AM-1241 (AM1241; AM 1241), an aminoalkylindole analog, is a novel, potent and selective cannabinoid (CB2) receptor agonist with potential analgesic effects (pain-killing). It exhibits 82-fold selectivity over the CB1 receptor and activates CB2 with a Ki of 3.4 nM.

Physicochemical Properties

Molecular Formula	C22H22IN3O3
Molecular Weight	503.33
Exact Mass	503.07
Elemental Analysis	C, 52.50; H, 4.41; I, 25.21; N, 8.35; O, 9.54
CAS #	444912-48-5
Related CAS #	444912-48-5
PubChem CID	10141893
Appearance	Solid powder
Density	1.3±0.1 g/cm3
Boiling Point	630.7±55.0 °C at 760 mmHg
Flash Point	335.2±31.5 °C
Vapour Pressure	0.0±1.8 mmHg at 25°C
Index of Refraction	1.693
LogP	3.41
Hydrogen Bond Donor Count	0
Hydrogen Bond Acceptor Count	4
Rotatable Bond Count	4
Heavy Atom Count	29
Complexity	613
Defined Atom Stereocenter Count	0
SMILES	O=C(C1=CC([N+]([O-])=O)=CC=C1I)C2=CN(CC3N(C)CCCC3)C4=C2C=CC=C4
InChi Key	ZUHIXXCLLBMBDW-UHFFFAOYSA-N
InChi Code	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
Chemical Name	(2-iodo-5-nitrophenyl)-[1-[(1-methylpiperidin-2-yl)methyl]indol-3-yl]methanone
Synonyms	AM-1241; AM 1241; AM1241; UNII-DLM851L3RD
HS Tariff Code	2934.99.9001
Storage	Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month
Shipping Condition	Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Biological Activity

Targets	CB2 ( Ki = 3.4 nM ); CB1 ( Ki = 280 nM )
ln Vitro	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.
ln Vivo	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]
Enzyme Assay	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% [3H]. 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.
Cell Assay	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 [3H]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 [3H]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.
Animal Protocol	Dissolved in DMSO; 0, 100, 300, 1000, 3000 μg/kg; i.p. injection Male Sprague-Dawley rats
References	[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.

Solubility Data

Solubility (In Vitro)	DMSO: ~101 mg/mL (~200.7 mM) Water: <1 mg/mL Ethanol: <1 mg/mL
Solubility (In Vivo)	2%DMSO+30%PEG 300+5%Tween 80+ddH2O: 5 mg/mL  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	1.9868 mL	9.9338 mL	19.8677 mL
	5 mM	0.3974 mL	1.9868 mL	3.9735 mL
	10 mM	0.1987 mL	0.9934 mL	1.9868 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.