Design, Synthesis, and Antitumor Activity of Erlotinib Derivatives

Nineteen erlotinib derivatives bearing different 1,2,3-triazole moieties were designed, synthesized, and evaluated for their potential against different cancer cell lines. The structures of the synthesized compounds were confirmed via 1H NMR, 13C NMR, and HR MS. Preliminary antitumor activity assay results suggested that some compounds showed remarkable inhibitory activity against different cancer cell lines including the corresponding drug-resistant ones. Among these compounds, 3d was the most promising one with an IC50 of 7.17 ± 0.73 μM (KYSE70TR), 7.91 ± 0.61 μM (KYSE410TR), 10.02 ± 0.75 μM (KYSE450TR), 5.76 ± 0.3 3 μM (H1650TR), and 2.38 ± 0.17 μM (HCC827GR). A preliminary mechanism study suggested that compound 3d suppressed cancer cell proliferation through the EGFR-TK pathway.

While such EGFR-TKIs can significantly improve the life quality and the median survival rate of patients and show better performance in terms of progression-free survival rate or objective response rate, such therapeutics also suffer problems such as drug resistance after a period of administration (Spaans and Goss, 2014;Karlsen et al., 2021;Zhao et al., 2021), and developing new EGFR-TK targeting chemical entities with reduced drug resistance but similar antitumor activity is highly desirable (Zhang et al., 2014;Maione et al., 2015;Tsubata et al., 2021).
We are interested in developing new chemical entities using known therapeutics as lead compounds (Mao LF. et al., 2020;Mao L. et al., 2020;Wang et al., 2020). Herein, we present our preliminary results on the preparation of new erlotinib derivatives, and the antitumor activity of the prepared compounds against different cancer cell lines. Successfully marketed drugs generally showed good druggability such as good pharmacokinetic property, ideal solubility or high activity, and drug discovery process could be effectively facilitated using a known drug as a lead compound.
Erlotinib ( Figure 1) is a classical EGFR-TKI approved for the treatment of advanced non-small-cell lung cancer (NSCLC) (Kim and Murren, 2002;Schettino et al., 2008). Compared with traditional chemotherapeutics, erlotinib can improve the median survival rate of patients and exhibit better performance in terms of progression-free survival rate, objective response rate, quality of life, and tolerability (Mathew et al., 2015).
In addition, erlotinib was also effective in the treatment of esophageal cancer (Choi et al., 2012). Esophageal cancer is a common malignant cancer with high morbidity and mortality, and 5-year survival rate for esophageal cancer patients is lower than that of lung cancer (Hiroshi et al., 2005;Wei et al., 2020). A study showed that 40-80% of esophageal cancer patients are diagnosed with high expression of EGFR (Hirsch et al., 2017), and erlotinib can be used as an adjuvant therapy for the treatment of esophageal cancer in combination with radiotherapy and chemotherapy (Sutter et al., 2005;Zhong et al., 2020). However, drug resistance and adverse reactions have become prominent issues after a period of erlotinib treatment (Sutter et al., 2010;Whitley et al., 2012), and development of new EGFR-TKIs is highly desirable to maintain the antitumor activity of the drug on the one hand, and to tackle the drug resistance problem on the other .
Nitrogen-containing heterocyclic compounds are widely present in nature and have played key roles in drug discovery (DeSimone et al., 2004;Costantino, 2006). Especially, 1,2,3triazole moieties are an important category of nitrogencontaining heterocycles in the design of biologically active molecules (Thomopoulou et al., 2015). 1,2,3-Triazole has a large dipole moment and can form a variety of non-covalent interactions with different functional groups. The structural characteristics of 1,2,3-triazoles make them ideal surrogates for amides, esters or carboxylic acids, and compounds bearing triazole moieties often showed broad-spectrum biological activities such as antibacterial (Röhrig et al., 2012;Mao et al., 2017), antimalarial, antifungal, antiviral (Hong et al., 2010), antituberculosis, and antitumor activities (Qi et al., 2020).
On the basis of these rationales, different 1,2,3-triazole moieties were introduced to erlotinib in an attempt to maintaining the antitumor activity of the parent drug and in some extent tackling the drug resistance problem of the drug ( Figure 2).

Chemistry
The preparation of the target compounds is illustrated in Scheme 1. Erlotinib (2) was obtained after the reaction of 4-chloro-6,7bis(methoxyethoxy)quinazolinone with 3-aminophenylacetylene (Chandregowda et al., 2007a, b, c). The target compounds 3a-3s were obtained via the click reaction of erlotinib with different azido compounds (Xu et al., 2016) (Table 1). The reaction conditions of these operations were mild, and the reactions were easy to carry out. The structures of the key intermediates and all target compounds were confirmed by nuclear magnetic resonance ( 1 H NMR and 13 C NMR) and high-resolution mass spectrometry (HR MS). The purity of the key compounds was checked with HPLC.

Suppression of Esophageal Cancer Cell
Lines and Non-Small-Cell Lung Cancer Cell Lines by Erlotinib-1,2,3-Triazole Derivatives A total of five tumor cell lines including three esophageal cancer cell lines (KYSE70, KYSE410, and KYSE450) and two lung cancer cell lines (H1650 and HCC827) were chosen for evaluating the anticancer activity of the newly synthesized erlotinib derivatives. Three types of esophageal cancer cell lines KYSE70TR, KYSE410TR, and KYSE450TR which were resistant to paclitaxel, and NSCLC cell lines HCC827GR and H1650TR, which were resistant to gefitinib and paclitaxel, respectively, were also tested for the comparison purpose. The resistance indices of these cell lines are shown in Table 2.
Frontiers in Pharmacology | www.frontiersin.org April 2022 | Volume 13 | Article 849364 3 for MTT assay to evaluate the toxicity of the compound against normal cells. As shown in Table 2, SHEE showed poor sensitivity to 3a, 3d, 3e, 3m, 3p, and 3r, with IC 50 values of over 50 μM. These preliminary results suggested that compounds 3d, 3m, and 3r may be used as lead compounds for the development of chemotherapeutic  Conditions: Growth inhibition was evaluated with MTT assay. The absorbance at 490 nm was measured using a microplate reader (Thermo). The results were from statistical analysis and were presented as mean ± SD.
Frontiers in Pharmacology | www.frontiersin.org April 2022 | Volume 13 | Article 849364 4 agents for esophageal cancer. Compound 3d was chosen for further study due to its good performance and easier preparation.
Next, 3d and erlotinib were tested for their activity against three drug-resistant esophageal cancer cell lines (KYSE70TR, KYSE410TR, and KYSE450TR) and two drug-resistant lung cancer cell lines (H1650TR and HCC827GR). The results expressed by IC 50 values are shown in Table 4. The preliminary results suggested that the inhibitory effect of erlotinib on drugresistant tumor cells was not significant and the IC 50 values were over 10 μM for all cases. In contrast, 3d showed good inhibitory effect on five drug-resistant tumor cell lines with IC 50 values of 7.17 ± 0.73 μM, 7.91 ± 0.61 μM, 10.02 ± 0.75 μM, 5.76 ± 0.33 μM, and 2.38 ± 0.17 μM, respectively.

Plate Clone Formation Assay
Plate clone experiments of 3d and erlotinib were also carried out to study the tumor response (Von Hoff et al., 1980), and the results   (Figure 3). The inhibition effects of 3d on both cancer cell lines and the corresponding drug-resistant ones were more significant than that of erlotinib.

Apoptosis in Esophageal Cancer Cell Lines Induced by Erlotinib-1,2,3-Triazole Derivatives
To clarify whether the inhibitory effects of these compounds on cell proliferation were related to apoptosis, compound 3d, which showed strong inhibitory effects on the proliferation of esophageal cancer cell lines, were chosen for further study. KYSE450 and KYSE450T cells were treated with DMSO or different concentrations of 3d and erlotinib for 48 h, the cells were stained with Annexin V and PI, and the proportion of apoptotic cells was detected with flow cytometry. The results are shown in Figure 4.
Results in Figure 4A showed that the proportion of KYSE450 apoptotic cells treated with 3d was 24.50% (5 μM) and 69.64% (10 μM), while the proportion of KYSE450TR apoptotic cells treated with 3d was 44.12% (5 μM) and 48.77% (10 μM). Results in Figure 4B showed that the proportion of KYSE450 apoptotic cells treated with erlotinib was 7.04% (5 μM) and 8.94% (10 μM), while the proportion of KYSE450TR apoptotic cells treated with erlotinib was 10.16% (5 μM) and 16.68% (10 μM). These preliminary results suggested that compound 3d could induce apoptosis of esophageal cancer KYSE450 cells and drug-resistant KYSE450TR cells in a concentration-dependent manner, and the performance of compound 3d on these two cells was better than that of erlotinib.

Erlotinib-1,2,3-Triazole Derivatives Trigger Apoptosis Through the Mitochondrial Pathway
To investigate whether the mechanism of 3d-induced KYSE450 cell apoptosis was related to mitochondrial apoptosis, protein electrophoresis was carried out to measure the protein levels of apoptosis-related marker proteins caspase-3, cytochrome-c, and PARP. The results are presented in Figure 5.
KYSE450 cells were treated with 3d for 6, 12, and 24 h, respectively. Analysis of total cell proteins showed that in  KYSE450 cells, the caspase-3 and cytochrome-c protein levels of 3d (4 μM) group were higher than those in the control at 0 h after administration. The results suggest that 3d can regulate KYSE450 cell apoptosis through the mitochondrial pathway. The cleaved PARP protein levels of the 3d group were higher than those of the control at 0 h after administration, suggesting that compound 3d may regulate KYSE450 apoptosis through DNA injury.

Erlotinib-1,2,3-Triazole Derivatives Induce Esophageal Cells Death via Arresting Cell Cycle
To assess whether the inhibitory effect of these compounds on the proliferation of esophageal cancer cells was related to cell cycle arrest, cancer cells were treated with DMSO or different concentrations of 3d and erlotinib. Then, the cell cycle phases were evaluated with flow cytometry (Figure 6). In comparison with the control, the 3d group showed that the ratio of KYSE450 cells in the G 0 /G 1 phase increased after 24 h with the increase in concentration (3.5, 7, 14, and 28 μM) in comparison with the control. However, the change in the ratio of S-phase cells and the ratio of G 2 /M-phase cells were not remarkable. When the cells were treated with erlotinib, the ratio of KYSE450 cells in the G 0 /G 1 phase increased significantly at 3.5 μM and increased further with the concentration (3.5, 7, 14, and 28 μM) but did not change significantly with the further increase in concentration. The ratio of S-phase cells and the ratio of G 2 /M-phase cells decreased significantly at 3.5 μM but did not change significantly when the concentration was further increased. Therefore, both compound 3d and erlotinib inhibited KYSE450 cells in the G 0 /G 1 phase.
For the drug resistance cell line KYSE450TR, the cell cycle results showed that the ratio of KYSE450TR cells in the G 0 /G 1 phase increased after treatment with compound 3d for 24 h with the increase in concentration (3.5, 7, 14, and 28 μM) in comparison with the control (Figure 7). However, the ratio of S-phase cells significantly reduced, and the ratio of G 2 /M-phase cells increased in a concentration-dependent manner. When cells were treated with erlotinib, the ratio of KYSE450TR cells in the G 0 /G 1 phase increased significantly at 3.5 μM and further increased with concentration (3.5, 7, 14, and 28 μM) but did not change significantly when the concentration was further increased. The ratio of S-phase cells decreased significantly at 3.5 μM, and the change was significant with the increase in concentration. The ratio of cells in the G 2 /M phase increased to a small degree with the increase in concentration. Therefore, compound 3d inhibited KYSE450TR cells in the G 2 /M phase, whereas erlotinib arrested KYSE450TR cells in the G 0 / G 1 phase.

Erlotinib-1,2,3-Triazole Derivatives Suppress Cancer Cell Proliferation Through EGFR-TK Pathway
To investigate whether the mechanism of 3d suppressing cancer cell proliferation was related to the EGFR-TK pathway, surface plasmon resonance (SPR) experiments were carried out to study the interaction between 3d and erlotinib with the EGFR. The results are presented in Figure 8. As these results showed, 3d can bind to the EGFR wild-type protein (H672-E410) and EGFR mutant protein (672-1210, L858R) with a K D (M) value of 6.88 × 10 −6 and 3.12 × 10 −6 , respectively. The K D (M) value for erlotinib and EGFR was 1.97 × 10 −6 . These preliminary results also suggested that 3d value between 3d and wild-type EGFR is 6.88 × 10 −6 . (B) Binding sensorgrams for 3d interaction with immobilized mutant protein of EGFR. The K D (M) value between 3d and mutant protein EGFR is 3.12 × 10 −6 . (C) Binding sensorgrams for erlotinib interaction with immobilized wild-type of EGFR. The K D (M) value between erlotinib and wild-type EGFR is 1.97 × 10 −6 .

CONCLUSION
In summary, erlotinib derivatives bearing 1,2,3-triazole moieties were designed, synthesized, and evaluated for their activity against different cancer cell lines. Several of these compounds exhibited remarkable antitumor activity than erlotinib against one or more cancer cell lines. Among these compounds, 3d demonstrated good cytotoxicity against all ten cancer cell lines. The underlying mechanisms of 3d-induced cancer cell deaths were mitochondrial apoptosis and cell cycle arrest. In addition, a preliminary study on the interaction between 3d and EGFR suggested that 3d can bind to both wild-type protein (H672-E410) and mutant protein (672-1210, L858R). Taking these results together, erlotinib-1,2,3-triazole derivative 3d could induce apoptosis and arrest cell cycle, and the combination of erlotinib and 1,2,3-triazole might be a successful strategy for the development of new EGFR inhibitors for cancer therapy.

EXPERIMENTAL PROTOCOLS Chemistry
All reagents and solvents were obtained from commercially available sources and were used as received. 1 H NMR and 13 C NMR spectra were acquired in DMSO-d 6 solution using a Bruker 600 spectrometer. Chemical shifts (δ) were given in parts per million with tetramethylsilane as internal reference. Coupling constants were expressed in hertz. High-resolution mass spectra (HRMS) measurements were carried out using a Bruker MicrOTOF-Q II mass spectrometer.

Cell Culture and Treatment
The human cancer cells H1650/H1650TR, HCC827/HCC827GR, KYSE70/KYSE70TR, KYSE410/KYSE410TR, and KYSE450/ KYSE450TR were cultured in the RPMI-1640 complete growth medium containing 100 U/mL of penicillin-streptomycin and 10% FBS. The cells were incubated at 37°C with 5% of CO 2 . The compounds were dissolved in DMSO to make a 50 mM stock solution and were diluted to the concentration of working solutions with the complete growth medium before administration.

MTT Assay for Cell Proliferation and Cytotoxicity
Cells were seeded in 96-well plates with densities of 2,200-2,500 cells/well in 100 μL. One day after seeding, the concentration of the test compounds between 0 and 50 μM, 0.1% DMSO was added to cells as control. Approximately 2200-2500 transfected cells in 100 μL were incubated in quintuplicate in 96-well plates. After 48 h, MTT was added and incubated in the plate for 1-4 h in the incubator. The absorbance at 490 nm was measured using a microplate reader (Thermo).

Culture Assay for Tumor Colony-forming Cells
Cells were seeded in 6-well plates with a density of 200 cells/well and cultured overnight for attachment. The cells were exposed to 3d or erlotinib of various concentrations (0, 2.5, 5, and 10 µM) separately for 10 days. Medium with or without compounds was changed every 48 h. When colony formation was visible, the medium was discharged. Then, the colonies were washed with cold PBS, fixed with 4% paraformaldehyde (PFA) for at least 30 min, and then stained with 0.2% crystal violet solution in 100% ethanol for 20 min.

Flow Cytometry Detection for Cell Apoptosis
Cell apoptosis analysis was carried out by flow cytometry using the Annexin V/PI apoptosis methods. Briefly, KYSE450/KYSE450TR (2 × 10 4 -3×10 4 /well) cells were incubated in 6-well plates for 48 h and then treated with 0.1% DMSO (as control), either compound 3d or erlotinib at various concentrations for 48 h, respectively. The cells were harvested and incubated with 250 μL of 1 × Annexin V binding buffer containing 5 μL of PI and 5 μL of FITC Annexin V (final concentration 1.8 μg/ml, Biolegend cat: 640945) for 15 min at room temperature in the dark. Then 200 µL of 1 × binding buffer was added for flow cytometry analysis (BD FACSCalibur ™ Flow Cytometer).

Western Blot Analysis
KYSE450 cells (3 × 10 5 /well) were incubated overnight in 10 square petri dishes and then treated with compound 3d at 4 μM for 0, 6, 12, and 24 h. Cells treated with 0.1% DMSO were used as control. Then, the cells were harvested, and total proteins were extracted. Total proteins were separated by 12% SDS polyacrylamide gel electrophoresis and transferred onto PVDF membranes. The membrane was blocked for 1 h, then incubated overnight with a 1: 1000 dilution of anti-caspase-3, anti-cytochrome-c, and anti-PARP, or 1 : 3,000 dilution of anti-β-action primary antibody at 4°C. Finally, 1 : 3,000 anti-rabbit secondary antibodies were incubated for 2 h at room temperature. Protein bands were developed by chemiluminescence.

Cell Cycle Analysis
KYSE450 and KYSE450TR cells were plated in 6-well plates with a density of 1 × 10 5 cells/well and cultured overnight to attach. The cells were treated with 3d or erlotinib at different concentrations (0, 3.5, 7, 14, and 28 μM) for 24 h. After trypsinization treatment, the cells were collected by centrifugation. The cell pellet was resuspended in 70% ethanol at −20°C for at least 3 h. The cells were washed with PBS and was re-suspended in 250 μL of 0.6% tricine with renease A for 1 h, then stained in PI (final concentration 1.8 μg/ml, Biolegend cat: 640945) for 15 min in the dark. Cells were re-transferred to the BD FACSCalibur ™ Flow Cytometer. All analyses were performed with FlowJo software v105.3.6.

EGFR Protein Affinity Was Determined by SPR
Surface plasmon resonance experiments were carried out to evaluate the interaction between 3d with the EGFR wild-type (0.468 μg/μL,