Novel Dual-Target Kinase Inhibitors of EGFR and ALK Were Designed, Synthesized, and Induced Cell Apoptosis in Non-Small Cell Lung Cancer

ALK-positive NSCLC coexisting with EGFR mutations is a frequently occurring clinical phenomenon. Targeting ALK and EGFR simultaneously may be an effective way to treat these cancer patients. In this study, we designed and synthesized ten new dual-target EGFR/ALK inhibitors. Among them, the optimal compound 9j exhibited good activity with IC50 values of 0.07829 ± 0.03 μM and 0.08183 ± 0.02 μM against H1975 (EGFR T790M/L858R) and H2228 (EML4-ALK) cells, respectively. Immunofluorescence assays indicated that the compound could simultaneously inhibit the expression of phosphorylated EGFR and ALK proteins. A kinase assay demonstrated that compound 9j could inhibit both EGFR and ALK kinases; thus, exerting an antitumor effect. Additionally, compound 9j induced apoptosis in a dose-dependent manner and inhibited the invasion and migration of tumor cells. All of these results indicate that 9j is worthy of further study.


Introduction
Non-small-cell lung cancer (NSCLC) is one of the most deadly cancers in the world [1] and accounts for more than 350 deaths every day [2]. NSCLC is the most common subtype of lung cancer, making up 80-85% of all lung cancer cases [3,4]. The epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK) family play a significant role in cell signal transduction, and their overexpression is implicated in the pathogenesis of numerous human solid cancers, especially NSCLC [5,6].
EGFR belongs to the ErbB family and is related to cell proliferation, survival, migration, adhesion, and differentiation [7,8]. So far, small molecule kinase inhibitors (EGFR-TKIs) have reached the fourth generation. The first generation of EGFR inhibitors, such as Gefitinib, Erlotinib and Icotinib, have exhibited a great deal of activity against NSCLCs with EGFR (L858R) mutations and EGFR deletions [9][10][11]. Second-generation EGFR inhibitors afatinib [12], dacomitinib [13], and neratinib [14] have been used for the treatment of the mutation, but suffer from a narrow therapeutic dose window. The third generation of EGFR-TKIs, for example, osimertinib, overcomes the clinical side effects of the second generation [15]. However, drug resistance develops after a period of use. As a result, fourthgeneration inhibitors were developed [16]. The ALK fusion gene is another important tumor driver gene found in NSCLC after mutations in the epidermal growth factor receptors [17]. ALK rearrangements have been detected in NSCLC, leading to the development of numerous ALK inhibitors to treat NSCLC that has ALK rearrangements [18]. A mere four years after the discovery of the EML4-ALK fusion protein in 2007, crizotinib [19], a

Chemistry
The synthetic route of target compounds 9a-9j is shown in Scheme 1. Nitration of 4bromo-2-methoxyaniline using KNO3 in H2SO4 yielded the corresponding nitro derivatives 2. The Suzuki coupling of 2 with 4-pyridineboronic acid afforded 3. Compound 3 reacted with (Boc)2O in the presence of 4-dimethylaminopyridine to form intermediate 4.

Chemistry
The synthetic route of target compounds 9a-9j is shown in Scheme 1. Nitration of 4-bromo-2-methoxyaniline using KNO 3 in H 2 SO 4 yielded the corresponding nitro derivatives 2. The Suzuki coupling of 2 with 4-pyridineboronic acid afforded 3. Compound 3 reacted with (Boc) 2 O in the presence of 4-dimethylaminopyridine to form intermediate 4.
N-methylation of 4, followed by the reduction with NaBH 4 , generated 5. By the hydrogenation of compound 5 in the presence of Pd/C, the carbon double bond and the nitro group were reduced simultaneously to obtain intermediate 6. Reaction with acryloyl chloride, deprotection of 6 gave the key intermediate 7, which reacted with the corresponding 8a-8j to obtain the target compounds 9a-9j.

Antiproliferative Activity against Human Lung Cancer Cells
On the basis of the synthesis, we used a CCK8 assay to screen the antiproliferative activity of the target compound in three cancer cell lines (H1975, EGFR T790M/L858R , H2228, EML4-ALK, and H522 cells), using osimertinib and brigatinib as references. The results are shown in Table 1

Antiproliferative Activity against Human Lung Cancer Cells
On the basis of the synthesis, we used a CCK8 assay to screen the antiproliferative activity of the target compound in three cancer cell lines (H1975, EGFR T790M/L858R , H2228,  EML4-ALK, and H522 cells), using osimertinib and brigatinib as references. The results are shown in Table 1. The in vitro biological activity results showed that most of the target compounds had a high inhibitory activity against tumor cells. Among them, compounds 9b, 9h, and 9j showed a good and relatively balanced antiproliferative activity for both the H1975 and H2228 cancer cell lines with IC 50 values of 0.0689 to 0.0782 µM against the H1975 cells, and 0.076 to 0.098 µM for the H2228 cells. Further analysis showed that the structure change of the compounds had a great impact on the activity. Compound 9a, cyclopropyl linked with a sulfoxide sulfur atom, showed a moderated activity with IC 50 0.4511 ± 0.07 µM against the H1975 cancer lines and 0.1214 ± 0.09 µM against H2228. It is interesting that the replacement of the R 2 group of 9a with methyl to obtain 9b allowed the activity to significantly improve (0.0689µM against the H1975 cancer lines and 0.0989 µM against the H2228 cells). Comparing the activity of 9b with that of 9c and 9d, it can be seen that the R 1 group also has a greater impact on the activity. Fluorine or chlorine atoms at the position of R 1 are not conducive to an improvement in activity (9c, R 1 = F, 0.2707 µM against H1975 and 0.1027 µM against H2228; 9d, R 1 = Cl, 0.1143 µM against H1975 and 0.089 µM against H2228). Compounds 9e-9g are ethyl linked with sulfoxide sulfur atoms and also exhibit a moderate to good antiproliferative activity with IC 50 values ranging from 0.06901 to 0.5495 µM against the H1975 cancer lines, and from 0.0928 to 0.1037 µM against H2228. In this series, when the R 1 group is a chlorine atom, there is no adverse effect on the activity. For example, 9g exhibited IC 50 values of 0.06901 µM against H1975 and 0.1037 µM against H2228 cells. The optimal antiproliferative activities (IC 50 values of 0.07829 ± 0.03 and 0.08183 ± 0.02 µM against H1975 and H2228 cancer cell lines, respectively) were provided by compound 9j, which features an iso-propyl as the R 2 group and a Cl atom as the R 1 position. Based on the above results, we can see that most of these target compounds could inhibit the proliferation of EGFR double mutant H1975 and H2228 cells effectively. Among them, compounds 9b, 9h, and 9j exhibited good and comparable activities. Considering cyclopropyl ring addresses multiple roadblocks that can occur during drug discovery, such as enhancing potency, reducing off-target effects, increasing metabolic stability, and so on [30], we chose 9j for the further study.

Safety Verification of Compound 9j
It is critical to evaluate the balance between the safety and efficacy of drugs for the development of new drugs; thus, we then conducted a cytotoxicity test on four types of nontumorigenic cell lines from different origins, including LO2 (human normal liver cells), HK2 (human kidney proximal convoluted tubule epithelial cells), HLF (human embryonic lung fibroblast), 293A (human embryonic kidney cells), and HUVEC (human umbilical vein endothelial cells). The results summarized in Table 2 showed that compound 9j exhibited very little apparent toxicity toward the five nontumorigenic cell lines (LO2, HK2, BJ, and HLF) and a good selectivity ratio range from 255.49 to 357.31, compared with 419.51 to 1058.5 for osimertinib. In addition, we evaluated the compound's safety in vivo by performing acute toxicity tests in mice, and 9j was injected intravenously at concentrations of 100 mg/kg, 150 mg/kg, and 300 mg/kg. In these three doses, no mouse death occurred, and HE staining of lung and heart tissues did not indicate toxicity either ( Figure S1). According to the results, we believe that the compound appears to be safe. tively) were provided by compound 9j, which features an iso-propyl as the R 2 group and a Cl atom as the R 1 position. Based on the above results, we can see that most of these target compounds could inhibit the proliferation of EGFR double mutant H1975 and H2228 cells effectively. Among them, compounds 9b, 9h, and 9j exhibited good and comparable activities. Considering cyclopropyl ring addresses multiple roadblocks that can occur during drug discovery, such as enhancing potency, reducing off-target effects, increasing metabolic stability, and so on [30], we chose 9j for the further study.
0.0041 ± 0.001 2.016 ± 0.008 / a The anti-proliferative activities of the compounds were assessed by the CCK8 assay. Data are averages of at least three independent determinants.

Safety Verification of Compound 9j
It is critical to evaluate the balance between the safety and efficacy of drugs for the development of new drugs; thus, we then conducted a cytotoxicity test on four types of nontumorigenic cell lines from different origins, including LO2 (human normal liver cells), HK2 (human kidney proximal convoluted tubule epithelial cells), HLF (human embryonic lung fibroblast), 293A (human embryonic kidney cells), and HUVEC (human umbilical vein endothelial cells). The results summarized in Table 2 showed that compound The anti-proliferative activities of the compounds were assessed by the CCK8 assay. Data are averages of at least three independent determinants.

Compound 9j's Activity on the EGFR and ALK Kinases
As compound 9j exhibited better antitumor properties in the cell experiments, we determined to study its kinase inhibitory activity. Four kinases (EGFR, EGFR L858R/T790M , ALK, and EML4-ALK) were tested using an ELISA assay. As shown in Figure 2A, the IC 50 for 9j against EGFR L858R/T790M and EML4-ALK was 35.7 ± 0.9 nM and 56.3 ± 0.3 nM, respectively. There was no effect on the wild-type EGFR or ALK kinases triggered by the compound. Consequently, the selection of the compound was good as both compounds balanced IC 50 against EGFR and ALK, respectively. In addition, the IC 50 values for the EGFR and ALK wild-type kinases were 236.16 ± 27 nM and 129.81 ± 15 nM, respectively, indicating a good selectivity and low toxicity. Following that, fluorescence assays were used to measure the phosphorylation of the EGFR and ALK protein affect with compound 9j at the cellular level. As shown in Figure 2B, the expression levels of the phosphorylated EGFR and ALK proteins decreased significantly after the compound 9j treatment for 2 to 4 h. Next, WB assay was used to test the protein expression of H1975 and H2228 cells at the concentration of 5, 10, and 100 nM. The results showed that the expression of p-EGFR and its downstream protein p-AKT, p-ALK, and its downstream protein p-ERK were inhibited after 3 h treatment of the compound ( Figure 2C). These results indicated that the compounds could not only inhibit the activity of the EGFR and ALK kinases, but also inhibit the expression of the p-EGFR, p-ALK, p-AKT, and p-ERK; thus, exerting antitumor effects.

Moleculardocking
To study the possible interaction between EGFR or ALK kinase with the target compounds, we performed the docking study with the crystal structure of kinase as templates (EGFR, PDB:3IKA, ALK, PDB, 2XB7) [31,32] and conducted to rationalize the inhibitory activity using Schrödinger suites. The binding mode between EGFR kinase and the compound 9j was shown in Figure 3A,B; like most kinase inhibitors, 9j occupies the ATPbinding pocket of the kinase, where isopropyl linked sulfoxide deep into the pocket and N-methylpiperidine to the solvent. Similar to Osimertinib, 9j exerts its inhibitory effect by forming covalent bonds with the Cys-797 site. In addition, 9j and Met-793 form two hydrogen bonds in the hinge region. N-methylpiperidine formed a salt bridge with Asp-800. These interaction sites facilitate the binding of 9j to EGFR kinase. In Figure 3C,D, compound 9j is the same as brigatinib, located near the hinge region of the ATP-binding pocket of the kinase; it forms a hydrogen bond to nitrogen on Met1199 through 2-aminopyrimidine fragments. N-methylpiperidine of 9j extends outward and forms a salt bond with Glu1210. These interactions suggest why 9j has an inhibitory effect on ALK.

Moleculardocking
To study the possible interaction between EGFR or ALK kinase with the target compounds, we performed the docking study with the crystal structure of kinase as templates (EGFR, PDB:3IKA, ALK, PDB, 2XB7) [31,32] and conducted to rationalize the inhibitory activity using Schrödinger suites. The binding mode between EGFR kinase and the compound 9j was shown in Figure 3A,B; like most kinase inhibitors, 9j occupies the ATPbinding pocket of the kinase, where isopropyl linked sulfoxide deep into the pocket and N-methylpiperidine to the solvent. Similar to Osimertinib, 9j exerts its inhibitory effect by forming covalent bonds with the Cys-797 site. In addition, 9j and Met-793 form two hydrogen bonds in the hinge region. N-methylpiperidine formed a salt bridge with Asp-800. These interaction sites facilitate the binding of 9j to EGFR kinase. In Figure 3C,D, compound 9j is the same as brigatinib, located near the hinge region of the ATP-binding pocket of the kinase; it forms a hydrogen bond to nitrogen on Met1199 through 2-aminopyrimidine fragments. N-methylpiperidine of 9j extends outward and forms a salt bond with Glu1210. These interactions suggest why 9j has an inhibitory effect on ALK.

Apoptosis Assay
The death of cells occurs by apoptosis, and the uncontrolled division and multiplication of cells causes cancer. Inhibiting cellular apoptosis is primarily responsible for cancer cell survival, since cellular apoptosis is negatively correlated with cell growth. We then verified the antitumor effect of compound 9j by inducing apoptosis through flow cytometry. As shown in Figure 4, the number of apoptotic cells gradually increased with the increase of the administration concentration. Compared with the control at 6.07%, the apoptotic rates of compound 9j at 10 nM, 50 nM, and 100 nM for 48 h were 12.7%, 26.8%, and 47.67%, respectively.

Apoptosis Assay
The death of cells occurs by apoptosis, and the uncontrolled division and multiplication of cells causes cancer. Inhibiting cellular apoptosis is primarily responsible for cancer cell survival, since cellular apoptosis is negatively correlated with cell growth. We then verified the antitumor effect of compound 9j by inducing apoptosis through flow cytometry. As shown in Figure 4, the number of apoptotic cells gradually increased with the increase of the administration concentration. Compared with the control at 6.07%, the apoptotic rates of compound 9j at 10 nM, 50 nM, and 100 nM for 48 h were 12.7%, 26.8%, and 47.67%, respectively.

Compound 9j Inhibited the Invasion and Migration of TUMOR Cells
The migration and invasion of tumor cells directly contribute to metastasis. Metastasis is the leading cause of death in cancer patients [33]. To determine the ability of 9j to inhibit tumor cell migration, the cell scratching assay was performed. Following scratching, phase contrast pictures were taken of the scratched and healing cell monolayers. As shown in Figure 5A, the scratch in the PBS group had a healing rate of about 70% after 48

Compound 9j Inhibited the Invasion and Migration of TUMOR Cells
The migration and invasion of tumor cells directly contribute to metastasis. Metastasis is the leading cause of death in cancer patients [33]. To determine the ability of 9j to inhibit tumor cell migration, the cell scratching assay was performed. Following scratching, phase contrast pictures were taken of the scratched and healing cell monolayers. As shown in Figure 5A, the scratch in the PBS group had a healing rate of about 70% after 48 h, and the healing rate clearly decreased following treatment with 9j. Further analysis of the transwell assay ( Figure 5B) revealed that 9j inhibited the H1975 cell proliferation and reduced its invasion ability in a concentration-dependent manner. All of these results indicate that 9j has a good ability to inhibit cell migration and invasion.

Chemistry General Methods
All the reagents used in the synthesis were obtained commercially and used without further purification, unless otherwise specified. The 1 H NMR and 13 C NMR spectra were recorded using TMS as the internal standard on a Bruker BioSpin GmbH spectrometer at 400 and 100 MHz, respectively. High-resolution mass spectra (HR-MS) were obtained using a Shimadzu LCMS-ITTOF mass spectrometer. The reactions were monitored by thinlayer chromatography (TLC) on glass-packed precoated silica gel plates, and visualized in an iodine chamber or with a UV lamp. Flash column chromatography was performed using silica gel (200-300 mesh) purchased from Qingdao Haiyang Chemical Co. Ltd. Chemical and solvents were of reagent grade and used as obtained from Alfa Aesar (Ward Hill, MS, USA) and Sigma-Aldrich (St. Louis, MO, USA) without further purification. The purity of all the final synthesised compounds were ≥95% as determined by high-performance liquid chromatography (HPLC) with a TC-C18 column (4.6 × 250 mm, 5 μm).

Chemistry General Methods
All the reagents used in the synthesis were obtained commercially and used without further purification, unless otherwise specified. The 1 H NMR and 13 C NMR spectra were recorded using TMS as the internal standard on a Bruker BioSpin GmbH spectrometer at 400 and 100 MHz, respectively. High-resolution mass spectra (HR-MS) were obtained using a Shimadzu LCMS-ITTOF mass spectrometer. The reactions were monitored by thinlayer chromatography (TLC) on glass-packed precoated silica gel plates, and visualized in an iodine chamber or with a UV lamp. Flash column chromatography was performed using silica gel (200-300 mesh) purchased from Qingdao Haiyang Chemical Co., Ltd. Chemical and solvents were of reagent grade and used as obtained from Alfa Aesar (Ward Hill, MA, USA) and Sigma-Aldrich (St. Louis, MO, USA) without further purification. The purity of all the final synthesised compounds were ≥95% as determined by high-performance liquid chromatography (HPLC) with a TC-C18 column (4.6 × 250 mm, 5 µm).

2-Methoxy-5-nitro-4-(pyridin-4-yl)aniline
N, N-Ditert-butyl(2-methoxy-4-(1-methyl-1,2,3,6-tetrahydropyridin-4-yl)-5nitrophenyl) Carbamate (5) To a solution of 4 (317.3 mg, 0.7 mmol), acetonitrile (5.0 mL) and iodomethane (0.2 mL, 2.5 mmol) were added. The mixture was stirred at 50 • C for 4 h. The mixture was concentrated under reduced pressure. The crude product was dissolved in MeOH (5.0 mL) and cooled to 0 • C. NaBH 4 (380.1 mg, 10.0 mmol) was slowly added. After the addition was complete, the cooling bath was removed and the mixture was stirred at room temperature for 1 h. The reaction was quenched by the slow addition of 1 N aqueous HCl. The MeOH was partially removed by vacuum, and the resulting residue was partitioned between EA and 1 N aqueous NaOH. Additional 1 N aqueous NaOH was added until the aqueous layer pH was >12. The mixture was extracted with EA and washed twice with 1 N aqueous NaOH. The organic layers were washed with brine, then dried over Na 2 SO 4 ; subsequently, after filtration, the solvent was concentrated under reduced pressure and the residue was purified by flash column chromatography on silica gel to give 5 as a light-yellow solid (310.1 mg, 64%). 1 Figure S6.
N-(5-Amino-4-methoxy-2-(1-methylpiperidin-4-yl) phenyl) Acrylamide (7) To a solution of 6 (974 mg, 2.2 mmol) in DCM (15.0 mL) and TEA (0.6 mL, 4.5 mmol), acryloyl chloride was added dropwise (244.4 mg, 2.7 mmol) at 0 • C. After stirring at 0 • C for 1 h. The reaction mixture was quenched with saturated sodium bicarbonate solution and extracted with DCM. The combined organic layers were washed with brine, then dried over Na 2 SO 4 ; in addition, after filtration, the solvent was concentrated under reduced pressure. The crude product (2.0 g, 4.1 mmol) was dissolved in DCM (5.0 mL). TFA (4.6 mL, 61.4 mmol) was added, and the mixture was stirred at room temperature overnight. The reaction mixture was basified with 6 N NaOH (aq) solution (pH 12) and extracted with DCM. The combined organic layers were washed with brine, then dried over Na 2 SO 4 ; subsequently, after filtration, the solvent was concentrated under reduced pressure and the residue was purified by flash column chromatography on silica gel to give 7 as a white solid (997 mg, 83%). 1 Figure S7.

CCK8 Assays
A total of 7000 cells per well were seeded in a 96-well plate with at least three technical replicates; then, exposed to different concentrations (10, 5, 1, 0.1, 0.05, 0.01 uM) of the test compounds for 72 h. After the treatment, CCK8 (Sigma, St. Louis, MO, USA) was added to each well under sterile conditions (to a final concentration of 10% of the total volume) and the plates were incubated for 2-4 h at 37 • C. Absorbance was measured at 450 nm in a microplate reader. The cytotoxic effects of each compound were expressed as IC 50 values, which represent the drug concentrations required to cause 50% tumour cell growth inhibition, and calculated with GraphPad Prism Software version 5.02 (GraphPad Inc., La Jolla, CA, USA).

Cell Apoptosis
To assay the number of apoptotic cells, cells were stained with allo-phycocyaninannexin V (#550475, BD Pharmingen, Becton Drive Franklin Lakes, NJ, USA) for 15 min; then, they were washed and resuspended in 1:500 propidium iodide (#81845, Sigma-Aldrich, St. Louis, Mo, USA) viability marker, and acquired on LSRFortessa. Data were analyzed with FlowJo software. Annexin V+ cells were considered as apoptotic cells.

Wound-Healing Assay
H1975 cells were plated in a six-well culture dish at 5 × 10 4 cells/dish and grown for 24 h, and thenonmigrated cells were scraped off the upper surface of the membrane with a 200 µL pipet. The medium was then replaced with a serum-free 1640 medium and treated with compound 9j at the indicated concentrations for another 24 h or 48 h. After the samples were washed with phosphate buffer solution (PBS), the cell images were immediately detected by a Zeiss LSM 570 laser scanning confocal microscope (Carl Zeiss, Jena, Germany).

In Vitro Cell Migration and Invasion Assay
Migration assay was performed using an 8 um pore Boyden chamber (24-well, #353097, Corning, NY, USA), and incubated in the presence or absence of compound 9j. A total of 50,000 cells were seeded in serum-free medium in the top chamber and allowed to migrate for 48 h toward the bottom chamber containing 10% fetal bovine serum medium. Cells were fixed and permeabilized with cold 70% ethanol and methanol, and stained with 0.4% crystal violet. The average number of migrating cells was evaluated from at least four independent microscope fields.

Molecular Docking
All the docking studies were performed by Schrödinger suites (Maestro v.9.0, Schrödinger Inc., New York, NY, USA). For ligand preparation, 3D structures of all the ligands were constructed using ChemBio3D ultra14.0 and then, the initial lowest energy conformations were calculated with ligPrep. For protein preparation, the X-ray crystal structures of kinases were downloaded from the RCSB PDB Bank (available online: http://www.rcsb.org/ (accessed on 2 February 2023)); then, Protein Preparation Wizard was used with default settings to pre-process and refine. For dockings, the grid center was placed at the centroid of the ligandbinding site; and then, Glide with SP protocol was used to perform the docking study. The docking poses were affirmed by PyMOL (www.pymol.org, accessed on 2 February 2023) and MOE 2018.01 [36].

Conclusions
In the process of cancer activation, it depends not only on a single signal pathway, but also on multiple interacting signal pathways. Single-target drugs regulate the main signal pathways of tumor; however, due to the complex relationship between signal pathways, it may also promote tumor cells to reactivate through other signal pathways, resulting in drug resistance. In addition, more and more cancer patients with two or more carcinogenic gene mutations are discovered in recent years. Based on the above, developing multi-target drugs is still one of the effective strategies for treating cancer.
ALK and EGFR are extremely effective targets in the process of tumor therapy. Targeting ALK and EGFR can effectively improve the quality of life and survival time of cancer patients. The currently used ALK inhibitors and EGFR inhibitors have produced drug-resistant mutations, affecting the clinical use of existing inhibitors. Dual-target inhibitors affect two different pathways of disease progression, often producing synergistic or enhancing effects and reducing the development of disease resistance.
To explore the application of multi-target drugs in anti-tumor, we synthesized ten new dual-target inhibitors targeting EGFR and ALK simultaneously with ceritinib as the lead molecular. The optimal compound 9j exhibited excellent activity for both EGFR T790M/L858R , EML4-ALK kinases, and inhibition activity against tumor cell proliferation. The other assay results for compound 9j, such as low toxicity to normal cells and effective inhibition of tumor cell migration and invasion, indicate that 9j is worthy of further study.