AdipoR1 (Kd = 1.8 μM); AdipoR2 (Kd = 3.1 μM)[1]
Adiponectin receptors AdipoR1 and AdipoR2.

AdipoRon hydrochloride, an orally active, selective AdipoR agonist, binds to AdipoR1 and AdipoR2 with Kds of 1.8 and 3.1 μM. AdipoRon (50 nM-50 μM) stimulates AMPK phosphorylation through AdipoR1 [1]. AdipoRon (50 μM) reduced TNF-α and TGF-β1 levels in L02 cells in a dose-dependent manner. AdipoRon has substantial and dose-dependent growth inhibition effects on macrophages [2]. AdipoRon therapy enhances heart functional recovery after reperfusion and suppresses apoptosis after MI [3]. AdipoRon causes vasodilation through a different mechanism than adiponectin and does not significantly reduce VSMC [Ca2+]i[4].

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AdipoRon hydrochloride is a selective agonist of AdipoR1 and AdipoR2. It activates AMPK signaling in cultured mammalian cells. The compound demonstrates antiproliferative effects and modulates metabolic pathways. It is orally active and exhibits anti-inflammatory and insulin-sensitizing activities.

AdipoRon (50 mg/kg, administered intravenously) significantly phosphorylates AMPK in the liver and skeletal muscle of wild-type mice, but not in the double-knockout Adipor1−/− or Adipor2−/− mice [1]. AdipoRon (0.02, 0.1, and 0.5 mg/kg, ig) prevented the structural deformation of the liver brought on by D-GalN challenge and reduced the hepatotoxicity that D-GalN induced in mice. At larger doses (0.1 and 0.5 mg/kg), AdipoRon's hepatoprotective effect is especially apparent [2]. In mice lacking APN, AdipoRon (50 mg/kg, orally administered) prevented increased cardiomyocyte apoptosis. In AMPK-DN mice, AdipoRon's anti-apoptotic effect is diminished but not completely eliminated [3].

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AdipoRon hydrochloride has demonstrated in vivo efficacy in multiple disease models. It alleviates memory impairment in septic mice, modulates AMPK signaling and alleviates airway inflammation in a murine asthma model, and ameliorates renal inflammation in diet-induced obese mice.

Binding assays. [1]
Surface plasmon resonance measurements were performed by a BIAcore X100 system and sensor chip SA (GE Healthcare). Human AdipoR1 and AdipoR2 were expressed with the baculovirus system, and purified to homogeneity. The AdipoR1 and AdipoR2 samples were then reconstituted into egg-phosphatidylcholine liposomes containing biotinyl phosphatidylethanolamine, as reported. Mouse full-length adiponectin was generated as previously described. AdipoR1 and AdipoR2 were immobilised onto a sensor chip SA to levels of 2,500-3,000 response units (RU) using standard immobilisation protocols (GE Healthcare). We used Rhodopsin receptor as control, and obserbed that AdipoRon indeed does not react Rhodopsin receptor at all. Experiments were carried out at 25 ˚C using running buffer (20 mM Hepes, pH 7.4, 200 mM NaCl, 10% glycerol, 0.05% (v/v) surfactant P20). Binding analyses were performed using a range of AdipoRon (0.49-31.25 µM) or adiponectin (1.5 ng-3.75 µg). Biacore X100 Evaluation Software was used to determine the equilibrium dissociation constant (KD) of the compound or proteins.

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3H-labelled AdipoRon binding assay.[1]
Tritium-labelled AdipoRon was made by a CRO company. AdipoRon was tritium labeled at the position indicated by the asterisk in the figure below. To the carboxylic acid solid (25 mg) was added 0.5 ml thionyl chloride and the suspension was carefully warmed to dissolve the solid. The mixture was stirred for 1 hr at room temperature and excess thionyl chloride was removed using a stream of nitrogen gas, and the residue was pumped dry under a vacuum for 30 min. The unlabelled amine dihydrochloride (35 mg) was dissolved in water (1 ml). Potassium carbonate (50 mg) was added and the free amine was extracted into dichloromethane (3 ml). This organic solution was dried using anhydrous sodium sulphate (5 mg). The suspension was filtered and the solvent was removed by rotary evaporation. The residue was pumped dry under a vacuum for 30 min. The free amine base was dissolved in dichloromethane (2 ml) : triethylamine (50 μl). The acid chloride was dissolved in dichloromethane and added to the solution of the amine base above. The mixture was stirred for 30 min to couple the acid chloride with the amine. The mixture was analysed using silica TLC plates eluting in CH2Cl2:MeOH:AcOH (95:5:0.1). This mixture was then purified using a Silica Sep-Pak (2 g), eluting with 3 x 2 ml dichloromethane, followed by CH2Cl2:MeOH:AcOH (95:5:0.1) 3 x 2 ml. The fractions 3 – 6 were combined and the solvent was removed under vacuum overnight to yield a colourless oil. Then the product was tritiated (296 MBq/mmol). The binding assay were performed according to the method described previously4,19-21, with slight modifications. The cells were incubated at 25˚C for 1 hr with binding buffer (ice-cold phosphate buffered sarine (PBS)) containing designated concentrations of 3H-labelled AdipoRon plus unlabeled competitors. The cells were then washed 10 times with PBS, lysed in 0.1 M NaOH, 0.1% SDS, and the cell-bound radioactivity was determined using -counter18,19,22. Nonspecific binding was determined using a 200-fold excess of unlabeled AdipoRon. Specific binding was calculated by subtracting nonspecific binding from the total binding.

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The AdipoR binding assay uses membranes from cells expressing recombinant AdipoR1 or AdipoR2. Radiolabeled adiponectin or a selective agonist is used as a tracer. Test compounds compete for binding, and EC50 values are determined from dose-response curves to establish agonist activity.

Cells and cell culture[2]
Immortalized normal human liver cells L02 and murine monocytic cell line RAW264.7 were used...
AdipoRon protects hepatocytes in vitro[2]
The hepatoprotective effects of AdipoRon were examined on L02 cell line in vitro, which might provide some clues for its activity and mechanism. The results showed that 5–50 μM AdipoRon pretreatment could attenuate the expression of TNF-α and TGF-β1, apparently in a dose-dependent manner (Fig. 2B), while little change appeared on the apoptosis or proliferation of hepatocytes by itself (Fig. 2A), which might implicate a hepatoprotective effect of AdipoRon, via suppression on proinflammatory...

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In cellular assays, cells expressing AdipoR1/R2 are treated with AdipoRon hydrochloride. AMPK activation is assessed by Western blot for phosphorylated AMPK. Glucose uptake is measured using radiolabeled 2-deoxyglucose. Cell viability is assessed using MTT assays.

50 mg/kg, p.o.Mice AMPK phosphorylation in vivo.[1]
To study AMPK phosphorylation in vivo, we injected 50 mg of AdipoRon per kg body weight intravenously into mice through an inferior vena cava catheter.

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In vivo studies use various animal models including septic mice, asthmatic mice, and diet-induced obese mice. AdipoRon hydrochloride is administered orally or systemically. Memory function, airway inflammation, renal inflammation, and metabolic parameters are assessed to evaluate therapeutic efficacy.

[1]. A small-molecule AdipoR agonist for type 2 diabetes and short life in obesity. Nature. 2013 Nov 28;503(7477):493-9.

[2]. Hepatoprotective effects of AdipoRon against d-galactosamine-induced liver injury in mice. Eur J Pharm Sci. 2016 Aug 9;93:123-131.

[3]. AdipoRon, the first orally active adiponectin receptor activator, attenuates postischemic myocardial apoptosis through both AMPK-mediated and AMPK-independent signalings. Am J Physiol Endocrinol Metab. 2015 Aug 1;309(3):E275-82.

[4]. Adiponectin Receptor Agonist, AdipoRon, Causes Vasorelaxation Predominantly Via a Direct Smooth Muscle Action. Microcirculation. 2016 Apr;23(3):207-20.

Adiponectin, secreted by adipocytes, binds to adiponectin receptors AdipoR1 and AdipoR2, exerting its antidiabetic effects by activating the AMPK and PPAR-α pathways, respectively. Obesity leads to decreased plasma adiponectin levels, subsequently triggering insulin resistance and type 2 diabetes. Therefore, orally active small molecules capable of binding to and activating AdipoR1 and AdipoR2 hold promise for improving obesity-related diseases, such as type 2 diabetes. This article reports the identification of an orally active synthetic small molecule AdipoR agonist. One compound, the AdipoR agonist (AdipoRon), can simultaneously bind to AdipoR1 and AdipoR2 in vitro. AdipoRon exhibits effects very similar to adiponectin in muscle and liver, such as activating the AMPK and PPAR-α pathways and improving insulin resistance and glucose intolerance in mice fed a high-fat diet, improvements that were completely lost in AdipoR1 and AdipoR2 double knockout mice. In addition, AdipoRon improved diabetes in the genetically obese rodent model db/db mice and extended the lifespan of db/db mice fed a high-fat diet. Therefore, oral active AdipoR agonists, such as AdipoRon, are a promising treatment for obesity-related diseases such as type 2 diabetes. [1]

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Adiponectin is an antidiabetic and antiatherosclerotic adipokines that plays a unique role in energy homeostasis. As an insulin-sensitizing hormone, adiponectin exerts a variety of biological effects via specific receptors (AdipoR1 and AdipoR2) by activating the AMP-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor (PPAR) α pathways. The oral active synthetic small molecule AdipoR agonist AdipoRon has shown very similar effects to adiponectin in vitro and in vivo, which may be a promising treatment for obesity-related diseases. Given the regulatory effects of adiponectin or AdipoRon on inflammatory responses and energy metabolism, they may have the potential to treat tissue damage. Therefore, this study explored its effects and possible mechanisms. In vitro studies on hepatocytes (L02) and macrophages (RAW264.7) showed that AdipoRon has protective and anti-inflammatory effects. These effects were validated in a mouse model of D-galactosamine (D-GalN)-induced acute liver injury: pretreatment with AdipoRon or bicyclol (positive control) repaired liver injury, characterized by significantly elevated serum and liver biomarkers (AST, ALT, MDA, and NOS). Furthermore, AdipoRon alleviated liver inflammation, manifested by reduced pro-inflammatory macrophage infiltration and decreased levels of tumor necrosis factor-α (TNF-α), transforming growth factor β1 (TGF-β1), interleukin-1β (IL-1β), and interleukin-6 (IL-6); simultaneously, AdipoRon also promoted AMPK activation through phosphorylation. Combined with liver histopathological results, these confirm that AdipoRon has a protective effect against D-GalN-induced liver injury, which may be attributed to its reduction of inflammation, inhibition of free radical reactions and enhancement of liver energy metabolism. [2]

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Adiponectin (APN) is a cardioprotective molecule. Decreased APN levels in diabetic patients exacerbate myocardial ischemia/reperfusion (MI/R) injury. Although APN administration has been shown to reduce MI/R injury in animal studies, several factors limit its clinical application. This study aimed to investigate whether AdipoRon (the first orally active APN receptor-binding molecule) can protect the heart from MI/R injury, and if so, to elucidate its mechanism of action. Wild-type (WT), APN knockout (APN-KO), and cardiomyocyte-specific AMPK dominant-negative (AMPK-DN) mice were treated with either the vector or AdipoRon (50 mg/kg, 10 minutes before myocardial infarction) and subjected to myocardial infarction/reperfusion (MI/R) injury (30 minutes/3–24 hours). Compared with the vector group, oral administration of AdipoRon significantly improved cardiac function and reduced cardiomyocyte apoptosis after ischemia in WT mice, as confirmed by DNA ladder formation, TUNEL staining, and caspase-3 activation assays (all P < 0.01). In APN knockout (APN-KO) or AMPK knockout (AMPK-DN) mice, MI/R-induced apoptosis was significantly enhanced. In APN-KO mice, the degree of reduction in MI/R damage by AdipoRon was similar to that in WT mice. In AMPK-DN mice, the anti-apoptotic effect of AdipoRon was partially inhibited but not completely lost. Finally, AdipoRon significantly reduced oxidative stress after ischemia, as evidenced by decreased NADPH oxidase expression and superoxide production. In summary, these results demonstrate for the first time that the orally effective APN receptor activator AdipoRon can effectively reduce ischemic cardiac injury, supporting APN receptor agonists as a promising new treatment for cardiovascular complications caused by obesity-related diseases such as type 2 diabetes. [3]

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Objective: AdipoRon is an adiponectin receptor agonist that has recently been proposed for the treatment of insulin resistance and hyperglycemia. Since adiponectin exerts an angioprotective effect through a NO-mediated signaling pathway, it was hypothesized that AdipoRon also has a similar potentially beneficial vasodilatory effect. Therefore, we investigated whether adiponectin can induce vasodilation and its mechanism of action. Methods: Vascular function of rat skeletal muscle arteries and mouse cerebral/coronary arteries was assessed using pressure and line tension assays. Results: qPCR confirmed the presence of adiponectin receptor mRNA expression in skeletal muscle, brain, and coronary arteries. Compound C (10 μM; AMPK inhibitor) did not block adiponectin-induced vasodilation. Inhibition of endothelium-dependent vasodilation using the L-NAME/Indomethacin/Apramine/TRAM-34 combination only slightly reduced adiponectin-mediated vasodilation in the brain and coronary arteries. Endothelial-removed cremasteric arteries showed a similar vasodilatory response to adiponectin to intact vessels, suggesting that adiponectin acts directly on vascular smooth muscle cells (VSMCs). K⁺ currents measured in VSMCs isolated from the mouse basilar artery and left anterior descending artery were unaffected by adiponectin. In cremasteric arteries, adiponectin induced vasodilation without a significant decrease in intracellular calcium ion concentration ([Ca²⁺]i) in VSMCs. Adiponectin itself induces vasodilation in intact cremasteric arteries, but fails to induce significant vasodilation in arteries without endothelial cells, consistent with the endothelium-dependent nature of adiponectin. Conclusion: Adiponectin exerts its vasodilatory effect through a different mechanism than adiponectin itself. The main mechanism by which adiponectin induces vasodilation is independent of endothelium-dependent relaxing factors, AMPK activation, K⁺ efflux-mediated hyperpolarization, and decrease in cytoplasmic [Ca²⁺]i. [4]

464.186

1781835-20-8

AdipoRon;924416-43-3

78243714

Off-white to light yellow solid

2

4

8

33

582

0

C1CN(CCC1NC(=O)COC2=CC=C(C=C2)C(=O)C3=CC=CC=C3)CC4=CC=CC=C4.Cl

TZVJQEGKRLDTHQ-UHFFFAOYSA-N

InChI=1S/C27H28N2O3.ClH/c30-26(28-24-15-17-29(18-16-24)19-21-7-3-1-4-8-21)20-32-25-13-11-23(12-14-25)27(31)22-9-5-2-6-10-22;/h1-14,24H,15-20H2,(H,28,30);1H

2-(4-benzoylphenoxy)-N-(1-benzylpiperidin-4-yl)acetamide;hydrochloride

AdipoRon hydrochloride; 1781835-20-8; 2-(4-Benzyoylphenoxy)-N-[1-(phenylmethyl)-4-piperidinyl]acetamidehydrochloride; 2-(4-benzoylphenoxy)-N-(1-benzylpiperidin-4-yl)acetamide;hydrochloride; AdipoRon?; 2-(4-Benzyoylphenoxy)-N-[1-(phenylmethyl)-4-piperidinyl]acetamide hydrochloride; C27H28N2O3.HCl; 924416-43-3 (free base);

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 (~107.5 mM; with ultrasonication)

Solubility in Formulation 1: ≥ 5 mg/mL (10.75 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 DMSO stock solution (50.0 mg/mL) to 400 μL of PEG300 and mix well; then add 50 μL of Tween-80 and mix well; finally add 450 μL of physiological saline and 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: ≥ 5 mg/mL (10.75 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 DMSO stock solution (50.0 mg/mL) to 900 μL of 20% SBE-β-CD saline and mix well.
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 3: 5 mg/mL (10.75 mM) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), Suspended solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of DMSO stock solution (50.0 mg/mL) to 900 μL of corn oil and mix well.

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