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Purity: ≥98%
SR8278 (SR-8278; SR 8278) is a novel, potent, synthetic and competitive Rev-Erbα antagonist with the potential for treating Duchenne muscular dystrophy (DMD). In mdx mice SR8278 increased lean mass and muscle function, and decreased muscle fibrosis and muscle protein degradation. SR8278 inhibits Rev-Erbα transcriptional repression with an EC50 of 0.47 μM. REV-ERBα is a member of the nuclear receptor superfamily that functions as a receptor for the porphoryin heme. REV-ERBα suppresses transcription of its target genes in a heme-dependent manner. SR8278 also blocks activity of Rev-Erbα agonist GSK 4112 in HEK293 cells. SR8278 increases expression of glucose-regulating genes, G6Pase and PEPCK in HepG2 cells.
Targets |
REV-ERBα(EC50 = 0.47 μM)
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ln Vitro |
SR8278 exhibits an EC50 of 0.47μM for REV-ERBα transcriptional inhibition inhibitory activity[1].SR8278 is structurally similar to the agonist but blocks the ability of the GSK4112 to enhance REV-ERBα-dependent repression in a cotransfection assay. Additionally, whereas GSK4112 suppresses the expression of REV-ERBα target genes involved in gluconeogenesis, SR8278 stimulates the expression of these genes. Thus, SR8278 represents a unique chemical tool for probing REV-ERB function and may serve as a point for initiation of further optimization to develop REV-ERB antagonists with the ability to explore circadian and metabolic functions[1].
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ln Vivo |
In 6-OHDA-lesioned mice, SR8278 (slow microinjection; 20 μg/mouse) restores emotion-related behaviors to their circadian rhythms and has antidepressant and anxiolytic effects in a circadian time-dependent manner [1]. ??Slow microinjection (SR8278; 20 μg/mouse) enriches R/N motifs recognized by REV-ERBα and NURR1 while restoring binding activity of both proteins to the tyrosine hydroxylase promoter [1].
Pharmacological Inhibition of REV-ERBα Activity with SR8278 Restored Circadian Mood-Related Behaviors in PD Mouse Models. SR8278 Microinjection Alters Remaining DAergic Neuron-Specific Transcription Levels of Rev-erbα and Nurr1 in the VTA. SR8278 Microinjection Restores Antagonistic Crosstalk of REV-ERBα and NURR1 Binding Activity to TH Promoter and TH Protein Levels in VTA at Dawn. SR8278 Treatment Induces Enrichments of REV-ERBα and NURR1 Binding Motifs at Dawn.[3] |
Enzyme Assay |
Stability [2]
Long-term (−80 °C for 10 days) and three freeze-thaw cycle stabilities of SR8278 in plasma were measured. Stabilities were expressed as the concentrations after different operations to the concentration at time zero. |
Cell Assay |
HEK293 cells were maintained in Dulbecco’s modified Eagles medium (DMEM) supplemented with 10% fetal bovine serum at 37 °C under 5% CO2. HepG2 cells were maintained and routinely propagated in minimum essential medium supplemented with 10% fetal bovine serum at 37 °C under 5% CO2. 24 h prior to transfection, HepG2 cells were plated in 96-well plates at a density of 15 × 103 cells/well. Transfections were performed using LipofectamineTM 2000 (Invitrogen). Sixteen h post-transfection, the cells were treated with vehicle or compound. 24 h post-treatment, the luciferase activity was measured using the Dual-GloTM luciferase assay system. The values indicated represent the means ± S.E. from four independently transfected wells. The experiments were repeated at least three times. The REV-ERBα and reporter constructs have been previously described [1].
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Animal Protocol |
Animal/Disease Models: 6-OHDA injured mice [1]
Doses: 20 μg/mouse Route of Administration: slow microinjection; 20 μg/mouse Experimental Results: The emotion-related behavioral defects of 6-OHDA injured mice were restored. Altered remaining DAergic neuron-specific REV-ERBα and Nurr1 transcript levels in the VTA. Restoration of REV-ERBα and NURR1 binding activity is associated with antagonistic crosstalk of TH promoter and TH protein levels in the VTA. Dawn induces enrichment of REV-ERBα and NURR1 binding motifs. Local Injection of SR8278 [3] The local injection of SR8278 into the midbrain towards the VTA was performed 3 h before each behavioral test under a dim red light. We followed the time-regiment, for handling animal care and slow microinjection of SR8278 to the VTA 3 h before behavioral tests, as shown previously. SR8278 was dissolved in ethanol to a concentration of 50 µg/µL. SR8278 (20 µg/mouse) or the corresponding vehicle (ethanol) was directly microinfused into the VTA using a 33-gauge injector cannula attached to a 10-µL Hamilton syringe at a rate of 0.1 µL/min. For the microinfusion, mice were anesthetized with sodium pentobarbital (50 mg/kg, i.p.), mounted on a stereotaxic apparatus, and unilaterally implanted with a 26-guage stainless steel cannula into the midbrain towards VTA (AP −3.2 mm, ML −0.5 mm, DV −3.5 mm). A 32-gauge dummy cannula was inserted into each guide cannula to prevent clogging. Once the jewelry screws were implanted in the skull as anchors, the whole assembly was affixed to the skull with resin cement.[3] Pharmacokinetic studies[2] Before SR8278 administration, a cannula was introduced into the jugular vein for injection of formulations and blood collection followed by previously published procedures. The rats were anesthetized by anesthesia cocktail (Ketamine 50 mg/mL, Xylazine 3.3 mg/mL and Acetopromazine 3.3 mg/mL) prior to the jugular vein surgery and administrated carprofen subcutaneously after the surgery [2]. Rats were dosed with SR8278 at 2 mg/kg through jugular vein cannula. Blood samples (200 μL) were withdrawn at 5, 10, 15, 30, 45 min, and 1, 1.5, 2, 4, and 6 h post dose from jugular vein cannula. The volume of blood removed at each sampling time was replaced with an equal volume of saline. And the blood samples were immediately centrifuged at 4 °C to separate the plasma fraction from the blood cells, and the plasma samples were stored at −80 °C until analysis.[2] |
ADME/Pharmacokinetics |
The PK parameters of SR8278 were obtained by the 3-compartmental model, using WinNonlin 3.3, and the results are shown in Table 4. The predicted concentrations for each time point were plotted in Fig. 5. As it is shown, the predicted concentrations are close to those detected values, which indicated the 3-compartment PK model fits the data very well. After i.v. administration, the initial concentrations (C0) of SR8278 in normal rats and diabetic rats were 2410.25 ± 202.36 vs. 3742.11 ± 1300.21 ng/mL, respectively. The low drug concentration in the plasma could be explained by its rapid distribution into other compartments, except the central compartment. The large distribution volumes (V) of SR8278 in normal rats and diabetic rats were 44401.58 ± 2106.63 and 43516.94 ± 22982.75 mL/kg, respectively. Moreover, it was also noted that the elimination half-lives (t1/2) of SR8278 were really short, which were only 0.17 ± 0.084 (normal rats) and 0.11 ± 0.04 h (diabetic rats), because of the fast elimination rate constant (K10). The AUC values of SR8278 were 608.33 ± 295.25 vs. 598.59 ± 276.92 ng·h/mL, in normal rats and diabetic rats, respectively. The limited blood exposure and fast clearance may represent a major problem in the clinical application of SR8278.[2]
The stabilities of SR8278 in plasma were evaluated at −80 °C for 10 days, and after three freeze-thaw cycles (Table 3). The recoveries of all samples were between 90.82 and 95.75% after various stability tests, which demonstrated that the SR8278 in plasma samples was stable under storage conditions. In addition, we also determined the stability of SR8278 in 50% methanol solution after 24 h storage at room temperature. The results showed that SR8278 was stable in 50% methanol solution for more than 24 h (Table 3), thus allowing adequate time for the completion of the assay. The good results of the method validation parameters, including recovery, viability, also demonstrated the stability of SR8278 during sample preparation and until analysis was completed. [2] The validated analytical method was used to identify the PK behaviors of SR8278 in normal rats and diabetic rats. The mean plasma concentration-time curves of SR8278 in the two groups of rats are shown in Fig. 5. Following administration of SR8278 (2 mg/kg, i.v.), the plasma concentrations of SR8278 in the diabetic rats were slightly higher than those in normal rats at each time point. However, these differences were not statistically significant, suggesting that diabetes has no effect on the in vivo behavior of SR8278.[2] |
References |
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Additional Infomation |
There is a relationship between circadian rhythm and metabolic disorders. The active agent, SR8278, could competitively bind to and inhibit the nuclear receptor, Rev-erb (a major modulator of mammalian circadian clock system), to regulate the metabolism in organisms. However, we had limited knowledge of the pharmacokinetic (PK) characteristics of SR8278. Here, we describe a sensitive and reproducible ultra-performance liquid chromatography-tandem mass spectrometric (UPLC-MS/MS) method to quantify SR8278 in vivo. The linearity range and the limit of quantification (LOQ) for SR8278 were 30-3000 ng/mL and 6 ng/mL, respectively. The inter-day and intra-day variability were within 10%. This UPLC-MS/MS method was successfully used to characterize the PK behaviors of SR8278 in normal and diabetic rats after intravenous (i.v.) injection at a dosage of 2mg/kg. No significant differences were observed in the PK parameters of SR8278 in normal and diabetic rats. Specifically, the values of areas under plasma concentration time curves (AUC), initial plasma concentrations (C0), elimination half-lives (t1/2), and clearances (CL) were 608.33 ± 295.25 vs. 598.59 ± 276.92 ng·h/mL, 2410.25 ± 202.36 vs. 3742.11 ± 1300.21 ng/mL, 0.17 ± 0.08 vs. 0.11 ± 0.04 h, 3330.83 ± 1609.48 vs. 3364.81 ± 1111.38 mL/kg·h for SR8278 in normal rats vs. diabetic rats, respectively. In conclusion, a UPLC-MS/MS method was successfully developed and validated for the first time, with a wide linearity range, low LOQ, small sample volume (10 μL), rapid analysis (4 min) and excellent recoveries (>80%). It was also used to clarify the PK characteristics of SR8378 in rats. The same PK behaviors of SR8278 in normal and diabetic rats showed that diabetes may have little or no effect on the disposition, metabolism and/or elimination in vivo, which may be of great importance for future clinical studies. [2]
Parkinson's disease is a neurodegenerative disease characterized by progressive dopaminergic neuronal loss. Motor deficits experienced by patients with Parkinson's disease are well documented, but non-motor symptoms, including mood disorders associated with circadian disturbances, are also frequent features. One common phenomenon is "sundowning syndrome," which is characterized by the occurrence of neuropsychiatric symptoms at a specific time (dusk), causing severe quality of life challenges. This study aimed to elucidate the underlying mechanisms of sundowning syndrome in Parkinson's disease and their molecular links with the circadian clock. We demonstrated that 6-hydroxydopamine (6-OHDA)-lesioned mice, as Parkinson's disease mouse model, exhibit increased depression- and anxiety-like behaviors only at dawn (the equivalent of dusk in human). Administration of REV-ERBα antagonist, SR8278, exerted antidepressant and anxiolytic effects in a circadian time-dependent manner in 6-OHDA-lesioned mice and restored the circadian rhythm of mood-related behaviors. 6-OHDA-lesion altered DAergic-specific Rev-erbα and Nurr1 transcription, and atypical binding activities of REV-ERBα and NURR1, which are upstream nuclear receptors for the discrete tyrosine hydroxylase promoter region. SR8278 treatment restored the binding activities of REV-ERBα and NURR1 to the tyrosine hydroxylase promoter and the induction of enrichment of the R/N motif, recognized by REV-ERBα and NURR1, as revealed by ATAC-sequencing; therefore, tyrosine hydroxylase expression was elevated in the ventral tegmental area of 6-OHDA-injected mice, especially at dawn. These results indicate that REV-ERBα is a potential therapeutic target, and its antagonist, SR8278, is a potential drug for mood disorders related to circadian disturbances, namely sundowning syndrome, in Parkinson's disease. [3] |
Molecular Formula |
C18H19NO3S2
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Molecular Weight |
361.47
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Exact Mass |
361.081
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Elemental Analysis |
C, 59.81; H, 5.30; N, 3.87; O, 13.28; S, 17.74
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CAS # |
1254944-66-5
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Related CAS # |
1254944-66-5
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PubChem CID |
53393127
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Appearance |
White to light yellow solid powder
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LogP |
3.538
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Hydrogen Bond Donor Count |
0
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Hydrogen Bond Acceptor Count |
5
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Rotatable Bond Count |
5
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Heavy Atom Count |
24
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Complexity |
473
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Defined Atom Stereocenter Count |
0
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InChi Key |
UIEBLUZPSFAFOC-UHFFFAOYSA-N
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InChi Code |
InChI=1S/C18H19NO3S2/c1-3-22-18(21)14-10-12-6-4-5-7-13(12)11-19(14)17(20)15-8-9-16(23-2)24-15/h4-9,14H,3,10-11H2,1-2H3
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Chemical Name |
ethyl 2-{[5-(methylsulfanyl)thiophen-2-yl]carbonyl}-1,2,3,4-tetrahydroisoquinoline-3-carboxylate
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Synonyms |
SR8278; SR 8278; ethyl 2-{[5-(methylsulfanyl)thiophen-2-yl]carbonyl}-1,2,3,4-tetrahydroisoquinoline-3-carboxylate; ethyl 2-(5-methylsulfanylthiophene-2-carbonyl)-3,4-dihydro-1H-isoquinoline-3-carboxylate; CHEMBL4754504; ethyl 2-(5-(methylthio)thiophene-2-carbonyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxylate; SR-8278
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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 (~276.64 mM)
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Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (6.92 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 (6.92 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 (6.92 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 | 2.7665 mL | 13.8324 mL | 27.6648 mL | |
5 mM | 0.5533 mL | 2.7665 mL | 5.5330 mL | |
10 mM | 0.2766 mL | 1.3832 mL | 2.7665 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.