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 compound with the structure 4-quinolone, substituted at positions 1, 3, 5, 6, and 7 with ethyl, carboxyl, amino, fluorine, and cyclohexylamino groups, respectively. It directly binds to and blocks the transcriptional activity of the active forkhead box protein O1 (Foxo1), but cannot bind to the Ser256 phosphorylated form of Foxo1. At low concentrations, it induces cell death and growth arrest in Burkitt lymphoma cell lines. It possesses various activities including hypoglycemic, anti-obesity, autophagy inhibition, antitumor, apoptosis induction, and Foxo1 inhibition. It is a quinoline monocarboxylic acid, quinolone, organofluorine compound, primary amino compound, secondary amino compound, and tertiary amino compound. Excessive glucose production by the liver through gluconeogenesis is partly responsible for elevated blood glucose levels in patients with type 2 diabetes mellitus (T2DM). The forkhead transcription factor forkhead box protein O1 (Foxo1) plays a crucial role in mediating the effects of insulin on hepatic gluconeogenesis. This study used a db/db mouse model to demonstrate the efficacy of orally administered small molecule Foxo1 inhibitors as treatments for type 2 diabetes mellitus (T2DM). We identified a series of compounds that bind to Foxo1 using mass spectrometry affinity screening, among which 5-amino-7-(cyclohexylamino)-1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (AS1842856) was identified. This compound effectively inhibits transcriptional activation of human Foxo1 and reduces glucose production by inhibiting the mRNA levels of glucose-6-phosphatase and phosphoenolpyruvate carboxykinase in rat hepatocyte lines. Oral administration of AS1842856 to diabetic db/db mice significantly reduced fasting blood glucose levels by inhibiting hepatic gluconeogenesis genes; however, administration of AS1842856 to normal mice had no effect on fasting blood glucose levels. Furthermore, AS1842856 also inhibited the increase in blood glucose levels after pyruvate injection in both normal and db/db mice. In summary, these findings suggest that FoxO1 inhibitors represent a new class of drugs that can be used to treat type 2 diabetes. [1]

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Objective: Oxidative stress-induced nucleus pulposus (NP) cell senescence is one of the important causes of intervertebral disc degeneration (IDD). This study aimed to investigate the role and mechanism of silencing information regulator 1 (SIRT1) in oxidative stress-induced rat NP cell senescence. Methods: Premature senescence of rat NP cells was induced by sublethal concentration of hydrogen peroxide (H2O2) (100 μM). SIRT1 was activated using SRT1720 (5 μM) to investigate its effect on NP cell senescence. FoxO1 and Akt were inhibited using AS1842856 (0.2 μM) and MK-2206 (5 μM), respectively, to investigate the role of the Akt-FoxO1-SIRT1 axis in rat NP cells. This study used resveratrol (20 μM) to pretreat rat nasopharyngeal cells. Resveratrol is a common antioxidant and an indirect activator of SIRT1. The aim was to investigate its role in aging rat nasopharyngeal cells. The results showed that the mRNA and protein levels of SIRT1 in H2O2-induced aging rat nasopharyngeal cells were reduced, while specific activation of SIRT1 could inhibit cell senescence. The Akt-FoxO1 pathway, as an upstream signaling pathway of SIRT1, may participate in the regulation of H2O2-induced senescence of rat nasopharyngeal cells by affecting the expression of SIRT1. In addition, resveratrol exerts an anti-aging effect in rat nasopharyngeal cells, which may be related to the Akt-FoxO1-SIRT1 axis. Conclusion: SIRT1 regulates oxidative stress-induced senescence of rat nasopharyngeal cells through the Akt-FoxO1 pathway, while resveratrol exerts an anti-aging effect by affecting this signaling pathway. [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.)