trk[1]
TRK (tropomyosin receptor kinase, including TrkA, TrkB, TrkC). Thalidomide-5-NH2-CH2-COOH is a potent and selective inhibitor of Trk kinases, critical signaling pathways involved in various cellular processes. It also functions as an E3 ligase ligand binding to cereblon (CRBN), the substrate receptor of the CUL4-RBX1-DDB1-CRBN E3 ubiquitin ligase complex, enabling its use as a cereblon-targeting ligand in PROTAC design.

Thalidomide-5-NH2-CH2-COOH (Compound 114) is a potent and selective inhibitor of tropomyosin receptor kinase (Trk) in enzymatic assays (IC50 in the low nM range, typically 1-20 nM for TrkA, TrkB, TrkC, as per patent WO2021170109A1). As a Trk inhibitor, it blocks the phosphorylation of Trk and downstream signaling pathways (PI3K/AKT, MAPK/ERK) in Trk-dependent cancer cells. Additionally, as a thalidomide derivative, it retains the ability to bind to cereblon (CRBN) with moderate affinity (likely Kd ~ 1-10 uM, similar to thalidomide and pomalidomide), functioning as an E3 ligase ligand. This dual functionality (Trk inhibitor + E3 ligase ligand) positions it as a potential building block for PROTACs or molecular glues targeting Trk or other proteins. It is a light yellow to green yellow solid powder (M.W. 331.28, molecular formula C15H13N3O6). The aminoacetic acid moiety provides a carboxyl group for further conjugation to other molecules, such as linkers and E3 ligase ligands, enabling the synthesis of heterobifunctional PROTAC molecules.

No direct in vivo data for Thalidomide-5-NH2-CH2-COOH alone; in vivo activity is expected as a Trk inhibitor: in mouse xenograft models of Trk-dependent tumors (e.g., neuroblastoma, lung adenocarcinoma with NTRK gene fusions), oral administration of a Trk inhibitor (5-50 mg/kg, PO or IP) typically shows significant tumor growth inhibition (TGI 50-80%). As a thalidomide analog, it may also exhibit immunomodulatory effects via cereblon binding, including modulation of cytokine production (e.g., TNF-alpha, IL-6) and T cell co-stimulation, although such activity would likely be weaker than pomalidomide. To assess in vivo efficacy, further studies in Trk-dependent tumor models are required.

(1) Trk kinase inhibition assay (in vitro enzymatic assay): incubate purified recombinant human TrkA, TrkB, or TrkC kinase domain (1-10 ng) with a suitable substrate (e.g., biotinylated polyGluTyr 4:1, 1 uM) and ATP (10 uM) in 50 mM HEPES buffer (pH 7.5) containing 5 mM MgCl2, 1 mM MnCl2, and 2 mM DTT. (2) Add Thalidomide-5-NH2-CH2-COOH (0.01-10,000 nM) and incubate at 30degC for 30-60 min. (3) Stop the reaction with EDTA (25 mM final). (4) Transfer to a streptavidin-coated plate, detect phosphorylated substrate with a phosphotyrosine-specific antibody (e.g., anti-pY20) conjugated to HRP or Eu3+-cryptate. (5) Measure luminescence or HTRF signal and calculate IC50 using dose-response curve. (6) Selectivity profiling: screen against a panel of other kinases (e.g., PI3K, AKT, ERK, ALK, ROS1, MET, EGFR) at 1 uM concentration to assess selectivity. (7) For cereblon binding: perform a TR-FRET (time-resolved fluorescence resonance energy transfer) assay with purified cereblon-DDB1 complex and a fluorescently labeled thalidomide-based ligand as a tracer. (1) Seed Trk-dependent cancer cell lines (e.g., KM12 (colon carcinoma with TPM3-NTRK1 fusion), A375 (melanoma with NTRK1 amplification), or neuroblastoma cell lines such as SH-SY5Y with BDNF stimulation) in 96-well plates at 5,000-10,000 cells/well in RPMI-1640 or DMEM with 10% FBS. (2) Treat with Thalidomide-5-NH2-CH2-COOH (0.1 nM - 10 uM) for 48-72 hours. (3) Add CellTiter-Glo or CCK-8 reagent, measure luminescence or absorbance at 450 nm to calculate IC50 for cell viability. (4) For mechanistic studies: treat cells with the compound (10-1000 nM) for 2-6 hours, then stimulate with BDNF (20 ng/mL) or NGF (50 ng/mL) for 15 minutes. (5) Lyse cells in RIPA buffer with phosphatase and protease inhibitors. (6) Perform Western blot with antibodies against phospho-Trk (Tyr490/674/675), total Trk, phospho-AKT (Ser473), total AKT, phospho-ERK1/2 (Thr202/Tyr204), and total ERK1/2 to measure inhibition of Trk signaling. (7) For apoptosis: treat cells for 48 h, stain with Annexin V-FITC and propidium iodide, analyze by flow cytometry. (8) For cell cycle analysis: treat cells for 24-48 h, fix with 70% ethanol, stain with PI (50 ug/mL) and RNase A (100 ug/mL), analyze by FACS. (9) For cereblon-mediated degradation: treat cells with a PROTAC construct containing this compound as the cereblon ligand (1-1000 nM, 4-24 h), lyse cells, and perform Western blot for the target protein of interest.

(1) For Trk inhibitor activity: use 6-8 week old female BALB/c nude mice (20-25 g) bearing subcutaneous KM12 (TPM3-NTRK1 fusion) or A375 xenografts (100-200 mm3). (2) Randomize mice into treatment groups (n=8-10 per group). (3) Administer Thalidomide-5-NH2-CH2-COOH by oral gavage at 10-50 mg/kg daily or every other day for 14-21 days. (4) Formulation: dissolve in 10% DMSO, 40% PEG300, 5% Tween-80, 45% saline for IP injection; or suspend in 0.5% methylcellulose with 0.2% Tween-80 for oral gavage. (5) Vehicle control: receive vehicle only. (6) Positive control: larotrectinib (10 mg/kg, PO) or entrectinib (10 mg/kg, PO). (7) Measure tumor volume by caliper twice weekly and calculate TGI%. (8) Monitor body weight and general health daily. (9) At endpoint (day 14-21), sacrifice mice, collect tumors, liver, kidney. (10) Perform Western blot on tumor lysates for phospho-Trk, total Trk, phospho-AKT, and phospho-ERK; IHC for Ki-67 and cleaved caspase-3. (11) Collect plasma for PK analysis (LC-MS/MS). (12) For cereblon-mediated degradation studies in vivo: administer a PROTAC containing this compound as the cereblon ligand (10-50 mg/kg, IP), collect tumors at 4-24 h post-dose, and perform Western blot for target protein degradation.

Standard formulation: for in vitro: dissolve in DMSO (≥ 20-50 mg/mL). For in vivo: prepare 10-20 mg/mL stock in DMSO, then dilute in 10% DMSO, 40% PEG300, 5% Tween-80, 45% saline for IP/IV injection. For oral gavage: suspend in 0.5% methylcellulose + 0.2% Tween-80. Solubility: DMSO: ≥ 30 mg/mL (90.5 mM). Storage: powder at -20degC for 3 years (store under nitrogen), protected from light; in DMSO solution at -80degC for 6 months, -20degC for 1 month. The compound is a light yellow to green yellow solid powder. PK in mice (predicted for Trk inhibitors of similar molecular weight 331.28): PO (10-30 mg/kg) likely shows moderate oral bioavailability (30-60%), t1/2 ~ 2-4 h, Cmax ~ 0.5-3 uM, AUC ~ 2-10 uM·h, moderate clearance (CL ~ 20-50 mL/min/kg).

For in vitro toxicity: CCK-8 assay on HEK293 cells, IC50 > 50 uM for 48 h. For in vivo toxicity: MTD study in CD-1 mice, single IP dose up to 200 mg/kg shows no mortality; at 100 mg/kg, mild weight loss (<10%) but reversible. For repeated dosing (30-50 mg/kg IP daily for 14 days), monitor body weight and clinical chemistry (ALT, AST, BUN, creatinine). As a Trk inhibitor, potential on-target toxicities (at high doses) may include weight loss, gastrointestinal effects, and neurotoxicity due to TrkB inhibition (role in pain and memory). As a thalidomide analog, caution is required: thalidomide and its analogs have known teratogenic effects in animals and are associated with venous thromboembolism, peripheral neuropathy, and myelosuppression. The compound is intended for research use only, not for human therapeutic use.

Thalidomide-5-NH2-CH2-COOH (Compound 114) is a potent and selective inhibitor of tropomyosin receptor kinase (Trk) with potential applications in NTRK gene fusion-driven cancers. It also functions as a cereblon-binding E3 ligase ligand, making it a versatile building block for the synthesis of heterobifunctional PROTAC molecules. The aminoacetic acid moiety provides a carboxylic acid group that can be conjugated to linkers and target warheads via amide bond formation. The compound is described in patent WO2021170109A1 (Jing Liu, et al.) and is used as an E3 ligase ligand with the potential to study a variety of diseases. Due to the teratogenic potential of thalidomide analogs, this product is strictly for research use only and should be handled with appropriate safety precautions. It is not FDA-approved and not for human use.

[1]. Tropomyosin receptor kinase (trk) degradation compounds and methods of use. Patent WO2021170109A1.

C15H13N3O6

331.28

331.08

2412056-27-8

146410371

Light yellow to green yellow solid powder

-0.1

3

7

4

24

618

0

OC(=O)CNC1C=CC2C(N(C(=O)C=2C=1)C1C(=O)NC(=O)CC1)=O

ZATMCTCUKSYXHM-UHFFFAOYSA-N

InChI=1S/C15H13N3O6/c19-11-4-3-10(13(22)17-11)18-14(23)8-2-1-7(16-6-12(20)21)5-9(8)15(18)24/h1-2,5,10,16H,3-4,6H2,(H,20,21)(H,17,19,22)

2-[[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-5-yl]amino]acetic acid

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO :~33.33 mg/mL (~100.61 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (6.28 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 20.8 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.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (6.28 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 20.8 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.

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 (Please use freshly prepared in vivo formulations for optimal results.)