SH2 domain-containing inositol 5'-phosphatase 2 (SHIP2) - Inhibitor. SHIP2 negatively regulates insulin signaling by hydrolyzing phosphatidylinositol-3,4,5-trisphosphate (PIP3) [1]
.
- IC50 values:
- Human SHIP2: 0.62 ± 0.02 μM [1]
.
- Mouse SHIP2: 0.34 ± 0.10 μM [1]
.
- Human SHIP1: 13 ± 2 μM (approximately 30-fold less potent than for SHIP2) [1]
.
- Human PTEN: >50 μM [1]
.
- Human synaptojanin: >50 μM [1]
.
- Human myotubularin: >50 μM [1]
.
- Ki values:
- Human SHIP2: 0.44 ± 0.19 μM (competitive inhibitor with respect to substrate Ins(1,3,4,5)P4) [1]
.
- Human SHIP1: 11 ± 1.4 μM [1]
.

Insulin-induced Akt phosphorylation in L6 myotubes is increased by AS1949490 (0-16 μM; 15 minutes) [1]. In L6 myotubes, AS1949490 (0-10 μM; 48) induces stress and promotes trophic activity [1]. AS1949490 (0-0.10 μM; 24-hour; L6 myotubes) inhibits the gluconeogenic process triggered by insulin [1]. In L6 myotubes, AS1949490 (10 μM; 48 hours) upregulates the GLUT1 gene to initiate oxygen switching [2].

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Enzyme Inhibition Kinetics: AS1949490 is a competitive inhibitor of SHIP2 with respect to the substrate Ins(1,3,4,5)P4. The Km for Ins(1,3,4,5)P4 with human SHIP2 was 44 ± 10 nM. Lineweaver-Burk analysis confirmed competitive inhibition with Ki of 0.44 μM [1]
.
- Selectivity Profile: AS1949490 demonstrated selectivity for SHIP2 over the closely related phosphatase SHIP1 (approximately 30-fold selectivity) and showed no inhibition of other intracellular phosphatases including PTEN, synaptojanin, and myotubularin at concentrations up to 50 μM [1]
.
- Akt Phosphorylation in L6 Myotubes: In L6 myotubes, AS1949490 (0.3-3 μM) enhanced insulin-induced phosphorylation of Akt (Ser473) in a concentration-dependent manner. At 1 nM insulin stimulation, 3 μM AS1949490 significantly increased Akt phosphorylation (P<0.001). However, AS1949490 did not enhance fetal bovine serum (FBS)-induced Akt phosphorylation, suggesting specificity for insulin signaling [1]
.
- Glucose Consumption: In L6 myotubes treated with AS1949490 (0.3-3 μM) and 1 nM insulin for 48 hours, glucose consumption increased in a concentration-dependent manner (P<0.01 to P<0.001) [1]
.
- Glucose Uptake: AS1949490 (0.3-3 μM) treatment for 48 hours significantly stimulated glucose uptake activity in L6 myotubes in a concentration-dependent manner (P<0.01 to P<0.001) [1]
.
- Gluconeogenesis in FAO Hepatocytes: In rat FAO hepatoma cells treated with AS1949490 (1-10 μM) and 1 nM insulin for 24 hours, gluconeogenesis was significantly decreased in a concentration-dependent manner (P<0.05 to P<0.001) [1]
.

The intracellular insulin signaling pathway is activated and the risk of diabetes in the diabetic retina is decreased by AS1949490 (300 mg/kg; Pathway; twice daily for 7 or 10 days) [1]. AS1949490 (300 mg/kg; 8-hour single dose).

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Acute Study in Normal Mice: Male ICR mice were fasted overnight and administered AS1949490 orally at 300 mg/kg. After 8 hours, liver tissue was collected for mRNA analysis. AS1949490 treatment significantly reduced hepatic mRNA levels of gluconeogenic genes: glucose-6 phosphatase (G6Pase) by approximately 50% (P<0.05) and phosphoenolpyruvate carboxykinase (PEPCK) by approximately 50% (P<0.01) [1]
.
- Chronic Study in Diabetic db/db Mice: Male db/db mice were treated with AS1949490 (300 mg/kg) orally twice daily for 7 or 10 days. After 7 days, plasma glucose was significantly reduced by 23% compared to vehicle (P<0.01), without affecting body weight or plasma insulin levels [1]
.
- Oral Glucose Tolerance Test (OGTT): After 10 days of treatment with AS1949490 (300 mg/kg twice daily), db/db mice were fasted overnight and subjected to OGTT (2 g/kg glucose orally). AS1949490 treatment significantly reduced fasting blood glucose by 37% at time -30 min (P<0.05) and significantly reduced the area under the curve (AUC) during 0-2 hours after glucose loading (P<0.05) [1]
.
- Hepatic Insulin Signaling: After 10 days of treatment with AS1949490 (300 mg/kg twice daily), liver tissue was collected and analyzed for GSK3β phosphorylation. AS1949490 significantly increased phosphorylation of GSK3β (Ser9) in the liver without changing total GSK3β protein levels, indicating enhanced insulin signaling [1]
.
- Pharmacokinetic Distribution: Pharmacokinetic analyses revealed that after oral administration, serum concentrations of AS1949490 were low, but the compound concentration was highest in the liver, suggesting hepatic targeting [1]
.

Malachite Green Phosphatase Assay: Purified recombinant phosphatases (100 ng) were incubated with substrate (Ins(1,3,4,5)P4 at 100 μM for SHIP2/SHIP1; PtdIns(3,4,5)P3 at 250 μM for PTEN, synaptojanin, and myotubularin) in reaction buffer (10 mM HEPES pH 7.25, 6 mM MgCl2, 0.1% CHAPS, 250 mM sucrose, 0.25 mM EDTA) in 384-well plates for 20 min at room temperature. Malachite green reagent was added, incubated for 25 min, and absorbance measured at 630 nm. IC50 values were calculated using regression analysis. Kinetic studies were performed with varying substrate concentrations at fixed inhibitor concentrations, and Ki values were determined from Lineweaver-Burk plots [1]
.
- Protein Expression and Purification: Human SHIP2 (residues 419-732), SHIP1 (399-714), mouse SHIP2 (421-733), human synaptojanin (492-856) catalytic domains, and full-length human myotubularin were cloned and expressed in E. coli with 6His tags, purified using immobilized metal affinity chromatography. Human PTEN with deletions (removing residues 2-6, 286-309, and 354-403) was expressed with GST tag in Sf-9 cells and purified with glutathione sepharose [1]
.

Western Blot Analysis [1]
Cell Types: L6 Myotubes
Tested Concentrations: 0, 4, 8 and 16 μM; ]. 1 nM (insulin)
Incubation Duration: 15 minutes
Experimental Results: Insulin-induced Akt phosphorylation increases in a dose-dependent manner.

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Western Blot Analysis[2]
Cell Types: L6 myotubes
Tested Concentrations: 10 µM
Incubation Duration: 48 hrs (hours)
Experimental Results: GLUT1 mRNA expression was increased in L6 myotubes, but GLUT4 mRNA expression was not.

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L6 Myotube Differentiation and Treatment: L6 myoblasts were seeded in 96-well plates and cultured until confluence. Differentiation to myotubes was induced by culturing for 5-6 days in DMEM containing 2% horse serum. Differentiated myotubes were serum-starved for 16 h in DMEM, then treated with AS1949490 for 15 min before stimulation with insulin or FBS for 10 min [1]
.
- Cell-Based ELISA for Phospho-Akt: After treatment, cells were fixed with 4% paraformaldehyde for 15 min, washed with TBS-T, permeabilized with 0.1% Triton X-100 for 10 min, and blocked with Blocking One solution for 1 h. Cells were incubated with Ser473 phospho-specific Akt antibody for 2 h at room temperature, then with HRP-conjugated secondary antibody. Signal was developed with TMB substrate and absorbance measured at 450 nm [1]
.
- Glucose Uptake and Consumption Assays: L6 myotubes were cultured for 48 h under differentiating conditions (2% horse serum in phenol red-free DMEM) with AS1949490 in the presence (glucose consumption) or absence (glucose uptake) of insulin. Glucose uptake and consumption were measured as previously described [1]
.
- Gluconeogenesis Assay in FAO Cells: Rat FAO hepatoma cells were seeded in 96-well plates and grown in DMEM with 10% FBS. Cells were treated with AS1949490 and 1 nM insulin for 24 h. After washing with PBS, glucose production buffer (glucose-free DMEM pH 7.4 with 20 mM sodium pyruvate, no phenol red) was added. After 6 h, glucose concentration in the medium was measured using Glucose CII-Test reagent [1]
.
- Western Blot Analysis: Cell lysates or liver tissue homogenates were separated by SDS-PAGE, transferred to PVDF membranes, and probed with antibodies against phospho-Akt (Ser473), total Akt, phospho-GSK3β (Ser9), or total GSK3β. Signals were detected with HRP-conjugated secondary antibodies and ECL systems, quantified using VersaDoc digital imaging system [1]
.
- mRNA Expression Analysis by RT-PCR: Total RNA was extracted from liver tissue using Isogen kit. cDNA was synthesized and real-time quantitative PCR was performed using SYBR green method with primers for G6Pase, PEPCK, and 18S rRNA (internal control). Relative mRNA levels were quantified using a PRISM 7900 Sequence Detector [1]
.

Animal/Disease Models: Male C57BL/KsJ Jcl-dbm mice and db/+db mice [1].
Doses: 300 mg/kg.
Route of Administration: oral; ; formulated ICR s) Inhibit gluconeogenesis and the expression of related genes [1]. twice (two times) daily for 7 or 10 days
Experimental Results: Decrease in plasma glucose (23% relative to vehicle). Reduction in fasting blood glucose (37% relative to vehicle) and area under the blood glucose concentration-time curve (AUC). Increases GSK3β phosphorylation in the liver without changing overall GSK3β protein levels.

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Animal/Disease Models: Male ICR mice (6 weeks old) [1]
Doses: 300 mg/kg
Route of Administration: po (po (oral gavage)) once for 8 hrs (hrs (hours)).
Experimental Results: PEPCK and G6Pase mRNA levels were diminished by approximately 50%.

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Animals:** Male ICR mice (6 weeks old) and male C57BL/KsJ Jcl-db/db mice (6 weeks old) were used. Animals were housed under standard conditions (controlled temperature, humidity, 12-h light-dark cycle) with free access to food and water [1]
.
- **Acute Study in Normal Mice:** ICR mice were fasted overnight and then administered AS1949490 (300 mg/kg) or vehicle (0.5% methylcellulose) orally. After 8 h, mice were euthanized and liver tissue collected for mRNA analysis [1]
.
- **Chronic Study in Diabetic Mice:** db/db mice were given AS1949490 (300 mg/kg) suspended in 0.5% methylcellulose or vehicle alone twice daily by oral gavage for 7 or 10 days. Blood samples were collected for plasma glucose and insulin measurements. Body weight was monitored throughout the study [1]
.
- **Oral Glucose Tolerance Test (OGTT):** After 10 days of treatment, db/db mice were fasted overnight. Blood glucose was measured at -30 min (before glucose load). Glucose solution (2 g/kg) was administered orally at time 0. Blood glucose was measured at 0.5, 1, 2, and 3 h after glucose loading. Area under the curve (AUC) was calculated [1]
.
- **Tissue Collection:** After the final treatment, liver tissue was collected, homogenized in lysis buffer, and analyzed for protein expression by Western blot [1]
.

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Animals: Male ICR mice (6 weeks old) and male C57BL/KsJ Jcl-db/db mice (6 weeks old) were used. Animals were housed under standard conditions (controlled temperature, humidity, 12-h light-dark cycle) with free access to food and water [1]
.
- Acute Study in Normal Mice: ICR mice were fasted overnight and then administered AS1949490 (300 mg/kg) or vehicle (0.5% methylcellulose) orally. After 8 h, mice were euthanized and liver tissue collected for mRNA analysis [1]
.
- Chronic Study in Diabetic Mice: db/db mice were given AS1949490 (300 mg/kg) suspended in 0.5% methylcellulose or vehicle alone twice daily by oral gavage for 7 or 10 days. Blood samples were collected for plasma glucose and insulin measurements. Body weight was monitored throughout the study [1]
.
- Oral Glucose Tolerance Test (OGTT): After 10 days of treatment, db/db mice were fasted overnight. Blood glucose was measured at -30 min (before glucose load). Glucose solution (2 g/kg) was administered orally at time 0. Blood glucose was measured at 0.5, 1, 2, and 3 h after glucose loading. Area under the curve (AUC) was calculated [1]
.
- Tissue Collection: After the final treatment, liver tissue was collected, homogenized in lysis buffer, and analyzed for protein expression by Western blot [1]
.

Tissue Distribution: Pharmacokinetic analyses showed that after oral administration, serum concentrations of AS1949490 were low, but the compound concentration was highest in the liver, suggesting hepatic targeting. Specific numerical pharmacokinetic parameters (half-life, Cmax, AUC) were not provided [1]
.

In Vivo Tolerability: During chronic treatment of db/db mice with AS1949490 (300 mg/kg twice daily for 10 days), no significant effects on body weight or food intake were observed, suggesting good tolerability at this dose [1]
.
- No Observed Toxicity: No toxic effects such as hypophagia or body weight reduction were observed at the doses tested [1]

[1]. Discovery and functional characterization of a novel small molecule inhibitor of the intracellular phosphatase, SHIP2. Br J Pharmacol. 2009 Oct;158(3):879-87.

[2]. Glucose metabolism activation by SHIP2 inhibitors via up-regulation of GLUT1 gene in L6 myotubes. Eur J Pharmacol. 2010 Sep 10;642(1-3):177-82.

Chemical Identity: AS1949490 is 3-[(4-chlorobenzyl)oxy]-N-[(1S)-1-phenylethyl]-2-thiophene-carboxamide. It was synthesized by Astellas Pharma Inc. The synthesis involves three steps: (1) O-alkylation of methyl 3-hydroxy-2-thiophene carboxylate with 4-chlorobenzyl chloride, (2) saponification to yield the carboxylic acid, and (3) amide coupling with (1S)-1-phenylethylamine [1]
.
- Discovery: AS1949490 was identified through high-throughput screening of an in-house chemical library using a malachite green phosphatase assay, representing the first reported small molecule inhibitor of SHIP2 [1]
.
- Mechanism of Action: By inhibiting SHIP2, AS1949490 prevents dephosphorylation of PIP3, leading to enhanced insulin signaling through the PI3K/Akt pathway. This results in increased Akt phosphorylation, enhanced glucose uptake and consumption in muscle cells, suppressed gluconeogenesis in hepatocytes, and improved glucose homeostasis in diabetic animals [1]
.
- Selectivity Rationale: SHIP2 is closely related to SHIP1 but has distinct physiological roles. The approximately 30-fold selectivity of AS1949490 for SHIP2 over SHIP1, and lack of inhibition of PTEN, synaptojanin, and myotubularin, supports its use as a tool to study SHIP2-specific functions [1]
.
- Therapeutic Potential: The study provides proof-of-concept that pharmacological inhibition of SHIP2 with a small molecule can improve insulin sensitivity and glucose homeostasis in type 2 diabetes models. AS1949490 represents a first-in-class SHIP2 inhibitor that could serve as a lead compound for further optimization [1]
.
- First-in-Class: This is the first report of a small molecule inhibitor of SHIP2, providing a valuable tool for elucidating the physiological and pathophysiological functions of SHIP2 [1]
.
- Dosing Information: In vitro studies used concentrations ranging from 0.3-10 μM. In vivo studies used oral dosing at 300 mg/kg (acute) or 300 mg/kg twice daily (chronic) [1]
.

C20H18CLNO2S

371.879

371.075

1203680-76-5

44473434

White to off-white solid powder

6.046

1

3

6

25

422

1

C[C@@H](C1=CC=CC=C1)NC(=O)C2=C(C=CS2)OCC3=CC=C(C=C3)Cl

RFZPGNRLOKVZJY-AWEZNQCLSA-N

InChI=1S/C20H18ClNO2S/c1-14(16-5-3-2-4-6-16)22-20(23)19-18(11-12-25-19)24-13-15-7-9-17(21)10-8-15/h2-12,14H,13H2,1H3,(H,22,23)/t14-/m0/s1

3-[(4-chlorophenyl)methoxy]-N-[(1S)-1-phenylethyl]thiophene-2-carboxamide

AS1949490 AS 1949490 AS-1949490

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 : ~50 mg/mL (~134.45 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (5.59 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 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 20.8 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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 (Please use freshly prepared in vivo formulations for optimal results.)