The following instruments were used: electronic balance (Hua Zhi Electronic Technology Co., Ltd.), 45–75 μM silica gel powder for column chromatography, silica gel GF254 for thin layer chromatography (TLC), UV lamp (model UV-IIB) for color development, Q Exactive HF LC-MS (Thermo Fisher Scientific) for mass spectrometry, Avance III 500 MHz liquid nuclear magnetic resonance spectrometer (TMS as internal standard, Bruker, Germany). All other reagents were analytically pure and used directly without further processing.

All molecular docking research were conducted on a Viglen Genie Intel (R) Core (TM) i5-7300HQ CPU @ 2.50 GHz running Windows 10 1909. AutoDockTools-1.5.6 and Pymol-1.7.0.0.win 32-py2.7 as software for molecular modeling. The X-ray co-crystallized structure of CDK4 and CDK6 were downloaded from the PDB data bank (http://www.rcsb.org/) and used for docking studies. The proteins were pretreated with Pymol to remove HOH and metal ions. Ligand structures were constructed using Chem3D. The docking results were visually inspected to determine the binding ability of the analyzed compounds at the active site.

The human cancer cell lines MDA-MB-231, MCF-7, A498, and Hela cells were purchased from the National Biomedical Laboratory (Beijing) and cultured in DMEM (KGM12800-500) or MEM medium (KGM41500-500) containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin, and the cells were incubated at 37°C and 5% CO₂ in a constant temperature incubator (Thermo Fisher Scientific, BB150). 0.25% trypsin was added when the confluence of cells was 70%–80% for digestion, passagework, and culture. For the next study, cells in the logarithmic growth phase and favorable growth state were selected.

Inoculated cells into 96-well culture plate with 1 × 10⁴ cells per well. After the cells had adhered, the medium in the 96-well plate was removed, and the cell culture medium with various concentrations of the compound WXJ-202 and the positive drug was added to the various groups, respectively. The cells were then treated for 24 h. 10 μL of methyl thiazolyl tetrazolium (MTT) solution was added to each well and continued to be incubated for 4 h. After the supernatant was removed, 100 μL of dimethyl sulfoxide (DMSO) was added to each well for color development. Each group’s absorbance (OD) was measured at 490 nm using a multifunctional microplate detector (BioTek, SYNERGY Neo2, United States).

The following formula was used to determine the inhibition rate based on the OD values obtained from a multipurpose microplate detector (BioTek, SYNERGY Neo2, United States). If the inhibition rate was ≤0%, it was recorded as 0%. I n h i b i t i o n   R a t e   % = 1 − E x p e r i m e n t a l   G r o u p   O D C o n t r o l   G r o u p   O D × 100 %

The effect of WXJ-202 on MDA-MB-231 cells was determined by morphological changes. MDA-MB-231 cells (1 × 10⁵ cells/well) were inoculated into 12-well plates and kept overnight at 37°C and 5% CO₂. After that, the cells were exposed to different concentrations of WXJ-202, and positive drugs, respectively. Images were taken with an inverted microscope (Gangnam XD-202).

The MDA-MB-231 and MCF-7 cells were plated in 12-well culture plates and cultivated for 24 h at 37°C with 5% CO₂, respectively. After aspirating and discarding the supernatant, the medium containing different concentrations of the compound WXJ-202 and the positive drug was added, respectively, and the mixture was incubated for 24 h. After collecting cells by centrifugation (1000 rpm, 5 min), cells were counted and inoculated at 10,000 cells per well in a 96-well plate that had been pre-coated with matrix gel (Corning Biocoat, 354248). The supernatant was removed after 1 h and 10 μL of MTT solution was added to continue the incubation for another 4 h. The supernatant was discarded and 100 μL of dimethyl sulfoxide (DMSO) was added to each well for color development. Each group’s absorbance (OD) values were then measured at 490 nm using a multifunctional microplate detector (BioTek, SYNERGY Neo2, United States) to calculate the adhesion rate.

MDA-MB-231 and MCF-7 cells were inoculated into 12-well cell culture plates at a density of 1.5 × 10⁵ cells/well, respectively. Overnight incubation allowed them to grow as a single cell layer. Scratch over the cell layer in the central portion of each well with a sterile pipette tip to create a scratch. Following that, cells were cleaned of cellular debris using sterile PBS. Cells were incubated with different concentrations of the compound WXJ-202 or positive drug in a complete medium. Images were taken at 0, 24 and 48 h using an inverted optical microscope (Gangnam XD-202). Observed the healing of scratches with time. The percentage of wound closure at 24 and 48 h was estimated from the closed area of migratory cells by using ImageJ software. The formula for calculating the scratch healing rate was as follows. S c r a t c h   H e a l i n g   R a t e   % = 0 h   S c r a t c h   A r e a − T i m e   P o i n t   S c r a t c h   A r e a 0 h   S c r a t c h   A r e a   × 100 %

The matrix gel (Corning Biocoat, 354248) was pre-thawed overnight at 4°C and diluted to 200 μg/mL using DMEM serum-free medium according to the instructions. 100 μL was wrapped on the bottom of the upper chamber surface of the Transwell and air-dried at 37°C and 5% CO₂ in an incubator for 4 h. Different concentrations of compound WXJ-202 and positive drug were added to MDA-MB-231 and MCF-7 cells, respectively, and the negative control concentration was 0 µg/ml. After 24 h, the cells were collected by centrifugation and prepared into 100 μL single cell suspension using serum-free medium containing 0.2% BSA and added dropwise to the upper chamber. Meanwhile, 650 µL of medium containing 10% fetal bovine serum (FBS) was added to the lower chamber as a chemotactic invasion inducer. After 24-48 hours of incubation, cells on the surface of the upper chamber of the Transwell were wiped off with a cotton swab. Cells that invaded the lower chamber of Transwell were fixed in paraformaldehyde, washed with PBS after 15 min, and stained with Giemsa or Crystal violet for 30 min. The staining solution was washed off with deionized water, dried, and placed under an inverted light microscope (Gangnam XD-202) to photograph randomly selected areas. Invasive cells were counted with ImageJ software.

The logarithmic growth phase MDA-MB-231 cells were inoculated into 6-well culture plates and MCF-7 cells were inoculated into 12-well plates, adding 1,000 cells of logarithmic growth stage to each well. The cells were evenly dispersed and incubated at 37°C in a 5% CO₂ incubator. After the cells were plastered, a culture medium containing different concentrations of compound WXJ-202 or positive control drug was added to each well, while a negative control group was set up. After 12 h of treatment, the medium was replaced with the normal medium, and the culture was continued for another 2 weeks with medium changes every 3 days. After the clones were formed, they were washed with PBS and fixed with 4% paraformaldehyde. The plates were then stained with Giemsa or Crystal violet, dried, and observed for colony formation. Finally, the well plates were inverted, photographed, and counted with ImageJ software.

The MDA-MB-231 cells were inoculated into 6-well plates at a density of 5 × 10⁵ cells per well and incubated at 37°C with 5% CO₂. The following day, different concentrations of compound WXJ-202 and positive drugs were added, respectively. After a 24 h treatment period, cells were collected by trypsinization without EDTA and centrifuged at 1,000 rpm for 10 min. After washing twice with PBS by gentle blowing, incubated with 10 μL Annexin V-FITC solution and 5 μL propidium iodide staining solution for 15 min at room temperature under light-proof conditions. The apoptosis rate of each group of cells was detected on a Guava flow cytometer (Guava easycyte 6HT 2L) and analyzed by Flowjo software.

The MDA-MB-231 cells were seeded in 6-well plates at 1 × 10⁶ cells per well. After the cells adhered, the serum was withdrawn for 24 h to synchronize the cells. A fresh culture medium containing different concentrations of compound WXJ-202 or positive drugs was replaced for 24 h treatment. The cells were digested with 0.25% trypsin and collected by centrifugation at 800 rpm for 5 min. 500 μL of pre-cooled phosphate buffered solution (PBS) was used to resuspend the cells twice, each time at 800 rpm for 5 min. PBS was discarded and the cells were fixed in 70% pre-cooled anhydrous ethanol at 20°C for 12–24 h. After being centrifuged and resuspended for 5 min, the fixed cells underwent three PBS washes. The supernatant was discarded and RNaseA was added for 30 min at 37°C. Finally, 500 μL of propidium iodide (PI) was added and stained for 30 min at room temperature and protected from light, and the cell cycle was measured using a flow cytometer (Guava easycyte 6HT-2 L) and analyzed using Flowjo software.

The MDA-MB-231 cells were inoculated in 6-well plates and treated with different concentrations of compound WXJ-202 or positive drugs after the cells adhered. After culturing in a cell incubator for 24 h, the cells of each group were collected. The cells were lysed with the cell lysis solution. After high-speed centrifugation, the supernatant was extracted and the protein was quantified by the BCA method. An appropriate amount of protein was taken, electrophoresed in 1 × SDS-PAGE buffer, and transferred to the methanol-pretreated PVDF membrane by the “wet transfer method.” The membrane was closed with 5% skimmed milk for 45 min, followed by adding primary antibody dilution (1:1,000 volumetric dilution): CDK4 (beyotime, AF6465), CDK6 (beyotime, AF0114), Cyclin D1 (ABclonal, A11022), E2F1 (beyotime, AF6756), Rb (beyotime, AF1564), p-Rb (beyotime, AF1135), Caspase-3 (beyotime, AF1213), Pro-caspase-3 (beyotime, AF1261), β-actin (beyotime, AA128). Incubated overnight at 4°C on a shaker. The next day, the membrane was washed 4 times with 1 × PBST, 5 min/time, and the corresponding secondary antibody diluent (volume dilution ratio 1:10,000) was added, and incubated at room temperature for 45 min. Washed the membrane 4 times with 1 × PBST, 5 min/time. Dropping luminescent liquid to develop on the imaging system. Data were processed using ImageJ software.

The breeding eggs were purchased from the Wufengshan Chicken Farm in Tongling, Anhui, China. All animal experiments followed the standards of the Animal Ethics Committee. Breeding eggs were incubated in a sterile environment at 37°C for 7 days, and then the air chamber was transferred to 1 cm from the umbilical blood vessel. The windows were sealed with sterile plastic wrap for later use. The cells were resuspended using a mixed suspension (1:1) of Matrigel and DMEM and adjusted them to 1.2-1.5 × 10⁶ cells/mL. resuspended cells were implanted into the vessels. After 4 days of implantation, a negative control group (DMSO), a positive control group and the experimental group were set up with 6 chicken embryos per group (n = 6). Different concentrations of WXJ-202 and positive drugs were added to the chicken embryo allantoic membrane, respectively. After 3 days of treatment with the drug, the tumors were removed, photographed, weighed, and recorded.

GraphPad Prism 9 software was used for the statistical analysis, and the data were expressed as the mean ± S.D of three independent experiments. The t-test was used to compare the data between the two groups. Statistics were considered significant for p values under 0.05.

2-((3-chloro-4-(pyridin-2-ylmethoxy) phenyl) amino)-7-cyclopentyl-N, N-dimethyl-7H-pyrrolo [2,3-d] pyrimidine-6-carboxamide (WXJ-202).

Under nitrogen protection, a 250 mL round bottom flask was charged with 2-chloro-7-cyclopentyl-N,N-dimethyl-7H-pyrrolo [2,3-d]pyrimidine-6-carboxamide(0.293 g, 1 mmol),3-chloro-4-(2-pyridylmethoxy) aniline (0.235 g, 1 mmol), Pd(OAc)₂ (0.0565 g, 0.25 mmol), Cs₂CO₃ (0.978 g, 3.0 mmol), XantPhos (0.289 g, 0.5 mmol) and 1,4-dioxane (25 mL), under the condition of 110°C, after stirring reaction for 5 h, monitoring should be until the raw material point disappears (developing solvent: dichloromethane: methanol = 40:1) under reduced pressure to remove the solvent to give a dark brown oil. Through rapid column chromatography separation (200–300 mesh silica gel, dichloromethane: methanol = 80: 1), the target product (0.278 g) was obtained as a pale-yellow solid with a yield of 56.6% (Figure 1). Hydrogen spectrum of compound WXJ-202 (Supplementary Figure S1): ¹H NMR (500 MHz, Chloroform-d) δ 8.62 (s, 1H), 8.59 (d, J = 4.8 Hz, 1H), 8.06 (d, J = 2.6 Hz, 1H), 7.75 (td, J = 7.7, 1.6 Hz, 1H), 7.67 (d, J = 7.8 Hz, 1H), 7.41 (d, J = 21.6 Hz, 1H), 7.26–7.20 (m, 2H), 6.94 (d, J = 8.9 Hz, 1H), 6.42 (s, 1H), 5.29 (d, J = 11.8 Hz, 2H), 4.73 (s, 1H), 3.15 (s, 6H), 2.65–2.46 (m, 2H), 2.14–2.00 (m, 4H), 1.67 (q, J = 6.1 Hz, 2H); Carbon spectrum of compound WXJ-202 (Supplementary Figure S2): ¹³C NMR (126 MHz, Chloroform-d) δ 164.10, 157.11, 155.41, 152.00, 151.71, 149.04, 148.87, 136.95, 134.70, 131.96, 123.13, 122.66, 121.25, 121.19, 118.24, 114.39, 112.43, 100.91, 71.84, 58.13, 30.15, 24.41; HR-MS m/z: 491.1965[M + H]⁺.

To investigate the binding of CDK4 and CDK6 to the compound WXJ-202, the program AutoDockTools-1.5.6 was used to perform a molecular docking study. The position of docking binding of compound WXJ-202 in CDK4 protein kinase (Figure 2A) indicated that compound WXJ-202 bound tightly to CDK4 and generated three hydrogen-bonding forces. The 2D interaction diagram of compound WXJ-202 with CDK4 protein kinase (Figure 2B) showed that these three hydrogen bonding forces were -CO- with LYS 35, -NH with ILE 12, -O- with ARG101. The location of compound WXJ-202 binding in CDK6 protein kinase (Figure 2C) revealed that the pyridine end of compound WXJ-202 extended into a groove on the outside of the pocket. The 2D interaction diagram of compound WXJ-202 with CDK6 protein kinase (Figure 2D) revealed that compound WXJ-202 had a significant interaction force with CDK6 protein kinase. This included strongly molecularly interacting hydrogen bonds, where -NH interacted with VAL 101 via hydrogen bonding and -CO- interacts with ASP163. The molecular docking results showed that both CDK4 and CDK6 were strongly bound to the compound WXJ-202.

In addition, we investigated the complexes formed by CDK4 and CDK6 with Cyclin D. Meanwhile, the binding conformations of WXJ-202 and Ribociclib in the binding pocket of CDK6 were superimposed for a more visual comparison. The results (Figures 2E, F) indicated that both Ribociclib and 202 maintained a similar conformation in the same binding pocket.

To explore the in vitro antitumor cell activity of compound WXJ-202, we performed MTT assays. The toxic effects of different concentrations of compound WXJ-202 on MDA-MB-231, A498, MCF-7, and Hela cell lines were examined using Abemaciclib, Ribociclib, and Imatinib as positive controls, respectively. The results are shown in Table 1. The results showed that after 24 h of treatment, compound WXJ-202 showed a progressive increase in inhibition of these 4 cell types compared to the control. The IC50 values calculated from the analysis are shown in Table 1. Compound WXJ-202 showed IC50 values of (9.31 ± 1.82 μM) on MDA-MB-231 cells, (7.16 ± 0.71 μM) on A498 cells, (5.76 ± 0.46 μM) on MCF-7 cells, and (3.64 ± 0.18 μM) on Hela cells, all superior to Ribociclib and Imatinib. Meanwhile, the IC50 of compound WXJ-202 on MDA-MB-231 cells (9.31 ± 1.82 μM) was lower than that of Imatinib (37.12 ± 3.18 μM), Ribociclib (34.96 ± 3.87 μM) and Abemaciclib (15.04 ± 4.53 μM).

Additionally, as demonstrated in Figure 3. By comparing the results of different concentrations of treatment at 0, 24 and 48 h, it was found that the morphology of MDA-MB-231 cells gradually rounded and was accompanied by loss of density at low, medium, and high concentrations (2.5, 10, and 40 μM) as the duration of action increased. The results indicated that the compound WXJ-202 disrupted cell morphology after acting on MDA-MB-231 cells.

Preliminary results from cytotoxicity assays indicated that compound WXJ-202 had cytotoxic potential in MDA-MB-231 cells and MCF-7 cells. Therefore, the effect of the compound WXJ-202 on the capacity of MDA-MB-231 cells and MCF-7 cells for adhesion, migration, and invasion was further investigated in this work. By examining the ability of compound WXJ-202-treated cells to adhere, we found that compound WXJ-202 inhibited the ability of MDA-MB-231 and MCF-7 cells to adhere most significantly compared to Ribociclib and Imatinib at the same concentration (Figures 4A–D). Compared with the control group, low, medium, and high concentrations of compound WXJ-202 all showed significant inhibition of the migratory ability of MDA-MB-231 and MCF-7 cells (Figures 4E–H). And this inhibition was stronger than the effect of Ribociclib and Imatinib at the same concentration. Cell invasion assays could assess whether compounds and positive agents could reduce the ability of tumor cells to degrade ECM proteins and invade. In this study, after treatment with 2.5, 10, 40 μM of compound WXJ-202 compared to the control, the number of MDA-MB-231 and MCF-7 cells invading the bottom surface of the transwell membrane was significantly reduced, cell density was significantly lower, cell invasion was significantly reduced, and dose-dependent (Figures 4I–L). The inhibition rates of invasive cells in the compound WXJ-202 low, medium and high concentration groups were (72.66 ± 2.34%, 18.33 ± 11.80%), (92.84 ± 3.46%, 56.11 ± 13.99%) and (97.88 ± 0.62%, 87.98 ± 4.73%), respectively, and the inhibition effects were all more significant than the same concentration of Ribociclib (95.50 ± 0.64%, 73.35 ± 3.45%) and Imatinib (91.30 ± 0.87%, 27.63 ± 14.47%). The above findings indicated that compound WXJ-202 significantly inhibited the adhesion, migration, and invasion of MDA-MB-231 cells and MCF-7 cells.

Next, we incubated different concentrations of compound WXJ-202 and positive drugs with MDA-MB-231 and MCF-7 cells to form clones, respectively. The experimental results (Figures 5A–D) showed that the proliferative capacity of MDA-MB-231 and MCF-7 cells decreased with increasing concentration of compound WXJ-202. When normalized to the control group, the proliferative capacities of the compound WXJ-202 at low, medium, and high doses were (63.89 ± 13.85%, 54.30 ± 9.16%), (19.04 ± 2.60%, 19.92 ± 6.82%), and (0.00 ± 0.00%, 7.08 ± 2.99%), respectively. Additionally, the number of cell clones was significantly reduced in the compound WXJ-202 5 μM and 10 μM treatment groups compared with the 10 μM positive drugs Imatinib (51.16 ± 3.36%, 53.56 ± 18.33%) and Ribociclib (41.17 ± 4.93%, 37.19 ± 6.42%) treatment groups. Thus, our experimental results showed that compound WXJ-202 significantly reduced the proliferative capacity of MDA-MB-231 and MCF-7 cells.

To determine the impact of compound WXJ-202 on apoptosis in breast cancer cells MDA-MB-231, the AnnexinV-FITC Apoptosis Detection Kit was used to detect MDA-MB-231 cells treated with compound WXJ-202. Our experimental results (Figures 6A, B) showed that the compound WXJ-202 significantly induced apoptosis, and the apoptosis was concentration-dependent. When the compound WXJ-202 concentration was varied from 2.5 μM to 40 μM, the apoptotic rate increased from 8.9% to 46.09%. Next, we used Western blot assay to verify the mechanism of apoptosis induction by compound WXJ-202. The results showed elevated expression levels of apoptosis-associated protein Caspase-3 and decreased expression levels of Pro-caspase-3 (Figures 8A, C, D). The above results indicated that compound WXJ-202 could induce apoptosis in MDA-MB-231 cells.

To further explore the mechanism by which the compound WXJ-202 exerts its anti-tumor effects, we analyzed the cell cycle changes after its action on MDA-MB-231 cells by flow cytometry. Compound WXJ-202 treated MDA-MB-231 cells at 2.5, 10 and 40 μM concentrations for 24 h. The ratios of the G0/G1 phase in the compound WXJ-202 group were (48.2 ± 4.8%), (51.9 ± 1.9%) and (75.8 ± 4.7%), respectively. In comparison to untreated cells [negative control group (42.9 ± 0.5%)], G1-phase cells in the compound WXJ-202 group grew by almost 33% at the 40 μM dose. The G0/G1 phase ratios in the positive control Ribociclib group (53.3 ± 2.3%) and the Imatinib group (56 ± 0.8%) were both lower than that of the compound WXJ-202 group at the same concentration (Figures 7A, B). The above experimental results indicated that compound WXJ-202 could significantly induce G0/G1 phase arrest in tumor cells and exert an anti-tumor activity.

Our experimental results proved that the compound WXJ-202 could induce apoptosis and cycle arrest in MDA-MB-231 cells. To investigate more deeply the mechanism of the above-mentioned effects, we performed Western blot assays. The results showed that compound WXJ-202 down-regulated the expression of MDA-MB-231 cell cycle-related proteins CDK4, CDK6, Cyclin D1, and E2F1, and the p-Rb/Rb ratio was reduced in a dose-dependent manner (Figures 8A, B, E). CDK4/6-Rb-E2F is a classic signaling pathway that regulates the cell cycle. As confirmed by our experimental results, when CDK4 and CDK6 were inhibited, the expression of p-RB and E2F1 was also reduced. Thus, the associated tumor cells were prevented from entering S-phase from G1-phase, thereby inhibiting the excessive proliferation of tumor cells and preventing the abnormal replication of tumor cells.

To further investigate the in vivo antitumor activity of compound WXJ-202, we constructed chicken embryo transplant tumor models with MDA-MB-231 and MCF-7 cells. After treatment with the compound WXJ-202 at concentrations of 0.5 μg/μL, 1 μg/μL, and 2 μg/μL, the angiogenesis rates were (76.08 ± 19.13%, 87.45 ± 1.57%), (54.12 ± 2.39%, 71.58 ± 4.12%), and (40.78 ± 9.94%, 59.03 ± 2.18%), respectively, with significantly enhanced ability to inhibit tumor angiogenesis and stronger effect than the positive drug Ribociclib at the same concentration (51.82 ± 8.62%, 69.83 ± 2.40%) (Figures 9A–D). Meanwhile, our experimental results showed that weights of chicken embryo tumors were (28.50 ± 6.57 mg, 33.83 ± 3.06 mg), (21.30 ± 3.39 mg, 23.17 ± 3.19 mg), and (11.70 ± 2.16 mg, 12.17 ± 2.79 mg) after treatment with low, medium, and high concentrations of compound WXJ-202, compared with the control group (35.70 ± 7.81 mg, 47.00 ± 2.37 mg), respectively (Figures 9E–H). Our experimental results showed that the tumor growth inhibition ability was enhanced with increasing concentration of compound WXJ-202 administration. In conclusion, compound WXJ-202 significantly inhibited the growth of MDA-MB-231 and MCF-7 cells in chicken embryo transplanted tumors, reflecting its excellent anti-tumor cell proliferation activity in vivo.