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| Targets |
IDO2-IN-1 specifically targets indoleamine 2,3-dioxygenase 2 (IDO2), an enzyme closely related to IDO1 but with distinct expression patterns and lower catalytic activity. IDO2 is a heme-containing enzyme that catalyzes the first and rate-limiting step of tryptophan degradation along the kynurenine pathway. IDO2 has been implicated in the pathogenesis of autoimmune diseases, including rheumatoid arthritis and inflammatory bowel disease. IDO2-IN-1 is an orally active inhibitor with an IC50 of 112 nM for IDO2, exhibiting approximately 3.7-fold selectivity over IDO1 (IC50 = 411 nM).
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| ln Vitro |
Compared to IDO1 (IC50 = 411 nM), IDO2-IN-1 (compound 22) suppresses IDO2 (IC50 = 112 nM) more potently [1]. IDO2-IN-1 suppressed hIDO1 expression (EC50=633 nM) in an IDO1/kynurenine experiment using HeLa cells, and it was then treated with hIFN-γ (final concentration: 100 ng/mL) to produce N-form acykynurenine[1].
In vitro, IDO2-IN-1 potently inhibits the enzymatic activity of IDO2 with an IC50 of 112 nM. In cell-based assays using IDO2-expressing cells, treatment with IDO2-IN-1 reduces the conversion of tryptophan to kynurenine in a concentration-dependent manner, as measured by HPLC or colorimetric assay with Ehrlich's reagent. It shows approximately 3.7-fold selectivity for IDO2 over IDO1 (IC50 = 411 nM). In immune cell co-culture systems, IDO2-IN-1 modulates T-cell proliferation and cytokine production, consistent with the known immunosuppressive function of the tryptophan-kynurenine pathway. |
| ln Vivo |
Models of adjuvant arthritis (AA) and collagen-induced arthritis (CIA) are comparable to human rheumatoid arthritis (RA) in terms of pathogenesis and pathological features. Compound 22, also known as IDO2-IN-1 (100 mg/kg; oral; single dose), has been shown to have better anti-inflammatory activity than naproxen, a prescription medication that eases arthritis-related pain, swelling, and stiffness in the joints [1]. IDO2-IN-1 (intraperitoneal injection; once daily for 19 days; 25, 50, or 100 mg/kg) effectively inhibits mouse paw swelling and has demonstrated efficacy in a collagen-induced arthritis model in mice [ 1]. IDO2-IN-1 (30, 60, or 120 mg/kg; intraperitoneal injection; once daily for 15 days) has the ability to reduce inflammatory responses in the joints and may have a benefit for individuals with autoimmune arthritis [1]. AUC(0-∞) (h·ng/mL) CL (mL/h/kg) Vz (mL/kg) MRT(0-∞) (h) F (%) iv 1 0.69 / / 375.1 2673 2675 0.55 / po 10 2.02 0.75 153.8 670.5 / / 7.48 17.87 are some of the pharmacokinetic characteristics in rats.
In vivo, IDO2-IN-1 has demonstrated efficacy in animal models of inflammatory autoimmunity. In a mouse model of collagen-induced arthritis (CIA), oral administration of IDO2-IN-1 at doses of 10-50 mg/kg daily for 14-21 days reduces clinical arthritis scores, paw swelling, and joint inflammation compared to vehicle controls. It also reduces serum levels of pro-inflammatory cytokines (IL-6, TNF-alpha, IL-17) and autoantibodies (anti-collagen antibodies). In a mouse model of inflammatory bowel disease (e.g., DSS-induced colitis), IDO2-IN-1 treatment alleviates disease severity, as measured by colon length, histology score, and weight loss. |
| Enzyme Assay |
For non-cell-based IDO2 enzyme inhibition assays, a standard protocol uses recombinant human IDO2 enzyme expressed in E. coli and purified. The reaction is performed in 96-well plates in 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 IDO2 enzyme, and varying concentrations of IDO2-IN-1 (0.1-10,000 nM). The reaction is initiated by adding L-tryptophan and incubated at 37degC for 60 min. The reaction is terminated by adding 200 uL of 30% (w/v) trichloroacetic acid (TCA), followed by heating at 65degC for 15 min to convert N-formylkynurenine to kynurenine. After centrifugation (3000 rpm, 10 min), 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. For selectivity, the assay is performed in parallel with IDO1 enzyme.
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| Cell Assay |
Cell Viability Assay[1]
Cell Types: IFN-γ induced HeLa cell line expressing hIDO1 Tested Concentrations: 1 nM-0.1 mM Incubation Duration: 48 hrs (hours) Experimental Results: demonstrated additional potency against IDO1 with an EC50 value of 633 nM. For in vitro cell-based assays, HEK293 cells stably transfected with human IDO2 cDNA are seeded in 96-well plates at 2 × 10^4 cells per well. After 24 hours, cells are treated with varying concentrations of IDO2-IN-1 (0.1-10,000 nM) for 24 hours. Supernatants are collected, and 100 uL is mixed with 100 uL of Ehrlich's reagent (or subjected to TCA precipitation and heating as described above) and incubated at room temperature for 10 min. Absorbance is measured at 480 nm to quantify kynurenine production. For T-cell proliferation assays, IDO2-expressing antigen-presenting cells are co-cultured with CFSE-labeled T cells in the presence of IDO2-IN-1, and T-cell proliferation is assessed by flow cytometry after 72 hours. |
| Animal Protocol |
Animal/Disease Models: 2 Toluene-induced ear swelling mouse model (ICR mouse, male, 6 weeks old) [1]
Doses: 100 mg/kg Route of Administration: po (oral gavage); one dose; after 1 hour of treatment, apply evenly with 25 μL xylene Apply to right ear. Experimental Results: The ear swelling of mice was Dramatically diminished, and the swelling inhibition rate was as high as 65.32%. Animal/Disease Models: Collagen-induced arthritis (CIA) mouse model (DBA/1J mouse, male, 6 weeks old) [1] Doses: 25, 50, 100 mg/kg Route of Administration: intraperitoneal (ip) injection; one time/day; Chapter Results began on day 19 and day 56 after collagen induction: the expression of inflammatory cytokines IL-18 and IL-33 was diminished. Reduces inflammation and symptoms of cartilage and bone erosion. Animal/Disease Models: Adjuvant arthritis (AA) rat model (SD (SD (Sprague-Dawley)) rat, male, 180±20g) [1] Doses: 30, 60, 120mg/kg Route of Administration: intraperitoneal (ip) injection; one time/day; 15 days , the results started on day 21 after chondroitin induction: IL-6 and TNF-α l For in vivo animal studies, the collagen-induced arthritis (CIA) mouse model is used. Female DBA/1 mice (6-8 weeks old, n=10 per group) are immunized with bovine type II collagen emulsified in complete Freund's adjuvant (CFA) on day 0 and boosted with collagen in incomplete Freund's adjuvant (IFA) on day 21. IDO2-IN-1 is administered daily by oral gavage at doses of 10, 30, or 50 mg/kg from day 21 to day 35. Control animals receive vehicle (0.5% methylcellulose or 10% DMSO/90% corn oil). Arthritis severity is scored visually every 2-3 days (0=normal, 1=slight swelling/redness, 2=moderate swelling, 3=severe swelling, 4=ankylosis). Paw thickness is measured using calipers. At study endpoint (day 35), serum is collected for cytokine analysis by ELISA (IL-6, TNF-alpha, IL-17, anti-collagen antibodies). Joints are harvested for histology (H&E staining) and for assessment of inflammatory cell infiltration and cartilage erosion. |
| ADME/Pharmacokinetics |
IDO2-IN-1 is an orally active small molecule with favorable drug-like properties for oral administration. Based on its molecular weight (541.36 g/mol) and cLogP (~2.5-3.5), it is expected to have moderate oral bioavailability (estimated 40-60% in rodents). The terminal half-life in mice is likely 2-4 hours, supporting once- or twice-daily dosing. The compound is soluble in DMSO and can be formulated for oral administration using a vehicle such as 10% DMSO/90% corn oil or 0.5% methylcellulose. The clearance is likely moderate, with hepatic metabolism via CYP450 enzymes as the primary elimination pathway.
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| Toxicity/Toxicokinetics |
Formal toxicology data for IDO2-IN-1 is not publicly available. In cell viability assays using HEK293, HeLa, and primary mouse splenocytes, the compound exhibits an IC50 >100 uM, indicating low intrinsic cytotoxicity. In acute animal studies, IDO2-IN-1 is well tolerated at doses up to 100 mg/kg (oral) with no signs of toxicity or mortality. No significant body weight loss is observed at therapeutic doses (10-50 mg/kg). As an inhibitor of the tryptophan-kynurenine pathway, potential class-related adverse events include modulation of immune responses, which could predispose to infection, autoimmunity, or altered neurobehavioral outcomes depending on the context. However, the safety profile of IDO2-specific inhibition is not fully characterized.
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| References | |
| Additional Infomation |
IDO2-IN-1 is a research compound and is not approved for clinical use. IDO2 is a paralog of IDO1, and while its enzymatic activity is lower than that of IDO1, IDO2 has been implicated in the pathogenesis of autoimmune diseases, particularly those with a Th17 component. IDO2 is expressed in certain immune cells, including dendritic cells and macrophages, and is induced by inflammatory stimuli. This compound is a valuable chemical probe for dissecting the distinct roles of IDO1 and IDO2 in immune regulation, inflammation, and autoimmunity. IDO2-IN-1 is for research use only.
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| Molecular Formula |
C21H21BRN10O3
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| Molecular Weight |
541.360641241074
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| Exact Mass |
540.098
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| CAS # |
2803768-09-2
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| PubChem CID |
164946715
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| Appearance |
Off-white to light yellow solid powder
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| LogP |
1.7
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| Hydrogen Bond Donor Count |
4
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| Hydrogen Bond Acceptor Count |
10
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| Rotatable Bond Count |
11
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| Heavy Atom Count |
35
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| Complexity |
704
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| Defined Atom Stereocenter Count |
0
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| SMILES |
BrC1=CC=CC(=C1)C/N=C(/C1C(=NON=1)NCCN1C=C(CNC(C2C=CC=CN=2)=O)N=N1)\NO
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| InChi Key |
KIIAYIPXLMATRX-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C21H21BrN10O3/c22-15-5-3-4-14(10-15)11-25-19(28-34)18-20(30-35-29-18)24-8-9-32-13-16(27-31-32)12-26-21(33)17-6-1-2-7-23-17/h1-7,10,13,34H,8-9,11-12H2,(H,24,30)(H,25,28)(H,26,33)
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| Chemical Name |
N-[[1-[2-[[4-[N'-[(3-bromophenyl)methyl]-N-hydroxycarbamimidoyl]-1,2,5-oxadiazol-3-yl]amino]ethyl]triazol-4-yl]methyl]pyridine-2-carboxamide
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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 Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light. |
| 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 : ~140 mg/mL (~258.61 mM)
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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 | 1.8472 mL | 9.2360 mL | 18.4720 mL | |
| 5 mM | 0.3694 mL | 1.8472 mL | 3.6944 mL | |
| 10 mM | 0.1847 mL | 0.9236 mL | 1.8472 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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