Design, Synthesis and Biological Evaluation of Syn and Anti-like Double Warhead Quinolinones Bearing Dihydroxy Naphthalene Moiety as Epidermal Growth Factor Receptor Inhibitors with Potential Apoptotic Antiproliferative Action

Our investigation includes the synthesis of new naphthalene-bis-triazole-bis-quinolin-2(1H)-ones 4a–e and 7a–e via Cu-catalyzed [3 + 2] cycloadditions of 4-azidoquinolin-2(1H)-ones 3a–e with 1,5-/or 1,8-bis(prop-2-yn-1-yloxy)naphthalene (2) or (6). All structures of the obtained products have been confirmed with different spectroscopic analyses. Additionally, a mild and versatile method based on copper-catalyzed [3 + 2] cycloaddition (Meldal–Sharpless reaction) was developed to tether quinolinones to O-atoms of 1,5- or 1,8-dinaphthols. The triazolo linkers could be considered as anti and syn products, which are interesting precursors for functionalized epidermal growth factor receptor (EGFR) inhibitors with potential apoptotic antiproliferative action. The antiproliferative activities of the 4a–e and 7a–e were evaluated. Compounds 4a–e and 7a–e demonstrated strong antiproliferative activity against the four tested cancer cell lines, with mean GI50 ranging from 34 nM to 134 nM compared to the reference erlotinib, which had a GI50 of 33 nM. The most potent derivatives as antiproliferative agents, compounds 4a, 4b, and 7d, were investigated for their efficacy as EGFR inhibitors, with IC50 values ranging from 64 nM to 97 nM. Compounds 4a, 4b, and 7d demonstrated potent apoptotic effects via their effects on caspases 3, 8, 9, Cytochrome C, Bax, and Bcl2. Finally, docking studies show the relevance of the free amino group of the quinoline moiety for antiproliferative action via hydrogen bond formation with essential amino acids.


Introduction
Over the past few decades, quinolones have transformed from a small and insignificant class of drugs primarily utilized for treating mild urinary tract infections to some of the most prescribed antibacterials globally [1][2][3][4]. An important different activity for quinolones has been investigated despite being well known as antibacterial. In the late

Introduction
Over the past few decades, quinolones have transformed from a small and insignificant class of drugs primarily utilized for treating mild urinary tract infections to some of the most prescribed antibacterials globally [1][2][3][4]. An important different activity for quinolones has been investigated despite being well known as antibacterial. In the late 1980s, quinolone derivatives held significant potency against eukaryotic Type II topoisomerases (topoisomerase II) and demonstrated cytotoxic activity against cancer cell lines. Hence, quinolinone derivatives are promising candidates for cancer treatment [5][6][7]. Several quinolone derivatives exemplified by voreloxin, AT-3639, and quarfloxin have already been used in clinics or in clinical trials [8,9].
In addition, quinolinone is an intriguing fused heterocyclic scaffold that is found in several FDA-approved and commercialized anticancer medications [10], including Neratinib (Nerlynx ® ), an EGFR-TK inhibitor [11] (Figure 1). Moreover, several quinolinone scaffold-containing compounds are potent EGFR inhibitors. Compound I has an EGFR IC50 of 0.0075 µ M [12], compound II has an EGFR IC50 of 5 nM [13], and compound III has an EGFR IC50 of 5.2 µ M [14,15] (Figure 1). Currently, 1,2,3-triazoles exhibit a diverse set of biological actions [16][17][18][19]. They possess various pharmacological and biological features, including anticancer action [20]. Additionally, the importance and applications of 1,2,3-triazole compounds have increased [21,22], after development of the reactions of organic azides with terminal alkynes under mild conditions catalyzed by Cu(I). Further, the regioselective formation of 1,2,3-triazoles using Cu-catalyzed [3 + 2] cycloaddition has proved to be the best example of click chemistry with extensive applications in organic and medicinal chemistry [23]. However, several investigations have shown that their biological features are attributable to the triazole moiety's making of hydrogen bonds, dipole-dipole, and stacking interactions, which warrant the development of stable complexes and, as a result, activate a cascade of metabolic activations such as apoptosis [24,25]. As a result, the antiproliferative activity of 1,2,3-triazole derivatives is explained by different mechanisms of action. Perihan and coworkers, for example, used a multi-target design technique to develop various 1,2,3-triazoles, one of which, compound IV (Figure 2), has been demonstrated to arrest the G2/M cell cycle and induce apoptosis in human cancer cells [26]. In addition, Khan and colleagues [27]  Currently, 1,2,3-triazoles exhibit a diverse set of biological actions [16][17][18][19]. They possess various pharmacological and biological features, including anticancer action [20]. Additionally, the importance and applications of 1,2,3-triazole compounds have increased [21,22], after development of the reactions of organic azides with terminal alkynes under mild conditions catalyzed by Cu(I). Further, the regioselective formation of 1,2,3-triazoles using Cu-catalyzed [3 + 2] cycloaddition has proved to be the best example of click chemistry with extensive applications in organic and medicinal chemistry [23]. However, several investigations have shown that their biological features are attributable to the triazole moiety's making of hydrogen bonds, dipole-dipole, and stacking interactions, which warrant the development of stable complexes and, as a result, activate a cascade of metabolic activations such as apoptosis [24,25]. As a result, the antiproliferative activity of 1,2,3-triazole derivatives is explained by different mechanisms of action. Perihan and coworkers, for example, used a multi-target design technique to develop various 1,2,3-triazoles, one of which, compound IV (Figure 2), has been demonstrated to arrest the G2/M cell cycle and induce apoptosis in human cancer cells [26]. In addition, Khan and colleagues [27] described novel diphenyl-1H-pyrazole-based acrylates linked to 1,2,3-triazole V (Figure 2) as prospective apoptosis-inducing cytotoxic agents. described novel diphenyl-1H-pyrazole-based acrylates linked to 1,2,3-triazole V (Figu 2) as prospective apoptosis-inducing cytotoxic agents. We recently reported on compound VI's design, synthesis, and antiproliferative a tivity ( Figure 2) [28]. Compound VI displayed a substantial antiproliferative activi against four cancer cell lines, with a mean GI50 = 0.23 µ M. Compound VI inhibited EGF activity with an IC50 of 0.11 µ M. The apoptotic mechanism demonstrated that compoun VI increased Caspase-3, Caspase-9, and Cytochrome-C levels in human (Panc-1) canc cells by 7.80, 19.30, and 13 times, respectively, compared to doxorubicin. Furthermore, V elevated Bax levels to 40-fold greater than normal untreated cells while decreasing an apoptotic Bcl-2 levels 6.3-fold.
Hybridization has emerged as a promising strategy in developing new drugs wi the potential to overcome cross-resistance and improve affinity and efficacy compare with the parent drugs [29,30]. Combining quinolinone with other anticancer pharmac phores may provide new candidates with great potency against drug-sensitive and dru resistant cancers.
Motivated by the facts presented here, we present the synthesis of a small set of qui oline-1,2,3-triazole hybrids that will be evaluated for antiproliferative activity. The new synthesized compounds consist of two scaffolds: Scaffold A (4a-e), which represents Sy like-quinoline derivatives, and Scaffold B, which represents Anti-like-quinoline deriv tives 7a-e, Figure 3. The two ligands of triazole and quinolinone moieties attached to th naphthalene core of Syn and Anti conformation-like represented diversity in the field biology. We recently reported on compound VI's design, synthesis, and antiproliferative activity ( Figure 2) [28]. Compound VI displayed a substantial antiproliferative activity against four cancer cell lines, with a mean GI 50 = 0.23 µM. Compound VI inhibited EGFR activity with an IC 50 of 0.11 µM. The apoptotic mechanism demonstrated that compound VI increased Caspase-3, Caspase-9, and Cytochrome-C levels in human (Panc-1) cancer cells by 7.80, 19.30, and 13 times, respectively, compared to doxorubicin. Furthermore, VI elevated Bax levels to 40-fold greater than normal untreated cells while decreasing anti-apoptotic Bcl-2 levels 6.3-fold.
Hybridization has emerged as a promising strategy in developing new drugs with the potential to overcome cross-resistance and improve affinity and efficacy compared with the parent drugs [29,30]. Combining quinolinone with other anticancer pharmacophores may provide new candidates with great potency against drug-sensitive and drugresistant cancers.
Motivated by the facts presented here, we present the synthesis of a small set of quinoline-1,2,3-triazole hybrids that will be evaluated for antiproliferative activity. The newly synthesized compounds consist of two scaffolds: Scaffold A (4a-e), which represents Syn-like-quinoline derivatives, and Scaffold B, which represents Anti-like-quinoline derivatives 7a-e, Figure 3. The two ligands of triazole and quinolinone moieties attached to the naphthalene core of Syn and Anti conformation-like represented diversity in the field of biology.
The antiproliferative activity of compounds 4a-e and 7a-e will be investigated using four cancer cell lines: A549 (epithelial cancer cell line), MCF-7 (breast cancer cell line), Panc-1 (pancreas cancer cell line), and HT-29 (colon cancer cell line). The most effective antiproliferative agents will be examined further for their potential inhibitory activity against EGFR as a target for their mechanistic action. Furthermore, the most potent derivatives will be tested for their ability to trigger apoptosis against caspases 3, 8, and 9, cytochrome c, Bax, and the anti-apoptotic Bcl2.  The antiproliferative activity of compounds 4a-e and 7a-e will be investigated using four cancer cell lines: A549 (epithelial cancer cell line), MCF-7 (breast cancer cell line), Panc-1 (pancreas cancer cell line), and HT-29 (colon cancer cell line). The most effective antiproliferative agents will be examined further for their potential inhibitory activity against EGFR as a target for their mechanistic action. Furthermore, the most potent derivatives will be tested for their ability to trigger apoptosis against caspases 3, 8, and 9, cytochrome c, Bax, and the anti-apoptotic Bcl2.
On the other hand, the same synthetic methodology was used to produce a new series of bis-quinolinone-1,2,3-triazole hybrids as more analogs for biological testing. The cycloaddition reaction of 4-azido-2-quinolinones 3a-e with highly symmetrical di-o-propargylated compound 6, obtained by propargylation of 1,5-dinaphthol (5), was promoted under an above similar condition, resulting in 76-84% yield of the corresponding symmetrical bisquinolinone triazole-based anti-like targets 7a-e (Scheme 2). The core architecture of these bis-quinolinone-triazoles and their diversity tempted us to study the anti-cancer properties of such compounds.
To confirm our results, we choose compound 7e which was assigned as [4,4 -(((naphthalene-1,5-diylbis(oxy))bis(methylene))bis(1H-1,2,3-triazole-4,1-diyl))-bis(1methylquinolin-2(1H)-one)]. Elemental analysis and all spectral data were acceptable with this proposed structure with chemical formula C 36 H 28 N 8 O 4 (m/z = 636). Through the interpretation of the various analyses that were conducted for compound 7e and all the previous compounds, it is undoubtedly clear that the proposed chemical composition is correct, and also, to prove the composition in another way, it was compared with spectral data for its analogous compound 4e (Table 1). This is claimed to be the reaction product of 1,8-bis(propargyloxy)naphthalene (2) with 4-azido-1-methyl-1H-quinolin-2one (3e). Additionally, the spectra are consistent with structure 7e but are very similar to those observed for sample 4e, for which 4e was assigned. The spectroscopic difference between these products is that, in 4e, C-4a' and C-8a' are nonequivalent, while in 7e, these two carbons are equivalent. However, in both compounds 4e and 2, C-8a' was assigned as co-resonant with another carbon which could not be observed separately. Therefore, we suspect that the correct structure for both products is 4e; see Table 1. In addition, all spectroscopic analyses of all quinolin-2-one rings are completely identical to what was previously published [37][38][39]. On the other hand, the novel synthesizing of naphthalene-bis-triazole-bis-quinolin-2(1H)-ones 7a-e was also confirmed on the bases of different spectral data. For example, we choose compound 7e which was assigned as [4,4′-(((naphthalene-1,5diylbis(oxy))bis(methylene))bis-(1H-1,2,3-triazole-4,1-diyl))bis(1-methylquinolin-2(1H)one)] ( Figure 6). To confirm our results, we choose compound 7e which was assigned as [4,4′-(((naphthalene-1,5-diylbis(oxy))bis(methylene))bis(1H-1,2,3-triazole-4,1-diyl))-bis(1-methylquinolin-2(1H)-one)]. Elemental analysis and all spectral data were acceptable with this proposed structure with chemical formula C36H28N8O4 (m/z = 636). Through the interpretation of the various analyses that were conducted for compound 7e and all the previous compounds, it is undoubtedly clear that the proposed chemical composition is correct, and also, to prove the composition in another way, it was compared with spectral data for its analogous compound 4e (Table 1). This is claimed to be the reaction product of 1,8-bis(propargyloxy)naphthalene (2) with 4-azido-1-methyl-1H-quinolin-2-one (3e). Additionally, the spectra are consistent with structure 7e but are very similar to those observed for sample 4e, for which 4e was assigned. The spectroscopic difference between these products is that, in 4e, C-4a' and C-8a' are nonequivalent, while in 7e, these two carbons are equivalent. However, in both compounds 4e and 2, C-8a' was assigned as co-resonant with another carbon which could not be observed separately. Therefore, we suspect that the   The mechanism for the obtained products 4a-e and 7a-e can be rationalized as, upon mixing (1 mmol) of terminal alkynes 2 and/or 6 with Cu(1), the salt of compound 8 would form. On the addition of (2 mmol) of azides 3a-e dissolved in DMF, a nucleophilic addition takes place to give the adduct 9, which undergoes nucleophilic attack to the N-negative charged on triple bond to give the adduct 10, which then further accepts H + and forms the intermediate 11, which then reacts with Cu(1) to give the final products 4a-e and 7a-e via further repeating the above steps as shown in Scheme 3 [23,40]. Scheme 3. Suggested mechanism for the formation of products 4a-e and 7a-e.

Antiproliferative Action
Cell Viability Assay A cell viability test was performed using MCF-10A (human mammary gland epithelial) cell line [41][42][43] to investigate the effect of 4a-e and 7a-e on normal cell lines. In this investigation, a concentration of 50 µ M of the studied compound is employed for four days, after which cell viability is assessed. The results showed that compounds 4a-e and 7a-e have no toxic effect and have more than 86% cell viability, as shown in Table 2.

Antiproliferative Assay
The newly synthesized compounds were tested for antiproliferative activity against four different types of cancer cells [44,45]: A-549 (epithelial cancer cell line), MCF-7 (breast cancer cell line), Panc-1 (pancreas cancer cell line), and HT-29 (colon cancer cell line). Erlotinib was used as the reference, and Table 2 displays the results of calculating the IC50 of each compound.
Generally, the newly evaluated compounds 4a-e and 7a-e displayed significant antiproliferative activity, with mean GI50 ranging from 34 nM to 134 nM compared to the reference erlotinib with a GI50 of 33 nM against the four tested cancer cell lines. Three compounds with the highest antiproliferative activity were identified: 4a and 4b with a Syn quinoline backbone structure (Scaffold A), and 7d with an Anti-quinoline backbone structure (Scaffold B), with GI50 values ranging from 34 nM to 54 nM.

Antiproliferative Action Cell Viability Assay
A cell viability test was performed using MCF-10A (human mammary gland epithelial) cell line [41][42][43] to investigate the effect of 4a-e and 7a-e on normal cell lines. In this investigation, a concentration of 50 µM of the studied compound is employed for four days, after which cell viability is assessed. The results showed that compounds 4a-e and 7a-e have no toxic effect and have more than 86% cell viability, as shown in Table 2. backbone), which have GI50 of 130 and 134 nM and are approximately 4-fold less potent than 4a, were greatly diminished when the free NH group in compound 4a was replaced with a methyl group. This finding highlights the significance of the free amino group at position-1 of the quinoline moiety for the antiproliferative action. Compound 4b (R1 = R2 = H, R3 = CH3) ranks second in efficacy against the cancer cell lines tested, with a GI50 of 45 nM, which is 1.4-fold less potent than erlotinib (GI50 = 33 nM). Compound 7b (R1 = R2 = H, R3 = CH3) shares the same substitution pattern as compound 4b, but was found to be 1.4 times less potent due to its anti-quinoline backbone structure. This finding emphasizes the impact of stereochemistry in the action of this class of organic compounds, with the syn derivatives being more active than the anti-derivatives. The same pattern becomes apparent when 4a (GI50 = 34 nM) and 7a (GI50 = 130 nM) are compared.
The nature and position of the substitution on the quinoline moiety were also studied. The GI50 of the 6-methyl quinoline derivative 4b (R1 = R2 = H, R3 = CH3) was found to be 1.8-fold more potent than that of the 6-methoxy derivative 4c (R1 = R2 = H, R3 = OCH3), indicating that the methyl group was better tolerated than the methoxy group. Finally, 8methyl quinoline derivative 4d (R1 = R3 = H, R2 = CH3) was found to be at least twofold less potent than that of the 6-methyl derivative 4b (R1 = R2 = H, R3 = CH3), demonstrating that the 6-position on the quinoline moiety was more tolerated than the 8-position.

Antiproliferative Assay
The newly synthesized compounds were tested for antiproliferative activity against four different types of cancer cells [44,45]: A-549 (epithelial cancer cell line), MCF-7 (breast cancer cell line), Panc-1 (pancreas cancer cell line), and HT-29 (colon cancer cell line). Erlotinib was used as the reference, and Table 2 displays the results of calculating the IC 50 of each compound.
Generally, the newly evaluated compounds 4a-e and 7a-e displayed significant antiproliferative activity, with mean GI 50 ranging from 34 nM to 134 nM compared to the reference erlotinib with a GI 50 of 33 nM against the four tested cancer cell lines. Three compounds with the highest antiproliferative activity were identified: 4a and 4b with a Syn quinoline backbone structure (Scaffold A), and 7d with an Anti-quinoline backbone structure (Scaffold B), with GI 50 values ranging from 34 nM to 54 nM.
Compound 4a (R 1 = R 2 = R 3 = H) was the most potent synthetic derivative, with a mean GI 50 of 34 nM compared to the reference erlotinib's GI 50 of 33 nM. Compound 4a inhibited the MCF-7 (breast cancer) cell line more effectively than erlotinib, with an IC 50 of 33 nM versus 40 nM. The antiproliferative effects of compounds 4e (R 1 = CH 3 , R 2 = R 3 = H, Syn quinoline backbone) and compound 7e (R 1 = CH 3 , R 2 = R 3 = H, Anti quinoline backbone), which have GI 50 of 130 and 134 nM and are approximately 4-fold less potent than 4a, were greatly diminished when the free NH group in compound 4a was replaced with a methyl group. This finding highlights the significance of the free amino group at position-1 of the quinoline moiety for the antiproliferative action.
Compound 4b (R 1 = R 2 = H, R 3 = CH 3 ) ranks second in efficacy against the cancer cell lines tested, with a GI 50 of 45 nM, which is 1.4-fold less potent than erlotinib (GI 50 = 33 nM). Compound 7b (R 1 = R 2 = H, R 3 = CH 3 ) shares the same substitution pattern as compound 4b, but was found to be 1.4 times less potent due to its anti-quinoline backbone structure. This finding emphasizes the impact of stereochemistry in the action of this class of organic compounds, with the syn derivatives being more active than the anti-derivatives. The same pattern becomes apparent when 4a (GI 50 = 34 nM) and 7a (GI 50 = 130 nM) are compared.
The nature and position of the substitution on the quinoline moiety were also studied. The GI 50 of the 6-methyl quinoline derivative 4b (R 1 = R 2 = H, R 3 = CH 3 ) was found to be 1.8-fold more potent than that of the 6-methoxy derivative 4c (R 1 = R 2 = H, R 3 = OCH 3 ), indicating that the methyl group was better tolerated than the methoxy group. Finally, 8-methyl quinoline derivative 4d (R 1 = R 3 = H, R 2 = CH 3 ) was found to be at least twofold less potent than that of the 6-methyl derivative 4b (R 1 = R 2 = H, R 3 = CH 3 ), demonstrating that the 6-position on the quinoline moiety was more tolerated than the 8-position.

EGFR Inhibitory Assay
Compounds 4a, 4b, and 7d, the most potent derivatives as antiproliferative agents, were tested for their efficiency as EGFR inhibitors [46,47] to understand how these substances affected the EGFR enzyme. According to Table 3, compounds 4a, 4b, and 7d significantly inhibited the activity of the EGFR enzyme, with IC 50 values ranging from 64 nM to 97 nM. Compound 4a, the most effective antiproliferative of all synthetic derivatives, showed higher potency than the standard drug erlotinib, with an IC 50 of 64 nM as opposed to erlotinib's IC 50 of 70 nM. Compounds 4b and 7d significantly inhibited EGFR, with IC 50 values of 93 and 97 nM, respectively, which were roughly 1.3-fold less effective than erlotinib. The outcomes of this assay supplemented cancer cell-based assay results, suggesting that EGFR-TK may be a viable target for these drugs' antiproliferative effects. The effects of derivatives 4a, 4b, and 7d on caspase-3 were studied using human epithelial cancer cell line (A-549) and compared to the reference drug doxorubicin [39,48]. The results showed that 4a, 4b, and 7d increased the level of active caspase-3 by 7-9 times when compared to control untreated cells, and that 4a, 4b, and 7d had remarkable overexpression of caspase-3 protein level (587.50 ± 4.50, 535.50 ± 4.50, and 485.50 ± 4.25 pg/mL, respectively) when compared to the reference doxorubicin (503.2 ± 4.50 pg/mL), as shown in Table 4. Compared with control untreated cells, the most active derivatives 4a and 4b increased the level of active caspase-3 by 9 and 8 times, respectively, and activated caspase-3 higher than doxorubicin, Table 4. The impact of compounds 4a and 4b on caspase-8 and caspase-9 was also assessed to clarify how compounds 4a and 4b induce apoptosis by activating the intrinsic or extrinsic route. The results showed that compound 4a increased caspase-8 and 9 levels by 6 and 19 times, respectively, while compound 4b showed a 4-and 18-fold increase in levels, respectively, compared to control cells. This indicates that both the intrinsic and extrinsic pathways were activated, with an effect that was more noticeable on the intrinsic pathway, because caspase-9 levels were higher, as shown in Table 4.
Effect of Compounds 4a, 4b, and 7d on Cytochrome C Level The concentration of Cytochrome c in a cell is crucial for activating caspases and starting the intrinsic apoptosis process [49]. The evaluation of hybrids 4a and 4b as inducers of Cytochrome c is summarized in Table 4. In the A-549 epithelial cancer cell line, hybrids 4a and 4b result in a 16-and 14-fold overexpression of Cytochrome c compared to the control. Accordingly, the results presented above show that Cytochrome c overexpression and the activation of the intrinsic apoptotic pathway by the investigated hybrids may be responsible for apoptosis.
Effect of Compounds 4a, 4b, and 7d on BaX and Bcl2 Levels The most effective hybrids 4a and 4b were further investigated for their impact on Bax and Bacl-2 levels against the A-549 epithelial cancer cell line, as shown in Table 5 [50]. The findings demonstrated that, compared to doxorubicin, 4a and 4b evoked a notable increase in Bax level. Compound 4a induction of Bax (298 pg/mL) was comparable to doxorubicin (276 pg/mL), 36 times higher than control untreated A-549 cancer cells, followed by compound 4b (284 pg/mL and 34-fold change). Finally, compound 4a (1.05 ng/mL), compound 4b (1.17 ng/mL), and doxorubicin (1.98 ng/mL) all reduced the level of the anti-apoptotic Bcl-2 protein in the A-549 cell line.

Molecular Docking Simulations
The EGFR is a recognized receptor that binds to the EGF outside of the cell membrane and is activated, leading to the receptor's phosphorylation. Cell survival, proliferation, and metabolism are mediated by phosphorylated EGFR. Dysfunction of the EGFR promotes uncontrolled cell development, which results in cell overgrowth and, ultimately, oncogenesis [51]. As a result, EGFR has been considered as a potential target for cancer therapy. Molecular docking analyses were carried out using the Glide software to better understand the interactions of promising compounds (4a, 4b, and 7d) with the EGFR target protein. In this methodology, Glide docking score, emodel, and Molecular mechanics with generalized Born and surface area solvation (MMGBSA) binding free energy (∆G Bind) were kept as support for the present work. ∆G Bind is a popular method to calculate the free energy of the binding of ligands to proteins. The minimal docking score and ∆G Bind needed for complex formation between ligand and protein show good binding affinity. More negative values suggest that the ligand is buried in the receptor cavity. The mean docking score for all three compounds is −6.98 kcal/mol, and the ∆G Bind is −67 kcal/mol, as shown in Table 6. Compound 4a, the most active EGFR inhibitor among the investigated compounds, also had the most significant docking score of −7.20 kcal/mol and a ∆G Bind of −75.62 kcal/mol when contrasted to Erlotinib (−9.07 and −84.86 kcal/mol, respectively). The binding interaction showed that compound 4a formed one hydrogen bond with Arg817 at a 2.21 A • bond length, while the central naphthyl ring formed a π-cation interaction with Lys721(4.08 A • ) in the EGFR kinase domain ( Figure 7A). The quinolin-2(1H)-one and triazole portions of compound 4a were shown to have substantial van der Waals contacts with Gly772 (−3.09 kcal/mol), Val702 (−3.95 kcal/mol), and Leu694 (−4.14 kcal/mol), which demonstrated that the molecule is entrenched within the active site.
On the other hand, Compounds 4b and 7d demonstrated hydrogen and hydrophobic interactions with Lys721, Met769, Phe771, and Lys405 residues in the EGFR kinase domain that were comparable to erlotinib, supporting its inhibitory activity towards EGFR. Lys721(4.08 A°) in the EGFR kinase domain ( Figure 7A). The quinolin-2(1H)-one and triazole portions of compound 4a were shown to have substantial van der Waals contacts with Gly772 (−3.09 kcal/mol), Val702 (−3.95 kcal/mol), and Leu694 (−4.14 kcal/mol), which demonstrated that the molecule is entrenched within the active site. On the other hand, Compounds 4b and 7d demonstrated hydrogen and hydrophobic interactions with Lys721, Met769, Phe771, and Lys405 residues in the EGFR kinase domain that were comparable to erlotinib, supporting its inhibitory activity towards EGFR.

Conclusions
In conclusion, a straightforward way to tether different quinolinones derivatives to 1,5-dinaphthol and 1,8-dinaphthol via 1,2,3-triazole linkers was elaborated based on Cucatalyzed [3 + 2] cycloaddition of o-propargyl units. In the course of the introduction of the propargyl groups, an interesting dependence of the regioselectivity on the substituent found at the OH groups of the naphthalene rings was observed as Syn and Anti isomerslike. By sequential [3 + 2] cycloadditions, it was possible to link two quinolinone moieties to the naphthalene skeleton. Remarkably, this cycloaddition occurred, and all products obtained were hitherto unknown. Pharmacological screening of novel products showed interesting and promising results as EGFR inhibitors with potential apoptotic antiproliferative action.

Conclusions
In conclusion, a straightforward way to tether different quinolinones derivatives to 1,5-dinaphthol and 1,8-dinaphthol via 1,2,3-triazole linkers was elaborated based on Cucatalyzed [3 + 2] cycloaddition of o-propargyl units. In the course of the introduction of the propargyl groups, an interesting dependence of the regioselectivity on the substituent found at the OH groups of the naphthalene rings was observed as Syn and Anti isomers-like. By sequential [3 + 2] cycloadditions, it was possible to link two quinolinone moieties to the naphthalene skeleton. Remarkably, this cycloaddition occurred, and all products obtained were hitherto unknown. Pharmacological screening of novel products showed interesting and promising results as EGFR inhibitors with potential apoptotic antiproliferative action.

Chemistry
General Details: See Appendix SA.

General Procedure for the Synthesis of Compounds 4a-e and 7a-e
A mixture of 1,8-bis(prop-2-yn-1-yloxy)naphthalene (2) or 1,5-bis(prop-2-yn-1-yloxy)naphthalene (6) (1.1 mmol) in 20 mL DMF, CuSO 4 .5H 2 O (0.4 mmol) and (0.4 mmol) of sodium ascorbate was stirred for 10 min at room temperature. To the above mixture, 4-azido compounds 3a-e (1.0 mmol) in 20 mL DMF were added dropwise. The reaction mixture was stirred at 50 • C for 24 h. After 14 hr, another portion of sodium ascorbate (0.4 mmol) was added to the reaction mixture to prevent the reversible process for Cu (+1). The reaction mixtures were monitored with TLC. After completion, the mixture was diluted with 100 gm ice, and the formed precipitate was filtered off and washed four times with cold water to give compounds 4a-e and 7a-e in excellent yields.

Biology
Appendix SA contains information on all biological experimental tests.

Docking Study
Molecular docking simulations were performed using MOE ® software within EGFR protein crystal structure with erlotinib as a co-crystallized ligand (PDB ID: 1M17). Docking protocol and other experimental details were used exactly as reported elsewhere [52][53][54]. See Appendix SA. Data Availability Statement: The study did not report any data.