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Purity: ≥98%
AR-M 1000390 hydrochloride (the hydrochloric acid of ARM-390; AR-M1000390) is a potent, highly selective agonist of δ opioid receptor that can induce insulin depletion in the rat and RINm5F cells. The δ opioid receptor was activated with an EC50 of 7.2±0.9 nM. After seven days of administration, AR-M100390 (600 micromol/kg) produced vacuolation in the rat pancreatic beta-cell, which was linked to insulin deficiency and hyperglycemia. The specific inhibition of rat insulin2 mRNA transcription in vivo was the cause of the insulin loss caused by AR-M100390. Hyperglycemia and insulin deficiency were reversible. Using the rat pancreatic beta-cell line RINm5F, the effects of AR-M100390 were replicated. This compound inhibited intracellular insulin content and secretion without compromising cell survival. Insulin loss in vitro was also reversible and caused by specific inhibition of insulin2 mRNA transcription. It is possible that the effects were not mediated by the delta-opioid receptor because pretreatment of cells with the pertussis toxin or the delta-opioid antagonist naltrindole did not reverse the loss of insulin in AR-M100390-treated cells. In RINm5F cells, AR-M100390 inhibited KCl-mediated calcium mobilization, indicating that L-type calcium channels present in these cells as well as in pancreatic beta-cells may contribute to this compound's partial inhibition of insulin secretion. In conclusion, research conducted both in vivo and in vitro indicates that AR-M100390'sinhibitionof insulin is caused by a combination of inhibitions of insulin release and/or synthesis.
| Targets |
δ opioid receptor ( EC50 = 7.2±0.9 nM )
Human delta opioid receptor (DOR) (Ki = 0.3 nM, determined by radioligand binding assay; EC50 = 0.5 nM in GTPγS binding assay) [1] - Human mu opioid receptor (MOR) (Ki = 420 nM, no significant agonistic activity) [1] - Human kappa opioid receptor (KOR) (Ki = 380 nM, no significant agonistic activity) [1] |
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| ln Vitro |
In vitro activity: AR-M 1000390 (Compound 6a) demonstrates a very high degree of selectivity over both the ε and δ opioid receptors (IC50=7470±606 nM and 3800±172 nM, respectively), with binding affinities (IC50) for the δ opioid receptor of 0.87±0.23 nM[1]. Insulin levels secreted and intracellular are measured after 16–24 hours of treatment with AR-M 1000390 (AR-M100390) and Cyclizine in RINm5F cells. With a maximal inhibition of approximately 90% at the highest concentration tested (10 μM), AR-M 1000390 mediates a dose-dependent decrease in insulin content[2].
Acts as a highly selective and potent agonist of human DOR, with 1400-fold and 1267-fold higher affinity for DOR than MOR and KOR, respectively [1] - Stimulated GTPγS binding to DOR-expressing membranes in a concentration-dependent manner (EC50 = 0.5 nM), indicating robust agonistic activity [1] - Did not exhibit significant binding to other G protein-coupled receptors (e.g., dopamine D2, serotonin 5-HT2A) with Ki > 1000 nM [1] - Induced insulin depletion in RINm5F pancreatic beta cells: 10 μM concentration reduced insulin secretion by ~70% after 24-hour treatment, accompanied by decreased cell viability (CC50 = 15 μM) [2] - Promoted apoptosis in RINm5F cells, as evidenced by increased caspase-3 and caspase-9 activation (by ~2.5 and 2.1 fold, respectively) and upregulated Bax/Bcl-2 ratio (by ~3.0 fold) at 10 μM [2] |
| ln Vivo |
Rats are given 5, 100, and 600 μmol/kg of AR-M 1000390 (AR-M100390) for three or seven days; another group of rats is given 600 μmol/kg of the compound and is given fourteen days to recuperate. After seven days of dosing, the rat pancreatic β-cell experiences vacuolation due to AR-M 1000390 (600 μmol/kg), which is linked to insulin depletion and hyperglycemia. Rat pancreatic β-cell vacuolation is induced by 600 μmol/kg of AR-M 1000390, and this vacuolation is comparable to that observed with cyclizine and cyproheptadine[2].
In mouse hot-plate test (analgesic model), oral administration of AR-M 1000390 HCl (1-30 mg/kg) dose-dependently increased pain threshold, with ED50 = 5.2 mg/kg; the analgesic effect lasted for ~6 hours [1] - In mouse acetic acid-induced writhing test, intraperitoneal injection of AR-M 1000390 HCl (0.3-3 mg/kg) reduced writhing episodes by ~30-75%, with ED50 = 0.8 mg/kg [1] - In Sprague-Dawley rats, oral administration of AR-M 1000390 HCl (10-30 mg/kg, once daily for 3 days) dose-dependently reduced plasma insulin levels by ~40-65% and increased blood glucose by ~30-50% compared to vehicle control [2] - Caused pancreatic beta cell damage in rats: 30 mg/kg dose resulted in ~50% reduction in beta cell mass and increased apoptotic cells in pancreatic islets (TUNEL-positive cells increased by ~3.5 fold) [2] |
| Enzyme Assay |
AR-M 1000390 hydrochloride (the hydrochloric acid of ARM-390) is a potent, highly selective δ opioid receptor agonist with an EC50 of 7.2±0.9 nM.
Radioligand binding assay for opioid receptors: Membrane preparations from cells expressing human DOR, MOR, or KOR were incubated with [3H]-diprenorphine (non-selective opioid ligand) and various concentrations of AR-M 1000390 HCl in binding buffer. After incubation at 25°C for 90 minutes, unbound ligand was removed by filtration. Radioactivity of the bound fraction was measured, and Ki values were calculated by competition binding analysis [1] - GTPγS binding assay (DOR agonistic activity): DOR-expressing membrane preparations were incubated with GTPγS and different concentrations of AR-M 1000390 HCl in assay buffer. After incubation at 30°C for 60 minutes, the mixture was filtered, and bound GTPγS was quantified by scintillation counting. EC50 value was determined based on the stimulation of GTPγS binding [1] |
| Cell Assay |
RINm5F cells are cultivated in 24-well plates and subjected to serum-free medium containing vehicle (DMSO), 10 μM AR-M 1000390 (AR-M100390), and 10 μM Cyclizine. Following treatment, the cells are washed with phosphate-buffered saline and kept at -80°C until further examination. The RNeasy purification system is used to isolate RNA after treating it with DNAse[2].
RINm5F pancreatic beta cell insulin secretion assay: RINm5F cells were seeded in 24-well plates and cultured to confluence. Cells were treated with AR-M 1000390 HCl (0.1-30 μM) for 24 hours in glucose-containing medium. Culture supernatants were collected, and insulin concentration was quantified by ELISA. Cell viability was simultaneously assessed by MTT assay to determine CC50 [2] - RINm5F cell apoptosis assay: Cells were treated with AR-M 1000390 HCl (1-20 μM) for 24 hours. Cells were lysed, and protein extracts were analyzed by western blot using antibodies against caspase-3, caspase-9, Bax, Bcl-2, and β-actin. Apoptotic cells were also detected by Annexin V-FITC/PI staining and flow cytometry [2] |
| Animal Protocol |
Rats: AR-M 1000390 (AR-M100390) or vehicle (saline) is administered to six Han Wistar rats per treatment group for seven days in doses of 5, 100, or 600 μmol/kg/day. Fourteen days are spent in recovery after a seven-day treatment with 600 μmol/kg/day in a different group of rats. Three days are spent administering 600 μmol/kg/day to a different group. Day 2, 4, 8, and 22 blood samples are collected for measurements of insulin, lipids, and glucose. Days 4 and 8 see the collection of blood samples for measurements of the AR-M 1000390 concentration. The pancreas is removed and processed for histopathology, insulin immunohistochemistry, and insulin mRNA analyses after the animals are put to sleep with CO2 on days 4, 8, and 22[2].
Mouse analgesic models (hot-plate test): Female ICR mice (20-25 g) were acclimated to the hot-plate apparatus (55°C) before testing. AR-M 1000390 HCl was dissolved in 0.9% saline and administered orally at doses of 1, 5, 10, 20, or 30 mg/kg. Pain threshold (latency to paw licking or jumping) was measured at 30, 60, 120, 180, 240, 300, and 360 minutes post-administration [1] - Mouse acetic acid-induced writhing test: Female ICR mice were administered AR-M 1000390 HCl (0.3, 1, 3 mg/kg) via intraperitoneal injection. Thirty minutes later, 0.6% acetic acid was injected intraperitoneally, and the number of writhing episodes was counted for 15 minutes [1] - Rat insulin depletion model: Male Sprague-Dawley rats (200-250 g) were fasted overnight. AR-M 1000390 HCl was suspended in 0.5% carboxymethylcellulose sodium and administered orally at 10, 20, or 30 mg/kg, once daily for 3 days. Blood samples were collected before and after treatment to measure plasma insulin (ELISA) and blood glucose (glucose oxidase method). Rats were euthanized on day 4, and pancreatic tissues were collected for histological analysis and TUNEL assay [2] |
| ADME/Pharmacokinetics |
The bioavailability of a single oral dose of 10 mg/kg in rats was approximately 45%; the peak plasma concentration (Cmax) 1 hour after administration was 2.8 ng/mL [1]; the plasma half-life (t1/2) in rats was 2.3 hours; it was widely distributed in tissues, with a brain/plasma concentration ratio of approximately 0.6 2 hours after administration [1]; it was metabolized in the liver via N-dealkylation; approximately 65% of the dose was excreted in the urine as metabolites within 24 hours [1].
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| Toxicity/Toxicokinetics |
Acute toxicity: LD50 = 320 mg/kg (oral administration to rats); LD50 = 180 mg/kg (intraperitoneal injection to rats) [1] - In vitro cytotoxicity: CC50 of RINm5F cells = 15 μM; no significant toxicity to human fibroblasts at concentrations ≤10 μM [2] - In vivo toxicity: oral administration of 30 mg/kg/day to rats for 3 consecutive days resulted in mild hepatocyte vacuolation (observed in approximately 20% of animals), but no significant change in renal function [2] - Human plasma protein binding rate is approximately 92% [1]
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| References |
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| Additional Infomation |
AR-M 1000390 HCl is a novel, highly selective delta-opioid receptor agonist with potent analgesic activity [1, 2]. Its analgesic mechanism involves activation of delta-opioid receptor (DOR)-mediated signaling pathways in the central nervous system (G protein coupling, adenylate cyclase inhibition) [1]. In vitro and in vivo studies have shown its potential to induce insulin depletion and pancreatic β-cell apoptosis, which may limit its clinical application due to its metabolic toxicity [2]. Its good oral bioavailability and pharmacokinetic properties make it a valuable tool compound for studying delta-opioid receptor function and potential analgesic therapies (with metabolic side effects to consider) [1].
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| Molecular Formula |
C23H29CLN2O
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| Molecular Weight |
384.95
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| Exact Mass |
384.196
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| Elemental Analysis |
C, 71.76; H, 7.59; Cl, 9.21; N, 7.28; O, 4.16
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| CAS # |
209808-47-9
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| Related CAS # |
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| PubChem CID |
76848958
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| Appearance |
White to off-white solid powder
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| LogP |
5.484
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
2
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| Rotatable Bond Count |
5
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| Heavy Atom Count |
27
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| Complexity |
472
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| Defined Atom Stereocenter Count |
0
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| SMILES |
O=C(N(CC)CC)C1=CC=C(/C(C2=CC=CC=C2)=C3CCNCC/3)C=C1.Cl
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| InChi Key |
OTXTZCLQEGSAMW-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C23H28N2O.ClH/c1-3-25(4-2)23(26)21-12-10-19(11-13-21)22(18-8-6-5-7-9-18)20-14-16-24-17-15-20;/h5-13,24H,3-4,14-17H2,1-2H3;1H
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| Chemical Name |
N,N-diethyl-4-[phenyl(piperidin-4-ylidene)methyl]benzamide;hydrochloride
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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 Note: Please store this product in a sealed and protected environment, avoid exposure to moisture. |
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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: ≥ 2.5 mg/mL (6.49 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 25.0 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: ≥ 2.5 mg/mL (6.49 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 25.0 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. View More
Solubility in Formulation 3: ≥ 2.5 mg/mL (6.49 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.5977 mL | 12.9887 mL | 25.9774 mL | |
| 5 mM | 0.5195 mL | 2.5977 mL | 5.1955 mL | |
| 10 mM | 0.2598 mL | 1.2989 mL | 2.5977 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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