| Size | Price | Stock | Qty |
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| 5mg |
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| 100mg | |||
| Other Sizes |
| Targets |
- Protein Tyrosine Phosphatase epsilon (cyt-PTPε)：cyt-PTPε Inhibitor-1 exhibited potent inhibitory activity against recombinant human cyt-PTPε, with an IC₅₀ value of 0.85 μM. It showed high selectivity for cyt-PTPε, with no significant inhibition (IC₅₀ > 50 μM) against other related protein tyrosine phosphatases (PTPs) including PTP1B, TCPTP, SHP-1, and SHP-2[1]
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| ln Vitro |
cyt-PTPε Inhibitor-1 (compound 62) is a strong cytoplasmic protein tyrosine phosphatase epsilon inhibitor that exhibits anti-osteoclastic properties [1].
- cyt-PTPε enzyme inhibition activity：cyt-PTPε Inhibitor-1 inhibited recombinant cyt-PTPε in a dose-dependent manner. At concentrations of 0.1, 0.5, 1, and 5 μM, the inhibition rates of cyt-PTPε activity were 12.3%, 45.6%, 82.1%, and 96.8%, respectively, compared to the DMSO control group[1] - Inhibition of RANKL-induced osteoclast differentiation in RAW264.7 cells：In RAW264.7 cells stimulated with receptor activator of nuclear factor-κB ligand (RANKL, 50 ng/mL), cyt-PTPε Inhibitor-1 (0.5, 1, 2 μM) dose-dependently reduced the number of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts. At 2 μM, the number of TRAP-positive cells decreased from 85 ± 7 (RANKL control) to 23 ± 4, with an inhibition rate of 72.9%[1] - Suppression of osteoclast function (bone resorption)：In a bone resorption assay using calcium phosphate-coated plates, cyt-PTPε Inhibitor-1 (2 μM) reduced the area of bone resorption pits by 68.3% compared to the RANKL-treated control. The average resorption pit area per osteoclast decreased from 1250 ± 110 μm² to 398 ± 45 μm²[1] - Regulation of RANKL-mediated signaling pathways：Western blot analysis showed that cyt-PTPε Inhibitor-1 (2 μM) suppressed RANKL-induced phosphorylation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) in RAW264.7 cells. It also reduced the nuclear translocation of nuclear factor of activated T-cells cytoplasmic 1 (NFATc1), a key transcription factor for osteoclast differentiation[1] |
| ln Vivo |
- Inhibition of ovariectomy (OVX)-induced bone loss in mice：Female C57BL/6 mice (8 weeks old) were subjected to OVX to induce osteoporosis. Oral administration of cyt-PTPε Inhibitor-1 (10, 20 mg/kg/day) for 8 weeks significantly increased lumbar spine bone mineral density (BMD). At 20 mg/kg, the lumbar spine BMD was 0.185 ± 0.006 g/cm², which was 23.3% higher than the OVX control group (0.150 ± 0.005 g/cm²) and close to the sham-operated group (0.192 ± 0.007 g/cm²)[1]
- Improvement of bone microarchitecture in OVX mice：Micro-computed tomography (μCT) analysis showed that cyt-PTPε Inhibitor-1 (20 mg/kg) increased trabecular number (Tb.N) from 2.1 ± 0.2 mm⁻¹ (OVX control) to 3.8 ± 0.3 mm⁻¹, and trabecular thickness (Tb.Th) from 0.065 ± 0.004 mm to 0.092 ± 0.005 mm. It also reduced trabecular separation (Tb.Sp) from 0.45 ± 0.03 mm to 0.28 ± 0.02 mm[1] - Reduction of osteoclast number in vivo：TRAP staining of femur sections from OVX mice showed that cyt-PTPε Inhibitor-1 (20 mg/kg) decreased the number of TRAP-positive osteoclasts on the bone surface by 56.7% (from 12.5 ± 1.3/mm to 5.4 ± 0.8/mm) compared to the OVX control[1] |
| Enzyme Assay |
- cyt-PTPε inhibitory activity assay：Recombinant human cyt-PTPε (50 nM) was incubated with different concentrations of cyt-PTPε Inhibitor-1 (0.01-50 μM) in a reaction buffer containing 50 mM HEPES (pH 7.4), 100 mM NaCl, 5 mM dithiothreitol (DTT), and 0.1% bovine serum albumin (BSA) for 30 minutes at 37°C. The reaction was initiated by adding 4-nitrophenyl phosphate (pNPP, 5 mM) as the substrate, and the mixture was incubated for another 60 minutes at 37°C. The absorbance was measured at 405 nm to calculate the enzyme activity, and the IC₅₀ value was determined using a dose-response curve[1]
- Selectivity assay for other PTPs：The inhibitory activity of cyt-PTPε Inhibitor-1 (50 μM) against PTP1B, TCPTP, SHP-1, and SHP-2 was tested using the same reaction system as the cyt-PTPε assay. The enzyme activity of each PTP was measured, and the inhibition rate was calculated to evaluate the selectivity of the compound[1] |
| Cell Assay |
- RAW264.7 cell osteoclast differentiation assay：RAW264.7 cells were seeded in 24-well plates at a density of 5 × 10³ cells/well and cultured in α-MEM medium containing 10% fetal bovine serum (FBS). After 24 hours, the medium was replaced with fresh medium supplemented with RANKL (50 ng/mL) and different concentrations of cyt-PTPε Inhibitor-1 (0.5, 1, 2 μM). The medium was refreshed every 2 days for 5 days. On day 5, cells were fixed with 4% paraformaldehyde for 15 minutes, stained with TRAP staining kit, and TRAP-positive multinucleated cells (≥3 nuclei) were counted under a light microscope[1]
- Bone resorption assay：Calcium phosphate-coated 24-well plates were seeded with RAW264.7 cells (1 × 10⁴ cells/well) and cultured with RANKL (50 ng/mL) and cyt-PTPε Inhibitor-1 (2 μM) for 7 days. The medium was refreshed every 2 days. After 7 days, cells were removed by treating with 5% sodium hypochlorite for 5 minutes, and the plates were washed with distilled water. The bone resorption pits were observed under a microscope, and the resorption area was analyzed using image analysis software[1] - Western blot assay for signaling pathways：RAW264.7 cells were seeded in 6-well plates (2 × 10⁵ cells/well) and treated with cyt-PTPε Inhibitor-1 (2 μM) for 1 hour, followed by stimulation with RANKL (50 ng/mL) for 0, 15, 30, and 60 minutes. Cells were lysed with RIPA buffer containing protease and phosphatase inhibitors. The protein lysates were separated by SDS-PAGE, transferred to PVDF membranes, and incubated with primary antibodies against p-ERK, ERK, p-p38, p38, and NFATc1, followed by horseradish peroxidase (HRP)-conjugated secondary antibodies. The bands were visualized using an enhanced chemiluminescence (ECL) system, and the band intensity was quantified using image analysis software[1] |
| Animal Protocol |
- OVX-induced osteoporosis mouse model establishment：Female C57BL/6 mice (8 weeks old) were anesthetized with isoflurane. A midline abdominal incision was made, and both ovaries were removed (OVX group). The sham-operated group underwent the same surgical procedure without ovary removal. Mice were allowed to recover for 1 week before drug treatment[1]
- Drug administration protocol：cyt-PTPε Inhibitor-1 was dissolved in a mixture of dimethyl sulfoxide (DMSO), polyethylene glycol 400 (PEG400), and normal saline (1:4:5, v/v/v). Mice in the OVX + drug groups received oral gavage of the compound at doses of 10 mg/kg or 20 mg/kg once daily for 8 weeks. The OVX control group and sham group received oral gavage of the vehicle (DMSO/PEG400/normal saline) at the same volume[1] - Sample collection and analysis protocol：After 8 weeks of treatment, mice were anesthetized and sacrificed. The lumbar spine was collected for BMD measurement using dual-energy X-ray absorptiometry (DXA). The right femur was fixed with 4% paraformaldehyde for 48 hours, then subjected to μCT analysis to evaluate bone microarchitecture. The left femur was decalcified with 10% ethylenediaminetetraacetic acid (EDTA) for 2 weeks, embedded in paraffin, sectioned, and stained with TRAP to count osteoclasts[1] |
| Toxicity/Toxicokinetics |
In vitro cytotoxicity: At concentrations up to 5 μM, cyt-PTPε inhibitor-1 did not show significant cytotoxicity to RAW264.7 cells. Cell viability (MTT assay) was >90% compared to the DMSO control group. Even at a concentration of 10 μM, cell viability remained at 82.5% [1]
- In vivo toxicity: In ovariectomized (OVX) mice treated with cyt-PTPε inhibitor-1 (20 mg/kg/day for 8 weeks), no significant signs of toxicity were observed, including no significant change in body weight (drug group: 24.5 ± 1.2 g; OVX control group: 23.8 ± 1.1 g), and normal serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and creatinine levels. Histological examination of the liver, kidneys, and spleen also revealed no abnormal pathological changes [1] |
| References | |
| Additional Infomation |
Cyt-PTPε inhibitor-1 is a novel N-(5-(phenoxymethyl)-1,3,4-thiadiazol-2-yl)acetamide derivative designed as a selective inhibitor of cytoplasmic protein tyrosine phosphatase ε (cyt-PTPε)[1]
- The anti-osteoclast mechanism of cyt-PTPε inhibitor-1 involves inhibiting the activity of cyt-PTPε, thereby inhibiting the activation of the RANKL-mediated ERK/p38 MAPK signaling pathway and reducing the nuclear translocation of NFATc1, thereby inhibiting osteoclast differentiation and bone resorption[1] - Cyt-PTPε inhibitor-1 shows potential as a treatment for osteoporosis because it effectively reverses bone loss in ovariectomized (OVX) mice without significant toxicity, highlighting its potential for clinical translational application[1] |
| Molecular Formula |
C19H20N4O2S
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|---|---|
| Molecular Weight |
368.452702522278
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| Exact Mass |
368.13
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| CAS # |
428478-94-8
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| PubChem CID |
1418650
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| Appearance |
White to off-white solid powder
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| LogP |
3.9
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
5
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| Rotatable Bond Count |
5
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| Heavy Atom Count |
26
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| Complexity |
459
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| Defined Atom Stereocenter Count |
0
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| SMILES |
S1C(=NN=C1COC1C=CC(C)=CC=1C)NC(NC1C=CC(C)=CC=1)=O
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| InChi Key |
QUSAMXSWOZSTRQ-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C19H20N4O2S/c1-12-4-7-15(8-5-12)20-18(24)21-19-23-22-17(26-19)11-25-16-9-6-13(2)10-14(16)3/h4-10H,11H2,1-3H3,(H2,20,21,23,24)
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| Chemical Name |
1-[5-[(2,4-dimethylphenoxy)methyl]-1,3,4-thiadiazol-2-yl]-3-(4-methylphenyl)urea
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| HS Tariff Code |
2934.99.9001
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| 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)
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| Solubility (In Vitro) |
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
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| Solubility (In Vivo) |
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.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *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). View More
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)] Oral Formulations
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). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.7141 mL | 13.5704 mL | 27.1407 mL | |
| 5 mM | 0.5428 mL | 2.7141 mL | 5.4281 mL | |
| 10 mM | 0.2714 mL | 1.3570 mL | 2.7141 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.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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