The study investigates its effects on several signaling pathways, including JAK1/STAT1/IRF1, p38/MSK1, and cellular ATP synthesis, but concludes the anti-inflammatory action is likely due to a combination of effects rather than inhibition of a single target [1].

When lipopolysaccharide (LPS) is applied to mouse microglial cell line BV2 cells, nitric oxide (NO) release and proinflammatory cytokine production are inhibited by bromisoval (BU). Interferon regulatory factor 1 (IRF1) expression and the phosphorylation of signal transducer and activator of transcription 1 (STAT1) induced by lipopolysaccharide (LPS) are inhibited by bromisoval. The NO release was not as substantially inhibited by the Janus kinase 1 (JAK1) inhibitor filgotinib as it was by Bromisoval; nevertheless, filgotinib virtually totally prevented LPS-induced STAT1 phosphorylation. The inhibitory impact of Bromisoval on LPS-induced NO was not affected by JAK1, STAT1, or IRF1 knockdown. Bromisoval and filgotinib work together to synergistically decrease NO release. Rotenone, an inhibitor of mitochondrial complex I, has a less significant inhibitory effect on the expression of pro-inflammatory mediators than bromisoval, but it does not prevent STAT1 phosphorylation or IRF1 expression. Rotenone and bromisoval both lower intracellular ATP (iATP) levels to a comparable degree. Rotenone and filgotinib together exhibited a potency comparable to that of bromisoval in inhibiting the release of NO in LPS-treated BV2 cells [1].

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In LPS-stimulated BV2 murine microglial cells, Bromisoval (100 μg/ml, ~450 μM) suppressed the release of nitric oxide (NO), as measured by the Griess reaction. This suppression was concentration-dependent (1 - 100 μg/ml). In contrast, dexamethasone (100 nM) did not suppress NO release in these cells [1].
Bromisoval (100 μg/ml) suppressed the LPS-induced mRNA expression of pro-inflammatory genes, including inducible NO synthase (iNOS), interleukin-1β (IL-1β), and interleukin-6 (IL-6), as determined by quantitative real-time RT-PCR (qPCR) 180 minutes after LPS stimulation [1].
Bromisoval (100 μg/ml) partially suppressed the LPS-induced phosphorylation of p38 MAP kinase and its substrate MSK1 at 30 minutes, and weakly suppressed STAT1 phosphorylation and IRF1 expression at 180 minutes, as shown by immunoblotting [1].
The JAK1 inhibitor filgotinib (1 μM) almost completely inhibited STAT1 phosphorylation, whereas Bromisoval (12.5 μg/ml) did not. A combination of Bromisoval (12.5 μg/ml) and filgotinib (1 μM) showed an additive inhibitory effect on NO release [1].
Knockdown of JAK1, STAT1, or IRF1 using specific siRNAs in BV2 cells did not abolish the inhibitory effect of Bromisoval on LPS-induced NO release, suggesting its primary mechanism in these cells is independent of this pathway [1].
Similar to rotenone (10 nM), a mitochondrial complex I inhibitor, Bromisoval (100 μg/ml) suppressed the LPS-induced increase in intracellular ATP (iATP) content after 2 hours and reduced cellular metabolic activity, as measured by WST1 assay after 45 minutes [1].
A combination of rotenone (10 nM) and the JAK1 inhibitor filgotinib (1 μM) suppressed LPS-induced NO release to a similar extent as Bromisoval (100 μg/ml) [1].
Bromisoval did not inhibit LPS-induced nuclear translocation of NFκB (p65) in BV2 cells, as shown by immunoblotting of nuclear fractions, nor did it affect NFκB-mediated transcription in a luciferase reporter assay [1].

Of each series, carbobromal and bromisoval (Bromvaletone) are the most powerful central depressants. Following intraperitoneal injection in male mice, the inhibitory activity (ISD50 value) and acute toxicity (LD50 value) of bromisoval were 0.35 (0.30-0.39) and 3.25 (2.89-3.62) mmol/kg, respectively [2].

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In a rat model of acute lung injury (ALI) induced by intratracheal instillation of E. coli, subcutaneous administration of Bromisoval (30 mg/kg, as a 1% solution in propylene glycol) in conjunction with the antibiotic meropenem (30 mg/kg) completely prevented death over a 48-hour observation period. Treatment with meropenem alone or meropenem plus dexamethasone (0.5 mg/kg) only slightly prevented death [1].

NFκB DNA Binding Assay: To assess the effect of Bromisoval on NFκB activity, nuclear extracts were prepared from BV2 cells stimulated with LPS (1 μg/ml) for 150 minutes in the presence or absence of Bromisoval (100 μg/ml) or dexamethasone. The DNA binding activity of NFκB p65 in these extracts was then determined using an ELISA-based TransAM NFκB p65 Activation Assay, following the manufacturer's protocol. The results showed that Bromisoval did not inhibit LPS-induced NFκB binding to its response element [1].

Acute Lung Injury (ALI) Model:** An *E. coli* suspension was instilled into the trachea of anesthetized male Wistar rats (8-9 weeks old) via a venous catheter. Immediately after bacterial instillation, rats received a single subcutaneous injection of the test compounds. The treatment groups were: control (no treatment), meropenem alone (30 mg/kg in saline), meropenem + Bromisoval (30 mg/kg; as a 1% solution in propylene glycol), and meropenem + dexamethasone (0.5 mg/kg; as a 0.05% solution in propylene glycol). Animal survival was monitored every 12 hours for 48 hours post-infection [1].

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Acute Lung Injury (ALI) Model: An E. coli suspension was instilled into the trachea of anesthetized male Wistar rats (8-9 weeks old) via a venous catheter. Immediately after bacterial instillation, rats received a single subcutaneous injection of the test compounds. The treatment groups were: control (no treatment), meropenem alone (30 mg/kg in saline), meropenem + Bromisoval (30 mg/kg; as a 1% solution in propylene glycol), and meropenem + dexamethasone (0.5 mg/kg; as a 0.05% solution in propylene glycol). Animal survival was monitored every 12 hours for 48 hours post-infection [1].

[1]. Effects of hypnotic bromovalerylurea on microglial BV2 cells. J Pharmacol Sci. 2017 Jun;134(2):116-123.

[2]. Comparison of the bromureide sedative-hypnotic drugs, bromvaletone (bromisoval) and carbromal, and their chloro analogues in mice. Clin Exp Pharmacol Physiol. 1976 Sep-Oct;3(5):443-7.

2-Bromo-N-carbamoyl-3-methylbutyramide is an N-acylurea, a urea compound in which one hydrogen atom is replaced by a 2-bromo-3-methylbutyryl group. It is both an N-acylurea and an organobromine compound. It has sedative and mild hypnotic effects, but may be toxic.

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Background: Bromisoval is a hypnotic drug that has been repurposed for study due to its anti-inflammatory effects. It has previously been shown to suppress LPS- or interferon-γ-induced phosphorylation of STAT1 and IRFs in microglia [1].
Mechanism of Action (Proposed): This study concludes that the marked anti-inflammatory effects of Bromisoval on BV2 cells are not solely due to NFκB inhibition or weak JAK1/STAT1/IRF1 inhibition. Instead, the effects are likely attributable to a synergistic combination of two actions: inhibition of the JAK1 pathway and a reduction in intracellular ATP (iATP) levels via suppression of mitochondrial metabolic activity. The combination of a JAK1 inhibitor and rotenone (which reduces iATP) mimicked the effect of Bromisoval [1].
Efficacy: In the rat ALI model, Bromisoval in combination with an antibiotic completely prevented death, showing superior efficacy to the antibiotic alone or in combination with the steroid dexamethasone [1].

C6H11BRN2O2

223.07

222

496-67-3

2447

White to off-white solid powder

1.504g/cm3

152 °C

1.514

1.692

2

2

2

11

170

0

BrC([H])(C(N([H])C(N([H])[H])=O)=O)C([H])(C([H])([H])[H])C([H])([H])[H]

CMCCHHWTTBEZNM-UHFFFAOYSA-N

InChI=1S/C6H11BrN2O2/c1-3(2)4(7)5(10)9-6(8)11/h3-4H,1-2H3,(H3,8,9,10,11)

2-bromo-N-carbamoyl-3-methylbutanamide

Isobromyl Bromaral Bromisoval bromovalerylureaBRN1773255 BRN 1773255 BRN-1773255

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 : ~300 mg/mL (~1344.87 mM)

Solubility in Formulation 1: ≥ 7.5 mg/mL (33.62 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (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 75.0 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: ≥ 7.5 mg/mL (33.62 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (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 75.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 7.5 mg/mL (33.62 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 75.0 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.)