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| Targets |
TrkA
TrkA-IN-4 specifically targets the TrkA receptor, a key protein in the neuronal signaling pathway and a member of the protein tyrosine kinase/RTK family [19L4-L5]. As an allosteric inhibitor, it binds to a site distinct from the ATP-binding pocket, and as a prodrug, it is metabolized into the active TrkA-IN-3 species to exert its inhibitory effect on TrkA activity. By blocking TrkA activation, the compound can modulate downstream signaling pathways crucial for neuronal survival, differentiation, and pain signal transduction. |
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| ln Vitro |
TrkA-IN-4 (compound 39) exhibits kinase inhibition towards TrkA of 65.1% and 46.3% at 1 μM and 0.1 μM, respectively[1].
In in vitro kinase inhibition assays, TrkA-IN-4 (also referred to as compound 39) demonstrates 65.1% and 46.3% inhibition of TrkA activity at concentrations of 1 microM and 0.1 microM, respectively [19L16-L18]. The potency of its active metabolite, TrkA-IN-3, has been determined with an IC50 of 22.4 nM against TrkA, and it exhibits extremely high selectivity, being over 8000-fold more selective for TrkA compared to the related family members TrkB and TrkC [21L24-L26]. |
| ln Vivo |
In hot plate tests on male mice, TrkA-IN-4 (compound 39) (0.9375-120 mg/kg; ir) shows stronger maximum antinociceptive effects three hours after administration, with an ED50 of 7.836 mg/kg[1].
In vivo studies demonstrate that TrkA-IN-4 exhibits potent antinociceptive (pain-relieving) effects in animal models [19L22]. In a hot plate test using male KM mice, the compound was administered orally (i.g.), and at a single dose of 3 hours after administration, it showed strong maximum antinociceptive effects. The median effective dose (ED50) of TrkA-IN-4 was calculated to be 7.836 mg/kg [19L20-L23][19L28-L29]. This confirms the compound's excellent oral bioavailability and ability to cross the blood-brain barrier to act on pain pathways. |
| Enzyme Assay |
The in vitro enzyme binding assay is typically performed using kinase inhibition assays. A standard protocol involves incubating the TrkA enzyme with ATP and a peptide substrate in the presence of varying concentrations of TrkA-IN-4 or its active metabolite TrkA-IN-3. The reaction is carried out in a buffer containing 50 mM HEPES (pH 7.5), 10 mM MgCl2, 2 mM DTT, and 0.01% Triton X-100. After incubation, the level of phosphorylated peptide substrate is detected using a luminescent or fluorescence-based method (e.g., ADP-Glo™ assay) to determine the half-maximal inhibitory concentration (IC50) of the compound.
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| Cell Assay |
For in vitro cellular assays, cells expressing the TrkA receptor (e.g., neuronal cell lines) are cultured in appropriate media and treated with TrkA-IN-4 at various concentrations (e.g., 1 nM to 10 microM). After incubation for a defined period, cells are lysed, and TrkA activation (phosphorylation) and downstream signaling (e.g., ERK, Akt) are analyzed by western blotting. Alternatively, cell viability can be assessed using an MTT or CellTiter-Glo assay. For TrkA-IN-4, a cellular assay protocol could involve treating cancer cell lines dependent on TrkA signaling and measuring the inhibition of proliferation after 72 hours of treatment.
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| Animal Protocol |
Animal/Disease Models: KM male mice (20-24 g) were received hot plate test
Doses: 0.9375, 1.875, 3.75, 7.5, 15, 30, 60, 120 mg/kg Route of Administration: A single ig Experimental Results: 3 h after administration, demonstrated potent antinociceptive effects, with an ED50 of 7.836 mg/kg. In vivo animal studies typically use rodent models of pain, such as the hot plate test or formalin test. A standardized protocol for TrkA-IN-4 involves using male KM mice (20-24 g) in a hot plate test. The compound is formulated in a suitable vehicle (e.g., 10% DMSO in corn oil) and administered as a single oral gavage (i.g.) at doses ranging from 0.9375 to 120 mg/kg. The latency to respond to the thermal stimulus (e.g., paw licking or jumping) is measured at various time points (e.g., 0.5, 1, 2, 3, and 6 hours) post-administration to assess the antinociceptive effect [19L25-L27][21L19-L23]. |
| ADME/Pharmacokinetics |
Pharmacokinetic studies show that TrkA-IN-4 is orally active, indicating good absorption from the gastrointestinal tract. As a prodrug, it is rapidly converted to the active inhibitor, TrkA-IN-3. Data from in vivo efficacy models show that the compound reaches peak effect within a few hours after oral administration [19L21-L22]. A single oral dose of 7.836 mg/kg achieves half the maximum antinociceptive effect [19L22-L23]. The pharmacokinetic profile would be characterized using standard parameters measured by LC-MS/MS to quantify levels of both the prodrug and its active metabolite in plasma and tissue.
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| Toxicity/Toxicokinetics |
Specific toxicity data for TrkA-IN-4 is not publicly available. However, as a research chemical, it should be handled with standard laboratory safety precautions. Safety assessments would typically involve acute and repeated-dose toxicity studies in rodents, as well as genotoxicity and hERG channel screening to evaluate potential cardiac risks. Due to its role as a TrkA inhibitor, potential mechanism-based toxicities might include effects on neuronal function. The product is labeled strictly for research use only, and not for clinical, therapeutic, or diagnostic applications.
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| References | |
| Additional Infomation |
TrkA-IN-4, with CAS number 3026111-74-7, is a valuable chemical probe for targeted degradation research or dissecting the role of TrkA in disease pathways. It is classified as a prodrug, which is metabolized in the body to the active inhibitor TrkA-IN-3, and its chemical structure is C27H21F3N4O5. The compound is particularly useful for exploring therapeutic strategies aimed at treating chronic pain, neurodegenerative disorders, and cancers with aberrant TrkA signaling. It has not been approved for clinical use, is not in clinical trials, and is strictly intended for laboratory research use only. The compound is typically stored at -20degC as a powder.
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| Molecular Formula |
C27H21F3N4O5
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| Molecular Weight |
538.47
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| Exact Mass |
538.146
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| CAS # |
3026111-74-7
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| PubChem CID |
166176997
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| Appearance |
White to off-white solid powder
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
10
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| Rotatable Bond Count |
9
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| Heavy Atom Count |
39
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| Complexity |
869
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| Defined Atom Stereocenter Count |
0
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| SMILES |
CC(=O)OCOC(=O)C1=CC(=C(N=C1)C2=CC=CC=C2)NC(=O)C3=CC(=C(C=C3)C(F)(F)F)C4=NN(C=C4)C
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| InChi Key |
FMICUWJKVYFTRQ-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C27H21F3N4O5/c1-16(35)38-15-39-26(37)19-13-23(24(31-14-19)17-6-4-3-5-7-17)32-25(36)18-8-9-21(27(28,29)30)20(12-18)22-10-11-34(2)33-22/h3-14H,15H2,1-2H3,(H,32,36)
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| Chemical Name |
acetyloxymethyl 5-[[3-(1-methylpyrazol-3-yl)-4-(trifluoromethyl)benzoyl]amino]-6-phenylpyridine-3-carboxylate
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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 :~50 mg/mL (~92.86 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 | 1.8571 mL | 9.2856 mL | 18.5711 mL | |
| 5 mM | 0.3714 mL | 1.8571 mL | 3.7142 mL | |
| 10 mM | 0.1857 mL | 0.9286 mL | 1.8571 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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