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 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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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]

(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]

[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]

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.)