DESIGN, SYNTHESIS, CRYSTAL STRUCTURE AND BIOLOGICAL EVALUATION OF NOVEL 4-ARYLAMINOQUINAZOLINE DERIVATIVES AS POTENT CYTOTOXIC AGENTS

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INTRODUCTION
Quinazoline is an important pharmacophore as a natural product, and mainly known for its wide range of biological activities [1][2][3]. Quinazoline compounds have a variety of biological activities such as antitumor, antimalaria, anti-inflammatory, antihypertensive, antivirus and antialzheimer's disease [4][5][6][7][8]. In molecular targeted therapy, quinazoline compounds play an important role which can selectively combine with active molecules in tumor cells to treat the related diseases [9,10]. For example, 4-arylaminoquinazoline is an early-developed epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKIs), which inhibits the growth of tumor cells by highly selective inhibition of epidermal growth factor receptor (EGFR) phosphorylation. Thus, it has been widely studied as an anticancer agent [11,12].
At present, some quinazoline drugs have been approved as antitumor drugs by the FDA, and widely used in clinical practice, especially in antitumor applications [13]. Since Gefitinib was used for targeted therapy of non-small cell lung cancer as the first generation EGFR-TKIs, it received extensive attention and small molecule targeted drugs had been greatly developed [14]. Currently listed drugs mainly include the first generation of EGFR-TKIs (such as Gefitinib [15], Erlotinib [16], Icotinib [17], etc), the second generation of EGFR-TKIs (such as Afatinib [18], Dacomitinib [19], Canertinib, etc) and the third generation of EGFR-TKIs (such as Osimertinib [20], Poziotinib, etc). However, with the using of small molecule targeted antitumor drugs for a long time, cancer cells would mutate, which resulted in certain drug resistance to the marketing drugs [21]. Moreover, because of the existence of the blood brain barrier, most small molecule inhibitors cannot enter the central nervous system, or be targeted for cancer cells with brain metastases.
As the third-generation EGFR-TKIs, Zorifertinib [22] not only avoids the drug resistance, but also penetrates the blood brain barrier to precisely target non-small cell lung cancer cells with brain metastases, so it is of great research significance. In view of the above situation, our group had synthesized several quinazoline derivatives in order to obtain new quinazoline drugs and studied their antitumor activity [23][24][25]. In this paper, we used Zorifitinib as the positive control drug, kept its core skeleton as 4-arylaminoquinazoline, and based on the reverse virtual screening of molecular docking and literature analysis. Pyrrolidine (used at 7-position of Cediranib quinazoline) and morpholine (used at 6-position of Gefitinib quinazoline) with similar molecular size were used to replace the heterocyclic ring at the 6-position of Zorifitinib quinazoline in order to produce better targeted binding. The phenyl ring substituent at the 4-position of quinazoline was modified by using different electron-withdrawing substituents and electron-donating substituents, and the density of electron cloud near the phenyl ring was adjusted, from which the substituent more suitable for the active pocket was selected, then designed and synthesized a series of novel 4-arylaminoquinazoline derivatives 5a(X/Y)~5c(X/Y), which were characterized by IR, 1 H NMR, MS, and the crystal structure of compound 5aX was characterized by X-ray to determine the spatial structure of the synthesized compounds. Furthermore, the physicochemical properties of the compounds were predicted using ADME data analysis, the inhibitory activity of these compounds on A549 cell line and NCI-H1975 cell line were tested in vitro by antitumor activity assay. Simultaneously, molecular docking analysis was performed to explore the possible binding mode between compound 5cX and EGFR kinase to analyze their mode of action.

Chemicals and instruments
All starting materials and reagents were obtained from commercial suppliers without further purification. The melting points of all compounds were determined on a Beijing Taike X-4 microscopy melting point apparatus and were uncorrected. 1 H NMR spectra were obtained on a Bruker Biospin 400 MHz spectrometer using TMS as the internal standard. IR spectra were recorded on a Bruker Platinum ART Tensor II FT-IR spectrometer. MS spectra were acquired on an Esquire-LC mass spectrometer (BrukerDal-tonics, USA) analytical system. The crystal data of compound 5aX was obtained by Bruker APEX-II CCD surface diffractometer. Both the human lung cancer cell line A549 and the human lung adenocarcinoma cell line NCI-H1975 were purchased from the Shanghai Academy of Life Sciences, Chinese Academy of Sciences, and passed on for cryopreservation by the Pharmacology Department of the Jiangsu Academy of Traditional Chinese Medicine. The inhibition rate data of compounds on cells were obtained from RT-6100 microplate reader.

Synthesis and characterization of pyrrolidine-1-carbonyl chloride (X)
In a round bottom flask (50 mL), the pyrrolidine (0.25 g, 3.52 mmol) was added to dichloromethane (5 mL), then triphosgene (0.86 g, 3.66 mmol) and pyridine (0.84 g, 10.87 mmol) were slowly added in the mixture at 0 °C. The mixture was stirred for 5 h at room temperature. After the completion, the dichloromethane was evaporated under reduced pressure to obtain X (0.42 g, yield: 89%) as a yellow solid. The next reaction was proceeded directly without further treatment.

Synthesis of morpholine-4-carbonyl chloride (Y)
In a round bottom flask (50 mL), the morpholine (0.20 g, 2.32 mmol) was added to dichloromethane (5 mL), then triphosgene (0.69 g, 2.33 mmol) and pyridine (0.55 g, 6.96 mmol) were slowly added in the mixture at 0 °C, then heated to room temperature and stirred for 5 h. After the completion, the dichloromethane was evaporated under reduced pressure to obtain Y (0.30 g, yield: 86%) as a yellow solid. And it was required to proceed directly to the next reaction without further treatment.

Crystal structure determination
The target compound 5aX was dissolved in the mixed solution of methanol and dichloromethane (VMeOH: VDCM = 2:1), and the solvent was slowly evaporated until regular needle-like crystals grow after 10 days. A colorless single crystal (C41H44N8O7) measuring 0.12 × 0.11 × 0.10 mm 3 was placed on a Bruker APEX-II CCD surface diffractometer. The data were collected by using MoKα radiation (λ = 0.71073 Å). In the range of 3.458° ≤ 2θ ≤ 58.850°, a total of 26819 independent diffraction points were scanned and collected while maintaining the temperature of 100 K, and 8417 of them could be used for calculation. The scheme was solved using the ShelXT structure, the structure was solved by using the inherent phase and optimized by the least square method of the ShelXL [26]  The data can be obtained from the Cambridge Crystallographic Data Centre via http://summary.ccdc.cam.ac.uk/ structure-summary-form.

In vitro antitumor activity assay
Cell cryovials were placed in a water bath at 37 °C, and shaken for 1-2 min until the cryovials were thawed. The supernatant was obtained after centrifuging (5 min) at 800 rpm. A small amount of culture medium containing 10% calf serum was added to it, and the cell mass was blown to uniformity with a pipette. Then, the obtained was transferred to a culture flask with 10-15 mL culture medium and keep in a 37 °C, 5% CO2 incubator. One day before the test, A549 cells were seeded in 96-well cell plates at 1000 cells per well. And H1975 cells were seeded in a 96-well cell plate at 2000 cells per well, and 80 µL of cell suspension was seeded in each well. The cell plate was placed in a 37 °C, 5% CO2 incubator for 12 h. On the day of the experiment, according to the compound arrangement diagram, 20 µL/well of the prepared compound working solution was added to the cell plate, and the cell plates were incubated at 37 °C in a 5% CO2 incubator for 72 h in the dark. After the incubation, 10 µL/well of CCK-8 reagent was added to the cell solution, and then placed in a 37 °C, 5% CO2 incubator for 1 h. The absorbance (OD) was measured with a microplate reader at a wavelength of 450 nm, and the inhibition ratio was calculated. The specific calculation formula is: IR% = (sample wells OD -control wells OD) / (control wells OD -blank wells OD) × 100%. According to the absorbance value, the IC50 value of the corresponding compounds were calculated and converted by the Bliss method.

Molecular docking study
The binding mode of compound 5cX was explained by selecting the protein structures of EGFR WT and EGFR L858R/T790M as receptors, which are retrieved from the RCSB protein database (RCSB.org) (PDB IDS: 2GS7 EGFR WT [27] and 5EDP EGFR L858R/T790M [28]) and the specific steps are as follows. First, water was removed from the receptor structures. Second, the hydrogens of crystal structures of the receptors and ligands were added, and the coordinate files of the individual proteins and ligands were generated into PDBQT files by using autodocktools-1.5.6 (The Scripps Research Institute, La Jolla, California, USA). Finally, the protein binding sites were covered by a grid box, after saving the file and performing molecular docking. The visual geometric simulation graph was drawn by PyMOL [29] after checking the docking results.

ADME properties
The drug properties of the compounds and the positive control drug were predicted by the Swiss ADME which is the free web tool to evaluate physicochemical properties, lipophilicity, water Solubility, pharmacokinetics, druglikeness and medicinal chemistry [30]. The predicted directions of drug properties are divided into six aspects: the pink area represents the optimal range for each property (lipophilicity: XLOGP3 between −0.7 and +5.0, size: MW between 150 and 500 g/mol, polarity: TPSA between 20 and 130 Å 2 , solubility: log S not higher than 6, saturation: fraction of carbons in the sp 3 hybridization not less than 0.25, and flexibility: no more than 9 rotatable bonds).
The structure of compound 5aX was further confirmed by single crystal X-ray diffraction analysis. The crystal structure of 5aX belongs to the monoclinic system which crystallized in space group P21/c. The molecular single crystal diagram, hydrogen bond diagram of compound 5aX and the unit cell stacking diagram are shown in Figure 1. Crystal data for the compound 5aX are given in Table 1. Hydrogen bond lengths (Å) and bond angles (°) of compound 5aX are given in Table 2. CCDC 2102043 contains the supplementary data for this paper. As shown in the data, the chemical bond lengths of compound 5aX are all within the normal range of single and double bonds, and the N4 atom has sp2 hybridization. The C-O bond near one end of the benzene ring is affected by the conjugation of the benzene ring, and the oxygen atom is biased to the side of the benzene ring. The carbonyl group has a strong electron withdrawing ability, while the nitrogen atom is biased towards the carbonyl side. Meanwhile, the bond angle is in the lowest energy state in the corresponding chemical environment, and the chemical structure is stable. Due to the conjugation of the benzene ring and the pyrimidine ring and the influence of steric hindrance, among the bond angles with N as the central atom, the bond angle of C14-N4-C15 is 128.4° (3)

Antitumor activity and structure-activity relationship
The antitumor activities of compounds against human lung cancer cells A549 and human lung adenocarcinoma cells H1975 were evaluated by CCK-8 method in vitro with Zorifertinib as the positive control, the inhibitory effects of the compounds against A549 and H1975 are shown in Table 3. For the human lung cancer cell line A549, part of the target derivatives showed a certain inhibitory activity. Among them, compound 5cX (R1 = pyrrolidine, R2 = 4-OCH3) had significant inhibitory activity, and its inhibition rate was 97.09%, which was better than that of Zorifertinib (the inhibition rate: 62.14%) and compound 5cX (R1 = pyrrolidine, R2 = 4-OCH3) was also better than compound 5aX (R1 = pyrrolidine, R2 = H, inhibition rate: 24.96%). The inhibition rate of compound 5bX (R1 = pyrrolidine, R2 = 2-CH3, 4-F) was 59.94%, which was close to that of the Zorifertinib (inhibition rate: 62.14%), and had a certain inhibitory effect. Most of the target derivatives were less sensitive to the drug-resistant cell line H1975, and the inhibition rate was mostly lower than that of Zorifertinib (49.60%). In order to further study the antitumor activity of compound 5cX in vitro on cell lines A549 and H1975, the half inhibitory concentration (IC50) of compound 5cX on A549 and H1975 was tested with Zorifertinib as a positive control and the results are shown in Table 4. For A549 cell line, compound 5cX (IC50 ＜ 2.5 µM) showed a more significant antitumor activity than Zorifertinib (IC50 = 31.08 µM). For H1975 cell line, the IC50 value of compound 5cX was less than 2.5 µM, and the inhibitory activity was also better than that of Zorifertinib (IC50 = 64.17 µM).

Molecular docking of compounds 5cX
In order to further clarify the binding mode of compound 5cX to EGFR (EGFR WT and EGFR L858R/T790M ), the binding mode of compound 5cX to EGFR was simulated by molecular docking. The PyMOL software was combined with EGFR WT protein (PDB Code: 2GS7) and EGFR L858R/T790M mutant protein (PDB Code: 5EDP). Molecular docking simulation results were shown in Figure 2. In the EGFR WT binding model, the Nitrogen atom at position 3 of quinazoline ring bound to the ALA-896 residue by hydrogen bonding, the ester group at position 6 of quinazoline formed hydrogen bonds with ASP-892 residue and LYS-855 residue, and the oxygen atom of the 7-position methoxy group was hydrogen-bonded to GLY-893. In the EGFR L858R/T790M binding model, the quinazoline ring is hydrogen-bonded to the MET-100 residue. Notably, the oxygen atom of the methoxy group on the 4-position bound to the LYS-52 residue and the ASP-162 residue through hydrogen bond, and it may result from the excellent inhibitory activity after the 4-position is substituted by p-methoxyaniline (compound 5cX). The specific physical and chemical parameters of target compounds are shown in Table 5 are the same as that of control drug Zorifertinib (0.55). It can be seen from the bioavailability radar chart (Figure 3) that the synthesized Zorifertinib derivatives had excellent physicochemical parameters, and the evaluated drug properties (such as polarity, lipophilicity, solubility, etc) were all within the reasonable range. Therefore, the synthesized derivatives could be considered as drug-like molecules, which were worthy for further study and exploration.

CONCLUSION
In this work, a series of novel 4-arylaminoquinazoline derivatives were synthesized, and the structure of compound 5aX was analyzed by single crystal X-ray diffraction. The antitumor activity of the target compounds in vitro was evaluated by CCK-8 method, with Zorifertinib as the positive control. The IC50 of compound 5cX on A549 and H1975 was tested and showed a good inhibitory effect on cell line A549 (IC50 ＜ 2.5 µM) and cell line H1975 (IC50 ＜ 2.5 µM), which is better than the positive control drug Zorifertinib (against A549: IC50 = 31.08 µM; against H1975: IC50 = 64.17 µM). The molecular docking of compound 5cX with EGFR WT and EGFR L858R/T790M showed better inhibitory activity by hydrogen bonding, and ADME prediction indicated that compounds had good physical and chemical parameters. Further studies on structural optimization and biological activities about these derivatives are still studied in our group and will be reported in the future.