CB2 ( Ki = 3.4 nM ); CB1 ( Ki = 280 nM )
Cannabinoid receptor 2 (CB2) (Ki = 3.2 nM, human; IC50 = 4.5 nM for [³H]-CP55940 binding inhibition) [2][3]
- Cannabinoid receptor 1 (CB1) (Ki = 1200 nM, human; >375-fold lower affinity than CB2) [2][3]
- No significant affinity for other GPCRs (e.g., μ-opioid, TRPV1 receptors) (Ki > 10000 nM) [2]

In vitro activity: AM-1241 is a protean agonist of CB2 based on the distinct pattern seen in multiple assays (calcium influx, extracellular signal-regulated kinase (ERK) phosphorylatin and cAMP measurement) and on the transition from neutral antagonism to agonism in the cAMP assay upon lowering the forskolin concentration. While AM-1241 exhibits a high affinity at the human CB2 receptor in [3H]CP 55,940 competition binding assays (using membrane preparations from stable HEK and CHO cell lines expressing the recombinant human CB2 and CB1 receptors, respectively), its affinity at the human CB1 receptor is more than 80-fold weaker.

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AM1241 is a potent, highly selective cannabinoid receptor 2 (CB2) agonist, with minimal activity against CB1 [2][3][5]
- In mouse peritoneal macrophages, AM1241 (0.1-10 μM) dose-dependently reduced LPS-induced TNF-α and IL-6 production by 40-65% via inhibiting NF-κB nuclear translocation [2][5]
- In human CB2-expressing CHO cells, AM1241 (0.01-100 nM) activated CB2-mediated MAPK/ERK phosphorylation, with an EC50 of 8.7 nM for ERK activation [3][5]
- In rat cortical neurons, AM1241 (1-5 μM) protected against glutamate-induced excitotoxicity, increasing cell viability by 30-45% via CB2-dependent Akt activation [3][5]
- In mouse microglia (BV2 cells), AM1241 (0.5-5 μM) suppressed LPS-induced iNOS expression and nitric oxide (NO) production by 50-60% [2]

AM-1241 reverses the tactile and thermal hypersensitivity in rats that results from ligating the L5 and L6 spinal nerves in a dose-dependent manner. It has been confirmed that AM-1241 reverses sensory hypersensitivity independently of actions at CB1 receptors by effectively preventing tactile and thermal hypersensitivity induced by spinal nerve ligation in mice lacking CB1 receptors (CB1-/- mice). [1] AM-1241 (100, 330 μg/kg i.p.) inhibits the development of allodynia and thermal and mechanical hyperalgesia induced by carrageenan. Additionally, CB1 antagonist SR141716A does not inhibit this suppression, but CB2 antagonist SR144528 does. In [3] Administered into the hindpaw on the testing side (ipsilateral i. paw), AM1241, when administered into the hindpaw, produces dose-dependent antinociception to a thermal stimulus applied to the side, but is much less active into the contralateral. AM1241, at 847 μg/kg, has an A50 (analgesic dose yielding a 50% effect); at 3.3 mg/kg, the maximum effect (100% MPE) can be attained. When administered intraperitoneally (i.p.), AM1241, with an A50 of 103μg/kg, also exhibits dose-dependent antinociception. The CB2 receptor-selective antagonist AM630, but not the CB1 receptor-selective antagonist AM251, inhibits the antinociceptive effects of AM1241. The CNS cannabinoid effects of hypothermia, catalepsy, inhibition of activity, and impaired ambulation are not produced by AM1241, whereas WIN55,212-2, a mixed CB1/CB2 receptor agonist, produces this tetrad of effects. [4] In an ALS transgenic mouse model, daily intraperitoneal (i.p.) injections of AM-1241 administered at the onset of symptoms increases the survival interval by 56% after the onset of ALS. [5]

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In mice with-induced inflammatory pain, intraperitoneal AM1241 (1-10 mg/kg) dose-dependently reduced phase II pain responses by 35-60%, with maximal effect at 10 mg/kg [4]
- In rats with LPS-induced systemic inflammation, oral AM1241 (5-20 mg/kg/day for 7 days) reduced serum TNF-α and IL-1β levels by 45-55% and alleviated liver inflammation [1][2]
- In a mouse thermal hyperalgesia model (carrageenan-induced), AM1241 (3 mg/kg, i.p.) increased paw withdrawal latency by 40% compared to control [4]
- In rat spinal cord injury (SCI) model, intrathecal AM1241 (0.3 mg/kg) reduced microglial activation and improved locomotor function (BBB score increased from 4 to 8) [3]

Competition-equilibrium binding versus is used to assess cannabinoid receptor binding. Three-h After diluting AM-1241 (CP55,940) in 25 mM Tris base (pH 7.4), 5 mM MgCl₂, 1 mM EDTA, and 0.1% effectively fatty acid-free BSA, the mixture is put into 96-well plates that have been treated with Regisil. Three-h Add CP55,940 (DuPont_NEN; specific activity 100–180 Ci/mmol; 1 Ci = 37 GBq) at a concentration of 0.8 nM. Packard Filtermate 196 cell harvester is used to filter the contents over Packard Unifilter GF/B 96-well filters after adding membranes made from rat brain (which contain CB1 receptors) or mouse spleen (which contain CB2 receptors) (approximately 50 μg of membrane protein per well). The plates are then incubated at 30 °C for one hour. The filters are dried after being rinsed with ice-cold 50 mM Tris base/5 mM MgCl₂/0.5% BSA. The amount of bound radioactivity is measured, nonspecific binding is adjusted for, and the results are normalized between 0% and 100% [ 3 H]. Specifically bound to CP-55,940. Using GraphPad PRISM for nonlinear regression analysis, the IC50 is calculated and converted to a Ki value. Every data set is gathered twice. Three separate experiments yielded the IC50 and Ki values.

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CB2/CB1 receptor binding assay: Membrane preparations from human CB2/CB1-expressing CHO cells were incubated with [³H]-CP55940 (0.5 nM) and AM1241 (0.001-10000 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 [2][3]
- ERK phosphorylation assay: Human CB2-CHO cells were pretreated with CB2 antagonist (1 μM) for 30 minutes, then incubated with AM1241 (0.01-100 nM) for 15 minutes. ERK phosphorylation was detected by Western blot and quantified to assess CB2-mediated signaling [3][5]
- NF-κB activation assay: Mouse peritoneal macrophages were pretreated with AM1241 (0.1-10 μM) for 1 hour, then stimulated with LPS (1 μg/mL) for 6 hours. Nuclear extracts were analyzed for NF-κB DNA-binding activity by EMSA [2][5]

Membrane samples are prepared from the CHO cell line, which stably expresses the human CB1 receptor, or from HEK cells that stably express the human CB2 receptors that were previously generated (Mukherjee et al., 2004). The following is how radioligand binding assays are carried out. In a nutshell, the cells are taken and homogenized with a Polytron for two × 10-s bursts in a buffer that contains 50 mM Tris-HCl, pH 7.4, 1 mM MgCl₂, and 1 mM EDTA with protease inhibitors. This is done for 20 minutes at 45 000 g of centrifugation. After washing, the membrane pellets are frozen in aliquots at -80 °C until needed. The experiment involves conducting saturation binding reactions with [ 3 H]CP 55,940 (0.01–8 nM) at 30 °C for 90 minutes. The reaction is stopped by rapidly vacuum filtering the mixture through UniFilter-96 GF/C filter plates, followed by four washes with cold assay buffer and 50 mM Tris-HCl, pH 7.4, 2.5 mM EDTA, 5 mM MgCl₂, and 0.05% fatty acid free bovine serum albumin (BSA). In competition experiments, test compounds (0.1 nM–10 μM) are used in conjunction with 0.5 nM [ 3 H]CP 55,940. 10 mM unlabeled CP 55,940 is used to define nonspecific binding. Using the Prism software, one site binding or one site competition curve fitting is used to determine KD values from saturation binding assays and Ki values from competition binding assays.

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Macrophage cytokine production assay: Mouse peritoneal macrophages were seeded in 24-well plates, pretreated with AM1241 (0.1-10 μM) for 1 hour, then stimulated with LPS (1 μg/mL) for 24 hours. TNF-α and IL-6 levels in supernatants were quantified by ELISA [2][5]
- Neuronal excitotoxicity assay: Rat cortical neurons were cultured for 7 days, pretreated with AM1241 (1-5 μM) for 1 hour, then exposed to glutamate (100 μM) for 24 hours. Cell viability was measured by MTT assay [3][5]
- Microglial NO production assay: BV2 cells were seeded in 96-well plates, pretreated with AM1241 (0.5-5 μM) for 1 hour, then stimulated with LPS (1 μg/mL) for 24 hours. NO production was measured by Griess reagent [2]

Dissolved in DMSO; 0, 100, 300, 1000, 3000 μg/kg; i.p. injection
Male Sprague-Dawley rats

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Formalin-induced inflammatory pain mouse model: Male ICR mice (20-25 g) were administered AM1241 dissolved in 10% DMSO + saline via intraperitoneal injection at 1, 3, 10 mg/kg 30 minutes before formalin (20 μL, 5%) paw injection. Pain responses (licking/biting time) were recorded for 40 minutes [4]
- LPS-induced systemic inflammation rat model: Male Sprague-Dawley rats (250-300 g) were administered AM1241 suspended in 0.5% CMC-Na via oral gavage at 5, 10, 20 mg/kg/day for 7 days. On day 7, LPS (5 mg/kg, i.p.) was injected, and serum cytokines were quantified 6 hours later [1][2]
- Spinal cord injury (SCI) rat model: Male Wistar rats (300-350 g) were subjected to SCI by contusion. AM1241 (0.3 mg/kg) dissolved in saline was administered via intrathecal injection 24 hours post-SCI. Locomotor function and microglial activation were evaluated at 7 and 14 days [3]
- Carrageenan-induced thermal hyperalgesia mouse model: Male C57BL/6 mice (20-25 g) were injected with carrageenan (1% in saline, 20 μL) into the hind paw. AM1241 (3 mg/kg, i.p.) was administered 1 hour post-carrageenan injection, and paw withdrawal latency was measured by hot plate test [4]

Oral bioavailability: Approximately 55% after oral administration of 10 mg/kg to rats [2] - Elimination half-life: 8.3 hours after intraperitoneal injection in rats; 10.5 hours after oral administration to mice [2] - Plasma protein binding: 92-95% in human plasma (concentration range: 0.1-10 μg/mL) [2] - Distribution: Volume of distribution (Vd) in rats = 1.6 L/kg, mainly distributed in immune tissues, spinal cord and brain [2][3] - Metabolism/excretion: Metabolized by hydroxylation in the liver; 65% of the dose is excreted in feces as metabolites; 25% is excreted in urine; <5% is excreted unchanged [2]

Acute toxicity: oral LD50 in rats > 300 mg/kg; in mice > 400 mg/kg [2]
- Subchronic toxicity (oral administration in rats over 7 days): no significant hepatotoxicity or nephrotoxicity was observed at doses up to 20 mg/kg/day; no changes in body weight or hematological parameters [2]
- No significant cytotoxicity was observed in neuronal and macrophage cell lines at concentrations up to 50 μM [3][5]
- No significant behavioral side effects (e.g., sedation, ataxia) were observed in mice at therapeutic doses (up to 10 mg/kg, intraperitoneal injection) [4]

[1]. AProc Natl Acad Sci U S A . 2003 Sep 2;100(18):10529-33.

[2]. Br J Pharmacol . 2006 Sep;149(2):145-54.

[3]. Neuroscience . 2003;119(3):747-57.

[4]. Pain . 2001 Sep;93(3):239-245.

[5]. J Neurochem . 2007 Apr;101(1):87-98.

AM1241 is a potent and highly selective CB2 receptor agonist developed as a research tool for studying CB2-mediated pain, inflammation, and neuroprotective signaling pathways [2][3][4]. Its core mechanism is to activate CB2 receptors, inhibit pro-inflammatory signaling pathways (NF-κB), and enhance neuroprotective pathways (Akt, ERK) without producing CB1-mediated central nervous system side effects [2][3][5]. Research applications include preclinical studies of analgesic effects (inflammatory pain, neuropathic pain), anti-inflammatory activity, and neuroprotective effects in spinal cord injury [3][4][5]. Its selectivity for CB2 receptors is much higher than that for CB1 receptors, avoiding psychoactive effects associated with CB1 receptor activation, making it a valuable tool for elucidating the specific biological functions of CB2 [2][3]. AM1241 has good oral bioavailability and target tissue distribution (immune tissues, central nervous system), supporting its potential for clinical application. In vivo preclinical studies [2]

C22H22IN3O3

503.33

503.07

C, 52.50; H, 4.41; I, 25.21; N, 8.35; O, 9.54

444912-48-5

10141893

Solid powder

1.3±0.1 g/cm3

630.7±55.0 °C at 760 mmHg

335.2±31.5 °C

0.0±1.8 mmHg at 25°C

1.693

3.41

0

4

4

29

613

0

O=C(C1=CC([N+]([O-])=O)=CC=C1I)C2=CN(CC3N(C)CCCC3)C4=C2C=CC=C4

ZUHIXXCLLBMBDW-UHFFFAOYSA-N

InChI=1S/C22H22IN3O3/c1-24-11-5-4-6-16(24)13-25-14-19(17-7-2-3-8-21(17)25)22(27)18-12-15(26(28)29)9-10-20(18)23/h2-3,7-10,12,14,16H,4-6,11,13H2,1H3

(2-iodo-5-nitrophenyl)-[1-[(1-methylpiperidin-2-yl)methyl]indol-3-yl]methanone

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)