Synthesis and In Vitro Antitumor Activity Evaluation of Gefitinib-1,2,3-Triazole Derivatives

In this study, 14 structurally novel gefitinib-1,2,3-triazole derivatives were synthesized using a click chemistry approach and characterized by 1H NMR, 13C NMR and high-resolution mass spectrometry (HRMS). Preliminary cell counting kit-8 results showed that most of the compounds exhibit excellent antitumor activity against epidermal growth factor receptor wild-type lung cancer cells NCI-H1299, A549 and NCI-H1437. Among them, 4b and 4c showed the most prominent inhibitory effects. The half maximal inhibitory concentration (IC50) values of 4b were 4.42 ± 0.24 μM (NCI-H1299), 3.94 ± 0.01 μM (A549) and 1.56 ± 0.06 μM (NCI-1437). The IC50 values of 4c were 4.60 ± 0.18 µM (NCI-H1299), 4.00 ± 0.08 μM (A549) and 3.51 ± 0.05 μM (NCI-H1437). Furthermore, our results showed that 4b and 4c could effectively inhibit proliferation, colony formation and cell migration in a concentration-dependent manner, as well as induce apoptosis in H1299 cells. In addition, 4b and 4c exerted its anti-tumor effects by inducing cell apoptosis, upregulating the expression of cleaved-caspase 3 and cleaved-PARP and downregulating the protein levels of Bcl-2. Based on these results, it is suggested that 4b and 4c be developed as potential new drugs for lung cancer treatment.


Introduction
Epidermal growth factor receptor (EGFR), encoded by the human epidermal receptor 1 (HER1, ErbB1) gene, is a cell surface receptor that belongs to the ERBB protein receptor family along with HER2/neu (ErbB2), HER3 (ErbB3) and HER4 (ErbB4).These receptor tyrosine kinase transmembrane proteins play a crucial role in regulating the proliferation, differentiation [1] and apoptosis of tumor cells.Studies have shown that inhibiting the activity and expression of tyrosine kinases can effectively suppress tumor growth and proliferation.Therefore, targeting EGFR has become a hot topic in the research of antitumor drugs.The United States Food and Drug Administration approved the first-generation EGFR tyrosine kinase inhibitors (TKIs)-gefitinib and erlotinib [2]-in 2003 and 2004, respectively.Initially approved for patients with advanced non-small cell lung cancer (NSCLC) who had not responded favorably to conventional chemotherapy, these agents demonstrated efficacy in a subset of participants during preliminary clinical trials [3,4].In 2004, several clinical studies [5,6] showed that the sensitivity of NSCLC to erlotinib or gefitinib is associated with specific activating mutations in the EGFR gene (Figure 1).In 2006, Japanese researchers [7] conducted a phase II clinical trial, using gefitinib as a first-line treatment for 16 patients with advanced NSCLC with EGFR mutations, achieving a response rate of 75%.Subsequent largescale phase III randomized controlled trials confirmed the superiority of EGFR-TKIs as the first-line treatment for patients with NSCLC with EGFR mutations.Compared to traditional chemotherapy, EGFR-TKIs demonstrated better progression-free survival, objective response rate, quality of life and tolerability.The use of targeted drugs has increased the median demonstrated be er progression-free survival, objective response rate, quality of life and tolerability.The use of targeted drugs has increased the median survival of patients from 4 months to over 40 months.These studies have further established the role of EGFR-TKIs for the first-line treatment of EGFR-mutant advanced NSCLC.EGFR-TKIs exhibit good inhibitory effects on EGFR-mutant lung cancer cells.However, their activity is weaker against wild-type than mutant lung cancer cells.We aimed to enhance the inhibitory activity against wild-type lung cancer cells by modifying the structure of EGFR-TKIs.

(gefitinib)
2 (erlotinib) The 1,2,3-triazole is a crucial class of nitrogen-containing heterocyclic compounds, consisting of a five-membered ring constructed from three nitrogen atoms and two carbon atoms [8].With a molecular formula of C2N3H3, 1,2,3-triazoles possess a unique planar and rigid structure, granting them the robust ability to intercalate with DNA.Additionally, they exhibit a large dipole moment, allowing for various non-covalent interactions with different biological targets such as hydrophobic, hydrogen bonding, van der Waals forces and dipole-dipole interactions [9].Moreover, the structural characteristics of 1,2,3triazoles permit them to serve as electronic equivalents of amides, esters, carboxylic acids and rigid analogs of olefins, endowing them with broad-spectrum biological activities [10].Consequently, they are often employed as crucial molecular building blocks for the synthesis of active compounds, including antibacterial [11], antimalarial [12], antifungal [13], antiviral [14], antitubercular [15] and anticancer agents [16,17], showcasing its widespread applications in the field of medicinal chemistry.
Numerous clinical drugs have demonstrated enhanced or novel biological activities through modification with 1,2,3-triazoles.For example, five erlotinib-conjugated 1,2,3-triazole derivatives were designed using a structure-based drug design strategy to explore novel IDO1 inhibitors.Among them, compound e with an o-bromobenzyl structure on the triazole ring showed the best IDO1 inhibitory activity (IC50 = 0.32 ± 0.07 µM) [18].In another study, the anti-HIV drug cabotegravir was modified by introducing a 1,2,3-triazole moiety to obtain compounds KJ1-KJ19.These compounds exhibited notable inhibitory effects on the proliferation of HepG2 liver cancer cells.Compounds KJ-5 and KJ-12 showed promising inhibitory potencies in particular.Furthermore, both KJ-5 and KJ-12 were found to induce apoptosis via the mitochondrial pathway and inhibit the invasion and migration of cancer cells.[19].By utilizing the structural features of the EGFR inhibitor erlotinib, compounds a1-a22 with erlotinib-1,2,3-triazole derivatives were obtained through click reactions [20].These compounds exhibited excellent inhibitory activity against five NSCLC cell lines, three esophageal squamous cell carcinoma cell lines and some chemotherapy-resistant tumor cell lines.Compound a12 showed the best performance (half maximal inhibitory concentration [IC50] < 10 M) and demonstrated strong antiproliferative and anticloning abilities against various tumor cells.Additionally, it induced apoptosis and cell cycle arrest in tumor cells.Compound 3l, targeting EGFR, exhibited binding to EGFR protein and downregulated the expression of phosphorylated extracellular signal-regulated kinase and Ki-67, demonstrating potential as an EGFR inhibitor [21].Moreover, compounds MMG-0358 and Vertex-AT, incorporating 1,2,3-triazole into the phenyl ring and aniline structure, were developed as indoleamine 2,3-dioxygenase-1 The 1,2,3-triazole is a crucial class of nitrogen-containing heterocyclic compounds, consisting of a five-membered ring constructed from three nitrogen atoms and two carbon atoms [8].With a molecular formula of C 2 N 3 H 3 , 1,2,3-triazoles possess a unique planar and rigid structure, granting them the robust ability to intercalate with DNA.Additionally, they exhibit a large dipole moment, allowing for various non-covalent interactions with different biological targets such as hydrophobic, hydrogen bonding, van der Waals forces and dipoledipole interactions [9].Moreover, the structural characteristics of 1,2,3-triazoles permit them to serve as electronic equivalents of amides, esters, carboxylic acids and rigid analogs of olefins, endowing them with broad-spectrum biological activities [10].Consequently, they are often employed as crucial molecular building blocks for the synthesis of active compounds, including antibacterial [11], antimalarial [12], antifungal [13], antiviral [14], antitubercular [15] and anticancer agents [16,17], showcasing its widespread applications in the field of medicinal chemistry.
Numerous clinical drugs have demonstrated enhanced or novel biological activities through modification with 1,2,3-triazoles.For example, five erlotinib-conjugated 1,2,3-triazole derivatives were designed using a structure-based drug design strategy to explore novel IDO1 inhibitors.Among them, compound e with an o-bromobenzyl structure on the triazole ring showed the best IDO1 inhibitory activity (IC 50 = 0.32 ± 0.07 µM) [18].In another study, the anti-HIV drug cabotegravir was modified by introducing a 1,2,3-triazole moiety to obtain compounds KJ1-KJ19.These compounds exhibited notable inhibitory effects on the proliferation of HepG2 liver cancer cells.Compounds KJ-5 and KJ-12 showed promising inhibitory potencies in particular.Furthermore, both KJ-5 and KJ-12 were found to induce apoptosis via the mitochondrial pathway and inhibit the invasion and migration of cancer cells.[19].By utilizing the structural features of the EGFR inhibitor erlotinib, compounds a1-a22 with erlotinib-1,2,3-triazole derivatives were obtained through click reactions [20].These compounds exhibited excellent inhibitory activity against five NSCLC cell lines, three esophageal squamous cell carcinoma cell lines and some chemotherapyresistant tumor cell lines.Compound a12 showed the best performance (half maximal inhibitory concentration [IC 50 ] < 10 M) and demonstrated strong antiproliferative and anticloning abilities against various tumor cells.Additionally, it induced apoptosis and cell cycle arrest in tumor cells.Compound 3l, targeting EGFR, exhibited binding to EGFR protein and downregulated the expression of phosphorylated extracellular signal-regulated kinase and Ki-67, demonstrating potential as an EGFR inhibitor [21].Moreover, compounds MMG-0358 and Vertex-AT, incorporating 1,2,3-triazole into the phenyl ring and aniline structure, were developed as indoleamine 2,3-dioxygenase-1 (IDO1) inhibitors.These compounds deeply penetrate the target protein of IDO1, forming hydrogen bonds with the ferrous ion in the heme of the target.The triazole-linked phenyl ring can penetrate a hydrophobic pocket surrounded by amino acids Cys129, Leu234 and Gly262.MMG-0358 and Vertex-AT both exhibit outstanding IDO1 inhibitory activity, with IC 50 values reaching 0.082 and 0.023 µM, respectively [22] (Figure 2).
Molecules 2024, 29, x FOR PEER REVIEW 3 of 17 (IDO1) inhibitors.These compounds deeply penetrate the target protein of IDO1, forming hydrogen bonds with the ferrous ion in the heme of the target.The triazole-linked phenyl ring can penetrate a hydrophobic pocket surrounded by amino acids Cys129, Leu234 and Gly262.MMG-0358 and Vertex-AT both exhibit outstanding IDO1 inhibitory activity, with IC50 values reaching 0.082 and 0.023 µM, respectively [22] (Figure 2).To identify the compounds with potent inhibitory effects on EGFR wild-type lung cancer cells, we modified gefitinib through a click reaction by introducing a 1,2,3-triazole moiety to its structure (Figure 3).We aimed to evaluate its inhibitory effects on wild-type lung cancer cells.Using gefitinib as a positive control, we employed the cell counting kit-8 (CCK-8) method to assess the in vitro antitumor activity against EGFR wild-type lung cancer cells NCI-H1299, A549 and NCI-H1437.Additionally, we investigated the impact on tumor cell clone formation, migration capability and apoptosis.

Chemistry
The synthetic strategy for the preparation of the target molecules is illustrated through a literature review [23][24][25] in Scheme 1.The key intermediate 2 was obtained through chlorination with 7-methoxy-6-(3-morpholinopropoxy)quinazolin-4(3H)-one as the raw material, and subsequent condensation with 3-aminophenylethynyl to yield To identify the compounds with potent inhibitory effects on EGFR wild-type lung cancer cells, we modified gefitinib through a click reaction by introducing a 1,2,3-triazole moiety to its structure (Figure 3).We aimed to evaluate its inhibitory effects on wild-type lung cancer cells.Using gefitinib as a positive control, we employed the cell counting kit-8 (CCK-8) method to assess the in vitro antitumor activity against EGFR wild-type lung cancer cells NCI-H1299, A549 and NCI-H1437.Additionally, we investigated the impact on tumor cell clone formation, migration capability and apoptosis.
(IDO1) inhibitors.These compounds deeply penetrate the target protein of IDO1, forming hydrogen bonds with the ferrous ion in the heme of the target.The triazole-linked phenyl ring can penetrate a hydrophobic pocket surrounded by amino acids Cys129, Leu234 and Gly262.MMG-0358 and Vertex-AT both exhibit outstanding IDO1 inhibitory activity, with IC50 values reaching 0.082 and 0.023 µM, respectively [22] (Figure 2).To identify the compounds with potent inhibitory effects on EGFR wild-type lung cancer cells, we modified gefitinib through a click reaction by introducing a 1,2,3-triazole moiety to its structure (Figure 3).We aimed to evaluate its inhibitory effects on wild-type lung cancer cells.Using gefitinib as a positive control, we employed the cell counting kit-8 (CCK-8) method to assess the in vitro antitumor activity against EGFR wild-type lung cancer cells NCI-H1299, A549 and NCI-H1437.Additionally, we investigated the impact on tumor cell clone formation, migration capability and apoptosis.

Chemistry
The synthetic strategy for the preparation of the target molecules is illustrated through a literature review [23][24][25] in Scheme 1.The key intermediate 2 was obtained through chlorination with 7-methoxy-6-(3-morpholinopropoxy)quinazolin-4(3H)-one as the raw material, and subsequent condensation with 3-aminophenylethynyl to yield

Chemistry
The synthetic strategy for the preparation of the target molecules is illustrated through a literature review [23][24][25] in Scheme 1.The key intermediate 2 was obtained through chlorination with 7-methoxy-6-(3-morpholinopropoxy)quinazolin-4(3H)-one as the raw material, and subsequent condensation with 3-aminophenylethynyl to yield intermediate 3. Finally, 1,2,3-triazole gefitinib derivatives were synthesized through a copper(I)-catalyzed azide-alkyne cycloaddition reaction.The structures of the key intermediates and all target compounds were confirmed through nuclear magnetic resonance spectroscopy ( 1 H NMR and 13 C NMR) and high-resolution mass spectrometry (HRMS).intermediate 3. Finally, 1,2,3-triazole gefitinib derivatives were synthesized through a copper(I)-catalyzed azide-alkyne cycloaddition reaction.The structures of the key intermediates and all target compounds were confirmed through nuclear magnetic resonance spectroscopy ( 1 H NMR and 13 C NMR) and high-resolution mass spectrometry (HRMS).Scheme 1. Synthesis route of gefitinib-1,2,3-triazole derivatives (compounds 4).

Compounds 4a-4n Suppress Cancer Cell Viability
The cell inhibitory effects of compounds 4a-4n on EGFR wild-type lung cancer cells NCI-H1299, NCI-H1437 and A549 were evaluated using the CCK-8 assay, with gefitinib as a positive control.The results showed that most compounds exhibited inhibitory effects on NCI-H1299, NCI-H1437 and A549 lung cancer cells, with compounds 4b and 4c showing the best performance.In the NCI-H1299 cell line, the IC50 values of 4b and 4c were 4.42 ± 0.24 µM and 4.60 ± 0.18 µM.In the NCI-H1437 cell line, the IC50 values of 4b and 4c were 1.56 ± 0.06 µM and 3.51 ± 0.05 µM.In the A549 cell line, the IC50 values of 4b and 4c were 3.94 ± 0.01 µM and 4.00 ± 0.08 µM.These values were be er than the inhibitory effects of gefitinib on the three cell lines (IC50 values = 14.23 ± 0.08 µM, 20.44 ± 1.43 µM, and 15.11 ± 0.05 µM, respectively)(Table 1).In the L02 cell line, the IC50 values of 4b and 4c were 20.25 ± 1.26 µM and 29.38 ± 5.53 µM.In our study, we observed that the inhibition rate of 4b and 4c at a concentration of 4 µM on normal hepatic cells L02 is less than 20%.However, the inhibition rate on lung cancer cells was found to reach 50%, indicating a certain selective effect of these two compounds on tumor cells (Table 2).The results indicated that the structural modification of gefitinib was successful, and after the introduction of benzyl or phenyl groups, the compounds exhibited significantly different activities.When the triazole linkage was a benzyl group, the activity was be er than that of the phenyl group.The presence of fluorine or bromine atoms in the ortho or meta position of the benzyl ring was favorable for enhancing the in vitro antitumor activity of the compounds.

Compounds 4a-4n Suppress Cancer Cell Viability
The cell inhibitory effects of compounds 4a-4n on EGFR wild-type lung cancer cells NCI-H1299, NCI-H1437 and A549 were evaluated using the CCK-8 assay, with gefitinib as a positive control.The results showed that most compounds exhibited inhibitory effects on NCI-H1299, NCI-H1437 and A549 lung cancer cells, with compounds 4b and 4c showing the best performance.In the NCI-H1299 cell line, the IC 50 values of 4b and 4c were 4.42 ± 0.24 µM and 4.60 ± 0.18 µM.In the NCI-H1437 cell line, the IC 50 values of 4b and 4c were 1.56 ± 0.06 µM and 3.51 ± 0.05 µM.In the A549 cell line, the IC 50 values of 4b and 4c were 3.94 ± 0.01 µM and 4.00 ± 0.08 µM.These values were better than the inhibitory effects of gefitinib on the three cell lines (IC 50 values = 14.23 ± 0.08 µM, 20.44 ± 1.43 µM, and 15.11 ± 0.05 µM, respectively)(Table 1).In the L02 cell line, the IC 50 values of 4b and 4c were 20.25 ± 1.26 µM and 29.38 ± 5.53 µM.In our study, we observed that the inhibition rate of 4b and 4c at a concentration of 4 µM on normal hepatic cells L02 is less than 20%.However, the inhibition rate on lung cancer cells was found to reach 50%, indicating a certain selective effect of these two compounds on tumor cells (Table 2).The results indicated that the structural modification of gefitinib was successful, and after the introduction of benzyl or phenyl groups, the compounds exhibited significantly different activities.When the triazole linkage was a benzyl group, the activity was better than that of the phenyl group.The presence of fluorine or bromine atoms in the ortho or meta position of the benzyl ring was favorable for enhancing the in vitro antitumor activity of the compounds.All values are given as means ± standard deviation (n = 3).

4b and 4c Induce Apoptosis in H1299 Cells
To elucidate whether the inhibitory effects of these compounds on cell proliferation and cytotoxicity are related to apoptosis, we conducted experiments with 4b and 4c which showed good inhibitory effects on the proliferation of the three cell lines.H1299 cells were treated with different concentrations of 4b and 4c for 48 h.The cells were stained with Annexin-V and propidium iodide (PI), and the proportion of apoptotic cells was detected by flow cytometry.As shown in Figure 4A, after treatment with compound 4b, the total apoptotic cell proportions of H1299 were 20.43 ± 1.72% (2 µM), 43.2 ± 2.4% (4 µM) and 77.4 ± 3.97% (8 µM).After treatment with compound 4c, the total apoptotic cell proportions of H1299 were 16.37 ± 0.57% (2 µM), 31.2 ± 2.36% (4 µM) and 51.77 ± 6.74% (8 µM).Compared with the normal control group, as the drug concentration increased the apoptotic proportion gradually increased (p < 0.01, p < 0.05).These results indicated that both 4b and 4c can significantly promote the apoptosis of H1299 cells in a concentration-dependent manner.
To further confirm the apoptosis-inducing activity of 4b and 4c in lung cancer cells, H1299 cells were treated with different concentrations of compounds 4b and 4c for 48 h, followed by 4 ,6-diamidino-2-phenylindole (DAPI) staining.Fluorescence microscopy was used to observe the cells and their representative images are shown in Figure 4B.Compared with the normal control group, H1299 cells treated with compounds 4b and 4c exhibited typical apoptotic features.As the drug concentration increased, nuclear staining intensified, and even nuclear condensation and fragmentation was observed.Therefore, the apoptosisrelated results suggest that both 4b and 4c can significantly promote apoptosis in human lung cancer cells (NCI-H1299) in a concentration-dependent manner.

4b and 4c Suppress Metastasis of H1299 Cells
Malignant proliferation forms the basis for the occurrence and development of tumors.When tumor cells proliferate to a certain extent, they may detach from the primary site and metastasize to other organs in the body [26].NSCLC is a highly metastatic cancer, and the low five-year survival rate is closely related to the high metastasis of lung cancer

4b and 4c Suppress Metastasis of H1299 Cells
Malignant proliferation forms the basis for the occurrence and development of tumors.When tumor cells proliferate to a certain extent, they may detach from the primary site and metastasize to other organs in the body [26].NSCLC is a highly metastatic cancer, and the low five-year survival rate is closely related to the high metastasis of lung cancer cells [27].Therefore, the metastasis of tumor cells is an important cause of death in cancer patients.
To investigate the inhibitory effects of compounds 4b and 4c on the migration of H1299 cells, cell scratch and transwell assays were employed.Figure 5A shows the results of the cell scratch assay, indicating that after treatment with compound 4b, the migration clo-sure rates of H1299 cells were 72.59 ± 2.12% (2 µM), 62.42 ± 5.50% (4 µM) and 49.79 ± 2.22% (8 µM).After treatment with compound 4c, the migration closure rates of H1299 cells were 53.26 ± 4.51% (2 µM), 40.04 ± 8.02% (4 µM) and 17.90 ± 3.46% (8 µM).The cell scratch closure rates in the compound-treated groups were significantly lower than those in the normal controlgroup and exhibited concentration dependence (p < 0.01, p < 0.05).Transwell migration experiment results, as shown in Figure 5B, indicated a significant reduction in the number of cells crossing the microporous membrane to reach the lower surface of the chamber in the compound-treated groups compared to the normal control group (p < 0.01).In summary, compounds 4b and 4c can inhibit the migration ability of H1299 cells in a concentration-dependent manner.

4b and 4c Inhibit the Clonogenic Ability of H1299 Cells
The colony formation assay was employed to assess the ability of adherent cells to proliferate and form cell colonies on a flat surface [28].It is an important method for detecting cell proliferation ability.In our study, the colony formation assay was used to further confirm that compounds 4b and 4c can inhibit the proliferation of H1299 cells (as shown in Figure 6).Cells were treated with low, medium and high concentrations (1, 2, 4

4b and 4c Inhibit the Clonogenic Ability of H1299 Cells
The colony formation assay was employed to assess the ability of adherent cells to proliferate and form cell colonies on a flat surface [28].It is an important method for detecting cell proliferation ability.In our study, the colony formation assay was used to further confirm that compounds 4b and 4c can inhibit the proliferation of H1299 cells (as shown in Figure 6).Cells were treated with low, medium and high concentrations (1, 2, 4 µM) of 4b and 4c for seven days.By counting the number of cell colonies, the analysis of the proliferation inhibition rate showed a significant reduction in the number of cell colonies in the compound-treated groups, and this effect was concentration-dependent.Even at the lowest concentration of 1 µM, compounds started to significantly inhibit the formation of H1299 cell colonies.Particularly at a concentration of 4 µM, the formation of colonies was almost completely inhibited, indicating that compounds 4b and 4c exert strong anti-proliferative effects on H1299 cells.
Molecules 2024, 29, x FOR PEER REVIEW 9 of 17 µM) of 4b and 4c for seven days.By counting the number of cell colonies, the analysis of the proliferation inhibition rate showed a significant reduction in the number of cell colonies in the compound-treated groups, and this effect was concentration-dependent.Even at the lowest concentration of 1 µM, compounds started to significantly inhibit the formation of H1299 cell colonies.Particularly at a concentration of 4 µM, the formation of colonies was almost completely inhibited, indicating that compounds 4b and 4c exert strong anti-proliferative effects on H1299 cells.

4b and 4c Trigger Apoptosis via the Apoptosis Signaling Pathway
We conducted an in-depth investigation into the mechanism by which compounds 4b and 4c induce apoptosis in cells.The B-cell lymphoma 2 (Bcl-2) protein family is located upstream in the apoptosis signaling pathway.Bcl-2 proteins play an anti-apoptotic role by inhibiting the release of cytochrome C (cyt-c) from the mitochondria.Many anticancer drugs induce apoptosis by downregulating Bcl-2 [29].When cell apoptosis occurs, the expression level of Bcl-2 is inhibited, cyt-c is released and, along with the apoptosis proteaseactivating factor, forms an apoptosome, initiating a cascade reaction of the caspase protein family.Initially, the apoptosome promotes the activation of caspase 9, which in turn activates caspase 3 forming the caspase 3 cleavage body.The caspase 3 cleavage body further activates the downstream PARP protein, ultimately promoting cell apoptosis [30].
Therefore, after treating H1299 cells with different concentrations of compounds for 48 h, cell proteins were extracted and Western blot analysis revealed a concentration-dependent decrease in the expression levels of Bcl-2, caspase 9, caspase 3 and PARP proteins in the groups treated with compounds 4b and 4c (Figure 7).Simultaneously, the expression levels of cleaved caspase 3 and cleaved PARP proteins increased.These results suggest that compounds 4b and 4c may effectively induce apoptosis in NSCLC cells through the Bcl-2/caspase3/PARP signaling pathway, and the apoptotic effect in the high drug concentration group was significantly stronger than that in the low drug concentration and normal control groups (p < 0.01, p < 0.05).
There is existing research indicating that matrix metalloproteinase 9 (MMP9) plays a crucial role in angiogenesis and cell migration [31].In NSCLC tissues, the protein expression level of MMP9 is significantly higher than that in normal adjacent tissues.This suggests a close correlation between the high expression of MMP9 and malignant metastasis of lung cancer, as well as a poor prognosis [32].To further investigate whether compounds regulate the migratory ability of NSCLC by modulating MMP9 protein levels, Western blot analysis showed a decrease in MMP9 protein expression with increasing drug concentration.Therefore, compounds 4b and 4c may inhibit the migratory ability of NSCLC cells by downregulating MMP9 protein expression.

4b and 4c Trigger Apoptosis via the Apoptosis Signaling Pathway
We conducted an in-depth investigation into the mechanism by which compounds 4b and 4c induce apoptosis in cells.The B-cell lymphoma 2 (Bcl-2) protein family is located upstream in the apoptosis signaling pathway.Bcl-2 proteins play an anti-apoptotic role by inhibiting the release of cytochrome C (cyt-c) from the mitochondria.Many anticancer drugs induce apoptosis by downregulating Bcl-2 [29].When cell apoptosis occurs, the expression level of Bcl-2 is inhibited, cyt-c is released and, along with the apoptosis proteaseactivating factor, forms an apoptosome, initiating a cascade reaction of the caspase protein family.Initially, the apoptosome promotes the activation of caspase 9, which in turn activates caspase 3 forming the caspase 3 cleavage body.The caspase 3 cleavage body further activates the downstream PARP protein, ultimately promoting cell apoptosis [30].
Therefore, after treating H1299 cells with different concentrations of compounds for 48 h, cell proteins were extracted and Western blot analysis revealed a concentrationdependent decrease in the expression levels of Bcl-2, caspase 9, caspase 3 and PARP proteins in the groups treated with compounds 4b and 4c (Figure 7).Simultaneously, the expression levels of cleaved caspase 3 and cleaved PARP proteins increased.These results suggest that compounds 4b and 4c may effectively induce apoptosis in NSCLC cells through the Bcl-2/caspase3/PARP signaling pathway, and the apoptotic effect in the high drug concentration group was significantly stronger than that in the low drug concentration and normal control groups (p < 0.01, p < 0.05).

Conclusions
This study was based on the structure of gefitinib.Fourteen structurally novel gefitinib derivatives were obtained through click reactions.Simultaneously, the synthesized 1,2,3-triazole derivatives were evaluated for their antitumor activity against wild-type lung cancer cells.The synthetic method was simple and rapid, and the compounds were characterized and confirmed structurally.The in vitro antitumor activity of the fourteen compounds was evaluated using three cancer cell lines.The results showed that compounds 4b and 4c exhibited strong antiproliferative activity in three NSCLC cell lines.Through cellular function and mechanism studies, it was found that compounds 4b and 4c downregulated the protein expression of Bcl-2, caspase 9 and MMP9, upregulated the protein expression of cleaved caspase 3 and cleaved PARP, inhibited tumor cell proliferation and migration and promoted cell apoptosis.This research lays the foundation for future in vivo experiments and clinical applications of these novel gefitinib derivatives.

Materials and Chemistry
All reagents and solvents obtained from commercially available source were used without further treatment. 1H NMR and 13 C NMR spectra were acquired in DMSO-d6 or Methanol-d4 solution with a Bruker 600 spectrometer (Bruker, Billerica, MA, USA).High- There is existing research indicating that matrix metalloproteinase 9 (MMP9) plays a crucial role in angiogenesis and cell migration [31].In NSCLC tissues, the protein expression level of MMP9 is significantly higher than that in normal adjacent tissues.This suggests a close correlation between the high expression of MMP9 and malignant metastasis of lung cancer, as well as a poor prognosis [32].To further investigate whether compounds regulate the migratory ability of NSCLC by modulating MMP9 protein levels, Western blot analysis showed a decrease in MMP9 protein expression with increasing drug concentration.Therefore, compounds 4b and 4c may inhibit the migratory ability of NSCLC cells by downregulating MMP9 protein expression.

Conclusions
This study was based on the structure of gefitinib.Fourteen structurally novel gefitinib derivatives were obtained through click reactions.Simultaneously, the synthesized 1,2,3-triazole derivatives were evaluated for their antitumor activity against wild-type lung cancer cells.The synthetic method was simple and rapid, and the compounds were characterized and confirmed structurally.The in vitro antitumor activity of the fourteen compounds was evaluated using three cancer cell lines.The results showed that compounds 4b and 4c exhibited strong antiproliferative activity in three NSCLC cell lines.Through cellular function and mechanism studies, it was found that compounds 4b and 4c downregulated the protein expression of Bcl-2, caspase 9 and MMP9, upregulated the protein expression of cleaved caspase 3 and cleaved PARP, inhibited tumor cell proliferation and migration and promoted cell apoptosis.This research lays the foundation for future in vivo experiments and clinical applications of these novel gefitinib derivatives.

Materials and Chemistry
All reagents and solvents obtained from commercially available source were used without further treatment. 1H NMR and 13 C NMR spectra were acquired in DMSO-d 6 or Methanol-d 4 solution with a Bruker 600 spectrometer (Bruker, Billerica, MA, USA).Highresolution mass spectra (HRMS) measurements were carried out using a Bruker Compact mass spectrometer.

Preparation of 4-(3-((4-chloro-7-methoxyquinazolin-6yl)oxy)propyl)morpholine
A mixture of Compound 1 (3.0 g, 9.0 mmol) in POCI 3 (10.0mL) was allowed to stir at 120 • C for 12 h.Progress of reaction was monitored by TLC.After completion, the reaction mixture was cooled to RT, diluted with cold water (300 mL) and allowed to stir for 10 min.The reaction mixture was adjusted to neutral pH with saturated sodium bicarbonate solution and extracted with dichloromethane.It was then dried with anhydrous magnesium sulfate, concentrated to afford 2 (2.0 g, 65%) as a yellow solid.Mp: 116-119 Aryl-azido (150 mg, 0.7 mmol) and 3 (250 mg, 0.56 mmol) were added to 15 mL mixed solvent (water/tert-butanol/THF = 1:1:1).Copper sulfate pentahydrate (14 mg, 0.1 mmol) and sodium ascorbate (23 mg, 0.1 mmol) were added, and the mixture was stirred at 85 • C for 12 h.After the completion of the reaction (monitored by TLC), the mixture was extracted with dichloromethane (15 mL × 3).The combined organic phase was washed successively with brine, dried over sodium sulfate and concentrated in vacuo.The residue was purified through column chromatography (CH 2 Cl 2 /MeOH = 20:1) to give the desired compound 4 as a crystalline powder.The nuclear magnetic resonance washed at least three times in PBS.The fluorescence microscope was used to observe the morphological changes of cell nuclei.5.3.5.Wound Healing Assay H1299 cells were seeded in a 6-well plate.When the cell confluency reached 95%, a wound line was scratched using a 200 µl pipette tip and then washed three times.Then, fresh medium containing 4b or 4c (2, 4 and 8 µM) was added and the plate was incubated at 37 • C with 5% CO 2 .All images were captured at 0 h and 24 h under an inverted microscope, and the quantification of scratches was analyzed using Image J (https://imagej.net/ij/, accessed on 4 January 2024).

Transwell Migration Assay
Transwell assay was used to determine the migration ability of H1299 cells.Cells were starved in serum-free RPMI 1640 medium for 24 h, then detached and resuspended in serum-free RPMI 1640 medium.5 × 10 5 cells/mL were inoculated into the upper chamber of a 24-well Transwell plate (Corning Inc., Corning, NY, USA), with a volume of 100 µL per well.Then, 100 µL serum-free medium containing 4b or 4c (2, 4 and 8 µM) was added to the upper chamber and the lower chamber was filled with 700 µL medium containing 20% FBS.After incubation for 24 h, H1299 cells on the upper membrane of the transwell were wiped off.The migrated cells were treated with 4% paraformaldehyde for 20 min, stained with 0.1% crystal violet for 20 min and washed three times with PBS.The number of H1299 cells that migrated to the underside of the membrane was counted under the inverted fluorescence microscope.Three randomly selected areas from each transwell were photographed and calculated using Image J.

Colony Formation Assay
H1299 cells were seeded in six-well plates at a density of 500 cells per well and treated with 4b or 4c (1, 2 and 4 µM) for seven days.Then, cells were per-fixed with 4% paraformaldehyde for 30 min, stained with 0.1% crystal violet for 15 min and then washed with pure water.After destain with water, the plates were allowed to dry at room temperature overnight.Image J was used to count the number of clone formations.

Western Blot Analysis
Firstly, H1299 cells were treated with the different concentrations of 4b and 4c at 4, 8 and 16 µM for 48 h.Then, cells were harvested using RIPA lysate (R0010, Solarbio, Beijing, China) and centrifuged at 12,000× g rpm for 15 min at 4 • C. Following that, the concentration of total protein was measured and taken from the supernatant.Protein was separated by 12% SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose filter membrane.After blocking in 5% milk for 2 h, the membranes were incubated with a specific primary antibody at 4 • C overnight.The next day, NC membranes were incubated for 1 h at room temperature with appropriate secondary antibodies and then protein bands were visualizing by chemiluminescence detection (ECL kit, Genview, Beijing, China).

Statistical Analyses
We performed statistical analyses using GraphPad Prism 9.5 software.Data were analyzed using one-way analysis of variance (ANOVA) followed by Dunnett's tests.Data were obtained from no fewer than three independent experiments.A p-value less than 0.05 was considered statistically significant.

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/molecules29040837/s1,The nuclear magnetic resonance spectroscopy ( 1 H NMR and 13 C NMR) and high-resolution mass spectrometry (HRMS) of compounds 4a-4n are Supplementary Materials provided as data.

Figure 1 .
Figure 1.The structures of gefitinib and erlotinib.The red parts represent the parent nucleus, and the black parts represent diverse substituents.

Figure 1 .
Figure 1.The structures of gefitinib and erlotinib.The red parts represent the parent nucleus, and the black parts represent diverse substituents.

Figure 4 .
Figure 4. Cell apoptosis induced by 4b and 4c in H1299 cells.(A) Apoptosis quantification detected by Annexin V-EGFP/PI staining.(B) Cell apoptosis morphological changes detected by DAPI staining.Red arrows indicate DAPI-positive (apoptotic) cells.Images represent at least three independent experiments.Data were mean ± SD. n = 3 for each concentration.NC, normal control.

Figure 4 .
Figure 4. Cell apoptosis induced by 4b and 4c in H1299 cells.(A) Apoptosis quantification detected by Annexin V-EGFP/PI staining.(B) Cell apoptosis morphological changes detected by DAPI staining.Red arrows indicate DAPI-positive (apoptotic) cells.Images represent at least three independent experiments.Data were mean ± SD. n = 3 for each concentration.NC, normal control.

Figure 5 .
Figure 5. Cell migration inhibition induced by 4b and 4c in H1299 cells.(A) In vitro cell scratch assays.Photographs of cells at 0 and 24 h after treatment with different concentrations of drugs (scale bar = 100 µm).(B) Transwell migration assays.Migration of cells to the lower surface of the chamber was determined by crystal violet staining.Data were mean ± SD. n = 3 for each concentration.NC, normal control.

Figure 5 .
Figure 5. Cell migration inhibition induced by 4b and 4c in H1299 cells.(A) In vitro cell scratch assays.Photographs of cells at 0 and 24 h after treatment with different concentrations of drugs (scale bar = 100 µm).(B) Transwell migration assays.Migration of cells to the lower surface of the chamber was determined by crystal violet staining.Data were mean ± SD. n = 3 for each concentration.NC, normal control.

Figure 6 .
Figure 6.Colony formation pictures of the H1299 treated with 4b and 4c.NC, normal control.

Figure 6 .
Figure 6.Colony formation pictures of the H1299 treated with 4b and 4c.NC, normal control.

Figure 7 .
Figure 7. Assessment of apoptosis and migration induced by compounds 4b and 4c.(A) Western blo ing results of the protein levels in H1299 cells treated with 4, 8 or 16 µM of 4b compounds.Numbers above each lane indicated the relative expression level of the protein.(B) Western blo ing results of the protein levels in H1299 cells treated with 4, 8 or 16 µM of 4c compounds.Numbers above each lane indicated the relative expression level of the protein.NC, normal control.

Figure 7 .
Figure 7. Assessment of apoptosis and migration induced by compounds 4b and 4c.(A) Western blotting results of the protein levels in H1299 cells treated with 4, 8 or 16 µM of 4b compounds.Numbers above each lane indicated the relative expression level of the protein.(B) Western blotting results of the protein levels in H1299 cells treated with 4, 8 or 16 µM of 4c compounds.Numbers above each lane indicated the relative expression level of the protein.NC, normal control.

Table 1 .
Cont.The bold values represent the IC 50 values of 4b and 4c with significant inhibitory effects.All values are given as means ± standard deviation.(n = 3) a Gefitinib was used as positive control in this study.

Table 2 .
Inhibitory rate of 4b and 4c in L02 cells.
(1.5 g, 4.5 mmol) and m-acetylenyl aniline (1.56 g, 13.4 mmol) in isopropanol (40 mL) was allowed to stir at 100 • C for 6 h.Reaction mixture was cooled to RT, diluted with cold water (50 mL) and allowed to stir at RT for 10 min.