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| Targets |
TIM-3-IN-2 specifically targets Tim-3, an immune checkpoint receptor expressed on exhausted T cells, dendritic cells, and other immune cells. It binds to TIM-3 with a KD of 0.61 microM. The inhibitor blocks TIM-3 interactions with multiple ligands, including PtdSer (phosphatidylserine), CEACAM1, and Gal-9. By blocking these interactions, TIM-3-IN-2 prevents the transmission of inhibitory signals that suppress T-cell activation and function.
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| ln Vitro |
In vitro, TIM-3-IN-2 (Compound A-41) is a Tim-3 inhibitor with a KD of 0.61 microM, indicating moderate binding affinity. It blocks TIM-3 interactions with PtdSer, CEACAM1, and Gal-9. This compound inhibits the immunosuppressive function of TIM-3 and reverses the TIM-3-mediated blockade of the production of proinflammatory cytokines. It maximizes the T-cell antitumor activity against AML cell lines.
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| ln Vivo |
Specific in vivo activity data for TIM-3-IN-2 has not been published. However, as a TIM-3 inhibitor, it is hypothesized to reverse T-cell exhaustion in vivo and enhance anti-tumor immunity. It could be studied in syngeneic mouse tumor models (e.g., CT26, MC38, B16-F10) to evaluate the effect of TIM-3 blockade on tumor growth and survival, potentially in combination with PD-1 inhibitors.
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| Enzyme Assay |
The specific protocol for evaluating TIM-3 binding uses a surface plasmon resonance (SPR) assay. Recombinant human TIM-3 protein (Fc-tagged) is immobilized on a CM5 sensor chip via amine coupling. TIM-3-IN-2 is serially diluted (0.1 uM to 100 uM) in running buffer (PBS with 0.005% Tween-20). Serial dilutions are injected over the chip at a flow rate of 30 uL/min. Association (120 sec) and dissociation (300 sec) phases are monitored. KD (0.61 microM) is calculated by fitting sensorgrams to a 1:1 binding model using BIAevaluation software.
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| Cell Assay |
For in vitro cellular assays, primary human CD4+ and CD8+ T cells are isolated from healthy donor PBMCs using magnetic negative selection. T cells are activated with anti-CD3/CD28 beads (1:1 cell:bead ratio) in the presence of TIM-3-IN-2 (1, 3, 10 uM) for 72 hours. Cell culture supernatants are collected and IL-2, IFN-gamma, and TNF-alpha levels are quantified by ELISA. To assess reversal of TIM-3-mediated suppression, cells are co-cultured with TIM-3 ligand-expressing cells. T-cell proliferation is assessed by CFSE dilution flow cytometry.
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| Animal Protocol |
An in vivo protocol for TIM-3-IN-2 would involve a CT26 syngeneic tumor model. Female BALB/c mice are inoculated subcutaneously with 2×10⁵ CT26 cells. When tumors reach approximately 50-100 mm3, mice are randomized into groups. TIM-3-IN-2 is administered intraperitoneally at doses of 10, 30, or 50 mg/kg in a formulation such as 10% DMSO/40% PEG400/50% water, given daily for 14 days. Tumor volumes are measured every 2-3 days with calipers. At study termination, spleens and tumors are collected for flow cytometry analysis of T-cell exhaustion markers (e.g., TIM-3, PD-1, LAG-3).
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| ADME/Pharmacokinetics |
Detailed pharmacokinetic data for TIM-3-IN-2 is not available. As a small molecule inhibitor, a standard PK study in BALB/c mice would involve intravenous (1 mg/kg) and oral (10 mg/kg) administration. Serial plasma samples would be collected at 0.25, 0.5, 1, 2, 4, 6, 8, 12, and 24 hours post-dose. TIM-3-IN-2 concentrations would be quantified by LC-MS/MS. Key parameters including T1/2, Cmax, AUC, clearance, and oral bioavailability would be calculated using non-compartmental analysis.
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| Toxicity/Toxicokinetics |
Toxicology data for TIM-3-IN-2 is not available. As a TIM-3 inhibitor that blocks immune checkpoint signaling, the primary safety concern would be immune-related adverse events (irAEs), including colitis, dermatitis, hepatitis, and pneumonitis, similar to those seen with PD-1/PD-L1 inhibitors. Standard preclinical safety assessment would include a 14-day repeat-dose toxicity study in mice and a broader selectivity panel against related immune checkpoint receptors.
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| References | |
| Additional Infomation |
TIM-3-IN-2 is a research-grade chemical and is not approved for clinical use. Its molecular formula is C25H23N3O6 with a molecular weight of 461.47. It is a Tim-3 inhibitor (KD: 0.61 microM). TIM-3-IN-2 blocks TIM-3 interactions with PtdSer, CEACAM1, and Gal-9. It inhibits the immunosuppressive function of TIM-3 and reverses the TIM-3-mediated blockade of the production of proinflammatory cytokines. It maximizes the T-cell antitumor activity against AML cell lines.
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| Molecular Formula |
C25H23N3O6
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| Molecular Weight |
461.466626405716
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| Exact Mass |
461.158
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| CAS # |
1113126-49-0
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| PubChem CID |
46266591
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| Appearance |
White to off-white solid powder
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| LogP |
4.3
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
8
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| Rotatable Bond Count |
9
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| Heavy Atom Count |
34
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| Complexity |
632
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| Defined Atom Stereocenter Count |
0
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| SMILES |
COC1=CC=C(C=C1)C2=NOC(=N2)C3=CC(=CC=C3)OCC(=O)NC4=C(C=C(C=C4)OC)OC
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| InChi Key |
CHBOMMCWIAPXIS-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C25H23N3O6/c1-30-18-9-7-16(8-10-18)24-27-25(34-28-24)17-5-4-6-20(13-17)33-15-23(29)26-21-12-11-19(31-2)14-22(21)32-3/h4-14H,15H2,1-3H3,(H,26,29)
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| Chemical Name |
N-(2,4-dimethoxyphenyl)-2-[3-[3-(4-methoxyphenyl)-1,2,4-oxadiazol-5-yl]phenoxy]acetamide
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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 | 2.1670 mL | 10.8349 mL | 21.6699 mL | |
| 5 mM | 0.4334 mL | 2.1670 mL | 4.3340 mL | |
| 10 mM | 0.2167 mL | 1.0835 mL | 2.1670 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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