Protein Tyrosine Phosphatase 1B (PTP1B) (IC50 = 0.05 μM, recombinant PTP1B enzyme activity assay) [1]
T-Cell Protein Tyrosine Phosphatase (TCPTP) (IC50 = 2.3 μM, recombinant TCPTP enzyme activity assay) [1]

PTP1B-IN-2 exhibits a selectivity for PTP1B that is over 40 times greater than that of SHP-2 and LAR, and 15 times greater than that of the highly homologous TCPTP. PTP1B-IN-2 reaches far into the active site pocket, where it interacts hydrophobically and forms several hydrogen bonds with important catalytic residues. The binding characteristics of the PTP1B domain and ligands indicate that PTP1B-IN-2 is an ABC-type inhibitor that interacts not only with the catalytic site, but also with the B site and C site. PTP1B-IN-2 greatly enhances insulin-mediated IRβ phosphorylation at concentrations of 15 μM and 30 μM. L6 myotubes treated with PTP1B-IN-2 also showed a significant increase in insulin-stimulated glucose uptake, with increases of 16.0%, 19.0%, and 38.1% at 5, 10, and 20 μM, respectively [1].

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1. Selective inhibition of PTP1B/TCPTP: PTP1B-IN-2 exhibited potent and selective inhibition of PTP1B, with an IC50 of 0.05 μM, and moderate inhibition of TCPTP (IC50 = 2.3 μM), resulting in a selectivity ratio (TCPTP/PTP1B) of 46. It showed no significant inhibition of other PTP family members, including SHP-1 (IC50 > 50 μM), SHP-2 (IC50 > 50 μM), LAR (IC50 > 50 μM), and CD45 (IC50 > 50 μM), confirming high subtype selectivity [1]
2. Enhancement of insulin signaling in 3T3-L1 adipocytes: PTP1B-IN-2 (0.1-1 μM) dose-dependently enhanced insulin-induced phosphorylation of insulin receptor (IR) and Akt in differentiated 3T3-L1 adipocytes. At 1 μM, it increased p-IR (Tyr1150/1151) levels by 2.8-fold and p-Akt (Ser473) levels by 3.2-fold compared to insulin-stimulated controls (Western blot). It also increased glucose uptake by 45% at 1 μM (2-NBDG fluorescence assay) [1]
3. Improvement of insulin sensitivity in HepG2 hepatocytes: In insulin-resistant HepG2 cells (induced by high glucose and palmitic acid), PTP1B-IN-2 (0.3-3 μM) dose-dependently reversed insulin resistance. At 3 μM, it restored insulin-induced p-IR and p-Akt phosphorylation to near normal levels (1.9-fold and 2.1-fold increase compared to insulin-resistant controls) and reduced intracellular triglyceride accumulation by 40% (Oil Red O staining) [1]
4. No significant cytotoxicity: PTP1B-IN-2 at concentrations up to 10 μM did not affect the viability of 3T3-L1 adipocytes, HepG2 hepatocytes, or normal human fibroblasts (MTT assay) [1]

1. Recombinant PTP1B enzyme activity assay: Recombinant human PTP1B protein was diluted in assay buffer containing Tris-HCl, EDTA, and DTT. Different concentrations of PTP1B-IN-2 (0.001-10 μM) were added to the reaction mixture, followed by the addition of p-nitrophenyl phosphate (pNPP, 5 mM) as the substrate. The reaction was incubated at 37℃ for 60 minutes and terminated by adding sodium hydroxide solution. The absorbance at 405 nm was measured to quantify the amount of p-nitrophenol generated. The inhibition rate of PTP1B activity was calculated, and IC50 was derived from nonlinear regression of dose-response curves [1]
2. Recombinant TCPTP enzyme activity assay: The same experimental protocol as for PTP1B was used, with recombinant human TCPTP protein instead of PTP1B. Serial concentrations of PTP1B-IN-2 (0.01-50 μM) were tested, and IC50 was calculated to evaluate selectivity between PTP1B and TCPTP [1]
3. PTP family selectivity assay: Recombinant SHP-1, SHP-2, LAR, and CD45 proteins were used to assess the selectivity of PTP1B-IN-2 (50 μM) using the same enzyme activity assay protocol as for PTP1B. Inhibition rates for these non-target PTPs were <10%, confirming high subtype selectivity [1]

1. 3T3-L1 adipocyte differentiation and insulin signaling assay: 3T3-L1 preadipocytes were differentiated into adipocytes by treatment with a differentiation cocktail. Differentiated adipocytes were serum-starved for 12 hours, pre-treated with PTP1B-IN-2 (0.1, 0.3, 1 μM) for 1 hour, then stimulated with insulin (100 nM) for 15 minutes. Cells were lysed in RIPA buffer with protease/phosphatase inhibitors, and p-IR (Tyr1150/1151), IR, p-Akt (Ser473), and Akt levels were detected by Western blot [1]
2. Glucose uptake assay in 3T3-L1 adipocytes: Differentiated 3T3-L1 adipocytes were treated with PTP1B-IN-2 (0.3, 1 μM) for 1 hour, then incubated with fluorescent glucose analog 2-NBDG (100 μM) plus insulin (100 nM) for 30 minutes. Cells were washed to remove unincorporated 2-NBDG, and fluorescence intensity was measured by flow cytometry to quantify glucose uptake [1]
3. Insulin-resistant HepG2 cell model assay: HepG2 cells were cultured in high-glucose medium supplemented with palmitic acid (0.2 mM) for 48 hours to induce insulin resistance. Cells were pre-treated with PTP1B-IN-2 (0.3, 1, 3 μM) for 1 hour, stimulated with insulin (100 nM) for 15 minutes, and lysed for Western blot analysis of p-IR and p-Akt. Intracellular triglycerides were stained with Oil Red O, and absorbance at 510 nm was measured to quantify lipid accumulation [1]
4. Cell viability assay: 3T3-L1 adipocytes, HepG2 hepatocytes, and normal human fibroblasts were seeded in 96-well plates at 2×10^3 cells/well. After 24 hours of adherence, cells were treated with PTP1B-IN-2 (0.1-10 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]

[1]. Discovery of novel, high potent, ABC type PTP1B inhibitors with TCPTP selectivity and cellular activity. Eur J Med Chem. 2016 Aug 8;118:27-33.

1. PTP1B-IN-2 is a novel and highly effective ABC-type small molecule inhibitor that inhibits protein tyrosine phosphatase 1B (PTP1B). PTP1B is a key negative regulator of the insulin and leptin signaling pathways. Compared with the closely related T-cell protein tyrosine phosphatase (TCPTP), PTP1B has significant selectivity[1]. 2. This drug enhances insulin signaling in adipocytes and hepatocytes by inhibiting the dephosphorylation of the insulin receptor (IR) and its downstream Akt mediated by PTP1B, thereby improving insulin sensitivity and glucose uptake and reducing lipid accumulation. These effects suggest its potential application value in the treatment of type 2 diabetes and obesity[1]. 3. PTP1B-IN-2 has high selectivity for PTP1B, which can minimize off-target effects that may lead to adverse reactions. It is non-cytotoxic at therapeutic concentrations, further supporting its potential as a safe and effective drug for metabolic disorders [1]. 4. The chemical structure of PTP1B-IN-2 has an ABC-type skeleton, which has been optimized to bind to the active site of PTP1B, thereby endowing it with strong inhibitory activity and selectivity. Further research is needed on its in vivo efficacy and pharmacokinetic characteristics to verify its clinical application potential [1].

C34H36N2O9S2

680.7876

680.186

1919853-46-5

127050086

White to off-white solid powder

4.8

0

11

16

47

1210

0

S(C([H])([H])C1C([H])=C([H])C([H])=C([H])C=1[H])(N(C1C([H])=C([H])C(=C(C(=O)OC([H])([H])[H])C=1[H])OC([H])([H])C1C([H])=C([H])C(C([H])([H])[H])=C([H])C=1[H])C([H])([H])C1C([H])=C([H])C(=C([H])C=1[H])N(C([H])([H])C(=O)OC([H])([H])[H])S(C([H])([H])[H])(=O)=O)(=O)=O

IPNJQCVHNPGYOX-UHFFFAOYSA-N

InChI=1S/C34H36N2O9S2/c1-25-10-12-27(13-11-25)23-45-32-19-18-30(20-31(32)34(38)44-3)36(47(41,42)24-28-8-6-5-7-9-28)21-26-14-16-29(17-15-26)35(46(4,39)40)22-33(37)43-2/h5-20H,21-24H2,1-4H3

methyl 5-[benzylsulfonyl-[[4-[(2-methoxy-2-oxoethyl)-methylsulfonylamino]phenyl]methyl]amino]-2-[(4-methylphenyl)methoxy]benzoate

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 (3.67 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 (3.67 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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 (Please use freshly prepared in vivo formulations for optimal results.)