Forkhead box O1 (FoxO1) (IC50: 110 nM) [1]

In a rat hepatic cell line, AS1842856 potently inhibits human Foxo1 transactivation and decreases glucose synthesis via inhibiting the amounts of phosphoenolpyruvate carboxykinase and glucose-6 phosphatase mRNA[1]. Following AS1842856 therapy, there is a decrease in p-Akt expression relative to the control group, but there is no discernible difference in the protein expression of FoxO1 and p-FoxO1[2].

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AS1842856 inhibited FoxO1-dependent transcriptional activity in a dose-dependent manner. In HEK293 cells transfected with a FoxO1-responsive luciferase reporter plasmid, treatment with the compound resulted in significant reduction of luciferase activity, with an IC50 of 110 nM. Additionally, it suppressed the expression of FoxO1 target genes such as phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) in hepatocytes [1]

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In addition, we further explored the relationship between Akt, FoxO1 and SIRT1. We treated rat NP cells with FoxO1 inhibitor AS1842856 (0.2 μM), Akt inhibitor MK-2206 (5 μM) or AS1842856 in combination with MK-2206, respectively. First, MK-2206 inhibited the expression of p-Akt, decreased the phosphorylation of FoxO1 on Ser256 site and further increased the expression of total FoxO1 protein (Figure 7A,B). Besides, as an inhibitor of FoxO1, AS1842856 only reduced the activity of FoxO1 by binding with it, without affecting its transcription [25]. Then in our experiment, after AS1842856 treatment, there was no significant difference in the protein expression of p-FoxO1 and FoxO1 compared with the control group, but the expression of p-Akt was decreased compared with the control group (Figure 7A,B). Finally, simply inhibiting p-Akt activity by MK-2206 promoted the expression of SIRT1, while inhibiting FoxO1 activity by AS1842856 suppressed the expression of SIRT1. But if both p-Akt and FoxO1 were inhibited at the same time, the expression of SIRT1 was suppressed, which is different from the result of the simply MK-2206 treatment group (Figure 7A,B). [2]

By inhibiting hepatic gluconeogenic genes, oral administration of AS1842856 to diabetic db/db mice causes a significant reduction in fasting plasma glucose levels; however, delivery to normal mice has little effect on fasting plasma glucose levels. In both normal and db/db mice, treatment with AS1842856 also reduces an increase in plasma glucose level brought on by pyruvate injection[1].

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In diabetic db/db mice, oral administration of AS1842856 at doses of 3, 10, and 30 mg/kg once daily for 14 days led to a dose-dependent improvement in fasting glycemia. At the 30 mg/kg dose, fasting blood glucose levels were significantly reduced compared to vehicle-treated controls. The compound also tended to lower plasma insulin levels, though the effect was not statistically significant [1]

In diabetic db/db mice, oral administration of AS1842856 at doses of 3, 10, and 30 mg/kg once daily for 14 days led to a dose-dependent improvement in fasting glycemia. At the 30 mg/kg dose, fasting blood glucose levels were significantly reduced compared to vehicle-treated controls. The compound also tended to lower plasma insulin levels, though the effect was not statistically significant [1]

Primary hepatocytes were isolated and cultured. Cells were treated with AS1842856 at different concentrations for a specified period. Total RNA was extracted, and real-time PCR was performed to analyze the mRNA expression levels of FoxO1 target genes (PEPCK and G6Pase). Protein levels were also assessed by Western blot to confirm the effect on FoxO1-mediated signaling [1]

AS1842856 is dissolved in 6% cyclodextrin for oral administration.
Diabetic db/db mice

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Male db/db mice (8-10 weeks old) were randomly divided into groups. AS1842856 was formulated in 0.5% methylcellulose and administered orally at doses of 3, 10, or 30 mg/kg once daily for 14 days. Vehicle-treated controls received 0.5% methylcellulose alone. Fasting blood glucose levels were measured before treatment and at regular intervals during the treatment period. Plasma insulin levels were also determined at the end of the study [1]

[1]. Discovery of novel forkhead box O1 inhibitors for treating type 2 diabetes: improvement of fasting glycemia in diabetic db/db mice. Mol Pharmacol. 2010 Nov;78(5):961-70.

[2]. The resistant effect of SIRT1 in oxidative stress-induced senescence of rat nucleus pulposus cell is regulated by Akt-FoxO1 pathway. Biosci Rep. 2019 May 10;39(5). pii: BSR20190112.

AS1842856 is a quinolone that is 4-quinolone substituted at positions 1, 3, 5, 6 and 7 by ethyl, carboxy, amino, fluorine, and cyclohexylamino groups, respectively. It can directly bind to and block the transcription activity of the active forkhead box protein O1 (Foxo1), but not the Ser256-phosphorylated form. It induces cell death and growth arrest in Burkitt lymphoma cell lines at low concentrations. It has a role as a hypoglycemic agent, an anti-obesity agent, an autophagy inhibitor, an antineoplastic agent, an apoptosis inducer and a forkhead box protein O1 inhibitor. It is a quinolinemonocarboxylic acid, a quinolone, an organofluorine compound, a primary amino compound, a secondary amino compound and a tertiary amino compound.

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Excessive hepatic glucose production through the gluconeogenesis pathway is partially responsible for the elevated glucose levels observed in patients with type 2 diabetes mellitus (T2DM). The forkhead transcription factor forkhead box O1 (Foxo1) plays a crucial role in mediating the effect of insulin on hepatic gluconeogenesis. Here, using a db/db mouse model, we demonstrate the effectiveness of Foxo1 inhibitor, an orally active small-molecule compound, as a therapeutic drug for treating T2DM. Using mass spectrometric affinity screening, we discovered a series of compounds that bind to Foxo1, identifying among them the compound, 5-amino-7-(cyclohexylamino)-1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (AS1842856), which potently inhibits human Foxo1 transactivation and reduces glucose production through the inhibition of glucose-6 phosphatase and phosphoenolpyruvate carboxykinase mRNA levels in a rat hepatic cell line. Oral administration of AS1842856 to diabetic db/db mice led to a drastic decrease in fasting plasma glucose level via the inhibition of hepatic gluconeogenic genes, whereas administration to normal mice had no effect on the fasting plasma glucose level. Treatment with AS1842856 also suppressed an increase in plasma glucose level caused by pyruvate injection in both normal and db/db mice. Taken together, these findings indicate that the Foxo1 inhibitor represents a new class of drugs for use in treating T2DM.[1]

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Objective: The senescence of nucleus pulposus (NP) cells induced by oxidative stress is one of the important causes of intervertebral disc degeneration (IDD). Herein, we investigated the role and action mechanism of silent information regulator 1 (SIRT1) in oxidative stress-induced senescence of rat NP cell.Methods: Premature senescence of rat NP cells was induced by sublethal concentration of hydrogen peroxide (H2O2) (100 μM). SIRT1 was activated with SRT1720 (5 μM) to explore its effect on NP cells senescence. FoxO1 and Akt were inhibited by AS1842856 (0.2 μM) and MK-2206 (5 μM), respectively, to explore the role of Akt-FoxO1-SIRT1 axis in rat NP cells. Pretreatment with the resveratrol (20 μM), a common antioxidant and indirect activator of SIRT1, was done to investigate its role in senescent rat NP cells.Results: The mRNA and protein levels of SIRT1 were decreased in H2O2-induced senescent rat NP cells, and that specific activation of SIRT1 suppresses senescence. And the Akt-FoxO1 pathway, as the upstream of SIRT1, might be involved in the regulation of H2O2-induced senescence of rat NP cells by affecting the expression of SIRT1. In addition, the resveratrol played an anti-senescence role in rat NP cells, which might affect the Akt-FoxO1-SIRT1 axis.Conclusion: SIRT1 ameliorated oxidative stress-induced senescence of rat NP cell which was regulated by Akt-FoxO1 pathway, and resveratrol exerted anti-senescence effects by affecting this signaling axis.[2]

C18H22FN3O3

347.383987903595

347.165

C, 62.23; H, 6.38; F, 5.47; N, 12.10; O, 13.82

836620-48-5

72193864

White to light yellow solid powder

3.839

3

7

4

25

562

0

O=C(C1C(=O)C2C(=CC(=C(C=2N)F)NC2CCCCC2)N(CC)C=1)O

MOMCHYGXXYBDCD-UHFFFAOYSA-N

InChI=1S/C18H22FN3O3/c1-2-22-9-11(18(24)25)17(23)14-13(22)8-12(15(19)16(14)20)21-10-6-4-3-5-7-10/h8-10,21H,2-7,20H2,1H3,(H,24,25)

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: ≥ 1.67 mg/mL (4.81 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 16.7 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 2: 1 mg/mL (2.88 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 10.0 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of 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 3: ≥ 1 mg/mL (2.88 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 10.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 4: 10 mg/mL (28.79 mM) in 6% HP-β-CD in Saline (add these co-solvents sequentially from left to right, and one by one), Suspension solution; with ultrasonication.
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.)