Protein expression and purification. DYRK272-479 with an N-terminal 6×His affinity tag and TEV protease cleavage site was cloned into pET28a vector. Sequence verified plasmid was transformed into E. coli BL21 (DE3) cell. Bacterial cultures were grown at 37 °C in LB medium to an OD600 of 0.8 before induced with 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) overnight at 25 °C. Cells were collected by centrifugation and suspended in the lysis buffer containing 20 mM HEPES, pH 7.5, 500 mM NaCl, 20 mM imidazole and 10% glycerol, and disrupted by sonication. The lysate was centrifuged at 15,000 r.p.m for 30 min twice at 4 °C. After centrifugation, the supernatant was initially purified using Ni-NTA column (GE Healthcare), and then eluted with lysis buffers supplemented with 300 mM imidazole. Eluent recombinant protein with the His6-tag was subsequently cleaved using TEV protease at 4 °C overnight. The cleaved protein was further purified using reverse affinity chromatography and size-exclusion chromatography in buffer containing 20 mM HEPES, pH 7.5, 250 mM NaCl. The pure protein was concentrated to 11.2 mg/mL and flash-frozen with liquid nitrogen for later usage.
Co-crystallization, data collection and structure determination. DYRK272-479 (10 mg/mL) was incubated with 1 mM YK-2-69 at 4 °C before crystallization. The protein-YK-2-69 mixture was then mixed in a 1:1 ratio with crystallization solution (0.1 M sodium citrate pH 5.5, 8% PEG3350) in a final drop size of 2 µL. The initial DYRK2-YK-2-69 crystals were grown at 4 °C by the hanging-drop vapor diffusion method and optimized by seeding. Cuboid-shaped crystals appeared after 2-7 days. Crystals were cryoprotected in the crystallization solution supplemented with 30% glycerol before frozen in liquid nitrogen. The X-ray diffraction data were collected at Shanghai Synchrotron Radiation Facility (SSRF) beamline BL19U. The diffraction data were indexed, integrated, and scaled using HKL-2000 (HKL Research). The structure was determined by molecular replacement using the published DYRK2 structure (PDB ID: 5LXD). A clear electron density was observed in the center of the ATP binding pocket after molecular replacement. YK-2-69 was fitted using the LigandFit program in Phenix. The structural model was further adjusted in Coot and refined using Phenix. The quality of the structural model was checked using the MolProbity program in Phenix. The crystallographic data and refinement statistics are summarized in Table S1.
Immunohistochemistry of prostate cancer patient samples. The prostate patient samples were obtained from Huai’an First People’s Hospital, Nanjing Medical University. Prostate cancer (PCa) tissues and adjacent normal prostate tissues were fixed in 4% formaldehyde solution and processed routinely for paraffin embedding. Sections were cut at around 4 µm thickness and placed on glass slides, and stained with DYRK2 antibody and Sheep anti-rabbit/mouse polymer. Add 2 drops of freshly prepared DAB solution to each sheet, and then Re-dyeing and dehydration seal. Sections were scanned by digital pathology scanner.
Cell culture. The PCa DU-145 cells were obtained from American Type Culture Collection (ATCC) and cultured in endotoxin-free RPMI1640 supplemented with 10% fetal bovine serum (FBS) (Gibco); RWPE-1 normal epithelial prostatic cells (ATCC) were cultured in KM supplemented with 10% FBS; PC-3 human PCa cells (ATCC), A549 human lung cancer cells (ATCC) were cultured in F-12K with 10%FBS, Panc-1 and HEK-293 cells (ATCC) were cultured in DMEM with 10%FBS; K562, HL-60, NCI-H460 and RPMI8226 were obtained from National Collection of Authenticated Cell Cultures and kept in RPMI1640 with 10% FBS; Breast tumor cell line, MDA-MB-231, was purchased from ATCC and cultivated in L-15 with 10% FBS. The pancreatic cancer cell line BXPC-3 was maintained in RPMI1640 with 10% FBS. All the cells are not among commonly misidentified cell lines, and were tested for mycoplasma contamination annually using a PCR Mycoplasma Detection Kit (G238, Applied Biological Materials Inc.). In order to prevent potential contamination, all the media were supplemented with Penicillin-Streptomycin (C0222, Beyotime) and Plasmocin prophylactic (ant-mpp, InvivoGen) according to the manufacturer’s instructions.
Cell proliferation assays. We seeded 5,000 cells/well DU-145, PC-3 cells on 96-well plate and treated with DMSO or DYRK2 inhibitor YK-2-69 for 72 h. Add 10 uL of CCK8 reagent to each well, mix lightly, and incubate the plates in an incubator at 37 ℃ with 5% CO2 for 1-4 h. The incubated cell culture plate was placed on the enzyme plate analyzer and the absorbance value was measured at 450 nm. 2,000 cells/well PCa cells (DU-145) were seeded and subjected to DYRK2 inhibitor YK-2-69 treatment for 5 days and the cell culture plate was placed on the enzyme plate analyzer every day and the absorbance value was measured at 450 nm.
Lentivirus production and infection. Lentivirus-induced DYRK2 KD was modified in DU-145 cells. In brief, 0.5 μg pMD2.G, 0.3 μg pMDLg/pRRE and 0.7 μg PrSV-Rev and 1.5 μg PLkO-shDYRK2 or PLkO-shNC were co-transfected into 293T cells in cell culture dish. The effectene transfection reagent packs lentiviruses. Lentivirus particles were collected at 48 h and 72 h after transfection and transferred directly into DU-145 cells containing 4 μg/mL polypropylene. PCa cells, including lentiviruses, were then rotated and inoculated for 90 min at 32 ℃ and 1200 rpm. Finally, 1 μg /mL puromycin was added to cultured PCa cells 48 h after rotation inoculation to select positive infected cells. The shRNA targeting oligo sequence:
Real-time quantitative PCR (RT-qPCR). DYRK2 KD cells or DU-145 cells were treated with vehicle control or YK-2-69 at indicated concentrations for 72 h. All the cells with total RNA isolated with the TRIZOL reagent was subjected to reverse transcription using the PrimeScriptTM RT reagent Kit (RR047Q, Takara). RT-qPCR reactions were performed with the THUNDERBIRDSYBR qPCR Mix (QPS201, TOYOBO) and primers listed in Table S2. Gene expression was calculated using the comparative DDCT method with the GAPDH for normalization.
Immunoprecipitation. We lyse the cells in PBS (20% Triton X-100, 10% CHAPS, and Protease Inhibitor Mixture (Roche) for 30 minutes at room temperature. After centrifugation for 5 minutes, the supernatant was incubated with 10 μL of anti-DYRK2 antibody (Santa Cruz Biotechnology Inc) for 2 hours at 4 °C. Rabbit IgG was used as a control. Incubate with antibody and supernatant with 2% BSA and 10 mg protein A-Sepharose beads (P1406, Sigma-Aldrich) at 4 °C overnight. On the next day, the protein was eluted three times with 0.1% PBST, resuspended in 2x SDS-PAGE loading buffer, and boiled for 5 min. The eluate was fractionated by SDS-PAGE.
Western blot analysis. DU-145, PC-3 and RWPE-1 cells were grown in T-75 flasks at 5 x 106 cells/mL and DU-145 cells were treated with vehicle or the specified YK-2-69 concentration for 72 h, and treated cells were harvested and lysed by sonication in receptor lysis buffer (RLB) containing 20 mM HEPES (pH 7.5), 500 mM NaCl, 1% Triton X-100, 1 mM DTT, 10% glycerol, phosphatase inhibitors (50 mM NaF, 1 mM Na2VO4), and protease inhibitor mix (Roche). Lysates were quantitated and 20 to 50 μg of protein lysates were boiled in SDS sample buffer, size fractionated by SDS-PAGE, and transferred onto a PVDF membrane (Immobilon). After blocking in 5% nonfat dry milk (or 3% BSA), membranes were incubated with the following primary antibodies overnight: Rb mouse monoclonal antibody (1:2000), Phospho-Rb rabbit monoclonal antibody (1:1000), CDK4 rabbit monoclonal antibody (1:1000), CDK6 mouse monoclonal antibody (1:2000), DYRK2 rabbit monoclonal antibody (1:1000), PARP rabbit monoclonal antibody (1:1000), RRS1 rabbit polyclonal antibody (1:1000), P21 rabbit monoclonal antibody (1:1000), P27 rabbit monoclonal antibody (1:1000), P53 rabbit polyclonal antibody (1:1000), c-MYC rabbit polyclonal antibody (1:1000), Phospho-c-MYC rabbit monoclonal antibody (1:1000), GAPDH mouse monoclonal antibody (1:20000), Vinculin rabbit polyclonal antibody (1:1000), Beta Tubulin mouse monoclonal antibody (1:3000). Following three washes in PBS-T, the blots were incubated with horseradish peroxidase-conjugated secondary antibody. Proteins were detected by electrochemiluminescence (Thermo Fisher Scientific, USA) and analyzed by Image J software.
Colony formation assays. Colony formation assays were performed with 5 × 102 cells, which were plated to a 24-well plate. Two weeks after initial plating, cells were fixed, stained with 0.1% crystal violet, and counted.
Migration and invasion assays. Cells were treated with vehicle or different concentrations (2, 4 and 8 μM) of YK-2-69 for 48 h, and equal numbers (2 × 104 cells per well) of the cells were seeded in FBS-free RPMI-1640 culture medium in the presence of vehicle or different concentrations of YK2-69 in the upper chambers of transwell inserts with an 8-mm pore size. The lower chambers were filled with 500 μL of medium supplemented with 10% FBS. Cells were allowed to invade the bottom chamber for 24 h. Non-invading or non-migrating cells in the upper surface were removed, and invaded or migrated cells on the lower surface were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet for 5 min. The stained cells were photographed and quantified.
Cell cycle and apoptosis assays. In this study, propidium iodide (PI) DNA staining was chosen to assess the cells located at G0/G1, S, and G2/M stages. For PI staining, 1×106 cells were collected, washed once with PBS, 70% ethanol was added, gently beaten, fixed at 4 ℃ for 12 h, centrifuged at 1000g for 5 min, and the cells were precipitated. Carefully suck out the supernatant and add 1 mL PBS and suspended in 0.5 mL Krishan’s buffer supplemented with 0.05 mg/mL PI, 0.1% trisodium citrate, 0.02 mg/mL ribonuclease A, and 0.3% NP-40, incubated at 37 ℃ for 30 min and then applied to flow cytometer directly. The samples were transferred onto ice before subjected to flow cytometry. Cell apoptosis was assayed by annexin V-FITC and PI. Cells were seeded at 1×106/well in 10% FBS–RPMI1640 into six-well plates and treated with test compounds for 24 h. The cells were then washed twice with cold PBS and resuspended in 1×binding buffer (0.1 M HEPES (pH 7.4), 1.4 M NaCl, 25 mM CaCl2) at a concentration of 1×106 cells/mL. A 100 μL volume of the solution (1×105 cells) was transferred to a 5 mL culture tube; 5 μL of FITC- Annexin V (BD, Pharmingen) and 5 µL PI were added to each tube. The cell suspension was gently vortexed and incubated for 30 min at room temperature (25 ℃) in the dark, and then 200 μL PBS was added to each tube. The apoptosis assay was carried out by flow cytometry (FACSVerse, BD, USA) at 488 nm excitation and the results were analyzed with FlowJo V10 software.
Structure-based virtual screening. The co-crystal structure of DYRK2 and LDN192960 was downloaded from RCSB Protein Data Bank (PDB code: 6K0J), then the disordered conformations, crystal water, and ligand in the co-crystal were removed. Next, protein was prepared by Prepare protein protocol in Discovery Studio (DS) 2020 and then the binding site of 6K0J was defined form PDB site records for further docking. Compounds of the Specs database (221,097 compounds) and in-house library (3,200 compounds) were first filtered based on Lipinski’s rule of five, Veber’s rule and pan assay interference compounds and then the remaining 195,483 compounds were subjected to structure-based virtual screening (DYRK2 PDB: 6K0J) via the Libdock protocol of DS2020. Next, the 9,696 ligands with a LibDock score of more than 125 were subjected to structure-based virtual screening via the CDOCKER protocol of DS2020. The 2,724 ligands with a “- CDOCKER INTERACTION ENERGY” of more than 55 were further clustered into 100 clusters by Find Diverse Molecules of DS2020. The 15 compounds (Figure S2) were selected through visual inspection and then validated in the DYRK2 inhibitory activity.
IC50 determination and kinase-inhibitor specificity profiling. Purified DYRK1A, DYRK1B, DYRK2, DYRK3 and DYRK4 protein were diluted to 10 mU in 50 mM Tris-HCl (pH7.5), 0.1 mM EGTA, 0.1% β-mercaptoethanol. A final volume of 25 μL kinase reaction buffer was constituted by base reaction buffer (20 mM HEPES (pH 7.5), 10 mM MgCl2, 1 mM EGTA, 0.02% Brij35, 0.02 mg/mL BSA, 0.1 mM Na3VO4, 2 mM DTT, 1% DMSO), 0.05 mM [33P-γATP] (10 Ci/L), 300 µM Woodtide (KKISGRLSPIMTEQ) substrate peptide and 10 mU DYRK2 protein. Tested compounds were serially diluted in DMSO with final concentration ranging from 0.1 nM to 10 μM and delivered to reaction mixture. The kinase reaction was allowed to run in room temperature for 3 h. The radioactivity was detected by filter-binding method. Kinase activity data were expressed as the percent remaining kinase activity in test samples compared to vehicle (DMSO). IC50 values and curve fits were obtained using GraphPad Prism 8.0 software.
Kinase inhibitor specificity profiling assays were carried out at Reaction Biology Corporation. YK-2-69 kinase specificity was determined against a panel of 370 protein kinases at the concentration 1 µM. The results are presented as an average of duplicate reactions.
Acute toxicity studies. To study the safety in vivo, seven-week ICR mice (weight 18 - 22 g), half male and half female, were randomly divided into one control group and three treatment groups (n = 10/group). Mice of treatment groups were administrated by oral at a dose of 2,500, 5,000, and 10,000 mg/kg, respectively. After one dose, the signs of toxicity were observed, and body weight was recorded once two days in 14 days.
Pharmacokinetic profiles. SD rats were used to determine the pharmacokinetic profiles of YK-2-69. SD rats were divided into intravenous and oral administration groups (n = 3/group). The dose of intravenous and oral administration group was 1 and 10 mg/kg, respectively. Blood samples of intravenous group were collected at 2 min, 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, and 12 h. and blood samples of oral administration group were collected at 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, and 24 h. The concentrations of YK-2-69 in serum were measured by LC/MS/MS.
In vivo antitumor activity. BABLc nude mice received subcutaneous injection of 1 × 107 DU-145 shVehicle cells and DU-145 KD cells in the right flank to establish shVehicle group (n = 6/group) and shDYRK2 group (n = 10/group), respectively. Tumor volumes and body weight were measured once two days. After 29 days, the shVehicle group mice were sacrificed for humane reasons, and tumor issues were weighed and taken photos. After 49 days, shDYRK2 group mice were sacrificed, and tissues were weighed and taken photos. Tumor tissues of each group were kept in -80 °C for further analysis.
BABLc nude mice received subcutaneous injection of 1 × 107 DU-145 cells in the right flank. When the average tumors reached the volumes of 100-150 mm3, the mice were randomly divided into control (n = 10/group), drug treatment groups (n = 10/group). YK-2-69 at doses of 100 and 200 mg/kg were given by oral every day. Palbociclib and enzalutamide were given to mice by oral at a dosage of 100 mg/kg/d and used as positive references for comparison, while control mice received the equal volume of saline. Tumor volumes and body weight were measured once two days. After 35 days, the mice in control group were sacrificed for humane reasons, and tumor issues were weighed and taken photos. After 49 days, mice of treatment groups were sacrificed, and tissues were weighed and taken photos. Tumor tissues of each group were kept in -80 °C for further analysis.
H&E and Ki67 Staining. Tumor tissues and normal tissues were fixed in 4% formaldehyde solution and processed routinely for paraffin embedding. Sections were cut at around 4 µm thickness and placed on glass slides, and counterstained with hematoxylin and eosin and anti-Ki-67.
RNA-seq sequencing. Total RNA samples were isolated from the YK-2-69 treated and DYRK2-KD cells respectively. RNA concentration was measured by NanoDrop 1000 (Thermo Fisher Scientific, Waltham, MA, USA), and RNA integrity was measured by BioAnalyzer (Agilent). According to Life Technologies guidelines, adding an appropriate amount of MIX1 or MIX2 to each RNA sample will result in approximately 1% of the total RNA-seq readings mapped to the 92 ERCC control sequences, assuming that the mRNA fragments are located at 2% of the total RNA. Using the Kapa chain mRNA-seq kit (Illumina) (Kapa Biosystems, Wilmington, USA), a library of 300 ng total RNA was constructed for each sample through 10 PCR amplification cycles. The library was purified using the AxyPrep MAG PCR purification kit (Thermo Fisher Scientific). Each library was quantified using a Qubit fluorometer (Life Technologies) and assessed for size distribution using 2100 BioAnalyzer (Agilent Technologies, Santa Clara, USA). Sequencing was performed using Truseq SR Cluster Kit V4-CBOt-HS (Illumina) on Illumina HiSeq 2500 (Illumina, San Diego, CA, USA) apparatus with a V4 chemically generated 51 bp single-ended read sequence. Each group contains 3-4 repeats and the corresponding control group keep the same number of repeats. Quality control of RNA-Seq reads was performed using FASTQC. The reads with low complexity or low quality were removed using Cutadapt. Trimmed reads were aligned to human genome reference (GRCh38) using STAR, and uniquely mapped reads were retained in the downstream analysis. RSEM was used to calculate the expression levels of genes. Differentially expressed gene (DEG) analysis was performed using DESeq2, P < 0.05 and 1.5-fold change was used as statistical significance. The IGV was used to show the distribution of readings in a particular transcript. Hierarchical clustering analysis was performed using R package ‘mclust’. Genome Set Enrichment Analysis (GSEA) were employed to calculate enrichment pathways based on the signature gene sets from the Molecular Biology Database (MSIGDB).
Synthesis of DYRK2 Inhibitors
General Methods. All chemicals were used as received without further purification unless stated otherwise. 1H and 13C NMR spectra were collected on BRUKER AV-300 or BRUKER AV-400 or BRUKER AV-500 spectrometer. Chemical shifts downfield from TMS, an internal standard, are shown in parts per million (ppm, δ). High Resolution Mass measurement was performed on Agilent QTOF 6520 mass spectrometer with electron spray ionization (ESI) as the ion source. Flash column chromatography was carried out using commercially available 200-300 mesh silica gel under pressure. The final compounds were analyzed with a Shimadzu 2020 series HPLC system using an Inertsil ODS-3 column (5 μm, 4.6 × 100 mm) with a solvent system consisting of H2O (mobile phase A) and MeOH (mobile phase B), and the purity of all the final compounds was found to be > 95%.
Synthesis of compound 12 and YK-2-69
The mixture of 6-aminonicotinaldehyde 16 (0.61 g, 5 mmol) and 1-isopropylpiperazine 17 (0.96 g, 7.5 mmol) in 1, 2-dichloroethane (40 mL) was stirred at room temperature for 2 h. Then NaBH(OAc)3 (2.12 g, 10 mmol) was added to the mixture and stirred at room temperature for 8 h. The reaction mixture was quenched by adding 1 M NaOH (50 mL) solution and extracted with DCM (40 mL) for three times. The DCM extract was dried by anhydrous Na2SO4 and the solvent was evaporated under reduced pressure. The residue was purified by chromatography on silica to provide 18 (0.95 g, 81%) as a white solid. 1H NMR (400 MHz, CDCl3) δ 7.88 (d, J = 2.2 Hz, 1H), 7.43 (dd, J = 8.4, 2.2 Hz, 1H), 6.48 (d, J = 8.4 Hz, 1H), 4.95 (s, 2H), 3.39 (s, 2H), 2.88 – 2.80 (m, 1H), 2.72 – 2.51 (m, 8H), 1.10 (d, J = 6.6 Hz, 6H).
The mixture of 6-aminonicotinic acid 19 (0.28 g, 2 mmol) and N,N'-carbonyldiimidazole (0.39 g, 2.4 mmol) in DMF (5 mL) was stirred at 70 ℃ for 10 min, then the mixture was stirred at room temperature. After 1 h, 1-ethylpiperazine 20 (0.46 g, 4.0 mmol) was added to the mixture and the mixture was stirred at room temperature overnight. The solvent was evaporated under reduced pressure and the residue was purified by chromatography on silica to provide 21 (0.40 g, 81%) as a white solid. 1H NMR (300 MHz, CDCl3) δ 8.19 – 8.17 (m, 1H), 7.57 – 7.54 (m, 1H), 6.51 – 6.48 (m, 0.9 Hz, 1H), 4.79 (s, 2H), 3.73 – 3.60 (m, 4H), 2.49 – 2.42 (m, 6H), 1.13 – 1.08 (m, 3H).
The mixture of 6-Bromobenzo[d]thiazole 22 (0.43 g, 2.0 mmol), 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bi(1,3,2-dioxaborolane) 23 (0.53 g, 2.1 mmol), Pd(dppf)Cl2 (22 mg, 0.06 mmol), and KOAc (0.59 g, 6.0 mmol) in DMF (10 mL) was stirred at 80 ℃ under Ar atmosphere. After 24 h, the mixture was filtered by diatomite, and the filtrate was evaporated under reduced pressure. The residue was purified by chromatography on silica to afford 24 (0.47 g, 90%) as a white solid. 1HNMR (300 MHz, CDCl3) δ 9.07 (s, 1H), 8.46 (s, 1H), 8.14 (d, J = 8.2 Hz, 1H), 7.94 (dd, J = 8.2, 1.1 Hz, 1H), 1.38 (s, 12H).
The mixture of 2,4-dichloro-5-fluoropyrimidine 25 (0.23 g, 1.4 mmol), Pd(PPh3)2Cl2 (21 mg, 0.03 mmol), Na2CO3 (0.27 g, 2.5 mmol), DME (10 mL), and H2O (0.25 mL) were stirred at 80 ℃ in three-neck flask under Ar atmosphere. Compound 24 (0.26 g, 1.0 mmol) dissolved in the DME (5 mL) was added to the mixture dropwise. After 16 h, the mixture was filtered by diatomite and the filtrate was evaporated under reduced pressure. The residue was purified by chromatography on silica to provide 26 (0.22 g, 82%) as a white solid. 1HNMR (300 MHz, CDCl3) δ 9.17 (s, 1H), 8.84 (d, J = 1.7 Hz, 1H), 8.58 (d, J = 3.1 Hz, 1H), 8.37 – 8.24 (m, 2H).
The mixture of compound 26 (133 mg, 0.5 mmol), compound 18 (141 mg, 0.6 mmol), Pd2(dba)3 (23 mg, 0.025 mmol), Xantphos (58 mg, 0.1 mmol), Cs2CO3 (326 mg, 1.0 mmol), and dioxane (5 mL) was stirred at 100 ℃ in the sealing tube under Ar atmosphere. The mixture was filtered by diatomite after 12 h, and the filtrate was evaporated under reduced pressure. The residue was purified by chromatography on silica to provide 10 (111 mg, 48%) as a white solid. 1H NMR (400 MHz, CDCl3) δ 9.15 (s, 1H), 8.78 (d, J = 1.6 Hz, 1H), 8.48 (d, J = 3.5 Hz, 1H), 8.38 (dd, J = 8.5, 0.8 Hz, 1H), 8.35 – 8.32 (m, 1H), 8.30 – 8.27 (m, 1H), 8.25 – 8.23 (m, 2H), 7.73 (dd, J = 8.6, 2.3 Hz, 1H), 3.50 (s, 2H), 2.68 – 2.44 (m, 9H), 1.06 (d, J = 6.5 Hz, 6H). HRMS (ESI) for C24H26FN7S [M+H]+: calcd. 464.2027; found, 464.2016
The mixture of 4-bromo-2-iodoaniline 27 (0.60 g, 2.0 mmol), sodium dimethyldithiocarbamate 28 (0.72 g, 4.0 mmol), Cu(OAc)2 (0.36 g, 2.0 mmol), K2CO3 (58 mg, 0.1 mmol) in DMF (10 mL) was stirred at 120 ℃. The mixture was filtered by diatomite after 12 h, and the filtrate was evaporated under reduced pressure. The residue was purified by chromatography on silica to provide 29 (0.44 g, 85%) as a white solid. 1H NMR (400 MHz, CDCl3) δ 7.69 (d, J = 1.9 Hz, 1H), 7.41 – 7.35 (m, 2H), 3.20 (s, 6H).
The synthetic procedure for compound 30 is similar to that of compound 24 and gave a yield of 88% as a yellow solid. 1H NMR (400 MHz, CDCl3) δ 8.06 (s, 1H), 7.73 (d, J = 7.8 Hz, 1H), 7.54 (d, J = 7.8 Hz, 1H), 3.22 (s, 6H), 1.35 (s, 12H).
The synthetic procedure for compound 31 is similar to that of compound 26 and gave a yield of 80% as a yellow solid. 1H NMR (400 MHz, CDCl3) δ 8.49 (d, J = 1.9 Hz, 1H), 8.45 (d, J = 3.6 Hz, 1H), 8.17 – 8.14 (m, 1H), 7.62 (d, J = 8.7 Hz, 1H), 3.27 (s, 6H).
The mixture of compound 31 (154 mg, 0.5 mmol), 21 (141 mg, 0.6 mmol), Pd2(dba)3 (23 mg, 0.025 mmol), BINAP (31 mg, 0.05 mmol), NaOtBu (96 mg, 1.0 mmol), and dioxane (5 mL) was stirred at 100 ℃ in the sealing tube under Ar atmosphere. The mixture was filtered by diatomite after 12 h, and the filtrate was evaporated under reduced pressure. The residue was purified by chromatography on silica to provide YK-2-69 (139 mg, 55%) as a white solid. 1H NMR (400 MHz, DMSO) δ 10.27 (s, 1H), 8.68 (d, J = 3.8 Hz, 1H), 8.52 (d, J = 2.0 Hz, 1H), 8.35 (d, J = 2.4 Hz, 1H), 8.31 (d, J = 8.7 Hz, 1H), 8.06 (d, J = 8.6 Hz, 1H), 7.86 (dd, J = 8.7, 2.4 Hz, 1H), 7.59 (d, J = 8.6 Hz, 1H), 3.60 – 3.48 (m, 4H), 3.21 (s, 6H), 2.40 – 2.36 (m, 4H), 1.01 (t, J = 7.1 Hz, 3H). 13C NMR (151 MHz, CDCl3) δ 170.6, 168.2, 155.8, 154.9 (d, J = 3.1 Hz), 154.0, 152.0 (d, J = 8.8 Hz), 151.1 (d, J = 256.0 Hz)147.5, 146.7 (d, J = 27.1 Hz), 137.9, 131.8, 127.3 (d, J = 7.5 Hz), 125.6 (d, J = 5.5 Hz), 124.6, 121.7 (d, J = 7.9 Hz), 118.6, 111.0, 52.2, 40.3, 11.9. HRMS (ESI) for C25H27FN8OS (M + H)+: calcd 507.2085; found, 507.2088.