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| Targets |
DO34 specifically targets the enzymes diacylglycerol lipase α (DAGLα) and β (DAGLβ). DAGLα is predominantly expressed in the brain, where it is the primary enzyme responsible for 2-AG synthesis in neurons. DAGLβ is more widely expressed in peripheral tissues and immune cells, playing a role in the immune system and other functions. By inhibiting both isoforms, DO34 effectively blocks the production of 2-AG, a full agonist of the cannabinoid receptors CB1 and CB2. This inhibition disrupts retrograde endocannabinoid signaling, which is crucial for modulating synaptic transmission, plasticity, and various neural processes. The selectivity of DO34 for DAGLα/β over other serine hydrolases is a key feature that minimizes off-target effects and makes it a powerful tool for dissecting the specific roles of these enzymes.
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| ln Vitro |
Using a real-time, fluorescence-based assay with a natural substrate, DO34 is a highly potent, selective, and centrally active inhibitor of DAGL expressed by recombinant human DAGLα. It has also been established that DO34, with an IC50 of 3–8 nM, is a strong inhibitor of DAGLβ[1].
DO34 demonstrates exceptional potency and selectivity in vitro. It inhibits DAGLα with an IC₅₀ of 6 nM, as determined using a real-time, fluorescence-based natural substrate assay with membrane lysates from HEK293T cells expressing recombinant human DAGLα. It also potently inhibits DAGLβ with an IC₅₀ ranging from 3 to 8 nM. This high level of potency, in the low nanomolar range, allows for effective inhibition of 2-AG synthesis at very low compound concentrations. The compound's in vitro activity is characterized by its ability to completely block the conversion of the substrate SAG to 2-AG. Its selectivity has been confirmed against other serine hydrolases, ensuring that its effects are specifically due to DAGL inhibition. DO34's mechanism is covalent and irreversible, leading to sustained inhibition of the enzyme. |
| ln Vivo |
Compound 39, DO34, inhibits the common cannabinoid CB1 receptor-mediated behavior of mice's fasting-induced refeeding. Compound 39, DO34, lowers 2-AG levels in the brain in a time- and dose-dependent manner [2]. The activation of complement CB1 is blocked by DO34. In MAGL-TKO mice, AM251 markedly raises basal PF-EPSCs; the DAGL inhibitor DO34 reverses AM251's impact [3].
In vivo, DO34 has been shown to be centrally active, effectively crossing the blood-brain barrier to inhibit DAGL in the brain. It has been demonstrated to reduce brain 2-AG levels in a dose- and time-dependent manner. One of the key phenotypic effects of DO34 in mice is its ability to impair fear extinction, a behavior that is typically mediated by cannabinoid CB1 receptor signaling. Furthermore, DO34 prevents fasting-induced refeeding in mice, which is a characteristic CB1-receptor-mediated behavior. Studies have shown that DO34 can block tonic CB1 activation, and it reverses the effects of AM251 (a CB1 inverse agonist) in MAGL-TKO mice. These in vivo studies highlight the crucial role of DAGL-mediated 2-AG synthesis in regulating behavior and central nervous system function. |
| Enzyme Assay |
The in vitro enzyme assay for DO34 typically uses a real-time, fluorescence-based natural substrate assay. In this setup, membrane lysates from HEK293T cells expressing recombinant human DAGLα are used as the enzyme source. The assay employs a fluorescently labeled substrate that mimics the natural substrate of DAGL. Upon cleavage by the active enzyme, the substrate releases a fluorophore, resulting in an increase in fluorescence that can be monitored in real-time. DO34 is incubated with the enzyme for a period, and the reaction is initiated by the addition of the substrate. The initial rate of fluorescence increase, which is proportional to enzyme activity, is measured for various concentrations of the inhibitor. A control without the inhibitor is used to determine 100% activity. The data is then plotted to generate a dose-response curve and calculate the IC₅₀ value.
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| Cell Assay |
In vitro cell-based assays for DO34 typically measure the production of 2-AG in cells that express DAGL, such as primary neurons or cell lines. Cells are treated with DO34 for a specified period, often in the presence of a stimulus that activates 2-AG production. Following treatment, the levels of 2-AG in the cell culture medium or cell lysates are quantified using liquid chromatography-tandem mass spectrometry (LC-MS/MS). This method provides a sensitive and specific measurement of the endocannabinoid. The reduction in 2-AG levels in the presence of DO34 is a direct measure of DAGL inhibition in a cellular context. These experiments are crucial for confirming that the biochemical inhibition observed in enzyme assays translates to a functional blockade of 2-AG synthesis in living cells.
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| Animal Protocol |
In vivo animal studies with DO34 are typically conducted in mouse models. One common protocol involves administering DO34 via intraperitoneal (i.p.) or oral (p.o.) injection to evaluate its effects on behavior and brain biochemistry. For example, to study fear extinction, mice are subjected to a fear conditioning paradigm where they learn to associate a tone with a foot shock. The next day, the mice are treated with DO34 and then exposed to the tone without the shock to measure the extinction of the fear response. To assess its effect on 2-AG levels, mice are treated with DO34 and sacrificed at various time points, and their brain tissue is harvested for 2-AG quantification by LC-MS/MS. These studies are essential for understanding the functional role of DAGL and 2-AG in vivo.
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| ADME/Pharmacokinetics |
DO34 has a molecular weight of 531.53 and is characterized by its central activity, indicating it can cross the blood-brain barrier. Its logP is 5.1, suggesting high lipophilicity, which is consistent with its ability to penetrate the CNS. The compound is soluble in DMSO at ≥100 mg/mL. Detailed pharmacokinetic parameters such as half-life, Cmax, and oral bioavailability are not extensively reported in public literature, but its efficacy in behavioral models after systemic administration confirms adequate exposure. Its irreversible, covalent mechanism of action suggests a long duration of effect, as new enzyme synthesis would be required to restore activity.
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| Toxicity/Toxicokinetics |
Comprehensive toxicity data for DO34 is not publicly available. As a potent, irreversible inhibitor of a key enzyme in the endocannabinoid system, there is potential for on-target effects, including those related to the disruption of normal endocannabinoid signaling. The compound is classified as a research tool and is not intended for human use. Standard laboratory safety precautions should be followed when handling DO34. Any future development as a therapeutic would require extensive preclinical toxicology studies to assess its safety profile, including genotoxicity, acute and chronic toxicity, and effects on the central nervous and immune systems.
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| References |
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| Additional Infomation |
DO34 is a state-of-the-art research tool for probing the biology of the endocannabinoid system. Its high potency and selectivity for DAGLα/β make it the inhibitor of choice for studying the physiological and pathological roles of 2-AG. It is widely used in neuroscience research to investigate the role of endocannabinoids in learning, memory, fear, pain, and reward. The compound is also used in immunology to study the role of DAGLβ in immune cell function. It has not entered clinical trials and does not have FDA approval for any indication.
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| Molecular Formula |
C26H28F3N5O4
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|---|---|
| Molecular Weight |
531.526836395264
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| Exact Mass |
531.209
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| CAS # |
1848233-58-8
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| Related CAS # |
DO34 analog;2098969-71-0
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| PubChem CID |
129188708
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| Appearance |
Off-white to light yellow solid powder
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| LogP |
5.1
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| Hydrogen Bond Donor Count |
0
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| Hydrogen Bond Acceptor Count |
9
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| Rotatable Bond Count |
6
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| Heavy Atom Count |
38
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| Complexity |
810
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| Defined Atom Stereocenter Count |
0
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| SMILES |
FC(OC1C=CC(=CC=1)C1=CN(C(N2CCN(C(=O)OC(C)(C)C)C[C@H]2CC2C=CC=CC=2)=O)N=N1)(F)F
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| InChi Key |
CQGMWUWJVBKTRM-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C26H28F3N5O4/c1-25(2,3)38-24(36)32-13-14-33(20(16-32)15-18-7-5-4-6-8-18)23(35)34-17-22(30-31-34)19-9-11-21(12-10-19)37-26(27,28)29/h4-12,17,20H,13-16H2,1-3H3
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| Chemical Name |
tert-butyl 3-benzyl-4-[4-[4-(trifluoromethoxy)phenyl]triazole-1-carbonyl]piperazine-1-carboxylate
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| Synonyms |
DO 34; DO-34; DO34
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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 : ≥ 100 mg/mL (~188.14 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 10 mg/mL (18.81 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 100.0 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: ≥ 10 mg/mL (18.81 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 100.0 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: ≥ 10 mg/mL (18.81 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 | 1.8814 mL | 9.4068 mL | 18.8136 mL | |
| 5 mM | 0.3763 mL | 1.8814 mL | 3.7627 mL | |
| 10 mM | 0.1881 mL | 0.9407 mL | 1.8814 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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