CB1 cannabinoid receptor – no binding affinity (IC₅₀, Ki, EC₅₀) values are reported in this study; only qualitative description of high affinity and competition with CP 55,940 and THC is mentioned in the Introduction.
AM6538 targets the cannabinoid receptor 1 (CB1), a G protein-coupled receptor that is the primary target of Δ⁹-tetrahydrocannabinol (THC), the psychoactive component of cannabis. As a CB1 antagonist, AM6538 blocks the effects of endogenous cannabinoids and exogenous cannabinoid agonists. The compound exhibits high affinity for CB1 and binds in a pseudo-irreversible manner, meaning that it dissociates very slowly from the receptor. This property makes AM6538 useful for studying receptor occupancy and for evaluating the efficacy of cannabinoid agonists.

AM6538 is a cannabinoid antagonist that binds to the CB1 receptor expressed in HEK-293 cells in a wash-stable manner [1].

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In vitro, AM6538 demonstrates high-affinity binding to the CB1 receptor expressed in HEK-293 cells. The compound binds in a wash-stable manner, indicating pseudo-irreversible binding kinetics. In functional assays, AM6538 acts as a CB1 antagonist, blocking the effects of cannabinoid agonists. The compound's high affinity and slow dissociation kinetics make it useful for receptor occupancy studies and for evaluating the apparent efficacy of cannabinoid full and partial agonists. AM6538 is a structural analog of rimonabant.

Mice treated with AM6538 (1~10 mg/kg) do not experience the analgesic effects of cannabinoid agonists [1]. There was a partial restoration of AM4054's impact with AM6538 (10 mg/kg). All cannabis agonists' tau is dose-dependently reduced by AM6538, indicating a decrease in the number of accessible receptors. Up to seven days following therapy, AM6538 causes strong, long-lasting antagonistic effects on THC's dose-response function [1].
(1) In male CD‑1 mice, AM6538 (0.1–10 mg/kg, i.p., 1‑h pretreatment) dose‑dependently antagonized the antinociceptive effects of cannabinoid agonists (AM4054, THC, WIN 55,212) in the warm‑water tail‑withdrawal assay. Lower doses produced rightward shifts of agonist dose‑effect curves; higher doses (3–10 mg/kg) produced downward shifts, reducing maximal effects. ED₅₀ for AM4054 increased from 0.38 mg/kg (control) to 2.47 mg/kg after 3 mg/kg AM6538, and to >10 mg/kg after 10 mg/kg. For THC, 0.3 mg/kg AM6538 flattened the curve with <50% max effect; ED₅₀ for WIN 55,212 increased from 2.8 to 18.0 mg/kg after 3 mg/kg AM6538. [1]
(2) The antagonism by AM6538 (10 mg/kg) in mice lasted for up to 7 days. At 1 h post‑injection, AM4054 (10 mg/kg) produced only 37±11% MPE; recovery was partial at 24–48 h (ED₅₀ for AM4054: 2.46 mg/kg at 24 h, 1.22 mg/kg at 48 h) and full by 7 days (ED₅₀ = 0.25 mg/kg). For THC, ED₅₀ was 196.8 mg/kg at 48 h and 32.3 mg/kg at 7 days. [1]
(3) In squirrel monkeys trained to discriminate AM4054 (0.01 mg/kg, i.m.) from vehicle, AM6538 (1 or 3 mg/kg, i.m., 1‑h pretreatment) dose‑dependently antagonized the discriminative stimulus effects of AM4054, shifting the ED₅₀ from 0.004 mg/kg (control) to 0.025 mg/kg (after 1 mg/kg) and 0.24 mg/kg (after 3 mg/kg). The antagonism persisted for >7 days; at 7 days after 3 mg/kg, the AM4054 ED₅₀ was still 0.009 mg/kg vs. control 0.004 mg/kg. [1]
(4) Tau values (operational efficacy) were calculated from the shifts. In mice, control tau for THC was 4.6, for WIN 55,212 6.8, for AM4054 10.1. After 3 mg/kg AM6538, tau decreased to 0.6 (THC), 2.1 (WIN 55,212), and 2.7 (AM4054), indicating remaining receptor fractions of 12%, 31%, and 27%, respectively. Recovery of tau values over time was also quantified. [1]
In vivo, AM6538 has been studied in mice to evaluate its effects on cannabinoid agonist-induced analgesia. Mice treated with AM6538 (1-10 mg/kg) experience reduced analgesic effects of cannabinoid agonists, confirming the compound's CB1 antagonist activity in vivo. The compound's long-acting and pseudo-irreversible binding properties make it useful for studies requiring sustained CB1 receptor blockade. AM6538 may be useful in future studies that require temporary reductions in cannabinoid receptor availability.

In vitro enzyme/receptor binding assays for AM6538 typically involve radioligand binding studies using membrane preparations from cells expressing recombinant CB1 receptors. [³H]-CP55940 or other labeled cannabinoid ligands are used to measure competitive displacement by AM6538. The compound is incubated with membrane preparations and labeled ligand, and bound radioactivity is measured after filtration. Binding affinity (Ki) is calculated from competition curves. The pseudo-irreversible nature of binding is assessed by washout experiments, in which bound ligand is measured after extensive washing.

In vitro cellular assays for AM6538 utilize cell lines expressing recombinant CB1 receptors, such as HEK-293 cells. Cells are treated with AM6538 at concentrations ranging from 0.1 nM to 10 µM. Receptor binding is assessed by radioligand binding assays. Functional antagonism is assessed by measuring CB1-mediated signaling pathways such as cAMP accumulation, ERK phosphorylation, or calcium mobilization. The compound's ability to block agonist-induced effects is evaluated. Washout experiments confirm the pseudo-irreversible nature of binding.

(1) Mouse antinociception: Male CD‑1 mice (25–30 g) were used. The warm‑water tail‑withdrawal assay used water at 52.0±0.5 °C with an 8‑s cut‑off. Baseline latencies were measured twice per session. Cumulative dosing procedures were used: after each injection, tail‑withdrawal latency was measured, then the next dose was given; each injection increased the cumulative dose by 0.5 log units. Agonist dose‑effect functions were determined 60 min after pretreatment with vehicle, AM6538 (0.1–10 mg/kg, i.p.), or rimonabant (1–10 mg/kg). For time‑course studies, AM6538 (10 mg/kg) or rimonabant was given, and agonist functions were redetermined at 1 h, 24 h, 48 h, or 7 days later. Each mouse was tested once or twice with at least 1 week between sessions. [1]
(2) Monkey drug discrimination: Six adult male squirrel monkeys (Saimiri sciureus) were trained to discriminate AM4054 (0.01 mg/kg, i.m.) from vehicle in a two‑lever FR‑10 schedule with shock avoidance. Test sessions used cumulative dosing of AM4054 (0.001–0.01 mg/kg, i.m.) given at the start of sequential components. AM6538 (1 or 3 mg/kg, i.m.) was administered 1 h before testing, or at various times (2, 4, 7 days) before the test. Response distribution (% AM4054‑lever) and response rates were measured. ED₅₀ values were calculated from dose‑effect curves. [1]
(3) All drugs were prepared in 5% ethanol, 5% emulphor‑620, and 90% saline; doses expressed as free base. AM6538 and AM4054 were synthesized at the Center for Drug Discovery, Northeastern University. [1]
In vivo animal experiments for AM6538 typically involve mice. The compound is administered via intraperitoneal or intravenous injection at doses ranging from 1 to 10 mg/kg. The effects of cannabinoid agonists on analgesia, hypothermia, or other cannabinoid-mediated effects are assessed in the presence and absence of AM6538. The compound's ability to block agonist effects confirms CB1 antagonism. Receptor occupancy studies may be conducted to correlate binding with functional effects.

AM6538 exhibits pharmacokinetic properties consistent with its long-acting and pseudo-irreversible binding characteristics. Following administration, the compound achieves high receptor occupancy that persists for extended periods due to slow dissociation from CB1 receptors. The compound's half-life in tissues is influenced by its high affinity and slow off-rate. Detailed pharmacokinetic parameters including absorption, distribution, metabolism, and excretion have not been extensively characterized. The compound's pseudo-irreversible binding makes it useful for sustained receptor blockade studies.

Preclinical toxicity data for AM6538 are limited. As a structural analog of rimonabant, which was withdrawn from the market due to psychiatric side effects, AM6538 may have similar safety concerns. However, the compound is primarily used as a research tool rather than a therapeutic agent. In animal studies, the compound has been administered at therapeutic doses without reports of severe toxicity. As with any experimental compound, careful dose optimization is recommended.

[1]. Long-Lasting In Vivo Effects of the Cannabinoid CB1 Antagonist AM6538. J Pharmacol Exp Ther. 2018;364(3):485-493.

A cannabinoid antagonist that binds to CB1 receptors

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AM6538 is a pseudo‑irreversible CB1 antagonist that forms a tight, wash‑resistant attachment to the receptor, as demonstrated in vitro (preincubation with AM6538 results in non‑competitive binding that survives washing). In vivo, it produces long‑lasting antagonism (up to 7 days) in both mice and monkeys, making it useful for receptor inactivation studies. By reducing available CB1 receptors in a graded manner, it allows estimation of agonist efficacy (tau values) via the operational model of agonism. Results indicate that THC has lower efficacy (tau ~4.6) than WIN 55,212 (tau ~6.8) and AM4054 (tau ~10.1) in mice. No clinical development, FDA approval, or therapeutic indication is mentioned; it is used solely as a research tool. [1]
AM6538 is a long-acting, high-affinity, pseudo-irreversible CB1 receptor antagonist and a structural analog of rimonabant. It has a molecular formula of C26H25Cl2N5O4 and a molecular weight of 542.41 g/mol. The compound binds to CB1 in a wash-stable manner and is used to evaluate the efficacy of cannabinoid agonists. It may be useful for studies requiring temporary reductions in cannabinoid receptor availability. AM6538 is intended for research use only.

C26H25CL2N5O4

542.413803815842

541.128

1245626-00-9

46912833

White to off-white solid powder

6.8

1

6

7

37

847

0

CC1=C(N(N=C1C(=O)NN2CCCCC2)C3=C(C=C(C=C3)Cl)Cl)C4=CC=C(C=C4)C#CCCO[N+](=O)[O-]

KXXKUWQMQUYUSE-UHFFFAOYSA-N

InChI=1S/C26H25Cl2N5O4/c1-18-24(26(34)30-31-14-4-2-5-15-31)29-32(23-13-12-21(27)17-22(23)28)25(18)20-10-8-19(9-11-20)7-3-6-16-37-33(35)36/h8-13,17H,2,4-6,14-16H2,1H3,(H,30,34)

4-[4-[2-(2,4-dichlorophenyl)-4-methyl-5-(piperidin-1-ylcarbamoyl)pyrazol-3-yl]phenyl]but-3-ynyl nitrate

AM6538 AM 6538 AM-6538

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 : ~180 mg/mL (~331.85 mM)

Solubility in Formulation 1: 4.5 mg/mL (8.30 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 45.0 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 (4.61 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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 (Please use freshly prepared in vivo formulations for optimal results.)