BML-190 (formerly IMMA; LM-4131; BML-190; LM4131; BML190; Indomethacin morpholinylamide), is a potent and selective cannabinoid CB2 receptor inverse agonist with potential anti-inflammatory activity. It exhibits 50-fold selectivity over the CB1 receptor and activates the cannabinoid CB2 receptor with a Ki of 435 nM.

Physicochemical Properties

Molecular Formula	C23H23CLN2O4
Molecular Weight	426.89
Exact Mass	426.134
Elemental Analysis	C, 64.71; H, 5.43; Cl, 8.30; N, 6.56; O, 14.99
CAS #	2854-32-2
Related CAS #	2854-32-2
PubChem CID	2415
Appearance	Light yellow to yellow solid powder
Density	1.3±0.1 g/cm3
Boiling Point	586.7±50.0 °C at 760 mmHg
Flash Point	308.6±30.1 °C
Vapour Pressure	0.0±1.6 mmHg at 25°C
Index of Refraction	1.625
LogP	2.99
SMILES	CC(N1C(C2=CC=C(Cl)C=C2)=O)=C(CC(N3CCOCC3)=O)C4=C1C=CC(OC)=C4
InChi Key	BJSDNVVWJYDOLK-UHFFFAOYSA-N
InChi Code	InChI=1S/C23H23ClN2O4/c1-15-19(14-22(27)25-9-11-30-12-10-25)20-13-18(29-2)7-8-21(20)26(15)23(28)16-3-5-17(24)6-4-16/h3-8,13H,9-12,14H2,1-2H3
Chemical Name	2-[1-(4-chlorobenzoyl)-5-methoxy-2-methylindol-3-yl]-1-morpholin-4-ylethanone
Synonyms	BML-190; IMMA; LM-4131; BML 190; LM4131; 2854-32-2; BML-190; 2-(1-(4-Chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl)-1-morpholinoethanone; Indomethacin morpholinylamide; IMMA; Ethanone, 2-[1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl]-1-(4-morpholinyl)-; CHEMBL72631; BML 190;BML190; LM 4131; Indomethacin Morpholinylamide
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 = 435 nM )
ln Vitro	The in vitro metabolism of an inverse agonist of the peripheral cannabinoid receptor (CB(2)), indomethacin morpholinylamide (BML-190), has been characterized using rat liver microsomal incubation. BML-190 was found to yield at least 15 metabolic products as identified by HPLC-MS/MS analysis. Four major phase one metabolic pathways either individually, or in combination, were proposed to account for the identified metabolic products: (1) loss of the p-chlorobenzyl group, (2) hydroxylation on the indole or on the morpholine ring, (3) morpholinyl ring opening, and (4) demethylation of the methoxyl group on the indole ring.[2] In vitro activity: BML-190 prefers CB2 receptors over CB1 receptors by a factor of 50. BML-190 enhances the forskolin-stimulated cAMP accumulation in HEK-293 cells that express the human CB2 receptor consistently. Cells expressing the CB2 receptor have lower basal levels of inositol phosphate production when exposed to BML-190. BML-190 at 10 μM reduces the accumulation of inositol phosphates by 38%. [1] An aminoalkylindole is BML-190. There are at least 15 metabolic products produced by BML-190. [2] LPS-induced NO and IL-6 production are inhibited by BML-190 in a concentration-dependent manner. Moreover, BML-190 prevents COX-2 induction and PGE2 synthesis brought on by LPS. [3]
Enzyme Assay	The aminoalkylindole BML-190 and diarylpyrazole AM251 ligands have previously been shown to bind to cannabinoid CB(2) and CB(1) receptors, respectively. In HEK-293 cells stably expressing the human CB(2) receptor, BML-190 and AM251 potentiated the forskolin-stimulated accumulation of cAMP. Moreover, the CB(2) receptor can interact productively with 16z44, a promiscuous G alpha(16/z) chimera. BML-190 and AM251 reduce the basal levels of inositol phosphate production in cells expressing the CB(2) receptor and 16z44. These results demonstrate that BML-190 and AM251 act as inverse agonists at the human CB(2) receptor acting via G alpha(i/o) and G alpha(q) family-coupled pathways.[1] Stock solutions of 15 mM BML-190 were prepared in DMSO and added in 2-µL aliquots as substrate to individual incubation aliquots. Rat liver microsomes containing 1.5 mg/mL of protein concentration were pre-incubated at 37 °C for 30 min. The 0.4-mL incubation aliquots contained 90 mM potassium phosphate (pH 7.4), 17 mM magnesium chloride, 7 mM NADPH, 17 mM glucose-6-phosphate, and 1.2 units of glucose-6-phosphate dehydrogenase. Incubation times ranged from 0.5 to 4 h. Incubations were halted by placing the incubation vials in an ice bath followed by the addition of an equal volume of methanol (0.2 mL). The quenched incubation mixtures were stored at −20 °C until analysis. Prior to HPLC separation, microsomal proteins were precipitated by centrifugation at room temperature, and the solvent was evaporated with a stream of nitrogen at 37 °C. The residual solution was applied to 6-mL SUPELCO C18 solid-phase extraction (SPE) columns pre-treated with water and methanol. The columns were washed with HPLC-grade water followed by elution with methanol. The effluents were again concentrated by a nitrogen stream at 37 °C to near dryness before reconstitution with methanol to 1 mL volumes for HPLC analysis.[2]
Cell Assay	For 20 to 24 hours, 293/CB2 cells are labeled with [3H]adenine (1 μCi/mL) in MEM containing 1% FBS. After 30 minutes at 37°C, labeled cells are challenged with 50 μM forskolin and the relevant BML-190, and the accumulation of cAMP is measured. Using transfection reagents, 2 × 105 293/CB2 cells are transiently transfected with 16z44 and/or pcDNA3 for IP assays. In order to produce IP, cells are labeled, challenged with BML-190, and assayed. For every data point, triplicates are run, and each ligand is tested in at least three different trials.
Animal Protocol
References	[1]. FEBS Lett . 2003 Feb 11;536(1-3):157-60. [2]. Eur J Pharm Sci . 2010 Sep 11;41(1):163-72. [3]. J Cell Biochem . 2001;81(4):715-23.
Additional Infomation	Delta(9)-Tetrahydrocannabinol (Delta(9)-THC) is the major psychoactive component of marijuana and elicits pharmacological actions via cannabinoid receptors. Anandamide (AEA) and 2-arachidonoyl-glycerol (2-AG) are endogenous ligands for cannabinoid receptors, which because of their structural similarities to arachidonic acid (AA), AEA, and 2-AG could serve as substrates for lipoxygenases and cyclooxygenases (COXs) that metabolize polyunsaturated fatty acids to potent bioactive molecules. In this study, we have compared the effects of Delta(9)-THC, AEA, 2-AG, and another cannabinoid agonist, indomethacin morpholinylamide (IMMA), on lipopolysaccharide (LPS)-induced NO, IL-6, and PGE(2) release from J774 macrophages. Delta(9)-THC, IMMA, and AEA diminish LPS-induced NO and IL-6 production in a concentration-dependent manner. 2-AG inhibits the production of IL-6 but slightly increases iNOS-dependent NO production. Delta(9)-THC and IMMA also inhibit LPS-induced PGE(2) production and COX-2 induction, while AEA and 2-AG have no effects. These discrepant results of 2-AG on iNOS and COX-2 induction might be due to its bioactive metabolites, AA and PGE(2), whose incubation cause the potentiation of both iNOS and COX-2 induction. On the contrary, the AEA metabolite, PGE(2)-ethanolamide, influences neither the LPS-induced NO nor IL-6 production. Taken together, direct cannabinoid receptor activation leads to anti-inflammatory action via inhibition of macrophage function. The endogenous cannabinoid, 2-AG, also serves as a substrate for COX-catalyzing PGE(2) production, which in turn modulates the action of CB2.[3]

Solubility Data

Solubility (In Vitro)

DMSO: 22~50 mg/mL (51.5~117.1 mM) Water: <1 mg/mL Ethanol: <1 mg/mL

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.86 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.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	2.3425 mL	11.7126 mL	23.4252 mL
	5 mM	0.4685 mL	2.3425 mL	4.6850 mL
	10 mM	0.2343 mL	1.1713 mL	2.3425 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.