS (ESI) data are provided. [1]
- Structure-activity relationship (SAR): Among the A series (salicylanilides), compounds with heavier halogen substituents (I > Br > Cl > F) at the 4-position of the B ring exhibited more potent anti-osteoclastogenic activities. A04 contains iodine at the 4-position of the B ring, contributing to its highest potency. The 5-fluoro substituent on the A ring also enhanced activity. [1]
- Comparison with other compounds: A04 (IC50 = 1.57 μM) was more potent than the positive controls 1a (NDMC101, IC50 = 10.0 μM), 1d (IC50 = 7.1 μM), and 5d (IC50 = 4.9 μM). [1]
- Mechanism of action: A04 inhibits RANKL-induced osteoclastogenesis by suppressing NFATc1 nuclear translocation. The study proposes that the anti-osteoclastogenic effect is mediated through the NFATc1 pathway. [1]

C13H8F2INO2

375.11

374.956

1912422-56-0

127043724

White to off-white solid powder

3.8

2

4

2

19

332

0

C(NC1=CC=C(I)C=C1F)(=O)C1=CC(F)=CC=C1O

IGRCFOGPIJEESA-UHFFFAOYSA-N

InChI=1S/C13H8F2INO2/c14-7-1-4-12(18)9(5-7)13(19)17-11-3-2-8(16)6-10(11)15/h1-6,18H,(H,17,19)

5-fluoro-N-(2-fluoro-4-iodophenyl)-2-hydroxybenzamide

NFATc1-IN-1; A04; 1912422-56-0; 5-Fluoro-N-(2-fluoro-4-iodophenyl)-2-hydroxybenzamide; NFATc1 inhibitor A04; orb1688505;

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)

DMSO : ~125 mg/mL (~333.24 mM)

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.)