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Article

Design, Synthesis, Antiproliferative Potency and In Silico Studies of Novel Alkynyl Quinazolines as Potential EGFR Inhibitors

by
Apostolia Gkoutzivelaki
1,
Sotiria-Iro Triantopoulou
2,
Lykourgos Chiniadis
3,
Alexandros Komiotis
4,
Charalampos Triantis
4,
Dimitri Komiotis
4,5,
Athanasios Papakyriakou
3,
Harris Pratsinis
3,* and
Stella Manta
1,*
1
Laboratory of Organic Chemistry, Department of Chemistry, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece
2
Institute of Nuclear & Radiological Sciences and Technology, Energy & Safety, National Centre for Scientific Research “Demokritos”, 15341 Athens, Greece
3
Institute of Biosciences & Applications, National Centre for Scientific Research “Demokritos”, 15341 Athens, Greece
4
Department of Pharmacy, Frederick University, Nicosia 1036, Cyprus
5
Department of Biochemistry and Biotechnology, School of Health Sciences, University of Thessaly, 41335 Larissa, Greece
*
Authors to whom correspondence should be addressed.
Int. J. Mol. Sci. 2026, 27(4), 1738; https://doi.org/10.3390/ijms27041738
Submission received: 14 January 2026 / Revised: 6 February 2026 / Accepted: 9 February 2026 / Published: 11 February 2026
(This article belongs to the Special Issue Nitrogen-Containing Heterocycles and Their Biological Applications)
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Versions Notes

Abstract

The epidermal growth factor receptor (EGFR) is a highly attractive and promising target for novel anticancer agents, particularly for non-small-cell lung cancer (NSCLC), due to its crucial role in regulating cell survival and proliferation. Despite the development of first-generation reversible inhibitors like Gefitinib and Erlotinib, acquired resistance necessitated the discovery of highly potent irreversible inhibitors effective against drug-resistant mutants. Molecular docking calculations utilizing both EGFR conformations identified five top-ranked compounds (QN012, QN017, QN019, QN022, and QN023) proposed for synthesis and biological evaluation. These in silico studies predicted high inhibitory activity against the active and inactive state of EGFR. Herein, we report the design, synthesis and biological evaluation of novel 4-anilino quinazoline derivatives, bearing various alkynyl substituents at position 6, expected to bind to the hinge Met793 residue of EGFR. The effects of the derivatives on various cancer cell lines in terms of cytotoxic/cytostatic activity, interference with cell cycle phase distribution, and suppression of EGFR phosphorylation set the basis for the design of more potent derivatives.

Graphical Abstract

1. Introduction

The epidermal growth factor receptor (EGFR) has been the focus of extensive scientific investigation and research due to its crucial role in regulating vital cellular processes such as cell survival, proliferation, differentiation, and migration [1]. Because of its significant involvement in these fundamental biological functions, EGFR has emerged as a highly attractive and promising target for the development of novel anticancer agents and therapeutic strategies. The EGFR has been extensively studied as an attractive target for the development of anticancer agents, due to its regulative role in important cellular processes, such as survival and proliferation [2,3]. The receptor possesses tyrosine kinase (TK) activity, normally activated through the binding of various ligands at the extracellular domain. The concomitant autophosphorylation of tyrosine residues in the intracellular domain stimulates various downstream signaling pathways, such as the RAS-RAF-MEK-ERK and the PI3K-AKT-mTOR [4,5]. Many types of human cancers, including lung, breast, head and neck, and brain cancers, are frequently associated with overexpression or mutations of EGFR [4,6]. Based on the above data, EGFR tyrosine kinase has become an attractive therapeutic target for cancer treatment, especially for non-small-cell lung cancer (NSCLC).
Extensive research efforts for the discovery of EGFR inhibitors led to the development of three generations of inhibitors, exemplified by the approved drugs Gefitinib [7,8], Afatinib [9], and Osimertinib [10], all sharing a central 4-anilinoquinazoline core. This is one of the most promising scaffolds due to its stability, ease of synthetic accessibility, and flexibility in structural modification and functionalization [11,12,13,14,15]. The lipophilicity of quinazoline derivatives facilitates their ability to traverse the blood–brain barrier, thereby contributing to their diverse biological activities, such as anti-inflammatory [16,17], anticancer [18,19,20], antidiabetic [2,3], antihypertensive [21], antimalarial [22], antimicrobial [23], antiviral [24], anticonvulsant [25,26], anticholinesterase [27] and antioxidant [28,29]. Concerning the first-generation reversible inhibitors Gefitinib and Erlotinib, although they offer great tolerability, acquired resistance appeared in more than 50% of patients, as a result of a secondary point mutation, T790M, which increases the affinity of the TK active site for ATP [30]. To overcome the resistance, the second- and third-generation EGFR-TKIs (such as afatinib and osimertinib, respectively) were developed, which enabled the irreversible binding to Cys797 residue in the ATP binding site. Despite the great therapeutic success of the third-generation EGFR inhibitors, the emergence of tertiary point mutations in EGFR, such as the EGFRC797S mutant, deprived the inhibitors of covalent bond formation by disturbing the corresponding binding with Cys797. Additionally, since irreversible inhibitors demonstrated better potency against EGFR T790M mutants than reversible inhibitors, a challenging target was to discover highly potent irreversible inhibitors of the drug resistant T790M mutants that spare EGFR WT activity to the greatest extent as a means to minimize mechanism-based toxicities [31].
Considering the above, the structure-based design of the compounds in this study was based on two X-ray crystal structures of the EGFR kinase domain in both active and inactive states. For the active state (active BLAminus state), the structure in complex with Erlotinib was utilized (PDB ID: 1M17) [32], while for the inactive state (SRC-inactive BLBplus state), the structure in complex with TAK-285 was used (PDB ID: 3POZ) [33]. As revealed by these crystal structures, both Erlotinib and TAK-285 are stabilized via a characteristic hydrogen bond between the N1 atom of their pyrimidine moiety and the backbone NH group of Met793. Building upon these structural insights, we designed a series of 6-alkynyl-4-anilinoquinazolines QN009–QN027 (Figure 1), bearing various alkynyl substituents at position 6 of the quinazoline pharmacophore, intended to mimic the hydrogen-bonding interaction of Erlotinib and TAK-285 with Met793, while potentially binding to both the active and inactive states of the EGFR kinase domain. We hypothesized that the quinazoline aromatic ring would occupy the lipophilic region I within the kinase active site, and we aimed to explore these aromatic substituents through structure–activity relationship studies to achieve a favorable methylthio-aryl interaction to enhance binding affinity. Such methionine-aromatic interactions play significant roles in stabilizing protein conformations [34] and in ligand–protein interactions [35]. To facilitate the selection of appropriate substituents, we employed computer-aided molecular docking calculations to predict the binding mode and affinity of each target compound. Based on the docking results for both the active and inactive conformations, we selected the five top-ranked candidates and herein, we report their synthesis and biological evaluation.
Ijms 27 01738 g001
Figure 1. Structures of the EGFR inhibitors Erlotinib, TAK–285, and the designed scaffold of 4,6-substituted quinazolines QN009–QN027. (R1 and R2 are shown in Table 1.)
Figure 1. Structures of the EGFR inhibitors Erlotinib, TAK–285, and the designed scaffold of 4,6-substituted quinazolines QN009–QN027. (R1 and R2 are shown in Table 1.)
Ijms 27 01738 g001
Table 1. Docking results of Erlotinib, TAK-285 and designed quinazolines for two states of EGFR, active and inactive. Predicted affinities are given as calculated binding free energies (ΔGcalc) and estimated inhibition constants (Kiest). n.d. indicates compounds for which the hydrogen-bonding interaction with Met793 was not detected in any docked pose. The two EGFR inhibitors and the most potent compounds are highlighted in bold. The codes of chemically synthesized compounds (see below, Scheme 1) are shown in parentheses.
Table 1. Docking results of Erlotinib, TAK-285 and designed quinazolines for two states of EGFR, active and inactive. Predicted affinities are given as calculated binding free energies (ΔGcalc) and estimated inhibition constants (Kiest). n.d. indicates compounds for which the hydrogen-bonding interaction with Met793 was not detected in any docked pose. The two EGFR inhibitors and the most potent compounds are highlighted in bold. The codes of chemically synthesized compounds (see below, Scheme 1) are shown in parentheses.
CompoundEGFR (Active State)EGFR (Inactive State)
ΔGcalc
(kcal/mol)		Kiest
(μM)		ΔGcalc
(kcal/mol)		Kiest
(μM)		R1R2
Erlotinib−7.07.2n.d.n.d.
TAK-285n.d.n.d.−9.80.06
QN009n.d.n.d.−8.60.52Ijms 27 01738 i001Ijms 27 01738 i002
QN010−7.81.8−8.30.90Ijms 27 01738 i003Ijms 27 01738 i004
QN011−7.53.2−7.91.64Ijms 27 01738 i005Ijms 27 01738 i006
QN012 (6e)−7.62.8−9.60.09Ijms 27 01738 i007Ijms 27 01738 i008
QN013−7.62.7−7.91.55Ijms 27 01738 i009Ijms 27 01738 i010
QN014−8.01.4n.d.n.d.Ijms 27 01738 i011Ijms 27 01738 i012
QN015−8.60.5n.d.n.d.Ijms 27 01738 i013Ijms 27 01738 i014
QN016−8.20.9−8.30.80Ijms 27 01738 i015Ijms 27 01738 i016
QN017 (6a)−8.50.6−8.70.43Ijms 27 01738 i017Ijms 27 01738 i018
QN019 (6b)−8.40.7−9.10.22Ijms 27 01738 i019Ijms 27 01738 i020
QN020−8.21.0−8.30.81Ijms 27 01738 i021Ijms 27 01738 i022
QN021−8.21.0−8.30.80Ijms 27 01738 i023Ijms 27 01738 i024
QN022 (6d)−8.21.1−9.00.25Ijms 27 01738 i025Ijms 27 01738 i026
QN023 (6c)−8.21.0−8.70.43Ijms 27 01738 i027Ijms 27 01738 i028
QN024n.d.n.d.n.d.n.d.Ijms 27 01738 i029Ijms 27 01738 i030
QN025−8.01.5−8.20.97Ijms 27 01738 i031Ijms 27 01738 i032
QN026−8.21.0−8.60.51Ijms 27 01738 i033Ijms 27 01738 i034
QN027−7.91.6−8.60.52Ijms 27 01738 i035Ijms 27 01738 i036

2. Results and Discussion

2.1. Molecular Docking

As established in our design rationale, in order to evaluate the binding potential of the designed 6-alkynyl-4-anilinoquinazolines (QN009–QN027), we performed molecular docking calculations using the EGFR kinase domain conformations. Docking was used as a preliminary screening tool to focus our synthetic efforts on derivatives predicted to form the essential hydrogen bond with the Met793 residue, and it was merely employed in this study to narrow down the number of compounds that would be synthesized and investigated experimentally. It should be noted that docking calculations may provide good indications about the bound pose of an inhibitor; however, estimation of the binding free energy lacks accuracy. Therefore, compounds with poor docking scores may still exhibit biological activity.
For the active state, the conserved Asp-Phe-Gly motif at the N terminus of the activation loop, the “DFG loop”, is at the “in-state” where the Asp side chain points toward the ATP-binding site (Figure 2). The αC-helix of the kinase domain is also at the “in-state”, with a characteristic salt bridge between β3-Lys and αC-Glu; the regulatory spine is assembled and the activation loop is extended. According to Modi and Dunbrack [36], this structure of EGFR is classified in the active BLAminus state. In the inactive state (Figure 3), the DFG motif remains “in” and the αC-helix is rotated outward, breaking the conserved β3-Lys–αC-Glu salt bridge and contributing to the disassembly of the hydrophobic regulatory spine, providing more space in the binding site. Our docking results for the compounds within the active and the inactive conformations of the EGFR kinase domain are shown in Table 1. Compound QN024 was not predicted to interact via the expected hydrogen bond with residue Met793, in either the active or inactive state of EGFR (designated with n.d. in Table 1). This was also the case for QN014 and QN015 but only for the inactive state of EGFR. Based on the docking results of the top-ranked compounds for both states of EGFR, we proposed the synthesis and biological evaluation of five compounds, namely QN012 (6e), QN017 (6a), QN019 (6b), QN022 (6d), and QN023 (6c).

2.2. Chemistry

Our synthetic effort involved the preparation of N-(3-chlorophenyl) and (4-fluorophenyl)-6-iodoquinazoline-4-amino analogs (5a,b) (Scheme 1). The reaction of commercially available anthranilic acid (1) with potassium iodide (KI) and hydrogen peroxide (H2O2) in the presence of acetic acid (CH3COOH) produced 2-amino-5-iodo-benzoic acid (2) in very good yield (84%). The Niementowski reaction [37] was employed for the quinazoline ring closure and the synthesis of the 3H-quinazolin-4-one core (3) was achieved using an excess of formamide (5 equiv) as a fusion accelerator at a reflux temperature (130–140 °C). Chlorination of compound 3 was then performed by SOCl2 catalyzed by DMF under reflux temperature, to furnish 6-iodo-4-chloroquinazoline (4), in very good yield (88%). Functionalization at C-4 of the pyrimidine ring of compound 4 by nucleophilic substitution of the chloride by 3-chloro or 4-fluoro anilines in i-PrOH gave the derivatives 5a,b. Finally, palladium catalyzed the Sonogashira coupling reaction [38] of 5a,b with several acetylenes, and furnished the desired disubstituted quinazoline derivatives 6a–e, in 80–90% yields. As docking studies suggested and in order to extract more detailed structure–activity relationships, diverse alkyne substituents R were selected, which included a phenyl ring substituted with chloro (6d), methyl (6a) and dimethyl (6b) or methoxyl groups (6c) and an alkynyl group (6e). In a typical experiment, compounds 5a or 5b were mixed with DMF; the appropriate alkyne, triethylamine (base), copper(I) iodide (CuI) (co-catalyst) and tetrakis(triphenylphosphine) palladium (0) (Pd(PPh3)4) (catalyst) were refluxed until the completion of the reaction. After removing all the volatile materials under vacuum, the solid obtained was further purified by flash chromatography to provide the target compounds 6a–d and 6e, respectively.

2.3. Biological Evaluation

2.3.1. Cytotoxic/Cytostatic Activity

The above synthesized compounds were tested for their cytotoxic/cytostatic activity against a panel of human cancer cell lines, corresponding to tumors of a variety of histopathologic origins. All cell lines are known to express wild-type EGFR; however, A431 cells are notorious for extreme EGFR overexpression due to gene amplification [39]. The A549 cell line, also reported to express high levels of EGFR [40], was chosen to represent non-small-cell lung cancer (NSCLC), a primary therapeutic target for EGFR inhibitors. Mammary adenocarcinoma MDA-MB-231 and prostate adenocarcinoma PC-3 cells have also been reported to express high EGFR levels [41,42]. A normal human dermal fibroblast strain was also used for comparative studies, as this cell type is also known to express EGFR [43]. Evaluation of cytotoxic/cytostatic activity was implemented based on the widely used MTT assay, and the IC50s of the compounds are shown in Table 2.
The five compounds exhibited medium-range IC50s, being substantially weaker than the doxorubicin hydrochloride used as the positive control. Compound 6c, bearing a 4-methoxyphenyl group, emerged as the most potent derivative in the series, particularly against A549 lung adenocarcinoma cells (IC50 = 1.5 μM), representing a 22-fold higher potency compared to its effect on normal fibroblasts (IC50 = 32.8 μM), which suggests that certain substitutions in this series can indeed lead to improved selectivity. It seems that a polar, electron-donating substituent at the para-position of the aromatic ring significantly enhances binding and biological response. Contrarily, compounds 6a (4-methyl) and 6b (2,5-dimethyl) showed moderate activity. Notably, the addition of a second methyl group in 6b resulted in a general decrease in cytotoxicity compared to 6a, which may be attributed to steric hindrance within the ATP-binding pocket. Analog 6e is the only derivative evaluated, carrying a fluorine at the para-position of the aniline. This structural change, combined with the 6-long pentyl chain, appears to impart a unique selectivity for certain cancer lines like PC-3 and MDA-MB-231. It may be hypothesized that those structural features may improve cellular permeability or interact differently with hydrophobic pockets in specific cell types. Ιt should be noted here, that the cytostatic activities (Table 2) cannot be directly correlated with the predicted binding affinity of the compounds (Table 1), due to low accuracy of molecular docking and/or to parameters (other than the binding to EGFR) affecting the cytotoxicity of the compounds. Although the newly synthesized quinazoline compounds, with the exception of 6c on A549 cells, exhibit comparable IC50 values in both cancerous and normal cells—indicating no selectivity and hence limited anticancer potential—they may serve as valuable scaffolds for the future design and development of more potent and selective derivatives.

2.3.2. Effects on the Cell Cycle

Exponentially proliferating epidermoid carcinoma A431 cells were used to assess the compounds’ effects on cell cycle phase distribution, following a 24 h treatment. The distribution of cell populations in G0/G1, S, and G2/M phases of the cell cycle, as well as sub-diploid events indicating apoptotic nuclei, are shown in Table 3.
All five compounds were found to inhibit cell proliferation by causing an accumulation of the cells at the G0/G1 phase and suppressing the S-phase percentage (and, to a lesser extent, the G2/M percentage). There was no significant effect on the sub-diploid events, supporting the idea that the five quinazoline derivatives act mainly as cytostatic agents. Although G0/G1 cell cycle arrest is not definitive evidence of EGFR inhibition, it is compatible with a mode of action through the EGFR kinase, as has been previously reported for Erlotinib [44] or other interventions targeting EGFR [45].

2.3.3. Effects on EGFR Activation

In an attempt to verify the ability of the five quinazoline derivatives to interfere with EGFR activation, we collected A549 cell lysates following pre-incubation of the cultures with the compounds (the highly potent EGFR kinase inhibitor AG1478 was included as a positive control) and subsequent activation of the receptor with EGF. Analysis of EGFR phosphorylation status with Western analysis (Figure 4) shows that all five quinazoline derivatives were capable to suppress EGFR phosphorylation due to EGF stimulation. Compound 6c was the most potent quinazoline regarding suppression of EGFR phosphorylation—although not reaching the potency of the positive control AG1478—while 6e was the weakest, in line with their cytostatic activity ranking on A549 cells (Table 2). A limitation of the current study is the absence of a cell-free EGFR kinase assay, allowing for direct mechanistic conclusions. Suppression of EGFR phosphorylation in the cellular context cannot exclude the possibility that the synthesized compounds may also interfere with other kinases upstream or downstream of EGFR kinase. Nevertheless, since the presence of other growth factors was minimized by serum starvation, suppression of EGFR phosphorylation triggered by EGF stimulation constitutes direct evidence of pathway interference, correlating well with the cytostatic activity of the compounds on A549 cells.

3. Materials and Methods

3.1. Chemistry

Melting points were recorded in a Mel-Temp apparatus and were uncorrected. Thin-layer chromatography (TLC) was performed on Merck precoated 60F254 plates (Merck, Darmstadt, Germany). Reactions were monitored by TLC on silica gel, with detection by UV light (254 nm). Flash column chromatography was performed using silica gel (240–400 mesh, Merck, Darmstadt, Germany). NMR spectra were recorded with an Agilent-500/54 or a Bruker DRX 500 MHz NMR spectrometer (Bruker Biospin, Rheinstetten, Germany), using DMSO-d6 as the solvent. All 1H (500 MHz) and 13C NMR (125 MHz) spectra were recorded with chemical shifts (ppm) and coupling constants (J) in hertz (Hz). UV–Vis spectra were recorded on a PG T70 UV–VIS spectrometer (PG Instruments, UK) and mass spectra were obtained on a ThermoQuestFinnigan AQA Mass Spectrometer (electrospray ionization) (San Jose, California, USA). DMF was stored over 3 Å molecular sieves. All reactions sensitive to oxygen or moisture were carried out under argon atmosphere.
Synthesis of 2-amino-5-iodobenzoic acid (2)
A mixture of 2-aminobenzoic acid (1) (10.98 g, 80.0 mmol), potassium iodide KI (13.30 g, 80.0 mmol) and acetic acid (100 mL) was cooled to 5 °C, followed by dropwise addition of hydrogen peroxide solution 30% w/w (9.0 mL, 88 mmol), and the reaction mixture was stirred for 2 h at room temperature, while we observed solid product formation. After completion of the reaction (EtOAc/Hexane 1/4), 65 mL of water was added and the mixture was chilled to 0–5 °C for 30 min followed by vacuum filtration. Solid was rinsed 4–5 times with cold water and was allowed to dry protected from light, in a stream of air to give iodo compound 2 (17.7 g, 84%). Rf = 0.21 (Hexane/EtOAc, 1:4) and mp (dec.): 219–221 °C. The chemical and physical properties were in full agreement with the data reported [46,47,48].
Synthesis of 6-iodoquinazolin-4(3H)-one (3)
A mixture of 2 (5.26 g, 20.0 mmol) and formamide (HCONH2) (6.4 mL, 162 mmol) was refluxed at 130–140 °C under vigorous steering. After 24 h, full consumption of the starting material with TLC was observed. The mixture was allowed to cool to room temperature and then an ice–water mixture (45 mL) was added. After stirring for 15 min, the mixture was filtered; the solid was rinsed 3 times with cold water and was allowed to dry protected from light, to afford compound 3 (4.14 g, 76%), as a brown solid. Rf = 0.26 (EtOAc/Hexane, 1:1) and mp (dec.): 273–275 °C. The chemical and physical properties were in full agreement with the data reported [17,49,50].
Synthesis of 6-iodo-4-chloroquinazoline (4)
A mixture of 6-chloroquinazoline-4 (3H)-one (3) (3.27 g, 12.0 mmol), SOCl2 (14.0 mL) and DMF (10 mL) was refluxed at 95 °C. After 1 h, a dark-brown solution emerged. The course of the reaction was checked by TLC and after 5 h of heating we observed the complete consumption of the starting material 3. The mixture was then allowed to cool to room temperature; then, the SOCl2 was removed in vacuo and the solid residue was obtained and dissolved in CH2Cl2 (50 mL). The solution was extracted sequentially with saturated aqueous solution of NaHCO3 (2 × 30 mL) and saturated brine (1 × 30 mL), dried over Na2SO4, and concentrated in vacuo. The brown solid was purified by column chromatography eluting with CH2Cl2 to give 4 as a yellowish solid (2.44 g, 70%). Rf = 0.22 (CH2Cl2) and mp: 188–191 °C. The chemical and physical properties were in full agreement with the data reported [49,50].
General procedure for the preparation of N-(3-chlorophenyl) and (4-fluorophenyl)-6-iodoquinazoline-4-amines (5)
Mixtures of 6-iodo-4-chloroquinazoline (4) (2.0 mmol), i-PrOH (8.0 mL), and appropriate anilines (2.2 mmol) in i-PrOH (2.0 mL) were refluxed at 95 °C for 1 h. The mixture was allowed to cool to room temperature, then cooled to 0–4 °C for 2 h and filtered under vacuum. Finally, the solid products were rinsed 3 times with isopropanol and dried to afford the corresponding derivatives 5a,b, in excellent yields (89% and 90%, respectively), as yellow solids.
Synthesis of N-(3-chlorophenyl)-6-iodoquinazoline-4-amine (5a)
An amount of 679 mg, 89%; mp: 219–221 °C; Rf = 0.57 (EtOAc/Hexane 1:1); λmax 397 nm (ε 4054); 1H NMR (500 MHz, DMSO-d6): δ 11.48 (s, 1H), 9.31 (s, 1H), 8.98 (s, 1H), 8.36 (d, 1H, J = 8.7 Hz), 7.95 (d, 1H), 7.77–7.75 (m, 2H), 7.53 (t, 1H, J = 8.1 Hz), 7.40 (d, 1H, J = 8.1 Hz); 13C NMR (125 MHz, DMSO-d6): δ 158.7, 152.2, 144.5, 140.3, 138.9, 133.4, 133.2, 130.9, 126.5, 124.3, 123.3, 123.1, 116.0, 94.5. ESIMS (m/z): 382.2 [M+H]+. Anal. Calcd. for C14H9ClIN3: C, 44.07; H, 2.38; Cl, 9.29; I, 33.26; N, 11.01%. Found: C, 44.45; H, 2.16; Cl, 9.01; I, 33.48; N, 11.33%.
Synthesis of N-(4-fluorophenyl)-6-iodoquinazoline-4-amine (5b)
An amount of 657 mg, 90%; mp: 275–277 °C; Rf = 0.50 (EtOAc/Hexane 1:1); λmax 397 nm (ε 4022); 1H NMR (500 MHz, DMSO-d6): δ 11.43 (s, 1H), 9.24 (s, 1H), 8.92 (s, 1H), 8.37 (d, 1H, J = 8.7 8.7Hz), 7.78–7.71 (m, 3H), 7.37–7.34 (m, 2H); 13C NMR (125 MHz, DMSO-d6): δ 162.5, 158.8, 152.2, 144.4, 133.6, 133.2, 127.1, 126.9, 123.1, 116.2, 115.9, 94.4; ESIMS (m/z): 366.05 [M+H]+. Anal. Calcd. for C14H9FIN3: C 46.05; H, 2.48; N, 11.51; F, 5.20; I, 34.75%. Found: C, 46.33; H, 2.22; N, 11.25; F, 5.48; I, 34.99%.
General procedure for the preparation of quinazoline derivatives (6a–e)
N-(3-Chlorophenyl) 5a or N-(4-fluorophenyl) 5b 6-iodoquinazoline-4-amine (0.20 mmol) was dissolved in a mixture of dry DMF (1.0 mL), triethylamine (0.60 mmol) and the appropriate terminal alkyne (0.40 mmol). CuI (0.02 mmol) and Pd(PPh3)4 (0.02 mmol) were added, and the reaction mixture was stirred at reflux temperature until completion of the starting material (60 to 90 min). The reaction mixture was concentrated under reduced pressure, and the crude residue was purified by flash chromatography on silica gel. The purified material was dried in vacuo to afford the corresponding derivative 6, in 65–82% yields, as a solid.
Synthesis of 4-N-(3-chlorophenyl)-6-(4-methylphenylacetylene) quinazoline (6a) QN017
An amount of 60.7 mg, 82%; mp: 200–202 °C; Rf = 0.17 (EtOAc/Hexane 1:4); λmax 397 nm (ε 5254); 1HNMR (500 MHz, DMSO-d6): δ 10.00 (s, 1H), 8.84 (s, 1H), 8.71 (s, 1H), 8.16 (s, 1H), 7.97 (d, 1H, J = 8.2 Hz), 7.90 (d, 1H, J = 8.3 Hz), 7.83 (d, 1H, J = 8.5 Hz), 7.53–7.51 (m, 2H), 7.43 (t, 1H, J = 8.0, 8.1 Hz), 7.31–7.29 (m, 2H), 7.20 (m, 1H), 2.44 (s, 3H, CH3); 13C NMR (125 MHz, DMSO-d6): δ 157.5, 155.5, 149.9, 141.1, 139.5, 135.9, 133.2, 131.8, 130.6, 130.0, 129.0, 126.7, 123.8, 121.8, 120.8, 120.7, 119.4, 115.7, 91.2, 88.8, 21.6; ESIMS (m/z): 370.90 [M+H]+. Anal. Calcd. for C23H16ClN3: C, 74.69; H, 4.36; N, 11.36; Cl, 9.58%. Found: C, 74.22; H, 4.61; N, 11.02; Cl, 9.17%.
Synthesis of 4-N-(3-chlorophenyl)-6-(2,5-dimethylphenylacetylene) quinazoline (6b) QN019
An amount of 50 mg, 65%; mp: 223–225 °C; Rf = 0.15 (EtOAc/Hexane 1:4); λmax 397 nm (ε 5326); 1HNMR (500 MHz, DMSO-d6): δ 10.02 (s, 1H), 8.82 (s, 1H), 8.70 (s, 1H), 8.14 (s, 1H), 7.99 (d, 1H, J = 8.6 Hz), 7.89 (d, 1H, J = 8.6 Hz), 7.84 (d, 1H, J = 8.0 Hz), 7.45 (t, 1H, J = 8.0, 8.1 Hz), 7.39 (m, 1H), 7.27–7.25 (m, 1H), 7.22–7.17 (m, 2H), 2.49, 2.31 (2s, 6H); 13C NMR (125 MHz, DMSO-d6): δ 157.6, 155.5, 149.9, 141.1, 137.2, 135.9, 135.6, 133.2, 132.4, 130.6, 130.4, 130.2, 130.1, 129.0, 126.5, 123.9, 122.0, 122.26, 120.9, 115.7, 92.9, 90.1, 20.8, 20.4; ESIMS (m/z): 384.20 [M+H]+. Anal. Calcd. for C24H18ClN3: C, 75.09; H, 4.73; N, 10.95; Cl, 9.23%. Found: C, 75.45; H, 4.35; N, 10.67; Cl, 9.48%.
Synthesis of 4-N-(3-chlorophenyl)-6-(4-methoxyphenylacetylene) quinazoline (6c) QN023
An amount of 60.2 mg, 78%; mp: 252–253 °C; Rf = 0.19 (EtOAc/Hexane 1:4); λmax 397 nm (ε 5152); 1HNMR (500 MHz, DMSO-d6): δ 10.00 (s, 1H), 8.83 (s, 1H, H-6), 8.72 (s, 1H), 8.16 (s, 1H), 7.97 (d, 1H, J = 8.0 Hz), 7.90 (d, 1H, J = 8.3 Hz), 7.84–7.83 (m, 1H), 7.44 (t, 1H, J = 8.0, 8.1 Hz), 7.20 (d, 2H, J = 7.9 Hz), 7.06–7.04 (m, 2H), 3.83 (s, 3H); 13C NMR (125 MHz, DMSO-d6): δ 160.3, 157.5, 155.4, 141.1, 135.8, 133.5, 133.2, 130.6, 129.0, 126.4, 123.8, 121.8, 121.1, 120.7, 115.1, 114.3, 91.3, 88.1, 55.8; ESIMS (m/z): 386.22 [M+H]+. Anal. Calcd. for C23H16ClN3O: C, 71.60; H, 4.18; N, 10.89; Cl, 9.19%. Found: C, 71.35; H, 4.45; N, 10.52; Cl, 9.53%.
Synthesis of 4-N-(3-chlorophenyl)-6-(3-chlorophenylacetylene) quinazoline (6d) QN022
An amount of 58.5 mg, 75%; mp: 230–232 °C; Rf = 0.23 (EtOAc/Hexane 1:4); λmax 397 nm (ε 5142); 1HNMR (500 MHz, DMSO-d6): δ 10.03 (s, 1H), 8.88 (s, 1H), 8.71 (s, 1H), 8.16 (s, 1H), 8.00 (dd, 1H, J = 8.3, 1.7 Hz), 7.89 (dd, 1H, J = 8.3, 1.8 Hz), 7.85 (d, 1H, J = 8.3 Hz), 7.56–7.52 (m, 1H), 7.49–7.43 (m, 3H), 7.35 (t, 1H, J = 8.6 Hz), 7.21 (dd, 1H, J = 7.9, 1.5 Hz); 13C NMR (125 MHz, DMSO-d6): δ 164.4, 160.5, 157.6, 155.7, 150.1, 141.1, 135.9, 133.2, 131.5, 130.6, 129.0, 128.3, 127.1, 123.8, 121.9, 120.8, 120.1, 118.6, 117.1, 115.6, 90.3, 89.6; ESIMS (m/z): 390.25 [M+H]+. Anal. Calcd. for C22H13Cl2N3: C, 67.71; H, 3.36; N, 10.77; Cl, 18.17%. Found: C, 67.28; H, 3.72; N, 10.48; Cl, 18.59%.
Synthesis of 4-N-(4-fluorophenyl)-6-(4-pentylphenylacetylene) quinazoline (6e) QN012
An amount of 65.5 mg, 80%; mp (dec.): 218–220 °C; Rf = 0.25 (EtOAc/Hexane 1:4); λmax 397 nm (ε 5064); 1HNMR (500 MHz, DMSO-d6): δ 9.98 (s, 1H), 8.80 (s, 1H), 8.62 (s, 1H), 7.94–7.88 (m, 2H), 7.79 (d, 1H, J = 8.6 Hz), 7.58–7.49 (m, 3H), 7.28–7.26 (m, 2H), 2.61–2.58 (m, 2H), 1.59–1.54 (m, 2H), 1.30–1.20 (m, 4H), 0.84 (t, 3H, J = 8.6 Hz); 13C NMR (125 MHz, DMSO-d6): δ 157.5, 155.5, 149.8, 144.3, 144.1, 135.9, 133.2, 131.8, 130.6, 129.3, 128.9, 126.6, 123.8, 121.9, 120.8, 119.6, 115.7, 91.3, 88.8, 35.5, 31.3, 30.8, 22.4, 14.4; ESIMS (m/z): 410.28 [M+H]+. Anal. Calcd. for C27H24FN3: C, 79.19; H, 5.91; N, 10.26; F, 4.64%. Found: C, 79.58; H, 5.48; N, 10.62; F, 4.98%.

3.2. Biological Evaluation

3.2.1. Cell Culture and Assessment of Cytotoxic/Cytostatic Activity

The following human cancer cell lines—derived from tumors of various histopathologic origins—were used for cytotoxicity evaluation: A431 from epidermoid carcinoma, A549 from lung adenocarcinoma, MDA-MB-231 from mammary adenocarcinoma, and PC-3 from prostate adenocarcinoma (all obtained from the American Type Culture Collection; ATCC, Rockville, MD, USA). To evaluate the compounds’ specificity against tumor cells, a normal human skin fibroblast cell strain from an adult donor [51] was employed. Cells were cultured at 37 °C, in the presence of 5% CO2 with 85% humidity, using Dulbecco’s minimal essential medium (DMEM) supplemented with penicillin (100 U/mL), streptomycin (100 μg/mL), and 10% (v/v) fetal bovine serum (all media from Life Technologies Europe BV, Thessaloniki, Greece), and sub-cultured using a trypsin (0.25%; Life Technologies Europe BV)–citrate (0.30%; Sigma, St. Louis, MO, USA) solution. Assessment of cytotoxicity was performed based on the widely used MTT assay [52] modified as follows: cells were plated in 96-well flat-bottomed microplates at a density of approximately 5000 cells/well, incubated overnight to ensure cell attachment, and serial dilutions of the quinazoline derivatives were added. After incubation for 72 h, the medium was replaced with a 1 mg/mL solution of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma) in serum-free, phenol-red-free DMEM; the cultures were incubated for a further 4 h and the MTT formazan was solubilized in 2-propanol (Sigma). The optical density of each well was assessed using a Fluostar Optima microplate reader (BMG Labtech, Offenburg, Germany) at a wavelength of 550 nm (reference wavelength 690 nm). Serial dilutions of vehicle (DMSO; Sigma) and doxorubicin hydrochloride (Sigma) were included as negative and positive controls, respectively. The results are expressed as IC50, i.e., the concentration bringing the optical density of treated cells to 50% in comparison to negative controls.

3.2.2. Flow Cytometric Cell Cycle Analysis

A431 cells were plated into 6-well plates at a density of 5 × 105 cells/well. After 24 h, and while cells were exponentially growing, they were treated with the quinazoline derivatives at the IC50 concentrations calculated for each compound by the MTT assay. Following incubation for 24 h, cells were trypsinized, fixed with 70% ethanol, and stored overnight at 4 °C. Then, cells were washed with Phosphate-Buffered Saline (PBS) and suspended in a propidium iodide (PI) staining solution containing 50 µg/mL PI, 10 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 10 µg/mL RNAse A in PBS. After incubation at 4 °C for 30 min, fluorescence was analyzed on a FACSCalibur (Becton Dickinson, San Jose, CA, USA) flow cytometer and DNA content was estimated using the ModFit LTTM Version 2 software (Verity Software House, Topsham, ME, USA).

3.2.3. Immunoblot Analysis

A549 cells were plated into 6-well plates at a density of 5 × 105 cells/well, left to grow until confluency, and then culture medium was changed to serum-free. After serum starvation for 48 h, cells were pretreated with the quinazoline derivatives (100 μM), the corresponding vehicle concentration (negative control), or the tyrphostin AG1478 (3 μM; Calbiochem-Merck KGaA, Darmstadt, Germany) as the positive control. The quinazoline derivative concentration was chosen so as to cover the whole range of cytostatic activities (IC50s) determined for A549 cells. EGF (100 ng/mL; Sigma) was added 45 min later (a sample pretreated with vehicle but without EGF was used to assess basal EGFR phosphorylation levels) and after 30 min cells were washed with Tris-Buffered Saline (TBS; 10 mM Tris-HCl pH 7.4, 150 mM NaCl) and lysed in a hot 2× SDS-PAGE sample buffer [125 mM Tris-HCl pH 6.8, 5% (w/v) SDS, 20% (v/v) glycerol, 125 mM β-mercaptoethanol, and 0.02% (w/v) bromophenol blue] supplemented with protease and phosphatase inhibitor cocktails (Sigma). Cell lysates underwent brief sonication, were clarified by centrifugation, and were subjected to SDS-PAGE in 7% (w/v) Bis-Tris polyacrylamide gels followed by protein transfer to PVDF membranes (Perkin Elmer-Thermo Fisher Scientific, Waltham, MA, USA). After blocking in 5% (w/v) non-fat milk in 0.05% Tween-20-supplemented TBS (TBS-T) for 60 min, membranes were incubated overnight either with anti-phospho-EGFR (Tyr1068) rabbit monoclonal antibody (D7A5; Cell Signaling Technology, Hertfordshire, UK) or with anti-EGFR rabbit polyclonal antibody (Cell Signaling Technology). After washes (thrice) in 5% non-fat milk in TBS-T, membranes were incubated with anti-rabbit horseradish peroxidase-conjugated antibody (Sigma) for 2 h, washed thrice, covered with Luminata Crescendo Western HRP substrate (Sigma), and immunoreactive bands were captured on a LAS-4000 luminescent image analyzer (Fujifilm Manufacturing USA Inc., Greenwood, SC, USA).

3.3. Computational Methods

The crystal structures of the epidermal growth factor receptor (EGFR) kinase domain were retrieved from the Protein Data Bank. The active conformation was obtained from PDB ID: 1M17 [32], while, for the inactive conformation, we used PDB ID: 3POZ [33]. Protein structures were prepared prior to docking by removing crystallographic water molecules, ions, and co-crystallized ligands. Polar hydrogen atoms were added, and Gasteiger partial charges were assigned using AutoDockTools v1.5.7 [53]. Grid maps for molecular docking were generated using AutoGrid4, by centering the grid box on the bound ligand in the ATP-binding site. The grid box dimensions were set to 60 × 60 × 60 grid points along the x, y, and z axes, respectively, with a uniform grid spacing of 0.375 Å, corresponding to a total search volume of 22.5 × 22.5 × 22.5 Å3. This grid size was selected to fully cover the ATP-binding pocket and adjacent subpockets, while allowing unrestricted rotational and translational freedom for all the ligands under study, including the bulkier and extended quinazoline derivatives. A total of 20 ligands were investigated, comprising 18 quinazoline derivatives and the two reference inhibitors, Erlotinib and TAK-285. The initial ligand geometries were generated using OpenEye’s OMEGA module [54], and then input files for docking were prepared using the standard protocol of AutoDockTools. All ligands were treated as fully flexible during docking, adjusting the number of rotatable bonds for each ligand.
Molecular docking was carried out using AutoDock v4.2.3 [53], employing the Lamarckian Genetic Algorithm (LGA). For each ligand, 100 independent docking runs were performed to ensure adequate conformational sampling. An initial population size of 150 individuals was used for each docking run; the maximum number of energy evaluations was set to 1.0 × 107, with a corresponding maximum of 27,000 generations. All other parameters were retained at their default values. Docked conformations were clustered based on a root-mean-square deviation (RMSD) tolerance of 2.0 Å, which is the default value and is appropriate to provide discrete clusters of bound poses for these compounds. For each ligand, the conformation that displayed a hydrogen bond between the quinazoline ring and the amide NH of Met793 was retained as the representative binding pose. Binding modes were visually inspected and figures were generated using the open-source PyMOL v2.5.

4. Conclusions

In summary, the 4-anilinoquinazoline core has proven to be an exceptionally robust and successful scaffold, serving as the foundation for three generations of approved EGFR inhibitors, including Gefitinib, Afatinib, and Osimertinib. The present study explored the integration of 6-alkynyl modifications into this established pharmacophore to develop novel derivatives (QN009–QN027) capable of targeting the EGFR kinase domain in both its active and inactive states. While our computational assessments identified compounds QN012, QN017, QN019, QN022, and QN023 as promising candidates, they exhibited medium-range IC50s, being substantially weaker than the reference compounds. We acknowledge that further structural optimization is required to bridge the potency gap and reach or overcome the inhibition levels exhibited by already known inhibitors. Specific modifications are currently in the preliminary stages of exploration based on our current findings, which serve as a significant starting point for the future design of more potent derivatives.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms27041738/s1.

Author Contributions

Conceptualization, H.P. and S.M.; synthetic methodology, A.G. and S.M.; computational and biological methodology, S.-I.T., L.C., A.P. and H.P.; validation, S.-I.T., L.C., A.P., H.P., A.G. and S.M.; investigation, S.-I.T., L.C., A.P., H.P., A.K., A.G. and S.M.; resources, D.K., A.P., A.K., C.T., H.P. and S.M.; data curation, A.P.; writing—original draft preparation, A.P., H.P. and S.M.; writing—review and editing, A.P., H.P. and S.M.; visualization, A.P., H.P. and S.M.; supervision, H.P. and S.M.; project administration, H.P. and S.M.; funding acquisition, D.K., A.P., A.K., C.T., H.P. and S.M. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data presented in this study are contained within the article or the Supplementary Material.

Acknowledgments

The authors acknowledge Angeliki Panagiotopoulou for her technical assistance and Maria Paravatou-Petsotas for helpful discussions during the whole project.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
DMEMDulbecco’s minimal essential medium
DMSOdimethyl sulfoxide
EGFepidermal growth factor
EGFRepidermal growth factor receptor
NSCLCnon-small-cell lung cancer
DMFN,N-Dimethylformamide
i-PrOHisopropanol
Pd(PPh3)4tetrakis(triphenylphosphine) palladium (0)
SOCl2thionyl chloride
EtOAcethyl acetate
CuIcopper(I) Iodide
CH3COOHacetic acid
MTT3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
PBSphosphate-buffered saline
TBStris-buffered saline

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Ijms 27 01738 sch001
Scheme 1. (i) KI, H2O2, CH3COOH, (ii) HCONH2, 130–140 °C, (iii) SOCl2, DMF, reflux temperature, (iv) 3-chloro or 4-fluoro anilines, i-PrOH, (v) Pd(PPh3)4, CuI, Et3N, terminal alkynes, DMF, reflux temperature.
Scheme 1. (i) KI, H2O2, CH3COOH, (ii) HCONH2, 130–140 °C, (iii) SOCl2, DMF, reflux temperature, (iv) 3-chloro or 4-fluoro anilines, i-PrOH, (v) Pd(PPh3)4, CuI, Et3N, terminal alkynes, DMF, reflux temperature.
Ijms 27 01738 sch001
Ijms 27 01738 g002
Figure 2. (A) X-ray crystal structure of EGFR kinase domain in the active state with bound Erlotinib, superimposed with its docked pose (white and yellow carbon sticks, respectively, with all N atoms in blue and O atoms in red). The conserved hydrogen-bonding interaction with Met793 (M769 in PDB ID 1M17) is indicated with dashed lines and the distance between heavy atoms in Å. (B–F) Bound poses of the selected quinazoline derivatives QN012 (B), QN017 (C), QN019 (D), QN022 (E) and QN023 (F) are shown in the same orientation as in panel (A).
Figure 2. (A) X-ray crystal structure of EGFR kinase domain in the active state with bound Erlotinib, superimposed with its docked pose (white and yellow carbon sticks, respectively, with all N atoms in blue and O atoms in red). The conserved hydrogen-bonding interaction with Met793 (M769 in PDB ID 1M17) is indicated with dashed lines and the distance between heavy atoms in Å. (B–F) Bound poses of the selected quinazoline derivatives QN012 (B), QN017 (C), QN019 (D), QN022 (E) and QN023 (F) are shown in the same orientation as in panel (A).
Ijms 27 01738 g002
Ijms 27 01738 g003
Figure 3. (A) X-ray crystal structure of EGFR kinase domain in the inactive state with bound TAK-285, superimposed with its docked pose (green and yellow carbon sticks, respectively, with all N atoms in blue and O atoms in red). The conserved hydrogen-bonding interaction with Met793 (orange C atoms) is indicated with dashed lines and the distance between heavy atoms in Å. (B–F) Bound poses of the selected quinazoline derivatives QN012 (B), QN017 (C), QN019 (D), QN022 (E) and QN023 (F) are shown in the same orientation as in panel (A).
Figure 3. (A) X-ray crystal structure of EGFR kinase domain in the inactive state with bound TAK-285, superimposed with its docked pose (green and yellow carbon sticks, respectively, with all N atoms in blue and O atoms in red). The conserved hydrogen-bonding interaction with Met793 (orange C atoms) is indicated with dashed lines and the distance between heavy atoms in Å. (B–F) Bound poses of the selected quinazoline derivatives QN012 (B), QN017 (C), QN019 (D), QN022 (E) and QN023 (F) are shown in the same orientation as in panel (A).
Ijms 27 01738 g003
Ijms 27 01738 g004
Figure 4. Western analysis of EGFR phosphorylation following pretreatment with the quinazoline derivatives and activation with EGF. A549 cell cultures, growth-arrested through serum deprivation, were pretreated with the five quinazoline derivatives (100 μM) or the corresponding concentration of vehicle (DMSO) as negative control, or AG1478 (3 μM) as positive control. Following EGFR activation with EGF (100 ng/mL) and incubation for 30 min, lysates were collected and subjected to Western analysis, as described in the Materials and Methods section. One representative experiment is shown. Densitometric analysis seen in the right part of the figure was performed following normalization of phosphorylated to total EGFR levels, setting the value for EGF/vehicle-treated samples as 100%.
Figure 4. Western analysis of EGFR phosphorylation following pretreatment with the quinazoline derivatives and activation with EGF. A549 cell cultures, growth-arrested through serum deprivation, were pretreated with the five quinazoline derivatives (100 μM) or the corresponding concentration of vehicle (DMSO) as negative control, or AG1478 (3 μM) as positive control. Following EGFR activation with EGF (100 ng/mL) and incubation for 30 min, lysates were collected and subjected to Western analysis, as described in the Materials and Methods section. One representative experiment is shown. Densitometric analysis seen in the right part of the figure was performed following normalization of phosphorylated to total EGFR levels, setting the value for EGF/vehicle-treated samples as 100%.
Ijms 27 01738 g004
Table 2. Cytotoxic/cytostatic activity of synthesized compounds.
Table 2. Cytotoxic/cytostatic activity of synthesized compounds.
CompoundIC50 (μM) 1
A431A549MDA-MB-231PC-3Normal
Fibroblasts
6a (QN017)40.0 (±14.7)24.0 (±5.6)83.4 (±19.4)49.6 (±11.5)40.1 (±9.3)
6b (QN019)63.2 (±3.9)42.5 (±10.3)114.9 (±28.0)87.1 (±21.2)121.5 (±29.6)
6c (QN023)27.2 (±3.5)1.5 (±0.5)63.3 (±14.7)33.8 (±7.9)32.8 (±7.6)
6d (QN022)26.4 (±6.4)12.7 (±1.7)61.5 (±15.0)172.9 (±42.1)51.0 (±12.4)
6e (QN012)32.7 (±11.8)50.5 (±18.6)56.6 (±20.8)13.1 (±4.8)27.0 (±9.9)
Doxorubicin hydrochloride0.013 (±0.005)0.132 (±0.011)0.103 (±0.045)0.345 (±0.133)0.258 (±0.101)
1 Experiments were performed in triplicate and the mean values (±standard deviation) are shown.
Table 3. Cell cycle phase distribution following treatment with the synthesized compounds.
Table 3. Cell cycle phase distribution following treatment with the synthesized compounds.
CompoundSub-Diploid (%)G0/G1 (%)S (%)G2/M (%)
6a (QN017)0.01 (±0.01) 175.69 (±0.79)19.29 (±0.68)5.02 (±0.35)
6b (QN019)0.03 (±0.02)90.72 (±0.86)8.28 (±0.10)1.00 (±0.82)
6c (QN023)0.00 (±0.00)81.02 (±0.63)13.86 (±0.28)5.12 (±0.39)
6d (QN022)0.05 (±0.08)79.44 (±0.13)15.31 (±0.48)5.25 (±0.43)
6e (QN012)0.05 (±0.04)65.54 (±0.54)25.00 (±0.79)9.47 (±0.31)
Control0.11 (±0.14)56.84 (±0.45)32.01 (±0.22)11.16 (±0.25)
1 Experiments were performed in triplicate and the mean values (±standard deviation) are shown.
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Gkoutzivelaki, A.; Triantopoulou, S.-I.; Chiniadis, L.; Komiotis, A.; Triantis, C.; Komiotis, D.; Papakyriakou, A.; Pratsinis, H.; Manta, S. Design, Synthesis, Antiproliferative Potency and In Silico Studies of Novel Alkynyl Quinazolines as Potential EGFR Inhibitors. Int. J. Mol. Sci. 2026, 27, 1738. https://doi.org/10.3390/ijms27041738

AMA Style

Gkoutzivelaki A, Triantopoulou S-I, Chiniadis L, Komiotis A, Triantis C, Komiotis D, Papakyriakou A, Pratsinis H, Manta S. Design, Synthesis, Antiproliferative Potency and In Silico Studies of Novel Alkynyl Quinazolines as Potential EGFR Inhibitors. International Journal of Molecular Sciences. 2026; 27(4):1738. https://doi.org/10.3390/ijms27041738

Chicago/Turabian Style

Gkoutzivelaki, Apostolia, Sotiria-Iro Triantopoulou, Lykourgos Chiniadis, Alexandros Komiotis, Charalampos Triantis, Dimitri Komiotis, Athanasios Papakyriakou, Harris Pratsinis, and Stella Manta. 2026. "Design, Synthesis, Antiproliferative Potency and In Silico Studies of Novel Alkynyl Quinazolines as Potential EGFR Inhibitors" International Journal of Molecular Sciences 27, no. 4: 1738. https://doi.org/10.3390/ijms27041738

APA Style

Gkoutzivelaki, A., Triantopoulou, S.-I., Chiniadis, L., Komiotis, A., Triantis, C., Komiotis, D., Papakyriakou, A., Pratsinis, H., & Manta, S. (2026). Design, Synthesis, Antiproliferative Potency and In Silico Studies of Novel Alkynyl Quinazolines as Potential EGFR Inhibitors. International Journal of Molecular Sciences, 27(4), 1738. https://doi.org/10.3390/ijms27041738

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