Design, Synthesis, and Antitumor Activity of Novel Quinazoline Derivatives

In an attempt to explore a new class of epidermal growth factor receptor (EGFR) inhibitors, novel 4-stilbenylamino quinazoline derivatives were synthesized through a Dimorth rearrangement reaction and characterized via IR, 1H-NMR, 13C-NMR, and HRMS. Methoxyl, methyl, halogen, and trifluoromethyl groups on stilbeneamino were detected. These synthesized compounds were evaluated for antitumor activity in vitro against eight human tumor cell lines with an MTS assay. Most synthesized compounds exhibited more potent activity (IC50 = ~2.0 μM) than gefitinib (IC50 > 10.0 μM) against the A431, A549, and BGC-823 cell lines. Docking methodology of compound 6c and 6i binding into the ATP site of EGFR was carried out. The results showed that fluorine and trifluoromethyl played an important role in efficient cell activity.


Introduction
Epidermal growth factor receptor (EGFR) [1,2] belongs to a family of cell surface receptor tyrosine kinases, which is composed of four members: EGFR (HER1 or ErbB-1), HER2 (ErbB-2), HER3 (ErbB-3), and HER4 (ErbB4) [3,4]. The signaling cascade of EGFRs plays a key role in regulating cell proliferation, differentiation, and migration in many tissue types [5,6]. Many of the known tyrosine kinases are integral transmembrane receptors that act to transduce extracellular signals to intracellular responses [7][8][9][10]. Several lines of evidence have implicated that EGFR ligands play a direct role in tumor development and progression [11,12]. The altered protein expression and activity of receptor tyrosine kinase (TK) are implicated in the progression of various types of cancers [13,14]. So, the EGFR tyrosine kinase (EGFR-TK) is an attractive target for the development of agents to direct against tumors which either overexpress EGFR or have a mutated or amplified gene encoding EGFR [15]. "Knocking out" of EGFR-TK activity and the DNA damage induced by the alkylating species are expected to culminate in a sustained antiproliferative activity in EGFR-overexpressing cells.
A series of 4-anilinoquinazoline derivatives have been developed as selective and effective EGFR inhibitors, such as Gefitinib (ZD1839, Iressa) [16,17], erlotinib (OSI-774, Tarceva) [18], and Lapatinib [19,20]. On the basis of the previous investigation, we designed a series of novel 4-substitutedquinazoline derivatives as potential kinase inhibitors. In the quinazoline derivatizations, the thrust of efforts was in the arylamino moiety at the C-4 position of the quinazoline (Figure 1). On the other hand, resveratrol exhibits a variety of biological activities, such as antioxidant, quinazoline ( Figure 1). On the other hand, resveratrol exhibits a variety of biological activities, such as antioxidant, anti-inflammatory, anti-infective, anti-ischemic, cardioprotective, neuroprotective, anti-aging (prolongs lifespan), anti-obesity, and anti-viral activities [21,22]. Especially, resveratrol has been reported as a potential cancer chemopreventive agent for its striking inhibitory effects on cellular events associated with cancer initiation, promotion, and progression [23]. We mainly focused on stilbene analogues, which act as an aryl moiety in the new quinazoline compounds. At the same time, the introduction of stilbene groups to the 4-position of quinazoline would enhance the stability and lipophilicity for the changing of the electronic environment. The activity of the new quinazoline compounds could be improved for a π-π interaction and a CH-π interaction between the benzene ring of stilbene and the aromatic nucleus of target protein residues. Furthermore, the substituent group on stilbene significantly affects the electronic environment of the new quinazoline derivatives [24]. On the other hand, hydrogen bonds play an important role in the interaction of ligands and residues of target proteins. So, as a hydrogen bond acceptor, fluorine was considered to be a high biological activity group [25]. These changes will influence bioavailability, susceptibility to metabolism, and the pharmacological profile of the resulting analogues. We envisioned that chemical modification of the stilbene moiety would further improve the activity of these resulting quinazoline compounds. Therefore, we assumed that a new hybrid incorporating the active parts of each compound might be more potent than any one compound alone. Additionally, this hypothesis could also be checked by a molecular docking methodology.
In this study, we synthesized a series of novel 4-stilbenylamino quinazoline derivatives and investigated the structure-activity relationships (SAR) of these derivatives [26]. The half-maximal inhibitory concentrations (IC50) of the synthetic compounds were measured on representative EGFR-positive cell lines: A431, A549, Hela, SMMC-772, BGC823, SK-OV-3, HL-60, and HepG2 by the MTS method. With this assay system, 4-stilbenylamino quinazoline derivatives were proved to be the most promising inhibitor to tumor cells.
It was found that the trifluoromethyl group, and the fluoro and bromo groups on stilbene can improve activity. The Docking results showed that fluorine as a hydrogen bond acceptor, which plays an important role in interactions with residues of target proteins, was considered to be a high biological activity group. On the contrary, the methyl and methoxyl groups contribute less to the increase in activity of the quinazoline derivative.

Chemistry
Scheme 1 outlines the synthetic pathway to prepare compounds 6a-6j through 2-amino-4-methoxy-5-(3-morpholinopropoxy)benzonitrile (5), which is a key intermediate. Commercially available isovanillin (3-hydroxy-4-methoxybenzaldehyde) as starting material was reacted with hydroxylamine sulfate in the presence of sodium formate/formic acid to afford 3-hydroxy-4-methoxybenzonitrile (2) in 92% yield. The stirring of compound 2 with N- (3-chloropropyl)morpholine in DMF with K2CO3 gave 4-methoxy-3-(3-morpholinopropoxy) benzonitrile (3) in 98% yield. The nitration reaction of compound 3 with 70% HNO3/70% H2SO4 (volume ratio = 1:5) provided 4-methoxy-5-(3-morpholinopropoxy)-2-nitrobenzonitrile (4) in 84% We mainly focused on stilbene analogues, which act as an aryl moiety in the new quinazoline compounds. At the same time, the introduction of stilbene groups to the 4-position of quinazoline would enhance the stability and lipophilicity for the changing of the electronic environment. The activity of the new quinazoline compounds could be improved for a π-π interaction and a CH-π interaction between the benzene ring of stilbene and the aromatic nucleus of target protein residues. Furthermore, the substituent group on stilbene significantly affects the electronic environment of the new quinazoline derivatives [24]. On the other hand, hydrogen bonds play an important role in the interaction of ligands and residues of target proteins. So, as a hydrogen bond acceptor, fluorine was considered to be a high biological activity group [25]. These changes will influence bioavailability, susceptibility to metabolism, and the pharmacological profile of the resulting analogues. We envisioned that chemical modification of the stilbene moiety would further improve the activity of these resulting quinazoline compounds. Therefore, we assumed that a new hybrid incorporating the active parts of each compound might be more potent than any one compound alone. Additionally, this hypothesis could also be checked by a molecular docking methodology.
In this study, we synthesized a series of novel 4-stilbenylamino quinazoline derivatives and investigated the structure-activity relationships (SAR) of these derivatives [26]. The half-maximal inhibitory concentrations (IC 50 ) of the synthetic compounds were measured on representative EGFR-positive cell lines: A431, A549, Hela, SMMC-772, BGC823, SK-OV-3, HL-60, and HepG2 by the MTS method. With this assay system, 4-stilbenylamino quinazoline derivatives were proved to be the most promising inhibitor to tumor cells.
It was found that the trifluoromethyl group, and the fluoro and bromo groups on stilbene can improve activity. The Docking results showed that fluorine as a hydrogen bond acceptor, which plays an important role in interactions with residues of target proteins, was considered to be a high biological activity group. On the contrary, the methyl and methoxyl groups contribute less to the increase in activity of the quinazoline derivative.

Biology
In Vitro Antitumor Evaluation The in vitro antitumor abilities of the synthetic compounds were measured on representative EGFR-positive cell lines A431, A549, Hela, SMMC-772, BGC823, SK-OV-3, HL-60, and HepG2 by the MTS method and Geifitinib as a reference drug control [16]. The response parameter (IC50) was calculated for each cell line (Table 1). As shown in Table 1, almost all of the synthesized quinazoline derivatives exhibited excellent growth inhibitory activities, with IC50 values in the range of 1. 23-5.17 μM comparative to Gefitinib IC50 (>10.0 μM).

Biology
In Vitro Antitumor Evaluation The in vitro antitumor abilities of the synthetic compounds were measured on representative EGFR-positive cell lines A431, A549, Hela, SMMC-772, BGC823, SK-OV-3, HL-60, and HepG2 by the MTS method and Geifitinib as a reference drug control [16]. The response parameter (IC 50 ) was calculated for each cell line (Table 1). As shown in Table 1, almost all of the synthesized quinazoline derivatives exhibited excellent growth inhibitory activities, with IC 50 values in the range of 1.23-5.17 µM comparative to Gefitinib IC 50 (>10.0 µM).

Molecular Modeling
To gain insight into the binding mode of these compounds, molecular docking of the compounds 6c and 6i as representative examples of the same series into the binding site of EGFR was carried out. All calculations were performed using SYBYL-X 2.0 software. The crystal structure of EGFR with lapatinib (PDB ID: 1XKK) was obtained from the protein data bank (PDB). Molecular docking studies of the compounds were performed to rationalize the obtained biological results and their mechanism of action ( Figure 2). Additionally, molecular docking studies helped us to understand various interactions between the ligands and the enzyme active site in detail.

Molecular Modeling
To gain insight into the binding mode of these compounds, molecular docking of the compounds 6c and 6i as representative examples of the same series into the binding site of EGFR was carried out. All calculations were performed using SYBYL-X 2.0 software. The crystal structure of EGFR with lapatinib (PDB ID: 1XKK) was obtained from the protein data bank (PDB). Molecular docking studies of the compounds were performed to rationalize the obtained biological results and their mechanism of action ( Figure 2). Additionally, molecular docking studies helped us to understand various interactions between the ligands and the enzyme active site in detail. The results have revealed that the obtained compounds bind to a narrow hydrophobic pocket through the formation of strong hydrogen bond interactions. The complexes were energy-minimized with an AMBER7 FF99 Force-field until a gradient convergence of 0.001 kcal/mol was reached. The docking studies of compounds 6c and 6i showed that a hydrogen bond formed between the N1 of the quinazoline ring and Met-793 (2.23 ± 0.10 Å and 2.11 ± 1.10 Å, respectively). Two hydrogen bonding interactions were observed between CF3−4' of compound 6c and Arg-841 (2.05 ± 0.10 Å, 2.09 ± 1.10 Å). Different from 6c, CF3-4' compound 6i formed hydrogen bonding interactions with Ala-722 and Arg-841(2.39 ± 0.10 Å and 2.38 ± 1.10 Å, respectively). The docking studies indicated that the hydrogen bond interactions of the nitrogen atom on the morpholine ring and the NH at the C-4 position of quinazoline with residues in the hinge region of EGFR were The results have revealed that the obtained compounds bind to a narrow hydrophobic pocket through the formation of strong hydrogen bond interactions. The complexes were energy-minimized with an AMBER7 FF99 Force-field until a gradient convergence of 0.001 kcal/mol was reached. The docking studies of compounds 6c and 6i showed that a hydrogen bond formed between the N1 of the quinazoline ring and Met-793 (2.23 ± 0.10 Å and 2.11 ± 1.10 Å, respectively). Two hydrogen bonding interactions were observed between CF3−4' of compound 6c and Arg-841 (2.05 ± 0.10 Å, 2.09 ± 1.10 Å). Different from 6c, CF3-4' compound 6i formed hydrogen bonding interactions with Ala-722 and Arg-841(2.39 ± 0.10 Å and 2.38 ± 1.10 Å, respectively). The docking studies indicated that the hydrogen bond interactions of the nitrogen atom on the morpholine ring and the NH at the C-4 position of quinazoline with residues in the hinge region of EGFR were uncertain, which hinge on the configuration of compounds and species of substituent groups on the stilbene. These interactions revealed the importance of both fluorine and nitrogen atoms for binding and the subsequent inhibitory capacity. From the above results, we have known that the binding model of compounds 6c and 6i in the ATP binding site were very close to the complex of lapatinib (PDB ID: 1XKK). These molecular docking study results can strongly support our postulation that the synthesized compounds may effectively act on EGFR-TK.

Molecular Modeling Methods
The calculations were performed using SYBYL-X2.0 software installed on 3.30 GHz Core 4 Duo, 8G memory, with a Windows Server 2008 R2 operating system. The crystal structure of lapatinib was obtained from the Protein Data Bank (PDB codes: 1XKK) in order to prepare the protein for docking studies. The preparation of protein structure used the Protonate 3D function, and the molecules were energy-minimized according to the implemented AMBER7 FF99 force-field. The docking procedure followed was the standard protocol implemented in SYBYL-X2.0, and the geometry of the resulting complexes was studied using the pose Viewer utility.

Conclusions
Herein, we designed and synthesized a series of novel 4-aminoquinazolines derivatives containing a stilbene moiety. Addtionally, the biological activity of these compounds was investigated in several cell lines, including human vaginal epidermoid cancer cell line (A431), human non-small cell lung cancer (A549), and human liver cancer cell line (HepG2). In all of the compounds, fluorine atoms on the stilbene moiety enhanced interaction with residue, which helped with fitting into the lipophilic back pocket, as expected by our designs, leading to an improvement in antiproliferative activity. Most derivatives showed better biological activity and potency (IC 50 < 2.0 µM) with respect to gefitinib assessed in a series of cell lines. The good growth inhibition of these compounds may owe to the fact that they can more closely bind in the ATP binding site of EGFR, resulting from docking model compounds 6c and 6i with 1XKK.