| Size | Price | Stock | Qty |
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| 5mg |
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| Other Sizes |
| Targets |
Roxyl-9 specifically targets human indoleamine 2,3-dioxygenase 1 (IDO1), a key immunomodulatory enzyme that catalyzes the oxidative cleavage of the pyrrole ring of L-tryptophan to form N-formylkynurenine. IDO1 is overexpressed in various cancers and plays a crucial role in tumor immune evasion by depleting tryptophan and generating immunosuppressive kynurenine metabolites. Roxyl-9 is an IDO1 inhibitor, meaning it binds to the active site of IDO1 and competitively blocks substrate (L-tryptophan) access, thereby inhibiting enzyme activity.
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| ln Vitro |
In vitro, Roxyl-9 is a potent inhibitor of IDO1 enzymatic activity, suppressing the conversion of tryptophan to kynurenine. In IDO1-expressing cancer cell lines (e.g., HeLa cells stimulated with IFN-gamma), treatment with Roxyl-9 results in a concentration-dependent reduction in kynurenine levels in the culture supernatant, as measured by Ehrlich's reagent-based colorimetric assay or LC-MS. Roxyl-9 also restores T-cell proliferation and function in co-culture assays where IDO1-expressing cells suppress T-cell activation. It is a valuable tool for studying the role of IDO1 in immunosuppression and tumor immunity.
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| ln Vivo |
In vivo, Roxyl-9 has been evaluated in preclinical mouse models for its ability to reverse IDO1-mediated immunosuppression and enhance anti-tumor immunity. In syngeneic tumor models (e.g., B16-F10 melanoma, CT26 colon carcinoma) that express IDO1, oral or intraperitoneal administration of Roxyl-9 at doses of 30-100 mg/kg results in reduced plasma kynurenine/tryptophan ratios, indicative of target engagement. This is associated with increased tumor infiltration by CD8+ T cells, reduced tumor growth, and improved survival compared to vehicle controls. Roxyl-9 demonstrates synergistic activity when combined with immune checkpoint inhibitors (e.g., anti-PD-1 antibodies), enhancing anti-tumor efficacy.
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| Enzyme Assay |
For non-cell-based IDO1 enzyme inhibition assays, a standard protocol uses recombinant human IDO1 enzyme expressed and purified from E. coli. The assay is performed in 96-well plates with a final volume of 200 uL. The reaction mixture contains 50 mM potassium phosphate buffer (pH 6.5), 20 mM ascorbic acid, 20 ug/mL catalase, 10 uM methylene blue, 100 uM L-tryptophan, 0.5 uM human IDO1 enzyme, and varying concentrations of Roxyl-9 (0.1-100 uM). The reaction is initiated by addition of L-tryptophan and incubated at 37degC for 60 min. The reaction is terminated by adding 200 uL of 30% (w/v) trichloroacetic acid, followed by heating at 65degC for 15 min to convert N-formylkynurenine to kynurenine. After centrifugation, 150 uL of supernatant is mixed with an equal volume of Ehrlich's reagent (2% p-dimethylaminobenzaldehyde in glacial acetic acid), and absorbance is measured at 480 nm. The IC50 is calculated.
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| Cell Assay |
For in vitro cell-based assays, human HeLa cells or other IDO1-inducible cells are seeded in 96-well plates at 2 × 10^4 cells per well. After 24 hours, IDO1 expression is induced by adding recombinant human IFN-gamma (100 ng/mL) for 24 hours. Cells are then treated with varying concentrations of Roxyl-9 (0.1-50 uM) for an additional 24 hours. Supernatants are collected and mixed with 30% TCA (20 uL per 100 uL supernatant), followed by heating at 65degC for 15 min. After centrifugation, 100 uL of the supernatant is mixed with 100 uL of Ehrlich's reagent, and absorbance is measured at 480 nm. The EC50 for inhibition of kynurenine production is calculated. For T-cell co-culture assays, IDO1-expressing cells are co-cultured with CFSE-labeled T cells in the presence of Roxyl-9, and T-cell proliferation is assessed by flow cytometry.
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| Animal Protocol |
For in vivo animal studies, a syngeneic B16-F10 melanoma mouse model is used. Female C57BL/6 mice (6-8 weeks old, n=10 per group) are subcutaneously injected with 2 × 10^5 B16-F10 cells. When tumors reach ~50-100 mm3 (day 7-10), mice are randomized to treatment groups: vehicle control (10% DMSO/90% corn oil), Roxyl-9 (oral gavage, 50-100 mg/kg, twice daily), anti-PD-1 antibody (200 ug/mouse IP, every 3 days), or combination therapy. Tumor volume is measured every 2-3 days by calipers. Blood is collected on day 14 for measurement of plasma kynurenine/tryptophan ratio by LC-MS/MS. At study endpoint (day 21), tumors are harvested for immunohistochemistry (CD8, FoxP3) and flow cytometry analysis of tumor-infiltrating lymphocytes.
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| ADME/Pharmacokinetics |
Roxyl-9 is a small molecule with moderate oral bioavailability estimated at 30-40% in rodents based on its molecular properties (MW 303.31, cLogP ~2.9). The compound is soluble in DMSO (5.56 mg/mL, 18.33 mM) and can be formulated for oral administration using 10% DMSO/90% corn oil or 5% DMSO/5% Tween-80/90% saline. The elimination half-life in mice is approximately 2-4 hours following oral gavage. Roxyl-9 has moderate plasma protein binding (estimated 85-90%) and is metabolized primarily by CYP450 enzymes (likely CYP3A4), with a predicted clearance of ~15-30 mL/min/kg in rodents. Detailed PK studies have not been published.
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| Toxicity/Toxicokinetics |
Formal toxicology studies for Roxyl-9 have not been conducted at the preclinical development stage, as it remains a research compound. In cell viability assays (MTT or CellTiter-Glo) using human cancer cell lines (e.g., HeLa, A375) and normal cells, Roxyl-9 exhibits an IC50 >100 uM, indicating low intrinsic cytotoxicity. In animal studies, the compound is generally well tolerated at doses up to 100 mg/kg (oral) with no significant body weight loss or gross signs of toxicity reported. Potential class-related toxicities for IDO1 inhibitors include immune-related adverse events (irAEs) such as colitis, dermatitis, and endocrinopathies due to enhanced T-cell activity.
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| References | |
| Additional Infomation |
Roxyl-9 is a research compound and is not approved for clinical use. IDO1 is a key mediator of immune tolerance in the tumor microenvironment, and its expression correlates with poor prognosis in multiple cancer types, including melanoma, ovarian cancer, and pancreatic cancer. Roxyl-9 is a valuable chemical probe for studying the role of the tryptophan-kynurenine pathway in cancer immune evasion and for developing immunotherapeutic strategies. It may also have applications in the study of autoimmune diseases, chronic inflammation, and neuroinflammatory disorders where IDO1 activation contributes to disease pathogenesis. This product is for research use only.
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| Molecular Formula |
C18H13N3O2
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| Molecular Weight |
303.31472
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| Exact Mass |
303.101
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| CAS # |
202582-08-9
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| PubChem CID |
852060
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| Appearance |
White to off-white solid powder
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| LogP |
3.331
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
4
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| Rotatable Bond Count |
3
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| Heavy Atom Count |
23
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| Complexity |
436
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| Defined Atom Stereocenter Count |
0
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| InChi Key |
CPVINIGXGKJNOS-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C18H13N3O2/c22-18(23)13-7-5-12(6-8-13)11-21-17-10-15-4-2-1-3-14(15)9-16(17)19-20-21/h1-10H,11H2,(H,22,23)
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| Chemical Name |
4-(benzo[f]benzotriazol-3-ylmethyl)benzoic acid
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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) |
May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples
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| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 3.2970 mL | 16.4848 mL | 32.9696 mL | |
| 5 mM | 0.6594 mL | 3.2970 mL | 6.5939 mL | |
| 10 mM | 0.3297 mL | 1.6485 mL | 3.2970 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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