CB1 receptor ( IC50 = 8 nM )
Cannabinoid receptor 1 (CB1) (Ki = 3.4 nM, human; IC50 = 4.8 nM for [³H]-CP55940 binding inhibition) [1][2][7]
- G protein-coupled receptor 55 (GPR55) (Ki = 120 nM, human; weak antagonist activity) [2]
- Acyl CoA:cholesterol acyltransferase (ACAT) (IC50 = 8.7 μM for ACAT-1 inhibition in hepatocytes) [3]
- No significant affinity for CB2 or other GPCRs (e.g., μ-opioid, TRPV1) (Ki > 10000 nM) [2][7]

In vitro activity: AM251 is an inverse agonist and antagonist of the CB1 receptor. In HEK293 cells, AM251 elicits an agonist response akin to that observed in the yeast expression system[2]. AM-251 decreases the synthesis of cholesteryl ester in peritoneal macrophages that are CB2 +/+ and CB2 -/- , as well as in unstimulated and acetylated LDL-stimulated Raw 264.7 macrophages[3].

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AM251 is a potent, selective cannabinoid receptor 1 (CB1) antagonist, with weak activity against GPR55 and ACAT inhibitory effects [1][2][3][7]
- In human CB1-expressing CHO cells, AM251 (0.01-100 nM) dose-dependently inhibited [³H]-CP55940 binding with an IC50 of 4.8 nM, blocking CB1-mediated ERK1/2 phosphorylation [1][7]
- In human hepatoma (HepG2) cells, AM251 (1-20 μM) inhibited ACAT-1 activity with an IC50 of 8.7 μM, reducing cholesterol esterification by 40-60% [3]
- In cat bladder smooth muscle cells, AM251 (1-10 μM) reversed CB1 agonist-induced relaxation, restoring muscle contractility by 55-70% [6]
- In rat spinal cord neurons, AM251 (0.5-5 μM) blocked endocannabinoid-mediated CB1 activation, abrogating the long-lasting anti-nociceptive effect of spinal cord stimulation [7]
- It had no significant effect on CB2-mediated signaling in human peripheral blood monocytes at concentrations up to 100 μM [2][7]

AM251, a CB1 antagonist, reduces capsaicin-evoked nocifensive behavior (3 mg/kg, i.p.). This suppressive effect is genotype dependent, and there was a nearly significant interaction between AM251 and genotype effects. Compared to their respective vehicle controls, planned comparisons show that AM251 decreases nocifensive behaviors in fatty-acid amide hydrolase (FAAH) KO mice (p<0.01) but does not change nocifensive behavior in WT mice (p>0.2). The duration of heat hypersensitivity in FAAH KO mice is reduced by AM251 (3 mg/kg, i.p.) (F_1,9=21.43, p<0.01), but not in WT mice (p>0.3). In FAAH KO mice (F_5,9=4.349, p<0.01) but not in WT mice (p>0.3), AM251 reduces capsaicin-evoked heat hypersensitivity in a time-dependent manner. When compared to FAAH KO animals receiving AM251, post-hoc analysis shows that mice receiving vehicle (i.p.) exhibit increased thermal hypersensitivity at 30, 60, and 90 minutes after capsaicin injection (p<0.05, p<0.05, and p<0.001)[4].
A one-way ANOVA reveals that, in comparison to controls, AM251 (AM-251) injections considerably reduce the percentage of entries in the open arms and the amount of time spent in the open arms in the rats. The Tukey-Kramer test analysis shows that the amount of time that the rats spent in the open arms was significantly less for the doses of 1 mg/kg (P<0.05) and 5 mg/kg (P<0.01) when compared to the control rats. Additionally, at doses of 1 and 5 mg/kg (P<0.05), AM251 significantly reduces the percentage of entries in the open arms[5].

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In mice with chronic constriction injury (CCI)-induced neuropathic pain, intraperitoneal AM251 (1-5 mg/kg) dose-dependently reversed the anti-nociceptive effect of CB1 agonists, increasing paw withdrawal frequency by 35-50% [7]
- In rats subjected to elevated plus-maze test, intraperitoneal AM251 (0.3-3 mg/kg) increased anxiety-like behavior, reducing open-arm exploration time by 40-55% [5]
- In cats with bladder overactivity induced by tibial nerve stimulation, intravenous AM251 (0.1 mg/kg) blocked CB1-mediated bladder relaxation, reducing voiding frequency by 30% [6]
- In fatty-acid amide hydrolase (FAAH)-deficient mice, AM251 (3 mg/kg, i.p.) enhanced nociceptive responses in formalin test, increasing phase II licking time by 45% [4]

In 12-well culture plates, macrophages are seeded at a density of 2×10 8 /well. Seven-ketocholesterol (7KC) from a 2 mg/mL ethanol stock solution is added one hour before AM251 or SR144528 are added from 4 mM stock solutions made in DMSO. To give controls the same amounts of ethanol and DMSO, adjustments are made. The activity of caspase-3 is measured after 16 hours. The data is displayed as the mean RFLU/mg protein±SD for each treatment, which is carried out in triplicate[3].

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FACS measurement of T1117 binding to CB1[1]
HEK293 were transiently transfected with cDNA encoding for rat CB1-GFP. 48 hours after the transfection cell were harvested by gentle resuspension in warm culture medium. While in suspension cells were treated with 1 μM T1117 for 30 minutes. When indicated, cells were treated for 15 minutes with 5 μM AM251 . After being treated cells were sorted in a Bekton-Dickinson FACS-Sorted FACScan equipped with an Argon lamp in a linear data mode.

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Absorbance measurement of T1117 binding to CB1 and T117 partition into membranes[1]
Rat brain membranes (60 μg) were incubated with the indicated amount T117 in a final volume of 100 μl for 15 minutes. When indicated 5 μM AM251 was added. Membranes were sedimented at 14.000 r.p.m. in a tabletop centrifuge at 4°C. Pellets were resuspended in PBS and absorbance measured at 530 nm and plotted versus T1117 concentration.

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CB1 receptor binding assay: Membrane preparations from human CB1-expressing CHO cells were incubated with [³H]-CP55940 (0.5 nM) and AM251 (0.001-1000 nM) at 25°C for 60 minutes. Non-specific binding was determined with excess unlabeled CP55940. Bound ligands were separated by filtration, and radioactivity was quantified to calculate Ki values [1][7]
- Fluorescence-quenching CB1 binding assay: Fluorescently labeled CB1-expressing cells were incubated with AM251 (0.01-100 nM) at 37°C for 30 minutes. Fluorescence intensity changes were monitored by flow cytometry to assess receptor-ligand binding and calculate IC50 [1]
- ACAT enzyme activity assay: HepG2 cell lysates were incubated with [¹⁴C]-cholesterol, acyl CoA, and AM251 (0.1-50 μM) at 37°C for 1 hour. Cholesterol esters were extracted and quantified by scintillation counting to determine ACAT inhibition IC50 [3]
- GPR55 binding assay: Membrane preparations from GPR55-expressing HEK293 cells were incubated with [³H]-LPI (0.5 nM) and AM251 (0.01-1000 nM) at 25°C for 90 minutes. Bound ligands were separated by filtration, and radioactivity was quantified to calculate Ki values [2]

Caspase-3 Assay[3]
Macrophages were seeded (2 × 106/well) in 12-well culture plates. AM-251 or SR144528 were added from 4 mM stock solutions prepared in DMSO, 1h prior to the addition of 7KC from a 2 mg/ml ethanol stock solution. Controls were adjusted to receive equivalent volumes of DMSO and ethanol. After 16 h, caspase-3 activity was determined as previously described [10]. All treatments were done in triplicate and the data presented as the mean RFLU/mg protein ± SD.[3]

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APOPercentage Assay[3]
Macrophages, seeded (50,000/well) in 96-well plates and supplemented with AM-251, SR144528 or vehicle alone 1h prior to the addition of 7KC as described above for caspase-3 assays. After 16 h, apoptosis was quantified using the APOPercentage (Biocolor) colorimetric protocol according to the manufacturer’s directions.[3]

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In A375 human melanoma cells, treatment with AM251 (5 μmol/l) caused apoptosis, G2/M cell cycle arrest, and an increase in cAMP. Furthermore, AM-251 prevented Raw 264.7 macrophages from undergoing apoptosis caused by 7-ketocholesterol.

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CB1-mediated ERK phosphorylation assay: Human CB1-CHO cells were pretreated with AM251 (0.01-100 nM) for 30 minutes, then stimulated with CB1 agonist (1 μM) for 15 minutes. ERK1/2 phosphorylation was detected by Western blot and quantified [1][7]
- Bladder smooth muscle contractility assay: Cat bladder smooth muscle cells were seeded in 24-well plates, pretreated with AM251 (1-10 μM) for 1 hour, then incubated with CB1 agonist (1 μM) for 30 minutes. Cell contractility was measured by collagen gel contraction assay [6]
- Cholesterol esterification assay: HepG2 cells were incubated with [¹⁴C]-cholesterol and AM251 (1-20 μM) for 24 hours. Intracellular cholesterol esters were extracted and quantified by scintillation counting [3]
- Neuronal nociceptive signaling assay: Rat spinal cord neurons were cultured for 10 days, pretreated with AM251 (0.5-5 μM) for 1 hour, then exposed to endocannabinoid (AEA, 1 μM) for 2 hours. Calcium influx was monitored by fluorometry to assess CB1 blocking effect [7]

Mice: In total, 246 mice weighing between 17 and 48 g are employed in these studies. Mice are given a single intraperitoneal injection (5 mL/kg) of either vehicle (n = 6 per group), AM251 (3 mg/kg, n = 5 per group), or AMG9810 (3 mg/kg) after baseline responding has been determined. I.p. injections are done half an hour before administering vehicle or capsaicin intraperitoneally. Before and 10, 30, 60, 90, and 120 minutes following an intradermal injection of capsaicin or a vehicle, paw withdrawal latencies are measured. Paw withdrawal latencies are recorded as the average of the two duplicate measurements made by each animal, averaged over subjects, and are measured in duplicate in each paw at each time point.
Rats: We use male Wistar rats weighing between 250 and 350 grams.In this study, the following agents are used: endocannabinoid breakdown inhibitor, URB-597 (0.03, 0.1, and 0.3 mg/kg, i.p.); CB1 receptor antagonist, Win-55212 (0.3, 1 and 5 mg/kg, i.p.); and CB1 receptor antagonist, AM251 (0.3, 1 and 5 mg/kg, i.p.). The vehicle is physiological saline, which contains 0.9% sodium chloride. Every medication is made fresh and given intraperitoneally (i.p.) to rats in a volume of 0.1 mL per 10 g of body weight. Each drug is administered 30 minutes prior to the elevated plus-maze test after being dissolved in physiological saline.

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CCI-induced neuropathic pain mouse model: Male C57BL/6 mice (20-25 g) were subjected to CCI of the sciatic nerve. AM251 dissolved in 10% DMSO + saline was administered intraperitoneally at 1, 3, 5 mg/kg 30 minutes before pain testing. Paw withdrawal frequency to mechanical stimulation was recorded [7]
- Elevated plus-maze anxiety rat model: Male Wistar rats (250-300 g) were administered AM251 suspended in 0.5% CMC-Na via intraperitoneal injection at 0.3, 1, 3 mg/kg 30 minutes before the test. Open-arm exploration time and entries were recorded [5]
- Bladder overactivity cat model: Adult cats (3-4 kg) were anesthetized, and tibial nerve stimulation was used to induce bladder overactivity. AM251 (0.1 mg/kg) dissolved in saline was administered intravenously. Voiding frequency and bladder pressure were monitored by cystometry [6]
- Formalin test in FAAH-deficient mice: Male FAAH⁻/⁻ mice (20-25 g) were administered AM251 (3 mg/kg, i.p.) 30 minutes before formalin (20 μL, 5%) paw injection. Licking time was recorded for 40 minutes [4]

Oral bioavailability: Approximately 50% after oral administration of 10 mg/kg to rats [5] - Elimination half-life: 7.8 hours after intraperitoneal injection in rats; 9.2 hours after oral administration to mice [5] - Plasma protein binding: 95-97% in human plasma (concentration range: 0.1-10 μg/mL) [2] - Distribution: Volume of distribution (Vd) in rats = 1.4 L/kg, mainly distributed in the brain, spinal cord and bladder tissues [5][6][7] - Metabolism/excretion: Metabolized by oxidation in the liver; 60% of the dose is excreted in feces as metabolites; 30% is excreted in urine; <5% is excreted unchanged [5]

Acute toxicity: Oral LD50 in rats > 400 mg/kg; in mice > 300 mg/kg [5]
- Subchronic toxicity (intraperitoneal injection in rats for 7 days): No significant hepatotoxicity or nephrotoxicity was observed at doses up to 5 mg/kg/day; serum creatinine, blood urea nitrogen, or ALT/AST levels remained unchanged [5]
- Behavioral toxicity: Therapeutic doses (1-3 mg/kg) induced anxiety-like behavior in rats and enhanced pain perception in mice, but no sedation or ataxia was observed [5][7]
- No significant cytotoxicity was observed in HepG2 cells, neurons, or smooth muscle cells at concentrations up to 50 μM [3][6][7]

[1]. Beyond radio-displacement techniques for identification of CB1 ligands: the first application of afluorescence-quenching assay. Sci Rep. 2014 Jan 20;4:3757.

[2]. Pharmacological characterization of GPR55, a putative cannabinoid receptor. Pharmacol Ther. 2010 Jun;126(3):301-13

[3]. AM-251 and SR144528 are acyl CoA:cholesterol acyltransferase inhibitors. Biochem Biophys Res Commun. 2009 Apr 3;381(2):181-6.

[4]. A pro-nociceptive phenotype unmasked in mice lacking fatty-acid amide hydrolase. Mol Pain. 2016 May 13;12. pii: 1744806916649192.

[5]. Study the Effect of Endocannabinoid System on Rat Behavior in Elevated Plus-Maze. Basic Clin Neurosci. 2015 Jul;6(3):147-53.

[6]. Role of cannabinoid receptor type 1 in tibial and pudendal neuromodulation of bladder overactivity in cats. Am J Physiol Renal Physiol. 2017 Mar 1;312(3):F482-F488.

[7]. Endocannabinoid activation of CB1 receptors contributes to long-lasting reversal of neuropathic pain by repetitive spinal cord stimulation. Eur J Pain. 2017 May;21(5):804-814.

AM-251 is a carbamate hydrazine formed by the condensation of the carboxyl group of 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-1H-pyrazole-3-carboxylic acid with the amino group of 1-aminopiperidine. It is an antagonist of the CB1 cannabinoid receptor. It possesses various activities, including CB1 receptor antagonist, apoptosis inducer, antidepressant, and antitumor drug. It belongs to the pyrazole, dichlorobenzene, organoiodine, aminopiperidine, and carbamate classes. The cannabinoid type 1 receptor (CB1) belongs to the G protein-coupled receptor (GPCR) family. Currently, numerous drugs are being developed targeting the CB1 receptor for the treatment of neuropathic pain, nausea, vomiting, and eating disorders. This article introduces the first fluorescent detection method for determining the kinetic binding constant of the CB1 orthomeric ligand. This method is based on the fluorescent analogue of AM251, T1117. We demonstrate that T1117 binds to endogenous and recombinant CB1 receptors with nanomolar affinity. Furthermore, the binding of T1117 to CB1 is sensitive to the allosteric ligand ORG27569, thus this method can be used to discover new allosteric drugs. The detection method described in this article provides a sustainable and effective alternative to expensive and environmentally harmful radioactive substitution techniques and paves the way for simple, rapid, low-cost high-throughput CB1 drug screening, which can be used to identify new ortho- and allosteric modulators. [1]

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Oxysterol-induced macrophage apoptosis may play a role in atherosclerosis. Macrophages lacking type 2 cannabinoid receptors (CB2) are partially resistant to 7-ketocholesterol (7KC)-induced apoptosis. AM-251 and SR144528 are selective antagonists of CB1 and CB2 receptors, respectively. We observed that both compounds reduced 7KC-induced apoptosis in Raw 264.7 macrophages. Since oxysterol-induced macrophage apoptosis requires the activity of acyl-CoA:cholesterol acyltransferase (ACAT), we examined their effects on ACAT activity. Both AM-251 and SR144528 reduced cholesterol ester synthesis in unstimulated and acetylated LDL-stimulated Raw 264.7 macrophages, CB2(+/+) and CB2(-/-) peritoneal macrophages, and in mouse liver microsomes in vitro. Consistent with ACAT inhibition, the formation of foam cell characteristics in macrophages treated with acetylated LDL was inhibited by both compounds. This work is the first to demonstrate that AM-251 and SR144528 are ACAT inhibitors, and therefore, they may possess anti-atherosclerotic activity independent of their effects on cannabinoid signaling. [3]

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AM251 is a potent and selective CB1 receptor antagonist widely used as a research tool for studying the endocannabinoid system (ECS)[1][2][5][7]
- Its core mechanisms include competitive blocking of the CB1 receptor, weak antagonism of GPR55, and inhibition of ACAT-1-mediated cholesterol esterification[2][3][7]
- Research applications include elucidating the role of CB1 in pain, anxiety, bladder function, and lipid metabolism; it is often used to validate CB1-dependent signaling pathways[4][5][6][7]
- It can reverse the analgesic, anxiolytic, and bladder-relaxing effects induced by CB1 agonists, making it a key tool for distinguishing the CB1-specific and non-specific effects of endocannabinoid system (ECS) modulators[5][6][7]
- Its high selectivity for CB1 relative to CB2 avoids off-target effects on immune cells, supporting its application in central and peripheral ECS research[2][7]

C22H21CL2IN4O

555.24

554.013

C, 47.59; H, 3.81; Cl, 12.77; I, 22.86; N, 10.09; O, 2.88

183232-66-8

2125

White to off-white solid powder

1.7±0.1 g/cm3

195-196℃

1.703

6.45

1

3

4

30

586

0

IC1C([H])=C([H])C(=C([H])C=1[H])C1=C(C([H])([H])[H])C(C(N([H])N2C([H])([H])C([H])([H])C([H])([H])C([H])([H])C2([H])[H])=O)=NN1C1C([H])=C([H])C(=C([H])C=1Cl)Cl

BUZAJRPLUGXRAB-UHFFFAOYSA-N

InChI=1S/C22H21Cl2IN4O/c1-14-20(22(30)27-28-11-3-2-4-12-28)26-29(19-10-7-16(23)13-18(19)24)21(14)15-5-8-17(25)9-6-15/h5-10,13H,2-4,11-12H2,1H3,(H,27,30)

1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-piperidin-1-ylpyrazole-3-carboxamide

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 (4.50 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.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (4.50 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 3: 1% DMSO +30% polyethylene glycol+1% Tween 80 : 8 mg/mL

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