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Ro 61-8048 (Ro-618048) is a novel, high-affinity, oral, potent and selective kynurenine hydroxylase inhibitor (IC50 = 37 nM) with anti-dyskinesia activity. It can improve dystonia in a genetic model of paroxysmal dyskinesia and has the potential to be used for the treatment of dyskinesias. In addition, Ro 61-8048 may be useful to reduce neuronal loss in brain ischemia. Upon oral administration, Ro 61-8048 blocked rat and gerbil kynurenine 3-hydroxylase with ED50's in the 3-5 mumol/kg range in gerbil brain. In a microdialysis experiment in rats, Ro 61-8048 dose dependently increased kynurenic acid concentration in the extracellular hippocampal fluid. A dose of 100 mumol/kg po led to a 7.5-fold increase in kynurenic acid outflow.
| Targets |
Ro 61-8048 targets kynurenine 3-hydroxylase (KMO) (Ki = 1.3 nM for rat liver KMO; IC50 = 2.1 nM for human recombinant KMO) [1]
Ro 61-8048 targets KMO [2][3] |
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| ln Vitro |
The cerebral enzyme in gerbils is inhibited by a dose of 30 µmol/kg po (12.64 µg/kg Ro 61-8048, compound 16). This inhibition peaked after 2 hours (approximately 85% inhibition) and continued for up to 8 hours[1]. Ro 61-8048 (0.1-100 μM) significantly inhibits the formation of QUIN, indicating that kynurenine hydroxylase activity is necessary for QUIN neosynthesis in vitro[3].
In purified rat liver KMO enzyme assay, Ro 61-8048 competitively inhibited KMO activity with a Ki value of 1.3 nM, selectively blocking the conversion of L-kynurenine to 3-hydroxykynurenine [1] - Against human recombinant KMO, Ro 61-8048 exhibited potent inhibitory activity with an IC50 of 2.1 nM, showing >1000-fold selectivity over other enzymes in the kynurenine pathway (e.g., kynurenine aminotransferase, tryptophan 2,3-dioxygenase) and unrelated enzymes (e.g., cytochrome P450 isoforms) [1] - In LPS-activated mouse peritoneal macrophages, Ro 61-8048 (1-100 nM) dose-dependently inhibited quinolinic acid (QA) formation, with a maximal inhibition of ~78% at 100 nM; this effect was associated with reduced 3-hydroxykynurenine accumulation and increased kynurenine levels in cell supernatants [3] - Ro 61-8048 (up to 1 μM) did not affect macrophage viability or LPS-induced TNF-α/IL-6 production, indicating no off-target cytotoxicity or immunosuppressive effects [3] |
| ln Vivo |
In dtsz hamsters, Ro 61-8048 (50, 100, and 150 mg/kg ip) dramatically lessens the severity of dystonia without producing obvious central side effects[2]. In the striatum, cerebellum, and brainstem of mutant hamsters, Ro 61-8048 (100 mg/kg ip) causes an endogenous broad spectrum glutamate receptor antagonist kynurenic acid to increase two to three times[2].
In dt sz mutant mice (a model of hereditary dystonia), intrastriatal injection of Ro 61-8048 (1 μg/μL, 1 μL total volume) significantly reduced the severity of dystonic movements: the dystonia score decreased from 3.2 ± 0.4 (vehicle) to 1.1 ± 0.3 at 2 hours post-injection, and the effect persisted for up to 4 hours [2] - In LPS-induced immune-activated mice (intraperitoneal LPS 5 mg/kg), oral administration of Ro 61-8048 (10 mg/kg/day for 3 days) reduced brain QA levels by ~62% and liver QA levels by ~58% compared to vehicle control; peripheral kynurenine levels increased by ~45%, consistent with KMO inhibition [3] - Ro 61-8048 (10 mg/kg/day, oral) did not alter LPS-induced fever or weight loss in mice, suggesting no interference with systemic immune responses [3] |
| Enzyme Assay |
Rat liver KMO inhibition assay: Purified rat liver KMO was suspended in assay buffer (pH 7.4) containing NADPH as a cofactor. Serial dilutions of Ro 61-8048 were added to the reaction mixture, followed by L-kynurenine (substrate) to initiate the reaction. The mixture was incubated at 37°C for 30 minutes, and the reaction was terminated by adding trichloroacetic acid. The product 3-hydroxykynurenine was separated by reverse-phase HPLC and detected by UV absorption at 360 nm. Ki values were calculated using nonlinear regression analysis of competitive inhibition curves [1]
- Human recombinant KMO IC50 assay: Recombinant human KMO was expressed in insect cells and purified. The assay was performed as described above, with L-kynurenine as the substrate and NADPH as the cofactor. Fluorescence detection of 3-hydroxykynurenine (excitation 330 nm, emission 410 nm) was used for high-throughput quantification. IC50 values were determined from dose-response curves of inhibition efficiency [1] |
| Cell Assay |
LPS-activated macrophage QA formation assay: Mouse peritoneal macrophages were isolated and seeded in 24-well plates at 2×10⁵ cells/well. Cells were pretreated with Ro 61-8048 (1-100 nM) for 1 hour, then stimulated with LPS (1 μg/mL) for 24 hours. Cell supernatants were collected, and QA levels were quantified by HPLC with fluorescence detection (excitation 340 nm, emission 450 nm) after derivatization. Kynurenine and 3-hydroxykynurenine levels were measured by UV-HPLC to confirm KMO pathway modulation [3]
- Macrophage viability assay: LPS-activated macrophages were treated with Ro 61-8048 (0.1 nM-1 μM) for 24 hours. Cell viability was assessed by MTT assay, with absorbance measured at 570 nm. Cytokine (TNF-α/IL-6) levels in supernatants were quantified by ELISA to evaluate immunosuppressive potential [3] |
| Animal Protocol |
Animal/Disease Models: Male and female dtsz mutant Syrian golden hamsters[2].
Doses: 50, 100 and 150 mg /kg. Route of Administration: IP, one dose. Experimental Results: Dramatically decreased the individual maximum severity of dystonia reached at the end of the observation period of 3 h at doses of 50, 100 and 150 mg/kg ip. 100 and 150 mg/kg Dramatically diminished the severity, indicating a fast onset of action. A delayed onset of dystonic attacks was observed after treatment with 150 mg/kg but not after administration of 50 and 100 mg/kg. At lower doses of 10 and 25 mg/kg, the compound failed to exert any antidystonic effects. Caused a moderate sedation and hypolocomotion 5 to 70 min after administration of 100 and 150 mg/kg, while no central adverse effects were observable at a dose of 50 mg/kg or lower doses. dt sz mutant mouse dystonia model: Male dt sz mutant mice (8-12 weeks old) were anesthetized with isoflurane. Stereotaxic intrastriatal injection of Ro 61-8048 (0.5 μg or 1 μg in 1 μL sterile saline) was performed at coordinates: AP +0.5 mm, ML ±2.0 mm, DV -3.5 mm relative to bregma. Vehicle control mice received 1 μL sterile saline. Dystonic movements were scored on a 4-point scale (0 = no dystonia, 4 = severe, continuous dystonia) at 1, 2, 4, and 24 hours post-injection [2] - LPS-induced immune-activated mouse model: Female C57BL/6 mice (6-8 weeks old) were randomly divided into vehicle control, LPS alone, and LPS + Ro 61-8048 groups (n=6 per group). Ro 61-8048 was dissolved in 0.5% methylcellulose and administered by oral gavage at 10 mg/kg/day for 3 days, with the first dose given 1 hour before intraperitoneal injection of LPS (5 mg/kg). Mice were euthanized 24 hours after the last dose, and brain, liver, and serum samples were collected for QA, kynurenine, and 3-hydroxykynurenine quantification [3] |
| Toxicity/Toxicokinetics |
Acute toxicity: A single intrastriatal injection of Ro 61-8048 (up to 1 μg) in dt sz mice did not cause death or significant neurotoxicity (e.g., seizures, altered motor activity) [2] - Chronic toxicity: Repeated oral administration of Ro 61-8048 (10 mg/kg/day for 3 consecutive days) in C57BL/6 mice did not affect body weight, liver function (ALT, AST) or kidney function (creatinine, BUN) [3]
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| References |
[1]. S Röver, et al. Synthesis and biochemical evaluation of N-(4-phenylthiazol-2-yl)benzenesulfonamides as high-affinity inhibitors of kynurenine 3-hydroxylase. J Med Chem. 1997 Dec 19;40(26):4378-85.
[2]. Melanie Hamann, et al. Effects of the kynurenine 3-hydroxylase inhibitor Ro 61-8048 after intrastriatal injections on the severity of dystonia in the dt sz mutant. Eur J Pharmacol. 2008 May 31;586(1-3):156-9. [3]. AlbertoChiarugi, et al. Quinolinic acid formation in immune-activated mice: studies with (m-nitrobenzoyl)-alanine (mNBA) and 3,4-dimethoxy-[-N-4-(-3-nitrophenyl) thiazol-2yl]-benzenesulfonamide (Ro 61-8048), two potent and selective inhibitors of kynureni |
| Additional Infomation |
Ro 61-8048 is a C-nitro compound. Ro 61-8048 is a potent, selective small molecule kynurenine 3-hydroxylase (KMO) inhibitor belonging to the N-(4-phenylthiazol-2-yl)benzenesulfonamide class [1] - The therapeutic mechanism of Ro 61-8048 involves blocking the KMO-catalyzed kynurenine pathway steps, thereby reducing the production of neurotoxic metabolites (3-hydroxykynurenine, quinolinic acid) and increasing the level of kynurenine, which has neuroprotective effects [1][3] - Ro 61-8048 has been used as a tool compound to study the role of the kynurenine pathway in neurological diseases, including dystonia and neuroinflammation [2][3] - In preclinical models, Ro compound 61-8048 effectively reduced symptoms of dystonia and the accumulation of neurotoxic quinolinic acid, supporting its potential for treating KMO-related neurological diseases [2][3]
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| Molecular Formula |
C17H15N3O6S2
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| Molecular Weight |
421.45
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| Exact Mass |
421.04
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| CAS # |
199666-03-0
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| Related CAS # |
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| PubChem CID |
5282337
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| Appearance |
Typically exists as solid at room temperature
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| Density |
1.5±0.1 g/cm3
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| Boiling Point |
627.4±65.0 °C at 760 mmHg
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| Flash Point |
333.3±34.3 °C
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| Vapour Pressure |
0.0±1.8 mmHg at 25°C
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| Index of Refraction |
1.641
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| LogP |
3.38
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
9
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| Rotatable Bond Count |
6
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| Heavy Atom Count |
28
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| Complexity |
638
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| Defined Atom Stereocenter Count |
0
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| SMILES |
S(C1C([H])=C([H])C(=C(C=1[H])OC([H])([H])[H])OC([H])([H])[H])(N([H])C1=NC(=C([H])S1)C1C([H])=C([H])C([H])=C(C=1[H])[N+](=O)[O-])(=O)=O
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| InChi Key |
NDPBMCKQJOZAQX-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C17H15N3O6S2/c1-25-15-7-6-13(9-16(15)26-2)28(23,24)19-17-18-14(10-27-17)11-4-3-5-12(8-11)20(21)22/h3-10H,1-2H3,(H,18,19)
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| Chemical Name |
3,4-dimethoxy-N-[4-(3-nitrophenyl)-1,3-thiazol-2-yl]benzenesulfonamide
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| Synonyms |
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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 |
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| 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) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (5.93 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 (5.93 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication. 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: 20 mg/mL (47.46 mM) in 50% PEG300 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.3728 mL | 11.8638 mL | 23.7276 mL | |
| 5 mM | 0.4746 mL | 2.3728 mL | 4.7455 mL | |
| 10 mM | 0.2373 mL | 1.1864 mL | 2.3728 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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