Design, synthesis, and biological evaluation of pyrido[2,3-d]pyrimidine and thieno[2,3-d]pyrimidine derivatives as novel EGFR^L858R/T790M inhibitors

Abstract

EGFR mutations have been identified in 20,000 reported NSCLC (non-small cell lung cancer) samples, and exon 19 deletions and L858R mutations at position 21, known as “classical” mutations, account for 85–90% of the total EGFR (epidermal growth factor receptor) mutations. In this paper, two series of EGFR kinase inhibitors were designed and synthesised. Among them, compound B1 showed an IC₅₀ value of 13 nM for kinase inhibitory activity against EGFR^L858R/T790M and more than 76-fold selectivity for EGFR^WT. Furthermore, in an in vitro anti-tumour activity test, compound B1 showed an effective anti-proliferation activity against H1975 cells with an IC₅₀ value of 0.087 μΜ. We also verified the mechanism of action of compound B1 as a selective inhibitor of EGFR^L858R/T790M by cell migration assay and apoptosis assay.

Keywords:

• NSCLC
• EGFR mutations
• EGFR^L858R/T790M
• antitumor
• derivatives

Introduction

Non-small cell lung cancer (NSCLC), the most common among lung cancers, is mainly caused by the overexpression or abnormal activation of the epidermal growth factor receptor (EGFR). EGFR mutations have been identified in 20 000 NSCLC samples, and exon 19 deletions and L858R mutations at position 21, known as “classical” mutations, account for 85–90% of all EGFR mutations^1–4. Therefore, EGFR tyrosine kinase inhibitor (TKI) therapy is recommended as the standard treatment for patients with advanced activating EGFR mutations⁴. However, first-generation EGFR-TKIs (gefitinib and erlotinib) induce T790M mutations after periods of use^5–8. T790M mutations spatially block the binding of TKIs to EGFR and increase receptor affinity for ATP, thereby reducing TKI efficacy.

To address drug resistance caused by T790M mutations, second-generation EGFR-TKIs, such as quinazoline-containing afatinib, have been developed^9–11. However, they lack clinical efficacy in patients with NSCLC harbouring T790M mutations because of their toxicity against the wild-type (WT).

To solve the problem of toxicity, third-generation irreversible EGFR-TKIs, including WZ4002, EGF816, CO-1686, AZD9291 (Tagrisso), and olmutinib (Olita), have been developed (Figure 1)^12–18. These inhibitors have high selectivity for EGFR^L858R/T790M kinase. In 2015, the U.S. Food and Drug Administration approved AZD9291 for the treatment of patients with EGFR^T790M mutations. In addition, olmutinib, developed by Hanmi Pharmaceuticals and Boehringer Ingelheim, was approved in South Korea in May 2016 for patients with locally advanced or metastatic EGFR T790M-positive NSCLC^19,20.

Figure 1. Structures of EGFR kinase inhibitors.

Results and discussion

Strategy for compound design

First, we conducted computer-simulated docking of olmutinib with the EGFRT790M protein (code: 3IKA). From the docking results, we surmised that the aminopyrimidine ring of olmutinib formed two hydrogen bonds of lengths 3.3 and 2.3 Å with the hinge region residue Met793 of EGFR^T790M. The thienopyrimidine structure was proximal to residue Met790. The end of the alkaline anilinecontaining side chain was close to the Leu718 residue and pointed towards the solvent region. One end of the acrylamide side chain (containing the Michael acceptor) extended into the back pocket and covalently bonded to the amino acid residue of Cys797, forming a 3.2 Å long hydrogen bond between the carbonyl oxygen and the amino acid residue of Cys797 (Figure 2).

Figure 2. The binding models of Omutinib with EGFR^T790M (PDB code: 3IKA). (A) (B) Docking of compound Omutinib with 3IKA; (C) 2D diagram of the interaction between Omutinib and 3IKA.

It was found that 4,5,6,7-tetrahydrothieno[3,2-c]pyridine is an important structural backbone and is widely used in a variety of drugs with good antibacterial and antitumor activities^21,22. The current study was designed to synthesise selective inhibitors of EGFR^L858R/T790M containing the structure of 4,5,6,7-tetrahydrothieno[3,2-c]pyridine with reference to the structure-activity relationship of similar drugs, combined with the molecular docking pattern of the control drug olmutinib.

Finally, based on the skeleton leap, two series of novel pyrido[2,3-d]pyrimidine and thieno[3,2-d]pyrimidine structures were synthesised as EGFR kinase inhibitors using olmutinib as the lead compound, and the structure-activity relationships of their counterparts were used as a reference. Through further studies, the most promising compound, B1, was screened; the overall design strategy is shown in Figure 3.

Figure 3. The design strategy of target compounds.

Chemistry

The synthesis of the target compounds A1–A15 is outlined in Scheme 1. Intermediate 2 was obtained by the synthesis of 2-aminonicotinic acid (1) with urea. Intermediate 2 was reacted with POCl₃ to obtain intermediate 3. Intermediate 3 was synthesised with 4,5,6,7-tetrahydrothieno[3,2-c]pyridine hydrochloride to synthesise intermediate 4. Intermediate 4 was reacted with 4-fluoro-2-methoxy-5-nitroaniline to obtain intermediate 5. Intermediate 5 was reacted with various aliphatic amines to obtain Intermediate 6a–h. Intermediate 6a–h was reduced with Fe, NH₄Cl to obtain intermediate 7a–h. Intermediate 7a–h was synthesised with various acyl chlorides to finally obtain the target compounds A1–A15.

Scheme 1. Reagents and conditions: (a) urea, 160 °C, 4–6 h; (b) POCl₃, 160 °C, 6 h; (c) 4,5,6,7-tetrahydrothieno[3,2-c]pyridine hydrochloride, CH₃OH/H₂O, K₂CO₃, rt, 4 h; (d) 4-fluoro-2-methoxy-5-nitroaniline, TsOH, EtOH, 90 °C, 3–5 h; (e) various aliphatic amines, K₂CO₃, DMF, 50 °C, 2–4 h; (f) Fe, NH₄Cl, EtOH/H₂O, 70 °C, 6 h; (g) various acyl chlorides, TEA, DMF, rt, 6 h.

The synthesis of the target compounds B1–B16 is outlined in Scheme 2. Intermediate 9 was obtained by the synthesis of ethyl 2-aminothiophene-3-carboxylate (8) with urea²³. Intermediate 9 was reacted with POCl₃ to obtain intermediate 10. Intermediate 10 was synthesised with different heterocyclic structures to synthesise intermediates 11a–e. Intermediates 11a–e were reacted with 4-fluoro-2-methoxy-5-nitroaniline to obtain intermediates 12a–e. Intermediate 12a–e reacted with various aliphatic amines to obtain intermediate 13a–g, Intermediate 13a–g was reduced by Fe, HCl to obtain intermediate 14a–g, Intermediate 14a–g reacted with various acyl chlorides to finally obtain the target compounds B1–B16.

Scheme 2. Reagents and conditions: (a) urea, 190 °C, 4–6 h; (b) POCl₃, 110 °C, 6 h; (c) different heterocyclic structures, EtOH/H₂O, K₂CO₃, rt, 4 h; (d) 4-fluoro-2-methoxy-5-nitroaniline, TsOH, EtOH, 150 °C, 3–5 h; (e) various aliphatic amines, K₂CO₃, DMF, 50 °C, 2–4 h; (f) Fe, HCl, EtOH/H₂O, 70 °C, 6 h; (g) various acyl chlorides, DIPEA, DMF, rt, 6 h.

Biological evaluation

EGFR tyrosine kinase inhibitory activities

The in vitro antitumor activity of pyrido[2,3-d]pyrimidines (A1–A15) and thieno[3,2-d]pyrimidines (B1–B16), and their inhibitory activity against various cancer cell lines, including NCI-H1975, A549, and NCI-H460, were evaluated by 3–(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay using AZD9291 and olmutinib as positive controls. Compounds inhibition activity of EGFR^L858R/T790M kinase as determined by ELISA, where activities are given as percentage inhibition at a concentration of 0.1 μM, and the results are summarised in Table 1.

Table 1. Cytotoxic activity studies^a.

				IC50^a (μM) ± SD			Inhibitory activity [%]
Compd	R^1	R^2	R^3	H1975	A549	H460	0.1 µM
A1				0.596 ± 0.075	21.901 ± 1.819	5.676 ± 0.239	19.2
A2				0.810 ± 0.058	1.251 ± 0.288	2.461 ± 0.146	22.5
A3				0.150 ± 0.045	0.393 ± 0.106	0.558 ± 0.099	36.0
A4				3.838 ± 0.856	>50	4.949 ± 0.475	6.0
A5				>50	>50	>50	–
A6				0.198 ± 0.055	21.924 ± 1.56	1.156 ± 0.065	19.2
A7				1.363 ± 0.143	1.423 ± 0.351	0.652 ± 0.019	23.4
A8				1.605 ± 0.201	7.013 ± 0.625	>50	10.0
A9				0.694 ± 0.106	>50	>50	19.4
A10				0.439 ± 0.089	7.905 ± 0.762	14.301 ± 1.378	28.8
A11				0.312 ± 0.057	23.880 ± 1.765	0.229 ± 0.013	23.9
A12				1.898 ± 0.409	>50	>50	7.6
A13				0.214 ± 0.050	>50	>50	23.5
A14				0.471 ± 0.067	>50	>50	30.3
A15				0.906 ± 0.236	5.314 ± 0.065	8.031 ± 1.087	2.0
B1				0.087 ± 0.016	1.508 ± 0.199	2.335 ± 0.268	90.3
B2				15.629 ± 1.03	>50	>50	–
B3				9.931 ± 0.866	27.853 ± 1.694	0.533 ± 0.060	19.5
B4				6.649 ± 0.587	1.960 ± 0.380	0.913 ± 0.083	25.6
B5				>50	7.061 ± 0.551	>50	–
B6				4.545 ± 0.374	>50	>50	6.7
B7				0.023 ± 0.003	0.441 ± 0.027	3.218 ± 0.173	96.2
B8				0.297 ± 0.024	0.440 ± 0.039	2.250 ± 0.196	34.6
B9				0.4185 ± 0.042	1.562 ± 0.243	1.317 ± 0.030	25.1
B10				3.072 ± 0.133	25.917 ± 2.13	>50	18.3
B11				0.106 ± 0.012	0.655 ± 0.056	0.239 ± 0.035	54.1
B12				>50	>50	>50	–
B13				37.509 ± 2.160	>50	>50	–
B14				0.120 ± 0.024	0.107 ± 0.031	1.844 ± 0.176	57.8
B15				0.165 ± 0.944	0.098 ± 0.019	1.315 ± 0.065	50.2
B16				0.126 ± 0.032	4.538 ± 0.378	6.480 ± 0.744	12.7
AZD9291				0.067 ± 0.035	0.379 ± 0.045	1.966 ± 0.071	98.7
Olmutinib				0.458 ± 0.045	4.219 ± 0.315	0.642 ± 0.048	98.4

^aThese values were the average obtained from three isolated experiments.

Unfortunately, compounds containing pyridopyrimidine-like structures exhibited less activity than those containing thienopyrimidine-like structures, likely due to the large parent nucleus structure of the compounds, which resulted in an inability to interact with protein amino acids deep in the lumen, giving rise to low cytotoxic activity. The A4, A5, A8, A12, and A15 compounds showed low activity against NCl-H1975, A549, and NCl-H460 cells. For example, for A5, the half maximal inhibitory concentration (IC₅₀) values in all three cells were greater than 50. And through examining the inhibition rate of EGFR^L858R/T790M, we found that the inhibition rates of the A series compounds were all below 36.0%, which laterally verified the above explanation.

B-series compounds containing thienopyrimidine-like structures exhibited high cytotoxic activity, particularly when substituted by N-methylpyrazole analogs (e.g. B7 and B1). Additionally, most B-series compounds showed higher activity against A549 and NCl-H1975 cells than against NCl-H460 cells, indicating that the compounds showed selective activity to cancer cells. Moreover, the activity against NCl-H1975 cells was higher than that against A549 cells, suggesting that these compounds were selective for EGFR^WT. Therefore, we focussed on B series compounds in this study.

The cellular activity of compounds to which N-methylpyrazole analogs were introduced was superior to that of compounds to which 4,5,6,7-tetrahydrothieno[3, 2-c]pyridine structures were introduced. The IC₅₀ values of compounds B2 and B8 in H1975 cells were 15.629 ± 1.03 and 0.297 ± 0.024 μM, respectively, and those in A549 cells were > 50 and 0.440 ± 0.039 μM, respectively, indicating that the introduction of N-methylpyrazole analog improved the activity of the compounds. In terms of activity towards A549 and H1975 cells, compound B9 was superior to B3. Compounds containing different side-chain lengths introduced to the acrylamide side chain or branched side chains had weaker activity than those containing halogen-containing side chains. This may be because long chains introduced at the end of the acrylamide side chain or long chains containing branched chains occupy a larger space, affecting the reactivity of the carbonyl group on the acrylamide side chain with amino acid residues. The Cl-atom introduced at the end of the acrylamide side chain contributed to the increased activity of the target compounds against A549 and NCI-H1975 cells. Compound B1 was more active than compounds B2 and B3. Similarly, compound B7 was more active than compounds B8, B9, and B10, suggesting that the modification of the acrylamide side chain enhances the activity of the compounds. In the cytotoxicity assays, the cellular activity of compounds with the N-methylpyrazole analog substitution was superior to that of compounds with the thienopiperidine structure(B1 vs B7, B2 vs B8).

When the N,N,N-trimethylethylenediamine substitution was introduced to the flexible chain, the activity was higher than that of compounds substituted with the N-methylpiperazine structure. For example, the IC₅₀ values of compounds B7 and B11 against H1975 cells were 0.023 ± 0.003 and 0.106 ± 0.012 μM, respectively, and against A549 cells were 0.441 ± 0.027 and 0.655 ± 0.056 μM, respectively. This may result from the deep penetration of the branched chain into the solvent region when the flexible chain is substituted by N,N,N-trimethylethylenediamine, which facilitates the formation of hydrogen bonds between the amide chain and amino acid residues.

In summary, we screened compounds B1 and B7 with superior activity. The cytotoxic IC₅₀ activity of B1 and B7 against H1975 cells was 0.087 ± 0.016 μM and 0.023 ± 0.003 μM, respectively, superior to that of olmutinib (IC₅₀ = 0.458 ± 0.045 μM). Compared with AZD9291 (IC₅₀ = 0.067 ± 0.035 μM), compound B7 was superior and B1 was slightly inferior. Compared with olmutinib (IC₅₀ = 4.219 ± 0.315 μM), B7 (IC₅₀ = 0.441 ± 0.027 μM) and B1 (IC₅₀ = 1.508 ± 0.199 μM) displayed greater toxicity against A549 cells. However, both values were lower than that of AZD9291 (IC₅₀ = 0.379 ± 0.045 μM).

To explore the effect of other small molecule substitutions on activity, we introduced morpholine, thiomorpholine, and tetrahydropyrrole substitutions and synthesised compounds B14–B16. Unfortunately, the activities of compounds B14, B15, and B16 on H1975 cells were inferior to those of B1 and B7. The experimental results revealed that compounds containing N-methylpyrazole analogs were superior to the other substituted compounds. Testing the inhibition rate of EGFR^L858R/T790M revealed that the inhibition rates of B1 and B7 reached 90.3% and 96.70%, respectively, which warranted further investigation.

Kinase inhibitory activity of the compounds

Compounds B1, B7, B11, and B14 with good inhibition rates were selected for further kinase activity studies, and A3, A10 was also tested for kinase activity in order to confirm whether the activity of the A series was as speculated. The EGFR kinase inhibitory activities of compounds A3, A10, B1, B7, B11, B14 and the two control drugs AZD9291 and olmutinib are summarised in Table 2. As speculated, the reason for the poor activity of compounds A3 and A10 was most probably that the spatial structure is too large, resulting in a poor binding ability to kinase and a significant decrease in activity. Compound B1 showed an IC₅₀ value of 13 nM for kinase inhibitory activity against EGFR^L858R/T790M and more than 76-fold selectivity for EGFR^WT in the kinase assay. Compound B1 was more selective for EGFR^L858R/T790M than the control drug AZD9291 and comparable to olmutinib. In contrast, compound B7 demonstrated an IC₅₀ value of 5.9 nM for kinase inhibitory activity against EGFR^L858R/T790M and was more than 16-fold selective for EGFR^WT. Therefore, compound B1 was superior to compound B7 in selectivity for EGFR^WT. The above results indicated that compounds B1 and B7 were selective inhibitors of EGFR^L858R/T790M. Moreover, the outcomes showed that all compounds had poor inhibitory activity against EGFR^{L858R/T790M/C797S} kinase. This suggested that compounds in this structural analog were more closely matched in spatial structure to kinases with EGFR^T790M mutations. Meanwhile, we studied the cytotoxicity of both compounds B1 and B7, assaying normal cell LO2, and found that both compounds had lower toxicity compared to the control drug, and the results were presented in Table 3.

Table 2. EGFR tyrosine kinase inhibitory activities.

Compd	EGFR IC50 (nM)^a
	L858R/T790M	WT	L858R/T790M/C797S	TL^b/WT selectivity
A3	349	>1000	ND	>2.9
A10	>1000	>1000	ND	>1.0
B1	13	996	>1000	76.6
B7	5.9	100	>1000	16.9
B11	173	>1000	>1000	>5.9
B14	63	>1000	>1000	>15.9
AZD9291	0.48	4.56	309	9.5
Olmutinib	12.5	>1000	>1000	>80

ND: Not determined; IC₅₀: half maximal inhibitory concentration; WT: wild-type.

^aThe values were obtained from the mean of three replicated assays.

^bTL represents EGFR^L858R/T790M.

Table 3. Cytotoxic activity studies^a.

Compound No.	IC50(μM)^a ±  SD
	B1	B7	AZD9291	Olmutinib
LO2	>40	>40	28.33 ± 0.42	21.93 ± 0.36

^aThese values were the average obtained from three isolated experiments.

Morphology assays of apoptotic cells

To further assess the antiproliferative and apoptotic effects of compounds B1 and B7 on A549 cells, we performed fluorescent staining experiments, as shown in Figure 4. In these experiments, the control group consisted of untreated A549 cells, and the experimental group consisted of A549 cells treated with compound B1 or B7 at concentrations of 1, 5, or 10 μM. Control cells were uniform and regular-shaped WT A549 cells. The inhibitory effect on A549 cells was enhanced by increasing compound concentrations. Compared with the control group, the number of A549 cells significantly decreased, the fluorescence intensity increased, cell edges sharpened, and cell fragments appeared with increasing compound concentration. Specifically, this effect was most pronounced at a compound concentration of 10 μM. These results indicate that B1 and B7 induce apoptosis in A549 cells in a concentration-dependent manner.

Figure 4. The cell morphology of B1 and B7 was observed using an orthotropic fluorescence microscope.

Cell migration inhibition ability

Cell migration is an important feature of cancer cell metastasis. The wound healing assay is a simple method to investigate the directional migration of cells in vitro. Therefore, we performed a wound-healing assay using compounds B1 and B7 to investigate their effect on A549 cell migration. A549 cells were treated with B1 or B7 at concentrations of 5 or 10 μM for 0, 24, and 48 h (Figure 5). The results revealed that at the same time points, wound healing was slower in the treated group than that in the blank control group, and wound healing inhibition increased with increasing drug concentrations. Furthermore, in the same period, the healing rate was slower in the B7 groups than in the B1 groups.

Figure 5. In vitro wound healing assays were performed on A549 cells with different concentrations of B1 and B7.

Based on the analysis of all the results shown above, we selected compound B1 with better selectivity for further investigation.

Effects of compound B1 on apoptosis

Considering the aforementioned experimental results, compound B1 was examined further. The apoptotic effect of compound B1 was further investigated by flow cytometry using annexin V and propidium iodide double staining. Figures 6 and 7 demonstrate the changes in H1975 cells treated with compound B1 at different concentrations (0, 0.1, 1, and 10 μM) for 48 h. As the concentration increased, the apoptotic effect of compound B1 on H1975 cells increased. The apoptosis rate of the blank control was 0.79%, whereas that of compound B1 reached 22.1% at 10 μM, suggesting that compound B1 is a potential EGFR inhibitor.

Figure 6. Effect of compound B1 on apoptosis.

Figure 7. Graph the apoptosis results of B1 by GraphPad Prism: mechanically damaged cell(Q1); necrotic cells(Q2); viable apoptotic cells(Q3); total apoptotic cells(Q2 + Q3).

Effect of compound B1 on the cell cycle

To investigate the anti-proliferative mechanism of compound B1, we performed an H1975 cell cycle blocking experiment using flow cytometry. Cell cycle arrest results were obtained after treatment with compound B1 at different concentrations (0.2, 1.0, and 5 μM) for 24 h. Figure 8 showed that with increasing concentrations, the cell cycle blocking effect on H1975 cells was more pronounced in the G₂/M phase. For compound B1, the percentage of G2/M-phase cells increased from 23.67% (blank control) to 24.41% at 0.2 μM, 27.68% at 1.0 μM, and 31.47% at 5 μM. However, the S phase not found significant changes. Which in turn inhibited H1975 proliferation, indicating that the most promising compound B1 was a potential EGFR inhibitor.

Figure 8. Effect of compound B1 on the cell cycle distribution of H1975 cell line for 24 h.

Molecular docking studies

To explore the binding mode of compound B1 and EGFR^T790M (code: 3IKA), a molecular docking simulation study was performed, and the docking results are shown in Figure 9. Figure 9(A,B) showed that B1 penetrated the EGFR^T790M protein lumen, the thienopyrimidine structure formed a bimolecular hydrogen bond with the hinge region Met793, and the side chain carbonyl group formed a hydrogen bond with Cys797. This may explain why B1 showed excellent inhibitory activity against the EGFR^L858R/T790M mutation. The side chain terminus containing the basic aliphatic amine is proximal to the ASP800 residue and points towards the solvent region. The three hydrogen bonds were 2.9 Å, 1.9 Å, and 2.5 Å in length, which is shorter than that observed in the control olmutinib (3.3 Å、2.3 Å and 3.2 Å), indicating a stronger interaction and possibly accounting for the superior inhibitory activity of B1 against H1975 cells. Figure 9(C) shows that apart from hydrogen bonding interaction, there are π-π stacking and hydrophobic interactions between the compound and amino acid residues, all of which contribute to the close combination of B1 and protein and improve the inhibition efficiency. The spatial conformation of B1 was remarkably similar to that of the control olmutinib with a greater degree of stacking, indicating that it can be well interacted with the protein, as shown in Figure 9(D).

Figure 9. The binding models of B1 with EGFR^T790M (PDB code: 3IKA). (A,B) Docking of compound B1 with 3IKA; (C) 2D diagram of the interaction between B1 and 3IKA; (D) B1 (Red) overlapping with olmutinib (yellow).

Conclusions

The most promising compound, B1, was selected from two series of novel structures of pyrido[2,3-d]pyrimidine and thieno[3,2-d]pyrimidine an EGFR kinase inhibitor. Compound B1 displayed higher cytotoxic activity against H1975 and A549 cells than olmutinib and slightly lower than AZD9291. The kinase inhibitory activity of B1 against EGFR^L858R/T790M exhibited an IC₅₀ value of 13 nM and a selectivity of over 76-fold for EGFR^WT. The mechanism of action of compound B1 was further examined using cell scratching, apoptosis, and cell cycle assays, revealing that compound B1 blocked the G₂/M phases of H1975 cells. Molecular docking experiments showed that compound B1 resembled olmutinib and other triple kinase inhibitors, and the structural position of compound B1 was essentially identical to that of olmutinib. Taken together, these results suggest that compound B1 is a potential EGFR^L858R/T790M kinase inhibitor.

Experimental section

Chemistry

The reagents and solvents were commercially purchased and no further purification was required. All reaction levels were monitored by TLC on silica gel plates (Yantai, Jiangyou). Thin-layer chromatography silica powders (200–300 mesh and 100–200 mesh, Qingdao, Haiyang) were used for column chromatographic separation and purification during the experiments. Compound melting points were determined on a melting point apparatus WRS-1C. ¹H-NMR and ¹³C-NMR were tested on a Bruker Avance III, 400 MHz spectrometer (Germany). Mass spectra (MS) were performed on a Waters high-resolution quadrupole time-of-flight tandem mass spectrometer (QTOF).

General procedure for the preparation of compounds (A1–A15)

At 0 °C, 7a–h (0.12 g, 0.24 mmol) and TEA (0.03 g, 0.3 mmol) were added to anhydrous DMF and various acyl chlorides (0.03 g, 0.33 mmol) were added slowly dropwise and the reaction was reacted at room temperature, and the progress of the reaction was monitored by TLC. At the end of the reaction, and then alkalised with aqueous potassium carbonate,water (20–30 ml) was added and extracted with DCM (30 ml × 3). The organic layers were combined, dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure to obtain the crude product. After purification by silica gel column chromatography (DCM:MeOH, 20:1), the target compounds (A1–A15) were obtained.

N-(5-((4–(6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl)pyrido[2,3-d]pyrimidin-2-yl)amino)-2-((2-(dimethylamino)ethyl)(methyl)amino)-4-methoxyphenyl)acrylamide(A1)

Yellow solid; yield: 38%. m.p.:179.6 − 180.5 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 10.11 (s, 1H), 8.99 (s, 1H), 8.77–8.74 (m, 1H), 8.28 (s, 1H), 7.97 (s, 1H), 7.36 (d, J = 4.8, 1H), 7.21–7.18 (m, 1H), 7.00 (s, 1H), 6.94 (d, J = 4.8, 1H), 6.61–6.52 (m, 1H), 6.26 (d, J = 16.9, 1H), 5.77 (d, J = 10.2, 1H), 4.86 (s, 2H), 4.02 (s, 2H), 3.88 (s, 3H), 3.13 (s, 4H), 2.96 (s, 2H), 2.69 (s, 3H), 2.31 (s, 6H). ¹³C NMR (101 MHz, DMSO-d₆) δ 165.61, 163.25, 163.13, 162.12, 158.45, 155.90, 135.26, 133.50, 133.29, 132.87, 126.58, 125.98, 124.09, 117.49, 117.35, 106.88, 105.47, 103.90, 60.29, 56.58, 54.81, 49.80, 48.71, 48.65, 46.32, 45.04, 43.09. HRMS (ES) calcd [M + Na]⁺ for C₂₉H₃₄N₈O₂S 581.2423, found 581.2429.

N-(5-((4–(6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl)pyrido[2,3-d]pyrimidin-2-yl)amino)-2–(4-(dimethylamino)piperidin-1-yl)-4-methoxyphenyl)propionamide(A2)

Yellow solid. yield 33%. m.p.:130.7 − 130.7 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.75 (s, 2H), 8.70 (s, 1H), 8.27 (d, J = 8.1, 1H), 7.90 (s, 1H), 7.37 (d, J = 5.0, 1H), 7.18 (dd, J = 7.4, 4.4, 1H), 6.99 (d, J = 4.9, 1H), 6.83 (s, 1H), 4.84 (s, 2H), 4.00 (d, J = 4.5, 2H), 3.86 (s, 3H), 3.13 (s, 2H), 3.04 (d, J = 10.7, 2H), 2.66 (t, J = 11.3, 2H), 2.43 (dd, J = 14.7, 7.4, 2H), 2.24 (s, 7H), 1.86 (d, J = 11.4, 2H), 1.65 (dd, J = 21.8, 11.4, 2H), 1.15 (t, J = 7.4, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 171.83, 165.58, 162.15, 158.44, 155.87, 135.21, 133.53, 133.31, 126.03, 125.48, 124.44, 124.09, 117.25, 106.88, 103.71, 61.89, 56.52, 51.78, 49.83, 48.63, 42.03, 30.09, 29.04, 25.10, 10.48. HRMS (ES) calcd [M + Na]⁺ for C₃₁H₃₈N₈O₂S 609.2736, found 609.2732.

N-(5-((4–(6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl)pyrido[2,3-d]pyrimidin-2-yl)amino)-2–(4-(dimethylamino)piperidin-1-yl)-4-methoxyphenyl)acrylamide(A3)

Yellow solid; yield: 36%; m.p.:116.4–117.3 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 9.06 (s, 1H), 8.80 − 8.73 (m, 2H), 8.28 (d, J = 8.1, 1H), 7.92 (s, 1H), 7.36 (d, J = 4.9, 1H), 7.18 (dd, J = 7.7, 4.2, 1H), 6.95 (d, J = 5.0, 1H), 6.84 (s, 1H), 6.71 (dd, J = 16.9, 10.2, 1H), 6.25 (d, J = 17.0, 1H), 5.76 (d, J = 10.2, 1H), 4.84 (s, 2H), 4.01 (s, 2H), 3.88 (s, 3H), 3.13 (s, 2H), 3.04 (d, J = 10.9, 2H), 2.66 (t, J = 11.3, 2H), 2.23 (s, 7H), 1.84 (d, J = 11.3, 2H), 1.68 (dd, J = 20.9, 10.4, 2H). HRMS (ES) calcd [M + Na]⁺ for C₃₁H₃₆N₈O₂S 607.2580, found 607.2567.

N-(5-((4–(6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl)pyrido[2,3-d]pyrimidin-2-yl)amino)-2-(dimethylamino)-4-methoxyphenyl)propionamide(A4)

Reddish brown solid; yield: 37%; m.p.:149.8–150.7 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.92 (s, 1H), 8.75 (d, J = 4.0, 1H), 8.62 (s, 1H), 8.27 (d, J = 8.1, 1H), 7.91 (s, 1H), 7.37 (d, J = 5.0, 1H), 7.18 (dd, J = 7.7, 4.4, 1H), 6.98 (d, J = 4.9, 1H), 6.82 (s, 1H), 4.84 (s, 2H), 4.01 (t, J = 4.8, 2H), 3.87 (s, 3H), 3.13 (s, 2H), 2.65 (s, 6H), 2.42 (q, J = 7.3, 2H), 1.12 (t, J = 7.4, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 172.05, 165.55, 162.16, 158.49, 155.84, 135.21, 133.53, 133.30, 126.01, 124.96, 124.08, 123.90, 117.21, 103.09, 56.50, 49.84, 48.58, 44.29, 29.84, 25.07, 10.44. HRMS (ES) calcd [M + Na]⁺ for C₂₆H₂₉N₇O₂S 526.2001, found 526.1987.

N-(5-((4–(6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl)pyrido[2,3-d]pyrimidin-2-yl)amino)-2-(dimethylamino)-4-methoxyphenyl)acrylamide(A5)

Reddish brown solid; yield:35%; m.p.:134.7–135.7 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 9.29 (s, 1H), 8.78 − 8.68 (m, 2H), 8.27 (d, J = 8.0, 1H), 7.92 (s, 1H), 7.36 (d, J = 5.0, 1H), 7.18 (dd, J = 7.8, 4.4, 1H), 6.94 (d, J = 5.0, 1H), 6.84 (s, 1H), 6.74 (dd, J = 16.9, 10.2, 1H), 6.24 (d, J = 17.0, 1H), 5.73 (d, J = 10.2, 1H), 4.84 (s, 2H), 4.01 (t, J = 4.7, 2H), 3.89 (s, 3H), 3.12 (s, 2H), 2.65 (s, 6H). HRMS (ES) calcd [M + Na]⁺ for C₂₆H₂₇N₇O₂S 524.1845, found 524.1836.

N-(5-((4–(6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl)pyrido[2,3-d]pyrimidin-2-yl)amino)-2–(4-ethylpiperazin-1-yl)-4-methoxyphenyl)propionamide(A6)

Yellow solid; yield: 33%; m.p.:187.8–188.9 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.75 (s, 2H), 8.71 (s, 1H), 8.28 (d, J = 8.1, 1H), 7.91 (s, 1H), 7.37 (d, J = 4.9, 1H), 7.19 (dd, J = 7.7, 4.3, 1H), 6.99 (d, J = 4.9, 1H), 6.87 (s, 1H), 4.85 (s, 2H), 4.00 (d, J = 4.6, 2H), 3.87 (s, 3H), 3.14 (s, 2H), 2.87 (s, 4H), 2.58 (s, 4H), 2.41 (dd, J = 14.1, 6.9, 4H), 1.14 (t, J = 7.4, 3H), 1.05 (t, J = 7.0, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 171.77, 165.62, 165.58, 162.13, 158.41, 155.88, 148.54, 148.35, 135.38, 135.22, 133.53, 133.30, 126.03, 125.51, 124.64, 124.09, 106.89, 103.85, 61.06, 56.55, 53.23, 52.14, 51.91, 49.84, 48.66, 48.63, 30.12, 25.10, 12.57, 10.46. HRMS (ES) calcd [M + H]⁺ for C₃₀H₃₆N₈O₂S 573.2760, found 573.2751.

N-(5-((4–(6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl)pyrido[2,3-d]pyrimidin-2-yl)amino)-2–(4-ethylpiperazin-1-yl)-4-methoxyphenyl)acrylamide(A7)

Yellow solid; yield: 35%; m.p.:80.6–81.5 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 9.07 (s, 1H), 8.77 (s, 1H), 8.28 (d, J = 8.0, 1H), 7.93 (s, 1H), 7.36 (d, J = 5.1, 1H), 7.20–7.16 (m, 1H), 6.95 (d, J = 5.1, 1H), 6.87 (s, 1H), 6.67–6.61 (m, 1H), 6.24 (d, J = 16.5, 1H), 5.76 (d, J = 10.2, 1H), 4.84 (s, 1H), 4.01 (t, J = 4.9, 2H), 3.89 (s, 3H), 3.13 (s, 4H), 2.87 (s, 4H), 2.58 (s, 4H), 2.41 (d, J = 7.1, 2H), 1.03 (d, J = 6.9, 3H). HRMS (ES) calcd [M + Na]⁺ for C₃₀H₃₄N₈O₂S 593.2423, found 593.2414.

N-(2–(4-acetylpiperazin-1-yl)-5-((4–(6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl)pyrido[2,3-d]pyrimidin-2-yl)amino)-4-methoxyphenyl)propionamide(A8)

Yellow solid; yield: 36%; m.p.:166.6–167.7 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.88 (s, 1H), 8.78–8.71 (m, 2H), 8.28 (d, J = 8.1, 1H), 7.92 (s, 1H), 7.37 (d, J = 5.0, 1H), 7.19 (dd, J = 7.0, 4.5, 1H), 6.99 (d, J = 4.7, 1H), 6.87 (s, 1H), 4.85 (s, 2H), 4.01 (d, J = 4.4, 2H), 3.87 (s, 3H), 3.64 (s, 4H), 3.14 (s, 2H), 2.82 (d, J = 19.9, 4H), 2.44 (dd, J = 14.6, 7.1, 2H), 2.06 (s, 3H), 1.15 (t, J = 7.3, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 171.99, 168.76, 165.57, 162.11, 158.37, 155.87, 135.24, 133.53, 133.29, 126.02, 125.74, 125.00, 124.10, 117.34, 106.93, 104.23, 56.56, 52.36, 51.79, 49.84, 48.62, 46.63, 41.72, 30.00, 25.08, 21.73, 10.47. HRMS (ES) calcd [M + Na]⁺ for C₃₀H₃₄N₈O₃S 609.2372, found 609.2365.

N-(2–(4-acetylpiperazin-1-yl)-5-((4–(6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl)pyrido[2,3-d]pyrimidin-2-yl)amino)-4-methoxyphenyl)acrylamide(A9)

Yellow solid; yield: 34%; m.p.:269.8–270.7 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 9.18 (s, 1H), 8.85 (s, 1H), 8.76 (d, J = 3.9, 1H), 8.29 (d, J = 8.2, 1H), 7.94 (s, 1H), 7.36 (d, J = 5.1, 1H), 7.20 (dd, J = 8.0, 4.3, 1H), 6.95 (d, J = 5.3, 1H), 6.89 (s, 1H), 6.73–6.66 (m, 1H), 6.26 (d, J = 16.9, 1H), 5.77 (d, J = 10.3, 1H), 4.85 (s, 2H), 4.02 (t, J = 5.0, 2H), 3.88 (s, 3H), 3.64 (s, 4H), 3.13 (s, 2H), 2.83 (d, J = 15.8, 4H), 2.05 (s, 3H). HRMS (ES) calcd [M + H]⁺ for C₃₀H₃₂N₈O₃S 585.2396, found 585.2397.

N-(5-((4–(6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl)pyrido[2,3-d]pyrimidin-2-yl)amino)-4-methoxy-2-thiomorpholinophenyl)propionamide(A10)

Yellow solid; yield: 37%; m.p.:98.3–99.4 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.82–8.69 (m, 2H), 8.29 (t, J = 7.8, 1H), 7.94 (d, J = 15.9, 1H), 7.37 (d, J = 5.0, 1H), 7.20 (td, J = 9.0, 4.4, 1H), 6.97 (dd, J = 12.6, 4.8, 1H), 4.84 (d, J = 6.0, 2H), 4.02 (d, J = 5.1, 2H), 3.88 (s, 3H), 3.10 (d, J = 24.4, 4H), 2.83 (s, 2H), 2.50 (s, 5H), 2.47–2.40 (m, 1H), 2.38 (d, J = 7.6, 1H), 1.23 (s, 1H), 1.12 (dd, J = 15.5, 7.8, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 172.50, 135.29, 133.55, 133.52, 133.29, 133.17, 126.01, 125.93, 124.93, 124.15, 124.09, 56.91, 56.58, 54.46, 54.44, 49.83, 29.19, 29.14, 28.10, 24.99, 10.27. HRMS (ES) calcd [M + Na]⁺ for C₂₈H₃₁N₇O₂S₂ 584.1878, found 584.1863.

N-(5-((4–(6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl)pyrido[2,3-d]pyrimidin-2-yl)amino)-4-methoxy-2-thiomorpholinophenyl)acrylamide(A11)

Yellow solid; yield: 38%; m.p.:188.6–189.5 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.84 (s, 1H), 8.71 (d, J = 8.5, 1H), 8.29 (t, J = 7.5, 1H), 7.95 (d, J = 18.4, 1H), 7.36 (d, J = 5.0, 1H), 7.21 (td, J = 8.5, 4.7, 1H), 6.96–6.90 (m, 1H), 6.76–6.53 (m, 1H), 6.26 (d, J = 17.7, 1H), 5.78 (d, J = 10.0, 1H), 4.84 (d, J = 5.3, 2H), 4.01 (s, 2H), 3.89 (s, 3H), 3.13 (s, 2H), 3.07 (d, J = 3.9, 2H), 2.83 (s, 2H), 2.50 (s, 6H). HRMS (ES) calcd [M + H]⁺ for C₂₈H₂₉N₇O₂S₂ 560.1902, found 560.1895.

N-(5-((4–(6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl)pyrido[2,3-d]pyrimidin-2-yl)amino)-4-methoxy-2-morpholinophenyl)propionamide(A12)

Yellow solid;. yield: 34%; m.p.:156.6–157.4 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.81 (s, 1H), 8.61 (s, 1H), 8.08 (s, 1H), 7.81 (s, 1H), 7.35 (d, J = 4.9, 1H), 6.99 (d, J = 4.9, 1H), 6.85 (s, 1H), 4.69 (s, 2H), 3.90 (t, J = 4.9, 2H), 3.85 (s, 3H), 3.78 (s, 4H), 2.97 (s, 2H), 2.83 (s, 4H), 2.44–2.38 (m, 2H), 1.13 (t, J = 7.4, 3H). HRMS (ES) calcd [M + Na]⁺ for C₂₈H₃₁N₇O₃S 568.2101, found 568.2094.

N-(5-((4–(6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl)pyrido[2,3-d]pyrimidin-2-yl)amino)-4-methoxy-2-morpholinophenyl)acrylamide(A13)

Yellow solid; yield: 35%; m.p.:185.6–186.7 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 9.12 (s, 1H), 8.70 (s, 1H), 8.09 (s, 1H), 7.83 (s, 1H), 7.34 (d, J = 5.0, 1H), 6.93 (d, J = 5.1, 1H), 6.87 (s, 1H), 6.69 (dd, J = 17.0, 10.3, 1H), 6.24 (d, J = 16.9, 1H), 5.76 (d, J = 10.3, 1H), 4.68 (s, 2H), 3.90 (t, J = 4.9, 2H), 3.87 (s, 3H), 3.79 (s, 4H), 2.95 (s, 2H), 2.84 (s, 4H). HRMS (ES) calcd [M + Na]⁺ for C₂₈H₂₉N₇O₃S 566.1945, found 566.1940.

N-(5-((4–(6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl)pyrido[2,3-d]pyrimidin-2-yl)amino)-2–(4-(2-hydroxyethyl)piperazin-1-yl)-4-methoxyphenyl)propionamide(A14)

Reddish brown solid; yield: 35%; m.p.:160.4–161.5 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.75 (s, 2H), 8.71 (s, 1H), 8.27 (d, J = 8.1, 1H), 7.91 (s, 1H), 7.37 (d, J = 5.0, 1H), 7.18 (dd, J = 7.7, 4.3, 1H), 6.99 (d, J = 4.9, 1H), 6.86 (s, 1H), 4.84 (s, 2H), 4.44 (s, 1H), 4.00 (d, J = 4.3, 2H), 3.87 (s, 3H), 3.55 (t, J = 6.0, 2H), 3.14 (s, 2H), 2.86 (s, 4H), 2.63 (s, 4H), 2.47 (d, J = 6.0, 2H), 2.44–2.38 (m, 2H), 1.15 (t, J = 7.4, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 171.78, 165.57, 162.13, 158.41, 155.88, 146.53, 135.22, 133.53, 133.30, 126.03, 125.52, 124.64, 124.09, 117.28, 106.90, 103.81, 60.77, 59.13, 56.54, 54.06, 51.94, 49.84, 48.65, 48.63, 30.12, 25.10, 10.47. HRMS (ES) calcd [M + Na]⁺ for C₃₀H₃₆N₈O₃S 611.2529, found 611.2527.

N-(5-((4–(6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl)pyrido[2,3-d]pyrimidin-2-yl)amino)-2–(4-(2-hydroxyethyl)piperazin-1-yl)-4-methoxyphenyl)acrylamide(A15)

Reddish brown solid; yield: 33%; m.p.:117.6–118.7 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 9.07 (s, 1H), 8.79–8.73 (m, 2H), 8.28 (d, J = 8.1, 1H), 7.92 (s, 1H), 7.36 (d, J = 5.0, 1H), 7.19 (dd, J = 7.6, 4.3, 1H), 6.95 (d, J = 5.0, 1H), 6.87 (s, 1H), 6.66 (dd, J = 16.7, 10.3, 1H), 6.24 (d, J = 17.0, 1H), 5.76 (d, J = 10.3, 1H), 4.84 (s, 2H), 4.43 (s, 1H), 4.01 (s, 2H), 3.89 (s, 3H), 3.54 (t, J = 5.5, 2H), 3.13 (s, 2H), 2.86 (s, 4H), 2.63 (s, 4H), 2.47 (d, J = 6.0, 2H). HRMS (ES) calcd [M + Na]⁺ for C₃₀H₃₄N₈O₃S 609.2372, found 609.2372.

General procedure for the preparation of compounds (B1-B16)

At 0 °C, 14a-14g (0.2 g, 0.4 mmol) and DIPEA (0.29 g, 2 mmol) were added to anhydrous DMF, and various acyl chlorides (0.08 g, 0.8 mmol) were added slowly dropwise and the reaction was reacted at room temperature, and the progress of the reaction was monitored by TLC. At the end of the reaction, and then alkalised with aqueous potassium carbonate, water (20–30 ml) was added and extracted with DCM (30 ml × 3). The organic layers were combined, dried over anhydrous sodium sulphate, filtered, and concentrated under reduced pressure to obtain the crude product. After purification by silica gel column chromatography (DCM:MeOH, 10–15:1), the target compounds (B1–B16) were obtained.

2-chloro-N-(5-((4–(6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl)thieno[3,2-d]pyrimidin-2-yl)amino)-2-((2-(dimethylamino)ethyl)(methyl)amino)-4-methoxyphenyl)acrylamide(B1)

Yellow solid; yield: 30%; m.p.:130.0–131.1 °C. ¹H NMR (400 MHz, CDCl₃) δ 10.18 (s, 1H), 9.51 (s, 1H), 7.58 (s, 1H), 7.29 (s, 1H), 7.12 (d, J = 4.9, 1H), 6.97 (d, J = 5.0, 1H), 6.92 (s, 1H), 6.79 (s, 1H), 6.65 (s, 1H), 5.88 (s, 1H), 5.07 (s, 2H), 4.23 (t, J = 5.1, 2H), 3.88 (s, 3H), 3.10 (s, 2H), 3.05 (t, J = 6.7, 2H), 2.68 (s, 3H), 2.39 (t, J = 6.7, 2H), 2.26 (s, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 171.28, 159.60, 158.00, 156.08, 144.90, 134.84, 133.92, 133.46, 133.19, 128.02, 127.78, 125.59, 122.97, 122.10, 120.66, 116.66, 110.63, 109.56, 104.15, 57.34, 56.07, 54.95, 48.21, 45.95, 45.33, 44.61, 29.71, 25.52. HRMS (ES) calcd [M + H]⁺ for C₂₈H₃₂N₇O₂S₂Cl 598.1826, found 598.1824.

(E)-N-(5-((4–(6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl)thieno[3,2-d]pyrimidin-2-yl)amino)-2-((2-(dimethylamino)ethyl)(methyl)amino)-4-methoxyphenyl)-4-methylpent-2-enamide(B2)

Yellow solid; yield: 35%; m.p.: 158.9–159.7 °C. ¹H NMR (400 MHz, CDCl₃) δ 9.51 (s, 1H), 9.36 (s, 1H), 7.60 (s, 1H), 7.09 (d, J = 5.0, 1H), 6.97 (dd, J = 14.1, 7.8, 2H), 6.92 − 6.83 (m, 2H), 6.72 (s, 1H), 6.25 (d, J = 15.3, 1H), 5.82 (d, J = 15.6, 1H), 5.06 (s, 3H), 4.22 (t, J = 4.8, 2H), 3.86 (s, 3H), 3.07 (dd, J = 12.6, 5.9, 4H), 2.66 (s, 4H), 2.47 (s, 6H), 1.13 (d, J = 6.7, 3H), 1.06 (d, J = 6.7, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 171.13, 164.00, 159.58, 156.13, 154.09, 151.17, 144.41, 134.62, 133.43, 133.22, 128.91, 127.59, 125.80, 122.79, 122.42, 120.66, 116.51, 111.14, 110.55, 103.98, 56.13, 53.85, 48.20, 45.97, 44.24, 43.80, 30.88, 30.83, 25.50, 21.78, 21.44. HRMS (ES) calcd [M + H]⁺ for C₃₁H₃₉N₇O₂S₂ 606.2685, found 606.2692.

N-(5-((4–(6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl)thieno[3,2-d]pyrimidin-2-yl)amino)-2-((2-(dimethylamino)ethyl)(methyl)amino)-4-methoxyphenyl)-3-methylbut-2-enamide(B3)

Yellow solid; yield: 34%; m.p.:188.4–189.4 °C. ¹H NMR (400 MHz, CDCl₃) δ 9.49 (d, J = 8.7, 2H), 7.53 (s, 1H), 7.27 (s, 1H), 7.09 (d, J = 4.9, 1H), 6.93–6.88 (m, 2H), 6.74 (s, 1H), 5.92 (s, 1H), 5.05 (s, 2H), 4.26 (t, J = 4.5, 2H), 3.85 (s, 3H), 3.09 (s, 2H), 2.94 (s, 2H), 2.68 (s, 3H), 2.42 (s, 2H), 2.34 (s, 6H), 2.23 (s, 3H), 1.92 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 171.34, 164.65, 159.55, 156.21, 150.60, 144.05, 134.48, 133.72, 133.03, 129.44, 127.42, 125.47, 122.80, 120.72, 120.10, 116.37, 110.50, 104.25, 57.05, 56.12, 48.32, 45.66, 45.21, 43.70, 27.47, 25.37, 19.91. HRMS (ES) calcd [M + H]⁺ for C₃₀H₃₇N₇O₂S₂ 592.2528, found 592.2517.

N-(5-((4–(6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl)thieno[3,2-d]pyrimidin-2-yl)amino)-4-methoxy-2–(4-methylpiperazin-1-yl)phenyl)acrylamide(B4)

Yellow solid; yield: 34%; m.p.:175.8–176.7 °C. ¹H NMR (400 MHz, CDCl₃) δ 9.53 (s, 1H), 8.62 (s, 1H), 7.55 (s, 1H), 7.26 (s, 1H), 7.11 (d, J = 5.0, 1H), 6.98 (d, J = 4.8, 1H), 6.91 (d, J = 5.9, 1H), 6.77 (s, 1H), 6.34 (dt, J = 17.0, 13.2, 2H), 5.76 (d, J = 9.7, 1H), 5.06 (s, 2H), 4.23 (t, J = 4.7, 2H), 3.86 (s, 3H), 3.09 (s, 2H), 2.92 (s, 4H), 2.62 (s, 4H), 2.40 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 171.27, 162.32, 159.61, 156.12, 144.29, 134.29, 133.47, 133.23, 132.40, 127.45, 126.86, 126.21, 125.67, 122.87, 120.66, 116.56, 110.60, 109.91, 103.41, 56.05, 55.99, 52.39, 48.24, 46.10, 45.94, 29.71, 25.49. HRMS (ES) calcd [M + H]⁺ for C₂₈H₃₁N₇O₂S₂ 562.2059, found 562.2058.

N-(2-((2-(dimethylamino)ethyl)(methyl)amino)-4-methoxy-5-((4-((1-methyl-1H-pyrazol-4-yl)amino)thieno[3,2-d]pyrimidin-2-yl)amino)phenyl)propionamide(B5)

Yellow solid; yield: 32%; m.p.: 102.2–103.1 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 9.68 (s, 1H), 9.63 (s, 1H), 8.47 (s, 1H), 8.08 (s, 1H), 8.03 (s, 1H), 7.58 (d, J = 5.8, 1H), 7.54 (s, 1H), 7.15 (d, J = 5.9, 1H), 6.96 (s, 1H), 3.79 (s, 3H), 3.76 (s, 3H), 3.01 (s, 2H), 2.64 (d, J = 29.6, 5H), 2.39 (s, 8H), 1.09 (t, J = 7.3, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 168.55, 158.46, 158.42, 154.19, 130.82, 130.74, 125.27, 122.58, 122.43, 119.71, 117.50, 110.86, 105.60, 56.33, 49.07, 44.93, 44.84, 43.00, 29.91, 10.38. HRMS (ES) calcd [M + H]⁺ for C₂₅H₃₃N₉O₂S 524.2556, found 524.2567.

N-(2-((2-(dimethylamino)ethyl)(methyl)amino)-4-methoxy-5-((4-((1-methyl-1H-pyrazol-4-yl)amino)thieno[3,2-d]pyrimidin-2-yl)amino)phenyl)acrylamide(B6)

Yellow solid; yield: 30%; m.p.:183.8–184.9 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 10.05 (s, 1H), 9.67 (s, 1H), 8.64 (s, 1H), 8.14–8.02 (m, 3H), 7.60 (d, J = 6.1, 1H), 7.54 (s, 1H), 7.15 (d, J = 5.8, 1H), 7.00 (s, 1H), 6.21 (d, J = 16.9, 1H), 5.73 (d, J = 10.3, 1H), 3.80 (s, 3H), 3.74 (s, 3H), 3.17 (s, 1H), 3.13 (s, 1H), 2.94 (s, 2H), 2.71 (s, 3H), 2.29 (s, 6H). ¹³C NMR (101 MHz, DMSO-d₆) δ 179.97, 163.08, 154.18, 132.74, 132.01, 130.70, 130.69, 129.14, 127.89, 127.80, 127.55, 126.63, 125.35, 122.61, 122.41, 119.76, 117.48, 56.32, 45.37, 42.76. HRMS (ES) calcd [M + H]⁺ for C₂₅H₃₁N₉O₂S 522.2400, found 522.2393.

2-chloro-N-(2-((2-(dimethylamino)ethyl)(methyl)amino)-4-methoxy-5-((4-((1-methyl-1H-pyrazol-4-yl)amino)thieno[3,2-d]pyrimidin-2-yl)amino)phenyl)acrylamide(B7)

Yellow solid; yield: 33%; m.p.:111.6–112.5 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 9.96 (s, 1H), 9.77 (s, 1H), 8.57 (s, 1H), 8.14 (s, 1H), 8.09 (s, 1H), 7.66 (d, J = 5.8, 1H), 7.60 (s, 1H), 7.18 (d, J = 5.8, 1H), 7.06 (s, 1H), 6.57 (s, 1H), 6.09 (s, 1H), 3.87 (s, 3H), 3.78 (s, 3H), 3.35 (s, 2H), 2.67 (s, 3H), 2.61 (s, 2H), 1.27 (dd, J = 15.9, 9.3, 6H). ¹³C NMR (101 MHz, DMSO-d₆) δ 168.34, 159.01, 157.99, 154.24, 148.99, 139.06, 133.40, 130.90, 126.01, 125.88, 123.47, 122.55, 122.44, 119.89, 117.78, 117.16, 111.16, 105.62, 56.52, 55.07, 55.06, 53.68, 44.26, 43.49. HRMS (ES) calcd [M + H]⁺ for C₂₅ H₃₀N₉O₂SCl 556.2010, found 556.2005.

(E)-N-(2-((2-(dimethylamino)ethyl)(methyl)amino)-4-methoxy-5-((4-((1-methyl-1H-pyrazol-4-yl)amino)thieno[3,2-d]pyrimidin-2-yl)amino)phenyl)-4-methylpent-2-enamide(B8)

Yellow solid; yield: 32%; m.p.:102.8–103.9 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 9.90 (s, 1H), 9.62 (s, 1H), 8.64 (s, 1H), 8.09 (s, 1H), 8.06 (s, 1H), 7.57 (d, J = 5.8, 1H), 7.52 (s, 1H), 7.15 (d, J = 5.9, 1H), 6.99 (s, 1H), 6.75 (dd, J = 14.7, 6.3, 1H), 6.07 (d, J = 15.3, 1H), 3.79 (s, 3H), 3.74 (s, 3H), 2.91 (s, 1H), 2.71 (s, 3H), 2.46 − 2.41 (m, 2H), 2.29 (s, 6H), 1.07 (s, 2H), 1.05 (s, 3H), 1.01 (d, J = 6.7, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 168.58, 163.56, 158.49, 155.06, 154.17, 150.55, 148.56, 139.49, 130.64, 127.97, 125.34, 122.64, 122.60, 122.42, 119.96, 119.67, 117.46, 110.83, 105.86, 57.09, 56.30, 45.59, 42.60, 30.58, 30.50, 21.89, 21.58. HRMS (ES) calcd [M + H]⁺ for C₂₈H₃₇N₉O₂S 564.2869, found 564.2855.

N-(2-((2-(dimethylamino)ethyl)(methyl)amino)-4-methoxy-5-((4-((1-methyl-1H-pyrazol-4-yl)amino)thieno[3,2-d]pyrimidin-2-yl)amino)phenyl)-3-methylbut-2-enamide(B9)

Yellow solid; yield: 32%; m.p.:118.8–119.7 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 9.67 (d, J = 24.9, 2H), 8.59 (s, 1H), 8.07 (d, J = 19.0, 2H), 7.62–7.47 (m, 2H), 7.14 (d, J = 5.6, 1H), 6.97 (s, 1H), 5.96 (s, 1H), 3.79 (s, 3H), 3.74 (s, 3H), 3.17 (s, 2H), 2.96 (s, 2H), 2.68 (s, 3H), 2.36 (s, 6H), 2.11 (s, 3H), 1.86 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 168.58, 164.70, 158.50, 150.80, 148.44, 139.37, 130.63, 127.85, 125.25, 122.59, 122.44, 120.00, 119.71, 117.73, 117.43, 110.82, 105.70, 73.11, 70.24, 56.31, 49.06, 45.18, 45.12, 42.66, 27.48, 19.85. HRMS (ES) calcd [M + H]⁺ for C₂₇H₃₅N₉O₂S 550.2713, found 550.2706.

(E)-N-(2-((2-(dimethylamino)ethyl)(methyl)amino)-4-methoxy-5-((4-((1-methyl-1H-pyrazol-4-yl)amino)thieno[3,2-d]pyrimidin-2-yl)amino)phenyl)but-2-enamide(B10)

Yellow solid; yield: 30%; m.p.: 148.0–149.1 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 9.76 (s, 1H), 9.69 (s, 1H), 8.54 (s, 1H), 8.11 (s, 1H), 8.06 (s, 1H), 7.61 (d, J = 5.8, 1H), 7.54 (s, 1H), 7.15 (d, J = 5.8, 1H), 6.96 (s, 1H), 6.77–6.71 (m, 1H), 6.39 (d, J = 14.9, 1H), 3.80 (s, 3H), 3.75 (s, 3H), 3.70–3.64 (m, 2H), 3.07 (s, 2H), 2.66 (s, 3H), 1.84 (dd, J = 14.2, 6.7, 6H), 1.23 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 168.53, 158.41, 154.17, 148.68, 144.60, 139.75, 139.53, 130.67, 126.92, 125.23, 123.92, 122.61, 122.41, 119.79, 117.46, 110.89, 105.48, 56.56, 56.33, 49.06, 42.94. HRMS (ES) calcd [M + H]⁺ for C₂₆H₃₃N₉O₂S 536.2556, found 536.2544.

2-chloro-N-(4-methoxy-5-((4-((1-methyl-1H-pyrazol-4-yl)amino)thieno[3,2-d]pyrimidin-2-yl)amino)-2–(4-methylpiperazin-1-yl)phenyl)acrylamide(B11)

Yellow solid; yield: 34%; m.p.:148.8–150.0 °C. ¹H NMR (400 MHz, CDCl₃) δ 9.94 (s, 1H), 9.48 (s, 1H), 7.85 (s, 1H), 7.44 (d, J = 9.8, 2H), 7.04 (s, 1H), 6.85–6.80 (m, 2H), 6.68 (s, 1H), 5.89 (s, 1H), 3.86 (s, 3H), 3.82 (s, 3H), 2.95 (s, 4H), 2.68 (s, 4H), 2.41 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 157.84, 156.59, 145.69, 145.67, 135.66, 133.05, 126.96, 126.12, 123.29, 121.71, 118.13, 117.71, 111.00, 110.77, 103.77, 55.95, 55.81, 52.44, 46.07, 39.17. HRMS (ES) calcd [M + H]⁺ for C₂₅H₂₈N₉O₂SCl 554.1853, found 554.1860.

N-(2-((2-(dimethylamino)ethyl)(methyl)amino)-4-methoxy-5-((4-morpholinothieno[3,2-d]pyrimidin-2-yl)amino)phenyl)propionamide(B12)

Brown liquid; yield: 33%; ¹H NMR (400 MHz, CDCl₃) δ 10.70 (s, 1H), 9.16 (s, 1H), 8.99 (s, 1H), 7.54 (s, 1H), 6.91 (d, J = 5.8, 1H), 6.66 (s, 1H), 3.97 (s, 4H), 3.87 (s, 3H), 3.66 (d, J = 6.5, 2H), 3.34 (s, 2H), 3.20 (s, 2H), 3.10 (d, J = 7.3, 2H), 2.84 (s, 5H), 2.67 (s, 6H), 1.25 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 203.38, 155.68, 144.68, 120.62, 116.93, 110.53, 103.06, 66.92, 56.29, 55.14, 53.86, 47.48, 44.29, 43.47, 42.11, 29.71, 17.40, 12.05, 10.23. HRMS (ES) calcd [M + H]⁺ for C₂₅H₃₅N₇O₃S 514.2600, found 514.2592.

N-(2-((2-(dimethylamino)ethyl)(methyl)amino)-4-methoxy-5-((4-morpholinothieno[3,2-d]pyrimidin-2-yl)amino)phenyl)acrylamide(B13)

Brown liquid; yield: 30%; ¹H NMR (400 MHz, CDCl₃) δ 9.88 (s, 1H), 9.46 (s, 1H), 7.54 (s, 2H), 7.22 (d, J = 6.1, 1H), 6.90 (d, J = 6.0, 2H), 6.74 (s, 1H), 5.67 (d, J = 10.2, 1H), 4.02 (s, 4H), 3.89 (s, 4H), 3.86 (s, 3H), 2.95 (s, 2H), 2.68 (s, 3H), 2.43 (s, 2H), 2.35 (s, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 163.04, 159.58, 156.05, 134.76, 132.35, 130.93, 128.85, 127.51, 125.90, 120.63, 116.65, 104.29, 67.05, 65.59, 56.93, 56.90, 56.06, 47.54, 44.99, 43.79. HRMS (ES) calcd [M + H]⁺ for C₂₅H₃₃N7O₃S 512.2444, found 512.2435.

2-chloro-N-(2-((2-(dimethylamino)ethyl)(methyl)amino)-4-methoxy-5-((4-morpholinothieno[3,2-d]pyrimidin-2-yl)amino)phenyl)acrylamide(B14)

Yellow solid; yield: 32%; m.p.:176.6–177.8 °C. ¹H NMR (400 MHz, CDCl₃) δ 10.12 (s, 1H), 9.44 (s, 1H), 7.57 (s, 1H), 7.22 (d, J = 6.0, 1H), 6.91 (d, J = 6.0, 1H), 6.79 (s, 1H), 6.61 (s, 1H), 5.85 (s, 1H), 4.02 (s, 4H), 3.88 (s, 7H), 3.09–3.02 (m, 2H), 2.67 (s, 3H), 2.43–2.37 (m, 2H), 2.27 (s, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 171.71, 159.57, 157.92, 155.96, 144.86, 134.75, 133.70, 127.94, 127.66, 122.08, 120.61, 116.83, 110.48, 109.37, 104.14, 67.03, 57.26, 56.06, 54.78, 47.52, 45.25, 44.61, 29.72. HRMS (ES) calcd [M + H]⁺ for C₂₅H₃₂N₇O₃SCl 546.2054, found 546.2048.

2-chloro-N-(2-((2-(dimethylamino)ethyl)(methyl)amino)-4-methoxy-5-((4-thiomorpholinothieno[3,2-d]pyrimidin-2-yl)amino)phenyl)acrylamide(B15)

Yellow solid; yield: 30%; m.p.: 68.5–69.8 °C. ¹H NMR (400 MHz, CDCl₃) δ 9.99 (s, 1H), 9.40 (s, 1H), 7.53 (s, 1H), 7.14 (d, J = 6.0, 1H), 6.93 (d, J = 5.9, 1H), 6.81 (s, 1H), 6.63 (s, 1H), 5.86 (s, 1H), 4.26 (s, 4H), 3.90 (s, 3H), 3.18 (s, 2H), 2.82 (s, 4H), 2.68 (s, 3H), 2.57 (s, 2H), 2.41 (s, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 171.61, 159.39, 157.89, 155.95, 145.06, 134.28, 133.48, 127.72, 122.39, 120.53, 117.07, 110.63, 109.59, 104.01, 56.77, 56.74, 56.14, 53.67, 53.60, 50.21, 44.79, 29.71, 27.37. HRMS (ES) calcd [M + H]⁺ for C₂₅H₃₂N₇O₂S₂Cl 562.1826, found 562.1819.

2-chloro-N-(2-((2-(dimethylamino)ethyl)(methyl)amino)-4-methoxy-5-((4-(pyrrolidin-1-yl)thieno[3,2-d]pyrimidin-2-yl)amino)phenyl)acrylamide(B16)

Yellow solid; yield: 35%; m.p.:155.0–156.2 °C. ¹H NMR (400 MHz, CDCl₃) δ 10.07 (s, 1H), 9.51 (s, 1H), 7.49 (s, 1H), 7.34 (d, J = 5.9, 1H), 6.82–6.75 (m, 2H), 6.62 (s, 1H), 5.84 (s, 1H), 3.93 (s, 4H), 3.87 (s, 3H), 3.07 (t, J = 6.5, 2H), 2.67 (s, 3H), 2.45–2.40 (m, 2H), 2.29 (s, 6H), 2.04 (s, 4H). ¹³C NMR (101 MHz, CDCl₃) δ 170.35, 157.90, 156.62, 156.26, 144.91, 134.41, 133.85, 128.14, 127.83, 121.87, 121.36, 115.11, 110.53, 109.77, 103.95, 57.21, 56.07, 54.63, 49.24, 45.20, 44.65. HRMS (ES) calcd [M + H]⁺ for C₂₅H₃₂N₇O₂SCl 530.2105, found 530.2090.

MTT assay

In vitro, antitumor activity of pyrido[2,3-d]pyrimidines (A1–A15) and thieno[3,2-d]pyrimidines (B1–B16) containing structural compounds and their inhibitory activity against various cancer cell lines, including NCI-H1975, A549 and NCI-H460 cells, were evaluated by MTT using AZD9291 and Olmutinib as positive controls.

In vitro enzymatic activity tests

The activities of three EGFR kinases (EGFR^L858R/T790M, EGFR^{L858R/T790M/C797S}, and EGFR^WT) were tested against the target compounds and the method was determined by enzyme-linked immunosorbent assay (ELISA).

Morphology assays of apoptotic cells

A549 cells were added to 6-well plates at a concentration of 3.6 × 10⁵/mL and incubated for 24h. Subsequently, cells were treated with different concentrations of compounds B1 and B7, respectively, and incubation was continued for 48h. A dual fluorescent staining solution containing AO/EB was added to each well for a few min. Cell morphology was observed by forward fluorescence microscopy and photographed.

Wound healing assays

A549 cells (3.6 × 10⁵/well) were treated with compounds B1 and B7 for 0 h, 24 h, and 48 h. Cells were observed by microscopy and photographed.

Cell apoptosis assay

Apoptosis was measured by flow cytometry using Annexin V/PI double staining by flow cytometry. H1975 cells at logarithmic growth stage were taken, cell counted, cell concentration adjusted, and inoculated into 6-well culture plates according to 3 × 10⁵/well, 5% CO₂, and incubated in a constant temperature incubator at 37 °C for 24 h. Cells were treated with different concentrations of compound B1. After 48 h of incubation. The cells were collected by trypsin digestion, washed 1 time with 1 ml/tube of PBS, and 200 μL of Annexin V-APC binding solution was added to resuspend the cells, and 5 μL of Annexin V-APC staining solution was added. The cells were incubated for 30 min at room temperature and protected from light. 10 μL of PI was added and incubated for 3 min at room temperature and protected from light. 200 μL of physiological saline was added, mixed well, and immediately measured by flow cytometry.

Cell cycle progression assay

H1975 cells were seeded in 6-well plates (3 × 10⁵/well) and cultured for 24 h, then treated with different concentrations (0.2, 1.0, 5 μM) of compounds and incubated at 37 °C for 24 h. Tumour cells were collected, washed twice with PBS (stored at 4 °C), and then fixed overnight in ice-cold 70% (v/v) ethanol, tumour cells were washed again with PBS and stained with PI (400 μL) for 30 min at room temperature in the dark. Finally, data acquisition and analysis were performed by using flow cytometry.

Molecular docking studies

The prepared crystal structure of EGFR^T790M (PDB code: 3IKA) was used for compound docking. First, protein preparation and optimisation was performed, mainly including the addition of hydrogen atoms and the removal of water molecules. Subsequently, the preparation of small molecules was performed, and finally, the docking was performed using AutoDock software, and the results were analysed using discovery studio 4.5 and PyMol 1.8.6 software.

Disclosure statement

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Additional information

Funding

This work was financially supported by the National Natural Science Foundation of China (Grant No. 81903469), Development Plan for Youth Innovation Team in Higher School of Shandong Province (Grant No. 2022KJ266), and the Graduate Student Research Grant from Weifang Medical University.