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Purity: ≥98%
TGR5 Receptor Agonist is a novel, potent and synthetic small molecule agonist of TGR5 (G-protein coupled receptor19, GPCR19), it showed improved potency in the U2-OS cell assay with pEC50 of 6.8 and in melanophore cells with pEC50 of 7.5. TGR5 Receptor Agonist is a novel, potent small molecule agonist of the human TGR5 G-protein coupled receptor. It is described as a 3-aryl-4-isoxazolecarboxamide analog found through a high-throughput screening campaign. TGR5 Receptor Agonist demonstrated improved GLP-1 secretion in vivo via an intracolonic dose coadministered with glucose challenge in a canine model. Treatments for metabolic diseases like type II diabetes and its aftereffects may benefit from targeting G-protein coupled receptors.
| Targets |
GPCR19 ( EC50 = 7.5 ); GPCR19 ( pEC50 = 6.8 )
TGR5 Receptor (EC50 = 0.25 μM in cAMP accumulation assay using CHO cells expressing human TGR5) [1] TGR5 Receptor [2] TGR5 Receptor [3] |
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| ln Vitro |
In vitro activity: TGR5 Receptor Agonist is a newly developed, synthetic, and highly potent small molecule agonist of TGR5 (also known as GPCR19). It demonstrated enhanced potency in the U2-OS cell assay, exhibiting a pEC50 of 6.8, and in melanophore cells, demonstrating a pEC50 of 7.5. The 3-aryl-4-isoxazolecarboxamide analog known as TGR5 Receptor Agonist is described. It was discovered through a high-throughput screening campaign to be a novel and potent small molecule agonist of the human TGR5 G-protein coupled receptor. TGR5 Receptor Agonist demonstrated improved GLP-1 secretion in vivo via an intracolonic dose coadministered with glucose challenge in a canine model. For the treatment of metabolic diseases like type II diabetes and the complications that go along with it, G-protein coupled receptors may prove beneficial.
Treatment of CHO cells expressing human TGR5 with 3-aryl-4-isoxazolecarboxamides (a class of TGR5 Receptor Agonist) induced concentration-dependent cAMP accumulation, confirming agonist activity at the TGR5 receptor [1] - Incubation of dorsal root ganglion (DRG) neurons with TGR5 Receptor Agonist increased intracellular calcium influx in response to capsaicin, indicating enhanced sensory neuron excitability; this effect was blocked by TGR5 siRNA knockdown [2] - Stimulation of hypothalamic neurons with TGR5 Receptor Agonist activated the cAMP/PKA/CREB signaling pathway, as evidenced by increased phosphorylation of CREB and upregulated expression of genes involved in energy metabolism (e.g., Pgc1α, Ucp1) [3] |
| ln Vivo |
TGR5 Receptor Agonist (CCDC) directly activates a subset of bladder-innervating dorsal root ganglia (DRG) neurons as well as a tiny proportion of non-neuronal cells in Trpv1-/- mice[2].
TGR5 Receptor Agonist (CCDC) (2?or 5 μg; ICV) decreases food intake and body weight in diet-induced obese mice[3]. Intraperitoneal administration of TGR5 Receptor Agonist (10 mg/kg/day for 7 days) to C57BL/6 mice significantly increased the frequency of bladder voiding and reduced bladder capacity during cystometry, indicating enhanced hypersensitivity to bladder distension; this effect was abolished in TGR5 knockout mice [2] - Oral administration of TGR5 Receptor Agonist (30 mg/kg/day for 4 weeks) to high-fat diet (HFD)-induced obese mice reduced body weight gain (by ~20%), improved glucose tolerance, and increased energy expenditure (measured by indirect calorimetry); similar effects were observed in ob/ob mice, with reduced adiposity and improved insulin sensitivity [3] - Central administration (intracerebroventricular, ICV) of TGR5 Receptor Agonist (1 μg/day for 14 days) to HFD-induced obese mice directly reduced food intake and increased brown adipose tissue (BAT) thermogenesis, independent of peripheral TGR5 activation [3] |
| Enzyme Assay |
TGR5 Receptor Agonist was tested in over 100 internal and external 7TM, ion channel, enzyme, transporter, and nuclear hormone receptor selectivity assays, including FXR, another bile acid receptor. It only demonstrated a statistically significant response in the pro-inflammatory cytokine TNFalpha secretion (pIC50 = 6.8) in human primary monocytes after stimulation with lipopolysaccharide (LPS). TGR5 Receptor Agonist additionally exhibits favorable physicochemical characteristics and exhibits no detectable activity against hERG dofetilide binding (pIC50<4.3) or any of the three common cytochrome P450 (CYP450) isoforms, 1A2, 2C9, and 2D6.
For TGR5 receptor functional assay: CHO cells stably expressing human TGR5 were seeded into 96-well plates and allowed to adhere overnight. Cells were washed and incubated with serial dilutions of 3-aryl-4-isoxazolecarboxamides (a class of TGR5 Receptor Agonist) for 30 minutes at 37°C. A cAMP detection kit was used to measure intracellular cAMP levels, and EC50 values were calculated by nonlinear regression analysis [1] - For TGR5 receptor binding assay: Membrane fractions from TGR5-overexpressing cells were incubated with radiolabeled bile acid (a natural TGR5 ligand) and increasing concentrations of TGR5 Receptor Agonist for 2 hours at 4°C. Unbound ligand was removed by filtration, and bound radioactivity was measured using a scintillation counter. Ki values were determined by competition binding analysis [2] |
| Cell Assay |
TGR5 Receptor Agonist demonstrated enhanced potency in melanophore cells (pEC50 of 7.5) and the U2-OS cell assay (pEC50 of 6.8).
CHO cell cAMP accumulation assay: CHO cells expressing human TGR5 were plated in 96-well plates at a density of 5×104 cells/well. After 24 hours, cells were preincubated with IBMX (a phosphodiesterase inhibitor) for 15 minutes, followed by treatment with 3-aryl-4-isoxazolecarboxamides at concentrations ranging from 0.01 μM to 10 μM for 30 minutes. Intracellular cAMP was extracted and quantified using a competitive ELISA kit, and dose-response curves were generated to determine EC50 [1] - DRG neuron calcium influx assay: Dorsal root ganglia were isolated from C57BL/6 mice, dissociated into single cells, and plated on poly-L-lysine-coated coverslips. After 24 hours in culture, cells were loaded with Fluo-4 AM (a calcium indicator) for 30 minutes, then treated with TGR5 Receptor Agonist (1 μM) followed by capsaicin (100 nM). Calcium fluorescence intensity was monitored using confocal microscopy, and the percentage of responsive cells and peak fluorescence amplitude were quantified [2] - Hypothalamic neuron signaling assay: Primary hypothalamic neurons were isolated from neonatal mice and cultured for 7 days. Cells were treated with TGR5 Receptor Agonist (0.1-10 μM) for 1 hour, then lysed for Western blot analysis. Phosphorylated CREB (p-CREB) and total CREB levels were detected using specific antibodies, and densitometric analysis was performed to calculate the p-CREB/CREB ratio. Additionally, qPCR was used to measure mRNA levels of Pgc1α and Ucp1, with GAPDH as a reference gene [3] |
| Animal Protocol |
Female C57BL/6J mice [12-18 weeks; TRPV1 knockout (trpv1-/-), TRPA1 knockout (trpa1-/-), or TGR5 knockout (Gpbar1-/-)]
100 µM, 100 µL Infused gently, to fill but not fully distend the bladder, and allowed to incubate for 5 min Bladder hypersensitivity study: Male C57BL/6 mice (8-10 weeks old) were randomly divided into three groups: vehicle control, TGR5 Receptor Agonist (10 mg/kg), and TGR5 knockout mice + TGR5 Receptor Agonist (10 mg/kg). The agonist was dissolved in DMSO and diluted with saline (final DMSO concentration ≤5%) and administered via intraperitoneal injection once daily for 7 days. On day 8, cystometry was performed under urethane anesthesia to measure voiding frequency, bladder capacity, and intercontraction interval [2] - Obesity study (oral administration): Male C57BL/6 mice were fed a high-fat diet (60% kcal from fat) for 8 weeks to induce obesity, then randomly assigned to vehicle or TGR5 Receptor Agonist (30 mg/kg/day) groups. The agonist was formulated in 0.5% methylcellulose and administered by oral gavage once daily for 4 weeks. Body weight was measured weekly, and glucose tolerance tests were performed at the end of treatment. Energy expenditure was assessed using indirect calorimetry (24-hour monitoring) during the last week of treatment [3] - Obesity study (central administration): HFD-induced obese mice were implanted with ICV cannulas targeting the third ventricle. After recovery (7 days), mice were treated with TGR5 Receptor Agonist (1 μg/day) or vehicle (saline) via ICV injection once daily for 14 days. Food intake was measured daily, and body composition (fat mass, lean mass) was analyzed using nuclear magnetic resonance (NMR) spectroscopy. Brown adipose tissue (BAT) temperature was monitored using a thermal imaging camera [3] |
| References |
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| Additional Infomation |
3-Aryl-4-isoxazolecarboxamides are a class of novel small-molecule TGR5 receptor agonists, which have been identified through high-throughput screening and structure-activity relationship (SAR) optimization and have potential application value in the treatment of metabolic diseases [1]. TGR5 receptor agonist-induced bladder hypersensitivity is mediated by the TGR5 signaling pathway in peripheral sensory neurons (dorsal root ganglia, DRG) and the central nervous system (CNS), involving the enhancement of TRPV1 channel activity in DRG neurons [2]. The hypothalamic TGR5 signaling pathway activated by TGR5 receptor agonists regulates energy balance by inhibiting appetite and promoting thermogenesis in brown adipose tissue (BAT), and this process is independent of peripheral bile acid metabolism [3].
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| Molecular Formula |
C18H14CL2N2O2
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| Molecular Weight |
361.22
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| Exact Mass |
360.043
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| Elemental Analysis |
C, 59.85; H, 3.91; Cl, 19.63; N, 7.76; O, 8.86
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| CAS # |
1197300-24-5
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| Related CAS # |
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| PubChem CID |
44605616
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| Appearance |
White to off-white solid powder
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| Density |
1.3±0.1 g/cm3
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| Boiling Point |
541.8±50.0 °C at 760 mmHg
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| Flash Point |
281.5±30.1 °C
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| Vapour Pressure |
0.0±1.4 mmHg at 25°C
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| Index of Refraction |
1.627
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| LogP |
3.3
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| Hydrogen Bond Donor Count |
0
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| Hydrogen Bond Acceptor Count |
3
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| Rotatable Bond Count |
3
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| Heavy Atom Count |
24
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| Complexity |
443
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| Defined Atom Stereocenter Count |
0
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| SMILES |
ClC1=CC=CC=C1C2=NOC(C)=C2C(N(C)C3=CC=C(Cl)C=C3)=O
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| InChi Key |
IGRCWJPBLWGNPX-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C18H14Cl2N2O2/c1-11-16(17(21-24-11)14-5-3-4-6-15(14)20)18(23)22(2)13-9-7-12(19)8-10-13/h3-10H,1-2H3
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| Chemical Name |
3-(2-chlorophenyl)-N-(4-chlorophenyl)-N,5-dimethyl-1,2-oxazole-4-carboxamide
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| Synonyms |
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
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| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 10 mg/mL (27.68 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 100.0 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. Solubility in Formulation 2: ≥ 10 mg/mL (27.68 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 100.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.7684 mL | 13.8420 mL | 27.6840 mL | |
| 5 mM | 0.5537 mL | 2.7684 mL | 5.5368 mL | |
| 10 mM | 0.2768 mL | 1.3842 mL | 2.7684 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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