| Size | Price | Stock | Qty |
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| 10mg |
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| 50mg |
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| 100mg |
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| 250mg | |||
| Other Sizes |
| Targets |
Diacylglycerol lipase (DAGL), the enzyme responsible for the synthesis of the endocannabinoid 2-arachidonoylglycerol (2-AG). [3]
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| ln Vitro |
The inhibition of cholinesterase may be the cause of RHC 80267's potentiating effect on acetylcholine: (1) RHC 80267 does not alter the response to carbachol, a carbamoyl derivative of acetylcholine that differs from acetylcholine only because it is resistant to cholinesterase hydrolysis; and (2) the cholinesterase inhibitor neostigmine mimics the effect of RHC 80267, which exhibits no additional effect when the maximum effective concentration of neostigmine is present. Analyzing RHC 80267's impact on cholinesterase activity in brain homogenates revealed that, within the same concentration range (i.e., 1–10 μM), RHC 80267 promotes acetylcholine relaxation while inhibiting this enzyme [2].
In endothelium-intact isolated human pulmonary arteries (hPAs) pre-incubated with RHC80267 (40 μM), the concentration-response curve (CRC) for the vasoconstrictor U46619 (a thromboxane A₂ analog) was shifted leftwards by a factor of 2.5, indicating an enhanced contractile response. This effect was not observed in endothelium-denuded hPAs. [3] In endothelium-intact isolated rat pulmonary arteries (rPAs) pre-incubated with RHC80267 (40 μM), the CRC for U46619 was shifted leftwards by a factor of 2.0, and the maximal contractile response (Rmax) to U46619 was significantly increased by approximately 20% compared to the control group. This effect was absent in endothelium-denuded rPAs. [3] In endothelium-intact isolated rPAs pre-incubated with RHC80267 (40 μM), the CRC for angiotensin II (ANG II) was shifted leftwards by a factor of 3.2, and the maximal contractile response (Rmax) to ANG II was significantly enhanced by approximately 30% compared to the control group. This modulatory effect was not observed in endothelium-denuded rPAs. [3] Pre-incubation with RHC80267 (40 μM) did not modify the vasoconstrictor effect of U46619 in endothelium-denuded hPAs and rPAs, nor did it modify the effect of ANG II in endothelium-denuded rPAs. [3] |
| ln Vivo |
In human and rat pulmonary arteries (hPA and rPA, respectively), angiotensin II (ANG II)-induced contraction is potentiated by RHC 80267 (40 μM) in an endothelium-dependent manner[3]. U46619 is a thromboxane A2 analog.
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| Enzyme Assay |
In Vitro Cholinesterase Activity Assay: Cholinesterase activity was determined using a modified Ellman's method. Rat brain homogenate (50 mg tissue per 1 mL of 0.05 M phosphate buffer, pH 7.2) was preincubated without or with different concentrations of RHC-80267 or neostigmine for 10 minutes at 37°C in 0.05 M phosphate buffer (pH 7.2) containing 0.25 mM DTNB (dithionitrobenzoate, Ellman's reagent). Acetylthiocholine (0.3 mM) was then added, and the change in absorbance was monitored at 412 nm using a spectrophotometer. The percent inhibition of cholinesterase activity was determined by comparing the Δ absorbance per minute values measured in the absence (control) and presence of the inhibitor. [2]
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| Cell Assay |
Preparation of Washed Human Platelets: Platelet-rich plasma was prepared from fresh human whole blood collected in citrate. Washed platelet suspensions were prepared by centrifugation and resuspended in Ca²⁺-free Krebs-Ringer bicarbonate buffer (pH 7.4) to a concentration of 3-5 × 10⁸ platelets/ml. [4]
Platelet Aggregation and Secretion Assay: Aggregation of platelet-rich plasma or washed platelets was measured in an aggregometer or lumi-aggregometer. Agonists (ADP, epinephrine, collagen, arachidonic acid, prostaglandin H₂) were added to stirred samples. RHC-80267 (dissolved in DMSO) or vehicle was added just prior to a 2-minute preincubation at 37°C. Aggregation was monitored by light transmission, and ATP release was measured simultaneously in the lumi-aggregometer. [4] [³H]Arachidonic Acid Release and Metabolism Assay: Washed platelets (3 × 10⁸/ml) were labeled with 2 μCi/ml [³H]arachidonic acid for 25 min at 30°C, then washed and resuspended. Labeled platelets were preincubated with 250 μM RHC-80267 or DMSO for 5 min at 37°C, then stimulated with 25 μg/ml collagen. Samples were taken at various time points and quenched in cold methanol. Lipids were extracted with chloroform and separated by thin-layer chromatography. Areas corresponding to arachidonic acid, its metabolites (TxB₂, 12-HETE, HHT), diacylglycerol, and monoacylglycerol were cut out and counted for radioactivity. Phospholipids were separated on SG-81 paper to determine radioactivity in each phospholipid class. [4] U-46619 Reversal Experiment: The same protocol as above was used, but U-46619 (5.7 μM) was added concurrently with collagen to platelets preincubated with or without 250 μM RHC-80267. Samples were taken after 2 minutes for lipid extraction and analysis. [4] |
| Animal Protocol |
Mesenteric Artery Contraction/Relaxation Protocol:** Male Wistar-Kyoto (WKY) rats (14 weeks old, 320-350 g) were anesthetized with diethyl ether and killed by decapitation. The superior mesenteric artery was removed and cleaned. Segments (approx. 2 mm long) were mounted in a wire myograph for isometric tension recording in physiological solution gassed with 95% O2-5% CO2, containing 10 μM indomethacin. After equilibration, vessels were set to a tension equivalent to that at 0.9 times the diameter at 100 mmHg. Endothelium integrity was tested with 1 μM acetylcholine. For experiments, arteries were preincubated with or without RHC-80267 (0.1-10 μM) for 30 minutes. Contraction was then evoked either by 100 mM KCl solution (for testing NO-dependent relaxation, with 1 μM phentolamine) or by 0.5 μM noradrenaline (for testing EDHF-type relaxation or relaxation to cromakalim, with 100 μM L-NOArg present). Once contraction reached a plateau, relaxations were evoked by cumulative addition of the relaxing agent (acetylcholine, carbachol, SNAP, or cromakalim). [2]
Mesenteric Artery Contraction/Relaxation Protocol: Male Wistar-Kyoto (WKY) rats (14 weeks old, 320-350 g) were anesthetized with diethyl ether and killed by decapitation. The superior mesenteric artery was removed and cleaned. Segments (approx. 2 mm long) were mounted in a wire myograph for isometric tension recording in physiological solution gassed with 95% O2-5% CO2, containing 10 μM indomethacin. After equilibration, vessels were set to a tension equivalent to that at 0.9 times the diameter at 100 mmHg. Endothelium integrity was tested with 1 μM acetylcholine. For experiments, arteries were preincubated with or without RHC-80267 (0.1-10 μM) for 30 minutes. Contraction was then evoked either by 100 mM KCl solution (for testing NO-dependent relaxation, with 1 μM phentolamine) or by 0.5 μM noradrenaline (for testing EDHF-type relaxation or relaxation to cromakalim, with 100 μM L-NOArg present). Once contraction reached a plateau, relaxations were evoked by cumulative addition of the relaxing agent (acetylcholine, carbachol, SNAP, or cromakalim). [2] |
| References |
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| Additional Infomation |
N-[6-[[(cyclohexylimino)oxomethyl]amino]hexyl]carbamate (cyclohexylimino) ester is a carbamate and also an organic nitrogen compound.
RHC80267 (O,O'-[1,6-hexanedylbis(iminocarbonyl)]dioxime cyclohexanone) is used in this study as a pharmacological tool to inhibit diacylglycerol lipase (DAGL), the enzyme that synthesizes 2-AG. Its application aims to elucidate the role of endogenously produced 2-AG in modulating vasoconstriction. The study's findings with RHC80267 support the hypothesis that U46619 and ANG II stimulate the rapid, endothelial-dependent synthesis of 2-AG via DAGL, which then acts as a negative feedback mechanism to attenuate vasoconstriction. In the experiments, a concentration of 40 μM RHC80267 was used, and tissues were pre-incubated with it for 30 minutes before the construction of concentration-response curves to vasoconstrictors. It was dissolved in DMSO (0.1% vol/vol). [3] |
| Molecular Formula |
C20H34N4O4
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|---|---|
| Molecular Weight |
394.52
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| Exact Mass |
394.257
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| CAS # |
83654-05-1
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| PubChem CID |
5063
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| Appearance |
White to off-white solid powder
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| Density |
1.2±0.1 g/cm3
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| Index of Refraction |
1.571
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| LogP |
3.12
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
6
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| Rotatable Bond Count |
11
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| Heavy Atom Count |
28
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| Complexity |
484
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| Defined Atom Stereocenter Count |
0
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| InChi Key |
RXSVYGIGWRDVQC-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C20H34N4O4/c25-19(27-23-17-11-5-3-6-12-17)21-15-9-1-2-10-16-22-20(26)28-24-18-13-7-4-8-14-18/h1-16H2,(H,21,25)(H,22,26)
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| Chemical Name |
(cyclohexylideneamino) N-[6-[(cyclohexylideneamino)oxycarbonylamino]hexyl]carbamate
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| Synonyms |
RHC80267 RHC-80267 RHC 80267
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| HS Tariff Code |
2934.99.9001
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| 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)
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| Solubility (In Vitro) |
DMSO : ~25 mg/mL (~63.37 mM)
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|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (6.34 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 25.0 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. Solubility in Formulation 2: ≥ 2.5 mg/mL (6.34 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 25.0 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. View More
Solubility in Formulation 3: ≥ 2.5 mg/mL (6.34 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.5347 mL | 12.6736 mL | 25.3473 mL | |
| 5 mM | 0.5069 mL | 2.5347 mL | 5.0695 mL | |
| 10 mM | 0.2535 mL | 1.2674 mL | 2.5347 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.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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