AdipoR1 (Kd = 1.8 μM); AdipoR2 (Kd = 3.1 μM)[1]

With Kds of 1.8 and 3.1 μM, AdipoRon is an oral AdipoR agonist that is both selective and active against AdipoR1 and AdipoR2. Through AdipoR1, AdipoRon (50 nM–50 μM) raises AMPK phosphorylation[1]. In L02 cells, AdipoRon (50 μM) attenuates TNF-α and TGF-β1 production in a dose-dependent manner. On macrophages, AdipoRon shows a notable and dosage-dependent proliferation suppression[2]. Treatment with AdipoRon dramatically reduces post-MI apoptosis and enhances cardiac functional recovery following reperfusion[3]. AdipoRon causes vasorelaxation and vasodilation through different pathways than adiponectin, all without significantly lowering VSMC [Ca2+]i[4].

In wild-type mice's skeletal muscle and liver, AdipoRon (50 mg/kg, iv) significantly phosphorylates AMPK, but not in Adipor1−/− or Adipor2−/− double-knockout mice[1]. AdipoRon (0.02, 0.1, and 0.5 mg/kg, ig) protects the hepatic architecture from distortion in response to D-GalN exposure and reduces the hepatotoxicity caused by D-GalN in mice. AdipoRon's hepatoprotective activity is most noticeable at higher dosages (0.1 and 0.5 mg/kg)[2]. AdipoRon (50 mg/kg, po) treatment rescues APN-deficient mice from enhanced cardiomyocyte apoptosis. In AMPK-DN mice, AdipoRon's antiapoptotic impact is diminished but not eliminated[3].

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.

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...

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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50 mg/kg, p.o.

Mice

[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 from adipocytes binds to adiponectin receptors AdipoR1 and AdipoR2, and exerts antidiabetic effects via activation of AMPK and PPAR-α pathways, respectively. Levels of adiponectin in plasma are reduced in obesity, which causes insulin resistance and type 2 diabetes. Thus, orally active small molecules that bind to and activate AdipoR1 and AdipoR2 could ameliorate obesity-related diseases such as type 2 diabetes. Here we report the identification of orally active synthetic small-molecule AdipoR agonists. One of these compounds, AdipoR agonist (AdipoRon), bound to both AdipoR1 and AdipoR2 in vitro. AdipoRon showed very similar effects to adiponectin in muscle and liver, such as activation of AMPK and PPAR-α pathways, and ameliorated insulin resistance and glucose intolerance in mice fed a high-fat diet, which was completely obliterated in AdipoR1 and AdipoR2 double-knockout mice. Moreover, AdipoRon ameliorated diabetes of genetically obese rodent model db/db mice, and prolonged the shortened lifespan of db/db mice on a high-fat diet. Thus, orally active AdipoR agonists such as AdipoRon are a promising therapeutic approach for the treatment of obesity-related diseases such as type 2 diabetes.[1]

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Adiponectin is an antidiabetic and antiatherogenic adipokine, which plays distinct roles in the balance of energy homoeostasis. As an insulin sensitizing hormone, adiponectin exerts multiple biological effects by the specific receptors (AdipoR1 and AdipoR2), through activation of AMP-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor (PPAR)α pathways. AdipoRon, an orally active synthetic small-molecule AdipoR agonist, shows very similar effects to adiponectin in vitro and in vivo, which could be a promising therapeutic approach for obesity-related disorders. In view of the regulatory effects of adiponectin or AdipoRon on inflammatory response and energy metabolism, they might be endowed a curative potential for tissue damage. Hence, its effects and possible mechanism were investigated. In vitro studies on hepatocytes (L02) and macrophages (RAW264.7) suggested a protective and anti-inflammatory potential of AdipoRon. The effects were verified in acute hepatic injury mice induced by d-galactosamine (D-GalN): hepatic lesions were restored by AdipoRon or bicyclol (positive reference drug) pretreatment, which were characterized by a significant increase in serological and hepatic biomarkers (AST, ALT, MDA and NOSs). Besides, AdipoRon attenuated the inflammation in the liver, characterized by the dwindling proinflammatory macrophage infiltration, as well as the shrinkage of tumor necrosis factor-α (TNF-α), transforming growth factor beta 1 (TGF-β1), interleukin-1 beta (IL-1β) and interleukin-6 (IL-6); meanwhile conversely promoted AMPK activation by phosphorylation. Combined with liver histopathology, these results demonstrated the hepatoprotective effects of AdipoRon against D-GalN-induced damage, which might be ascribed to the attenuation of inflammation, inhibition of free radical reactions, as well as enhancement of liver energy metabolism.[2]

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Adiponectin (APN) is a cardioprotective molecule. Its reduction in diabetes exacerbates myocardial ischemia/reperfusion (MI/R) injury. Although APN administration in animals attenuates MI/R injury, multiple factors limit its clinical application. The current study investigated whether AdipoRon, the first orally active molecule that binds APN receptors, may protect the heart against MI/R injury, and if so, to delineate the involved mechanisms. Wild-type (WT), APN knockout (APN-KO), and cardiomyocyte specific-AMPK dominant negative (AMPK-DN) mice were treated with vehicle or AdipoRon (50 mg/kg, 10 min prior to MI) and subjected to MI/R (30 min/3-24 h). Compared with vehicle, oral administration of AdipoRon to WT mice significantly improved cardiac function and attenuated postischemic cardiomyocyte apoptosis, determined by DNA ladder formation, TUNEL staining, and caspase-3 activation (all P < 0.01). MI/R-induced apoptotic cell death was significantly enhanced in mice deficient in either APN (APN-KO) or AMPK (AMPK-DN). In APN-KO mice, AdipoRon attenuated MI/R injury to the same degree as observed in WT mice. In AMPK-DN mice, AdipoRon's antiapoptotic action was partially inhibited but not lost. Finally, AdipoRon significantly attenuated postischemic oxidative stress, as evidenced by reduced NADPH oxidase expression and superoxide production. Collectively, these results demonstrate for the first time that AdipoRon, an orally active APN receptor activator, effectively attenuated postischemic cardiac injury, supporting APN receptor agonists as a promising novel therapeutic approach treating cardiovascular complications caused by obesity-related disorders such as type 2 diabetes.[3]

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Objective: AdipoRon, an adiponectin receptor agonist, was recently proposed for treating insulin resistance and hyperglycemia. As adiponectin is vasoprotective via NO-mediated signaling, it was hypothesized that adipoRon similarly exerts potentially beneficial vasodilator effects. We therefore examined if adipoRon induces vasorelaxation and via what contributing mechanisms. Methods: Vascular function was assessed in skeletal muscle arteries from rats and cerebral/coronary arteries from mice using pressure and wire myography. Results: Using qPCR, mRNA for adiponectin receptors was demonstrated in skeletal muscle, cerebral and coronary arteries. AdipoRon-caused vasorelaxation was not abolished by compound C (10 μM; AMPK inhibitor). Inhibition of endothelium-dependent relaxation with combinations of l-NAME/indomethacin/apamin/TRAM-34 only slightly reduced adipoRon-mediated vasorelaxation in cerebral and coronary arteries. EC-denuded cremaster arteries showed similar relaxant responses to adipoRon as in intact vessels, suggesting adipoRon directly impacts VSMCs. K(+) currents measured in VSMCs isolated from mouse basilar and LAD arteries were not altered by adiopRon. In cremaster arteries, adipoRon induced vasorelaxation without a marked decrease in VSMC [Ca(2+)]i . Adiponectin, itself, caused vasodilation in intact cremaster arteries while failing to cause significant dilation in EC-denuded arteries, consistent with endothelium dependency of adiponectin. Conclusions: AdipoRon exerts vasodilation by mechanisms distinct to adiponectin. The dominant mechanism for adipoRon-induced vasorelaxation occurs independently of endothelium-dependent relaxing factors, AMPK activation, K(+) efflux-mediated hyperpolarization and reductions in cytosolic [Ca(2+)]i .[4]

C27H28N2O3

428.52

428.209

C, 75.68; H, 6.59; N, 6.54; O, 11.20

924416-43-3

AdipoRon hydrochloride;1781835-20-8

16307093

Off-white to light yellow solid powder

1.2±0.1 g/cm3

645.3±55.0 °C at 760 mmHg

344.1±31.5 °C

0.0±1.9 mmHg at 25°C

1.632

4.14

1

4

8

32

582

0

SHHUPGSHGSNPDB-UHFFFAOYSA-N

InChI=1S/C27H28N2O3/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)

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

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 (5.83 mM) (saturation unknown) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
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: ≥ 2.08 mg/mL (4.85 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 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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Solubility in Formulation 3: ≥ 2.08 mg/mL (4.85 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 20.8 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: ≥ 2.08 mg/mL (4.85 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 20.8 mg/mL clear DMSO stock solution to 900 μL corn oil and mix evenly.

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