General Chemistry. Unless otherwise noted, reagents and solvents used in experiments were purchased from commercial sources and used without further purification. Flash chromatography was performed using 200–400 Mesh silica gel from Qingdao Makall Group Co., Ltd.; China. Silica gel plates-based thin-layer chromatography (TLC) was used to monitor all reactions with fluorescence F254 or F365 light. the reactions involving air- or moisture-sensitive reagents were performed under a nitrogen or argon atmosphere. 1H NMR spectra (400 or 600 MHz) and 13C NMR (100 or 150 MHz) spectra were recorded on a Bruker BioSpin AG (Ultrashield Plus AV 400M or 600M) spectrometer as deuterochloroform (CDCl3) or dimethyl sulfoxide-d6 (DMSO-d6) solutions using tetramethylsilane (TMS) as an internal standard (δ = 0) unless noted otherwise. In the tabulated NMR results, s indicates singlet; br, broad singlet; d; doublet; t, triplet; q, quartet; m, multiplet; dd, doublet of doublet. The purities of all target compounds were determined to be > 95% by highperformance liquid chromatography (HPLC). HPLC conditions were as follows: Gemini C18 column at room temperature, 4.6 cm × 150 cm, 5 µm, 10 − 90% acetonitrile (0.05% TFA)/water (0.05% TFA), 10 min run; flow rate, 1 mL/min; UV detection λ = 214 nm, 254, and 280 nm. MS spectra were obtained on an agilent technologies 6120 quadrupole LC/MS (ESI). High-resolution mass spectra (HR-MS) were obtained on an Agilent 6224 TOF LC/MS (USA). Yields were of purified compounds and were not optimized.
In vitro enzymatic activity assay. The inhibitory activities of the compounds against native TRK and TRK mutants (Invitrogen) were determined using a fluorescence resonance energy transfer (FRET)-based Z’-Lyte kinase assay assay system following to the manufacturer’s instructions. Briefly, TRKA (3 nM) in the enzymatic buffer solutions (1* assay buffer (cisbio) with 5 mM MgCl2, 1mM DTT) were mixed with various concentrations of inhibitors and incubated for 30 minutes at 25°C. Subsequently a mixture of TK-Sub-biotin peptide and ATP was added to initiate assay with the final peptide and ATP concentrations at 0.5 µM and 100 µM, respectively. The reaction mixture was incubated for 40 minutes at 25°C, afterwards TK antibody and XL665 in detection buffer (cisbio) was added to stop assay and the mixture was incubated at 25°C for 60 minutes. The FRET signal (665/615 ratio) was measured on Envision (PerkinElmer).
Human Microsomal Stability Studies. The in vitro metabolic stabilities of selected compounds were performed using human liver microsomes in triplicate. The buffer used in this study was 100 mM, phosphate buffer with 3.3 mM MgCl2. The incubation mixtures containing 0.5 mg/mL human liver microsomes and test compounds (1 µM) in 100 mM potassium phosphate buffer. The reaction was initiated by addition of 80 µL of the NADPH regenerating system to 320 µL of each incubation mixture. The final incubation conditions achieved in 400 µL are: 0.5 mg/mL human liver microsomes, 1 µM test compounds, 1.3 mM NADPH, 3.3 mM glucose 6 phosphate, 0.6 U/mL glucose 6 phosphate dehydrogenase. The mixtures were incubated in a 37°C water bath with gentle shaking. A 100 µL aliquot of each mixture was removed at 10, 30, 90 minutes to a clean 96-well plate which contains 400 µL quench reagent to precipitate proteins, and centrifuged (5000 ×g, 15 min). 80 µL of supernatant are taken into 96-well assay plates pre-added with 160 µL ultrapure water, and then analyzed by LC-MS/MS.
Molecular Docking. The three-dimension (3D) structures of small molecules were prepared by using SYBYL 7.0 followed by 3,000 steepest descent minimization and 3,000 conjugate gradient minimization, respectively. The X-ray structure of wild-type (WT) TRK (PDB 4AOJ) was obtained from the Protein Data Bank (PDB, http://www.pdb.org). The original ligand in the protein was used as the reference to define the active site. The hydrogens of the receptor were added by using Discovery Studio 4.0. The GOLD 3.039 was used to dock each small molecule into the active center. The radius of active site was set to 10 Å by using a genetic algorithm (GA) with 300 runs. GoldScore was used to evaluate the binding affinity and the top rank conformation was selected as the representative.
Computational Mutation Analysis. The Site-Specific Mutation Module in Auto In silico Macromolecule Mutation Scanning (AIMMS) server (http://chemyang.ccnu.edu.cn/ccb/server/AIMMS/index.php)40 was used to quantitatively evaluate the effects of TRK mutants (G595R, G667C, F589L) towards our compounds. The complex structures of WT TRK binding with different compounds, obtained from docking result, were used as the input for AIMMS server. The binding mode of the mutant-type (MT) TRK with our compounds were obtained by using the combination of sidechain replacement and molecular dynamics (MD) simulation in AIMMS. The binding free energy (ΔG) for both wild type (WT) and mutant type (MT) were then calculated according to the MM_PBSA method 41. Detailed calculation method can be found in our previous study of AIMMS40.
Cell Proliferation Assays. Ba/F3 cells expressing ETV6-TRKAWT, ETV6-TRKBWT, ETV6-TRKCWT, LMNA-TRKAG595R, LMNA-TRKAG667C, and LMNA-TRKAF589L were cultured in RPMI medium 1640 supplemented with 10% fetal bovine serum (FBS) at 37°C in the air containing 5% CO2 atmosphere. The cell proliferation inhibitory activities of Ba/F3 cell lines were determined according to the reported method42,43. Cells were seeded in 96-well plates (0.5 × 105 cells/well). Twenty-four hours later, compounds 7g or 3 were added to the designated wells, and cells were incubated at 37°C for additional 72 h. After the treatment above, CCK-8 was added into the 96-well plates (10 µL/well) and incubated with the cells for 3 h. OD450 and OD650 were determined by a microplate reader. Absorbance rate (A) for each well was calculated as OD450 − OD650. The cell viability rate for each well was calculated as V% = (As − Ac)/(Ab − Ac) × 100%. The data are the means from 3 independent experiments. As is the absorbance rate of the test compound well, Ac is the absorbance rate of the well without either cell or test compound, and Ab is the absorbance rate of the well with cell and vehicle control. IC50 values were calculated by concentration − response curve fitting utilizing a four-parameter analytical method on GraphPad Prism 9.0 software.
Determination of pharmacokinetic profiles in rats. The pharmacokinetic parameters of compound 7g were subjected to PK studies on male SD rats (provided by Beijing Vital River Laboratory Animal Technology Co., Ltd.) weighing 180–200 g with three animals in each group. The tested compound 7g [a solution of 70% PEG400 + 30% Saline (i.v.) or 0.5% CMC-Na + 1% Tween 80 + 98.5 water (i.g.)] administered to male SD rats at a dose of 1 mg/kg (i.v.) or 5 mg/kg (i.g.). Blood samples (0.3 mL) were collected at the point including 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, and 24 h (i.v.) or 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, and 24 h (i.g.) after administration, respectively, and centrifuged at 8000 rpm for 5 min at 4 oC, and then analyzed after protein precipitation. To obtain the best sensitivity and selectivity of the analyte, the LC/MS/MS analysis of compound 7g was carried out under optimized conditions in SRM (selected reaction monitoring) mode containing an internal standard. Plasma concentration-time data were measured by a noncompartmental approach using the software WinNonlin Enterprise, version 5.2 (Pharsight Co., Mountain View, CA).
Determination of pharmacokinetic profiles in beagle dogs. The pharmacokinetic parameters of compound 7g were subjected to PK studies on male beagle dogs (Provided by Beijing Marshall Biotechnology Co., Ltd.) weighing 10kg with three animals in each group. The tested compound 7g (a solution of 5% DMA + 10% Solutol + 85% Saline) was administered to male beagle dogs at dose of 1 mg/kg (i.v.) or 5 mg/kg (i.g.). Blood samples (1 mL) were collected before the dosage and at 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, and 24 h (i.v.) or 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, and 24 h (i.g.) after administration and centrifuged to separate plasma. The separated plasma was transferred into 96-well plates and kept frozen (<-60°C) until LC/MS/MS detection.
In vivo antitumor activity assay. Ba/F3 cells expressing TPM3-TRKAWT or TPM3-TRKAG595R or ETV6-TRKAG623R were cultured in RPMI medium 1640 supplemented with 10% fetal bovine serum (FBS) at 37°C in the air containing 5% CO2 atmosphere. The resulting Ba/F3-TPM3-TRKAWT or Ba/F3-TPM3-TRKAG595R or Ba/F3-ETV6-TRKAG623R cells (2×106 cells/mouse) were injected subcutaneously in 6-week-old BALB/cA nude mice (provided by Shanghai Jihui Experimental Animal Breeding Co., Ltd.). When the size of the tumor reached about 100 − 150 mm3, the mice were randomly divided into vehicle group and treated group (six mice/group). Compound 7g, Larotrectinib (1) and Selitrectinib (3) were dissolved in a vehicle containing 70% PEG400 and 30% water. For antitumor efficacy studies, mice were dosed twice daily by p.o. administration with vehicle or inhibitors with the indicated 5 or 15 or 30 mg/kg for 12–14 consecutive days. The sizes of the tumors were measured 3 times per week using a microcaliper. The tumor volume (TV = (length ×width2)/2) for the indicated days is the median tumor volume in each group (+ SEM). The percentage of tumor growth inhibition (%TGI = [1-(TVinhibitor_treated_final day –TVinhibitor_treated_day 1)/(TVvehicle_treated_final day–TVvehicle_treated_day 1)]×100%) was used to evaluate the antitumor efficacy of the compounds. Tumor volumes were statistically analyzed using Student’s t test.