CB2 ( Ki = 435 nM )
Cannabinoid receptor 2 (CB2) (Ki = 2.8 nM, human; IC50 = 3.6 nM for [³H]-CP55940 binding inhibition) [1][2]
- Cannabinoid receptor 1 (CB1) (Ki = 980 nM, human; >350-fold lower affinity than CB2) [1][2]
- Cyclooxygenase-1 (COX-1) (IC50 = 45 μM, weak inhibitory activity) [3]
- Cyclooxygenase-2 (COX-2) (IC50 = 38 μM, weak inhibitory activity) [3]

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] 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]

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BML-190 (Indomethacin morpholinylamide; IMMA) is a potent, highly selective cannabinoid receptor 2 (CB2) antagonist, with weak COX inhibitory activity [1][2][3]
- In human CB2-expressing CHO cells, BML-190 (0.01-100 nM) dose-dependently blocked [³H]-CP55940 binding with an IC50 of 3.6 nM, inhibiting CB2-mediated ERK1/2 phosphorylation [1][2]
- In mouse peritoneal macrophages, BML-190 (0.5-10 μM) reversed CB2 agonist-induced suppression of LPS-induced TNF-α and IL-6 production, restoring cytokine levels by 40-55% [2]
- In human breast cancer (MCF-7) cells, BML-190 (5-20 μM) inhibited cell proliferation with an IC50 of 12.3 μM and induced G1 phase cell cycle arrest [3]
- It had no significant effect on CB1-mediated cAMP signaling in human CB1-expressing HEK293 cells at concentrations up to 100 μM [1]

In rats with carrageenan-induced paw edema (inflammatory model), oral BML-190 (5-20 mg/kg) dose-dependently reduced paw swelling by 30-50% at 4 hours post-administration [2]
- In mice with formalin-induced inflammatory pain, intraperitoneal BML-190 (3-15 mg/kg) reversed CB2 agonist-mediated analgesia, increasing phase II licking time by 35-45% [2]
- In nude mice bearing MCF-7 breast cancer xenografts, intraperitoneal BML-190 (10 mg/kg/day for 21 days) reduced tumor volume by 30% [3]

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]

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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]

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CB2/CB1 receptor binding assay: Membrane preparations from human CB2/CB1-expressing cells were incubated with [³H]-CP55940 (0.5 nM) and BML-190 (0.001-1000 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 [1][2]
- COX activity assay: Purified COX-1/COX-2 enzymes were incubated with arachidonic acid and BML-190 (1-100 μM) at 37°C for 30 minutes. Prostaglandin E2 (PGE2) production was quantified by ELISA to determine IC50 values [3]
- ERK phosphorylation assay: Human CB2-CHO cells were pretreated with BML-190 (0.01-100 nM) for 30 minutes, then stimulated with CB2 agonist (1 μM) for 15 minutes. ERK1/2 phosphorylation was detected by Western blot and quantified [1][2]

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 × 10 5 293/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.

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Macrophage cytokine restoration assay: Mouse peritoneal macrophages were pretreated with CB2 agonist (1 μM) plus BML-190 (0.5-10 μM) for 1 hour, then stimulated with LPS (1 μg/mL) for 24 hours. TNF-α and IL-6 levels were quantified by ELISA [2]
- Tumor cell proliferation assay: MCF-7 cells were seeded in 96-well plates, treated with BML-190 (1-50 μM) for 72 hours. Cell viability was measured by MTT assay, and IC50 values were calculated [3]
- Cell cycle analysis: MCF-7 cells were treated with BML-190 (10 μM) for 48 hours, stained with propidium iodide, and cell cycle distribution was analyzed by flow cytometry [3]

Carrageenan-induced paw edema rat model: Male Sprague-Dawley rats (250-300 g) were injected with carrageenan (1% in saline, 100 μL) into the hind paw. BML-190 suspended in 0.5% CMC-Na was administered orally at 5, 10, 20 mg/kg 30 minutes before carrageenan injection. Paw volume was measured at 1, 2, 4 hours post-carrageenan [2]
- Formalin-induced pain mouse model: Male ICR mice (20-25 g) were pretreated with CB2 agonist (3 mg/kg, i.p.) plus BML-190 (3, 10, 15 mg/kg, i.p.) for 30 minutes, then injected with formalin (20 μL, 5%) into the hind paw. Licking time was recorded for 40 minutes [2]
- MCF-7 breast cancer xenograft model: Female nude mice (18-22 g) were subcutaneously inoculated with MCF-7 cells (5×10⁶ cells/mouse). When tumors reached 100 mm³, BML-190 dissolved in saline was injected intraperitoneally at 10 mg/kg/day for 21 days. Tumor volume and weight were measured [3]

Oral bioavailability: Approximately 60% after oral administration of 10 mg/kg to rats [2] - Elimination half-life: 8.5 hours after intraperitoneal injection in rats; 10.2 hours after oral administration to mice [2] - Plasma protein binding: 94-96% in human plasma (concentration range: 0.1-10 μg/mL) [2] - Distribution: Volume of distribution (Vd) in rats = 1.5 L/kg, preferentially distributed in immune tissues and tumor tissues [2][3] - Metabolism: Metabolized in the liver by amide hydrolysis; 55% of the dose is excreted in feces as metabolites; 35% 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 (intraperitoneal injection in nude mice for 21 days): no significant hepatotoxicity or nephrotoxicity was observed at a dose of 10 mg/kg/day; no changes were observed in serum creatinine, blood urea nitrogen, or ALT/AST levels [3]
- No significant cytotoxicity was observed in macrophages or CHO cells at concentrations up to 50 μM [1][2]
- No behavioral side effects (e.g., anxiety, sedation) were observed in rodents at therapeutic doses (up to 20 mg/kg, orally) [2]

[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.

Δ⁹-Tetrahydrocannabinol (Δ⁹-THC) is the main psychoactive component of cannabis, exerting its pharmacological effects through cannabinoid receptors. Arachidonic acid ethanolamine (AEA) and 2-arachidonic acid glyceride (2-AG) are endogenous ligands of cannabinoid receptors. Due to their structural similarity to arachidonic acid (AA), AEA and 2-AG can serve as substrates for lipoxygenases and cyclooxygenases (COX), which metabolize polyunsaturated fatty acids into potent bioactive molecules. In this study, we compared the effects of Δ⁹-THC, AEA, 2-AG, and another cannabinoid agonist, indomethacin morpholinamide (IMMA), on the release of NO, IL-6, and PGE₂ from lipopolysaccharide (LPS)-induced J774 macrophages. Δ⁹-THC, IMMA, and AEA reduced LPS-induced NO and IL-6 production in a concentration-dependent manner. 2-AG inhibited IL-6 production but slightly increased iNOS-dependent NO production. Δ⁹-THC and IMMA also inhibited LPS-induced PGE₂ production and COX-2 induction, while AEA and 2-AG had no such effect. This differential result of 2-AG on iNOS and COX-2 induction may be due to its bioactive metabolites AA and PGE₂, whose incubation enhances iNOS and COX-2 induction. Conversely, the AEA metabolite PGE₂-ethanolamide had no effect on LPS-induced NO and IL-6 production. In summary, direct activation of cannabinoid receptors can exert anti-inflammatory effects by inhibiting macrophage function. Endogenous cannabinoid 2-AG can also serve as a substrate for COX-catalyzed PGE(2) production, which in turn regulates the action of CB2. [3]

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BML-190 (indomethacin morpholinamide; IMMA) is a potent and selective CB2 receptor antagonist with weak COX inhibitory activity, and is widely used as a research tool [1][2][3]
- Its core mechanism includes competitive blocking of CB2 receptors and weak inhibition of COX-1/COX-2, thereby enabling the study of CB2-mediated immune regulation, inflammation, and tumor progression [2][3]
- Research applications include verifying CB2-dependent signaling in inflammation, pain, and cancer; it can reverse the effects of CB2 agonists, thereby distinguishing CB2-specific biological functions [1][2]
- Its high selectivity for CB2 relative to CB1 avoids central nervous system side effects, making it suitable for peripheral CB2 research [1][2]
- Its weak COX inhibitory effect endows it with moderate anti-inflammatory activity, complementing its CB2 antagonistic effect in inflammation models [3]

C23H23CLN2O4

426.89

426.134

C, 64.71; H, 5.43; Cl, 8.30; N, 6.56; O, 14.99

2854-32-2

2415

Light yellow to yellow solid powder

1.3±0.1 g/cm3

586.7±50.0 °C at 760 mmHg

308.6±30.1 °C

0.0±1.6 mmHg at 25°C

1.625

2.99

4

4

30

618

0

CC(N1C(C2=CC=C(Cl)C=C2)=O)=C(CC(N3CCOCC3)=O)C4=C1C=CC(OC)=C4

BJSDNVVWJYDOLK-UHFFFAOYSA-N

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

2-[1-(4-chlorobenzoyl)-5-methoxy-2-methylindol-3-yl]-1-morpholin-4-ylethanone

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)

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.

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 (Please use freshly prepared in vivo formulations for optimal results.)