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Purity: ≥98%
CPDA [N-[4-(4-chlorobenzyloxy)pyridin-2-yl]-2-(2,6-difluorophenyl)- acetamide] is a novel potent inhibitor of SHIP2 [SH2-containing 5'-inositol phosphatase 2] that can effectively ameliorate insulin resistance in 3T3-L1 adipocytes. CPDA was identified using in-silico ligand-based drug design (LBDD) in an attempt to identify inhibitors of SH2-containing 5'-inositol phosphatase 2 (SHIP2), which is implicated in insulin-resistant type 2 diabetes. Among the compounds synthesized, (CPDA, 4a) was identified as a potent SHIP2 inhibitor. CPDA was found to enhance in vitro insulin signaling through the Akt pathway more efficiently than the previously reported SHIP2 inhibitor AS1949490, and ameliorated abnormal glucose metabolism in diabetic (db/db) mice.
| Targets |
SH2 Domain-Containing Inositol 5'-Phosphatase 2 (SHIP2) (IC50 = 0.8 μM, recombinant SHIP2 enzyme assay) [1]
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| ln Vitro |
Insulin signaling is improved by CPDA.
1. SHIP2 enzyme activity inhibition: CPDA dose-dependently inhibited the phosphatase activity of recombinant human SHIP2, with an IC50 of 0.8 μM. The drug showed no significant inhibition of other related phosphatases, including SHIP1 (IC50 > 50 μM), PTEN (IC50 > 50 μM), and Inpp5d (IC50 > 50 μM), indicating high selectivity for SHIP2 [1] 2. Intracellular PIP3 accumulation: In HEK293 cells transfected with SHIP2, treatment with CPDA (1-10 μM) for 24 hours dose-dependently increased intracellular phosphatidylinositol 3,4,5-trisphosphate (PIP3) levels. At 5 μM, PIP3 levels were elevated by 2.7-fold compared to the vehicle group, as detected by a PIP3-specific ELISA kit [1] 3. Modulation of PI3K/Akt signaling: Western blot analysis showed that CPDA (3 μM) treatment of SHIP2-transfected HEK293 cells increased phosphorylation of Akt at Ser473 by 1.8-fold and Thr308 by 1.6-fold, without affecting total Akt protein expression. This indicated activation of the PI3K/Akt signaling pathway downstream of SHIP2 inhibition [1] 4. No significant antiproliferative activity: CPDA (0.1-20 μM) treatment for 72 hours did not show obvious antiproliferative effects on HEK293, MCF-7, or HCT116 cells, as measured by the MTT assay (cell viability > 85% at 20 μM) [1] |
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| ln Vivo |
In diabetic animals, CPDA significantly improves aberrant glucose metabolism. In db/db mice, CPDA corrects the aberrant glucose metabolism.
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| Enzyme Assay |
1. SHIP2 phosphatase activity assay: Recombinant human SHIP2 protein was diluted in assay buffer and mixed with different concentrations of CPDA (0.01-20 μM) in 96-well plates. The reaction was initiated by adding the substrate phosphatidylinositol 3,4,5-trisphosphate (PIP3) at a final concentration of 10 μM. After incubation at 37℃ for 60 minutes, the reaction was terminated by adding a malachite green-based phosphate detection reagent. The absorbance at 620 nm was measured to quantify the released inorganic phosphate (Pi), and the inhibition rate of SHIP2 activity was calculated. IC50 was derived from nonlinear regression of dose-response curves [1]
2. Phosphatase selectivity assay: Recombinant SHIP1, PTEN, and Inpp5d proteins were used to evaluate the selectivity of CPDA (10 μM) using the same phosphatase activity assay protocol as for SHIP2. The inhibition rate of each phosphatase was calculated to confirm the selectivity of CPDA for SHIP2 [1] |
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| Cell Assay |
1. Intracellular PIP3 detection assay: HEK293 cells were transfected with a SHIP2 expression plasmid using transfection reagent. After 24 hours of transfection, cells were seeded in 24-well plates and treated with CPDA (1, 3, 5, 10 μM) or vehicle for 24 hours. Cells were lysed in ice-cold lysis buffer, and intracellular PIP3 levels were measured using a PIP3-specific ELISA kit according to the manufacturer's protocol [1]
2. Western blot assay for PI3K/Akt signaling: SHIP2-transfected HEK293 cells were treated with CPDA (0.5, 1, 3 μM) for 24 hours. Cells were lysed in RIPA buffer containing protease and phosphatase inhibitors. Total protein was separated by SDS-PAGE, transferred to PVDF membranes, and probed with antibodies against p-Akt (Ser473), p-Akt (Thr308), total Akt, and GAPDH (loading control). Chemiluminescent signals were detected and quantified using imaging software [1] 3. Cell viability assay: HEK293, MCF-7, and HCT116 cells were seeded in 96-well plates at a density of 2×10^3 cells/well. After 24 hours of adherence, cells were treated with CPDA (0.1-20 μM) for 72 hours. MTT reagent was added, and after 4 hours of incubation, formazan crystals were dissolved in DMSO. Absorbance at 570 nm was measured to calculate cell viability [1] |
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| Animal Protocol |
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| References | |||
| Additional Infomation |
1. CPDA is a small molecule inhibitor designed and synthesized to target SHIP2. SHIP2 is a phosphatase that negatively regulates the PI3K/Akt signaling pathway by dephosphorylating PIP3 to PIP2. Its mechanism of action includes inhibiting SHIP2 phosphatase activity, leading to intracellular PIP3 accumulation, which in turn activates the PI3K/Akt pathway [1]. 2. CPDA has a high selectivity for SHIP2 compared to other phosphatases (SHIP1, PTEN, Inpp5d), minimizing off-target effects, making it a valuable tool compound for studying the biological function of SHIP2 and the PI3K/Akt signaling pathway. However, it did not show antiproliferative activity in the tested cancer cell lines, indicating that its potential as an antitumor drug is limited [1]. 3. CPDA belongs to the 4-substituted 2-pyridine-2-amide class of compounds, and its chemical structure has been optimized to bind to SHIP2 and inhibit its activity. Its development laid the foundation for further modifications to improve efficacy, pharmacokinetic properties and potential therapeutic applications [1]
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| Molecular Formula |
C₂₀H₁₅CLF₂N₂O₂
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| Molecular Weight |
388.8
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| Exact Mass |
388.079
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| Elemental Analysis |
C, 61.79; H, 3.89; Cl, 9.12; F, 9.77; N, 7.21; O, 8.23
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| CAS # |
1415834-63-7
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| Related CAS # |
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| PubChem CID |
71089297
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| Appearance |
Light yellow to yellow solid powder
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| LogP |
4.846
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
5
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| Rotatable Bond Count |
6
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| Heavy Atom Count |
27
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| Complexity |
467
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| Defined Atom Stereocenter Count |
0
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| SMILES |
O=C(NC1=NC=CC(OCC2=CC=C(Cl)C=C2)=C1)CC3=C(F)C=CC=C3F
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| InChi Key |
GISJHCLTIVIGLX-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C20H15ClF2N2O2/c21-14-6-4-13(5-7-14)12-27-15-8-9-24-19(10-15)25-20(26)11-16-17(22)2-1-3-18(16)23/h1-10H,11-12H2,(H,24,25,26)
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| Chemical Name |
N-[4-[(4-chlorophenyl)methoxy]pyridin-2-yl]-2-(2,6-difluorophenyl)acetamide
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| Synonyms |
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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 |
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| 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) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (6.43 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 (6.43 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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.5720 mL | 12.8601 mL | 25.7202 mL | |
| 5 mM | 0.5144 mL | 2.5720 mL | 5.1440 mL | |
| 10 mM | 0.2572 mL | 1.2860 mL | 2.5720 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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