Quinoline-Based Molecules Targeting c-Met, EGF, and VEGF Receptors and the Proteins Involved in Related Carcinogenic Pathways

The quinoline ring system has long been known as a versatile nucleus in the design and synthesis of biologically active compounds. Currently, more than one hundred quinoline compounds have been approved in therapy as antimicrobial, local anaesthetic, antipsychotic, and anticancer drugs. In drug discovery, indeed, over the last few years, an increase in the publication of papers and patents about quinoline derivatives possessing antiproliferative properties has been observed. This trend can be justified by the versatility and accessibility of the quinoline scaffold, from which new derivatives can be easily designed and synthesized. Within the numerous quinoline small molecules developed as antiproliferative drugs, this review is focused on compounds effective on c-Met, VEGF (vascular endothelial growth factor), and EGF (epidermal growth factor) receptors, pivotal targets for the activation of important carcinogenic pathways (Ras/Raf/MEK and PI3K/AkT/mTOR). These signalling cascades are closely connected and regulate the survival processes in the cell, such as proliferation, apoptosis, differentiation, and angiogenesis. The antiproliferative biological data of remarkable quinoline compounds have been analysed, confirming the pivotal importance of this ring system in the efficacy of several approved drugs. Furthermore, in view of an SAR (structure-activity relationship) study, the most recurrent ligand–protein interactions of the reviewed molecules are summarized.


Introduction
Quinoline 1 is an important and versatile nucleus recurrent in several natural and pharmacologically active molecules. Due to the presence of nitrogen, which withdraws electrons by resonance, this fused aromatic scaffold is an electron-deficient ring system (Figure 1), displaying weak tertiary base properties. Quinoline reacts with similar behaviour of pyridine and allows both nucleophilic and electrophilic substitution reactions. From a biological point of view, the nontoxic effect to humans, via inhalation and oral absorption, makes the 1-aza-naphthalene an interesting scaffold to be studied for the development of more selective drugs [1].
Quinoline and its related derivatives exhibit a broad spectrum of pharmacological activities from antibacterial, antifungal, antimalarial, anthelmintic, local anaesthetic, antipsychotic, and anticancer [2]. Especially in the research of new anticancer agents, quinoline is one of the most important scaffolds in drug discovery. Indeed, over the last few years, an increase in the publication of papers about quinoline derivatives possessing antiproliferative properties has been observed. This trend can be justified by the "druggability", versatility, and accessibility of the quinoline nucleus [3]. Quinoline and its related derivatives exhibit a broad spectrum of pharmacological activities from antibacterial, antifungal, antimalarial, anthelmintic, local anaesthetic, antipsychotic, and anticancer [2]. Especially in the research of new anticancer agents, quinoline is one of the most important scaffolds in drug discovery. Indeed, over the last few years, an increase in the publication of papers about quinoline derivatives possessing antiproliferative properties has been observed. This trend can be justified by the "druggability", versatility, and accessibility of the quinoline nucleus [3].
Furthermore, different quinoline small molecules, acting as protein kinases inhibitors, have been approved by the Food and Drug Administration (FDA) for clinical uses [4,5]. In this contest, bosutinib, a potent inhibitor of Abl, was approved in 2012 for the treatment of Philadelphia chromosome-positive chronic myelogenous leukaemia (CML) [6]; lenvatinib, an inhibitor of RET and VEGFR has been employed, since 2015, for the treatment of differentiated thyroid cancers [7]; and neratinib and cabozantinib were authorized for the clinical treatment respectively in 2017 and 2012 [8,9].
Due to the importance of quinoline nucleus in the medicinal chemistry field, the main purpose of this review is to provide an overview of the most relevant anticancer quinoline molecules described in the literature, in the last decade. In particular, the attention has been focused on quinoline drugs and experimental active derivatives, able to interfere with c-Met (mesenchymalepithelial transition factor), VEGF, and EGF receptors and the correlated proteins of the intracellular signalling pathways. The interest on these targets rises from their crucial involvement in several carcinogenic degenerations. In fact, closely connected by each other, they regulate the survival mechanisms in the cell, such as proliferation, apoptosis, differentiation, and angiogenesis.
In this light, the evaluation of the antiproliferative data and the analysis of most frequent ligandprotein interactions of quinoline-based compounds could support, in a drug discovery process, the search for new and more efficacious multi-targets drugs.

EGFR, c-MET, and VEGFR Signal Pathways in Cancer Cell
Aberrant receptor tyrosine kinases (RTKs) are deeply involved in cancer progression, and for this reason represent the key oncology therapeutic targets. Among the most studied, in this review the attention has been focused on three crucial growth factor receptors: c-Met, EGFR, and VEGFR [10].
C-Met is a tyrosine kinase receptor belonging to the MET (MNNG HOS transforming gene) family, and is normally expressed on the surfaces of liver, pancreas, prostate, kidney, muscle, and bone marrow cells, where mediates tissue regeneration and wound healing [11,12]. The endogenous ligand of c-Met is HGF/SF (ligand hepatocyte growth factor/scatter factor), that induces signal Furthermore, different quinoline small molecules, acting as protein kinases inhibitors, have been approved by the Food and Drug Administration (FDA) for clinical uses [4,5]. In this contest, bosutinib, a potent inhibitor of Abl, was approved in 2012 for the treatment of Philadelphia chromosome-positive chronic myelogenous leukaemia (CML) [6]; lenvatinib, an inhibitor of RET and VEGFR has been employed, since 2015, for the treatment of differentiated thyroid cancers [7]; and neratinib and cabozantinib were authorized for the clinical treatment respectively in 2017 and 2012 [8,9].
Due to the importance of quinoline nucleus in the medicinal chemistry field, the main purpose of this review is to provide an overview of the most relevant anticancer quinoline molecules described in the literature, in the last decade. In particular, the attention has been focused on quinoline drugs and experimental active derivatives, able to interfere with c-Met (mesenchymal-epithelial transition factor), VEGF, and EGF receptors and the correlated proteins of the intracellular signalling pathways. The interest on these targets rises from their crucial involvement in several carcinogenic degenerations. In fact, closely connected by each other, they regulate the survival mechanisms in the cell, such as proliferation, apoptosis, differentiation, and angiogenesis.
In this light, the evaluation of the antiproliferative data and the analysis of most frequent ligand-protein interactions of quinoline-based compounds could support, in a drug discovery process, the search for new and more efficacious multi-targets drugs. From a molecular point of view, all the quinoline-based inhibitors of c-Met interact with the kinase domain of the receptor in the cytosolic side, involving amino acids residues from 1078 to 1345 [13,30]. As an example, in Figure 4a, the crystal structure of c-Met kinase domain in complex with foretinib (PDB id: 3LQ8) is reported: the ligand binds to c-Met in an extended conformation occupying the ATP-binding site and the adjacent deep hydrophobic pocket. The cyclopropane-1,1dicarboxiamide framework interferes with the Phe 1223 of the DFG motif, determining the disruption of the catalytic conformation (from DFG-in to DFG out) and the reorganization of the activation loop, which almost entirely envelops the ligand (Figure 4a). In the cavity the reoriented Phe 1223 forms a ππ stacking with the central fluorophenyl ring, the quinoline nitrogen links with a hydrogen bond the backbone amide of Met 1160 and also the dicarboxamide moiety forms additional hydrogen bonds with Lys 1110 and Asp 1222 (Figure 4b) [30][31][32]. From a molecular point of view, all the quinoline-based inhibitors of c-Met interact with the kinase domain of the receptor in the cytosolic side, involving amino acids residues from 1078 to 1345 [13,30]. As an example, in Figure 4a, the crystal structure of c-Met kinase domain in complex with foretinib (PDB id: 3LQ8) is reported: the ligand binds to c-Met in an extended conformation occupying the ATP-binding site and the adjacent deep hydrophobic pocket. The cyclopropane-1,1-dicarboxiamide framework interferes with the Phe 1223 of the DFG motif, determining the disruption of the catalytic conformation (from DFG-in to DFG out) and the reorganization of the activation loop, which almost entirely envelops the ligand (Figure 4a). In the cavity the reoriented Phe 1223 forms a π-π stacking with the central fluorophenyl ring, the quinoline nitrogen links with a hydrogen bond the backbone amide of Met 1160 and also the dicarboxamide moiety forms additional hydrogen bonds with Lys 1110 and Asp 1222 (Figure 4b) [30][31][32].
Considering cabozantinib and foretinib as lead compounds, for the design of new quinoline derivatives selectively acting on c-Met, SAR studies highlight the 4-phenoxyquinoline and an aromatic moiety, connected each other by a linker, as the fundamental pharmacophoric portions in the modulation of c-Met tyrosine kinase activity ( Figure 5). Generally, the linker should have two important features: (1) the distance of five atoms (the so-called "five atoms regulation") between the 4-phenoxyquinoline moiety and the aromatic one; (2) the presence of donor and/or acceptor hydrogen-bond groups and at least one amide. These general features are also shown in the structure of cabozantinib and foretinib, in which there is a cyclopropane-1,1-dicarboxiamide framework as linker [33].
Considering cabozantinib and foretinib as lead compounds, for the design of new quinoline derivatives selectively acting on c-Met, SAR studies highlight the 4-phenoxyquinoline and an aromatic moiety, connected each other by a linker, as the fundamental pharmacophoric portions in the modulation of c-Met tyrosine kinase activity ( Figure 5). Generally, the linker should have two important features: (1) the distance of five atoms (the so-called "five atoms regulation") between the 4-phenoxyquinoline moiety and the aromatic one; (2) the presence of donor and/or acceptor hydrogen-bond groups and at least one amide. These general features are also shown in the structure of cabozantinib and foretinib, in which there is a cyclopropane-1,1-dicarboxiamide framework as linker [33]. In the box the binding cavity of the receptor occupied by the quinoline small molecule is illustrated (the activation loop is represented with transparent surface) [31]; (b) interactions of foretinib with amino acid residues of the c-Met active site [32].  In the last few years, a lot of more active and selective quinoline derivatives have been synthesized and biologically evaluated by modifying lead compounds ( Table 1). The most representative modifications are: (1) at position C-7 of quinoline ring system, generally with the introduction of appropriate substituents to improve solubility, (2) at the linker portion [34], that has been modified by different cyclic/acyclic 5-atoms-analogues with similar electronic features:  against the more responsive human cancer cell lines and the IC50 values (concentration of the compound producing 50% of inhibition activity) against the c-Met receptor, (bold data show the values of target compounds lower than that of the positive control in the corresponding experiment).   [35] compound producing 50% of inhibition activity) against the c-Met receptor, (bold data show the values of target compounds lower than that of the positive control in the corresponding experiment).   [36] values of target compounds lower than that of the positive control in the corresponding experiment).  1.1 ± 0.21 [37] values of target compounds lower than that of the positive control in the corresponding experiment).             1.35 [50] 5-         8.92 [54] In order to extrapolate important general information about the interactions between ligands and amino acid residues in the active site, the binding mode of the previous cabozantinib/foretiniblike derivatives has been explored through molecular docking simulations, (  8.92 [54] In order to extrapolate important general information about the interactions between ligands and amino acid residues in the active site, the binding mode of the previous cabozantinib/foretiniblike derivatives has been explored through molecular docking simulations, ( Figure 6 or condensed with other ring systems, exhibit inhibitory activity on PI3K/AkT/mTOR pathway 8.92 [54] In order to extrapolate important general information about the interactions between ligands and amino acid residues in the active site, the binding mode of the previous cabozantinib/foretiniblike derivatives has been explored through molecular docking simulations, ( Figure 6 or condensed with other ring systems, exhibit inhibitory activity on PI3K/AkT/mTOR pathway and several molecules are currently in clinical trials. For example, omipalisib (GSK2126458) is a quinoline derivative, substituted at C-6 with a 2-(methoxy)-3-benzenesulfonamide pyridinyl moiety, [54] Molecules 2020, 25, 4279 11 of 41 In this regard, after treatment with interesting quinoline compounds, in Table 1 are reported the IC 50 values (concentration of the compound producing 50% of cell growth inhibition) evaluated against the more responsive human cancer cell lines and the IC 50 values (concentration of the compound producing 50% of inhibition activity) against the c-Met receptor, (bold data show the values of target compounds lower than that of the positive control in the corresponding experiment).
In order to extrapolate important general information about the interactions between ligands and amino acid residues in the active site, the binding mode of the previous cabozantinib/foretinib-like derivatives has been explored through molecular docking simulations, ( Figure 6). These studies pointed out π-π stacking interactions between the quinoline ring and Tyr 1159 and the formation of a hydrogen bond between the lone pair of the quinoline nitrogen and Met 1160 , playing a pivotal role in the stabilization of the ligand-protein binding. Other important interactions occur through hydrogen bonds between the O and N atoms of the different linker regions and amino acids residues as Asp 1222 and Lys 1110 [35,36,38,40,41,48,51].  [36]; (c) 7 [38]; (d) 9 [40]; (e) 10 [41]; (f) 16 [48]; (g) 19 [51].
In 2011, Wang and co-workers designed a relatively unexplored quinoline chemotype, identifying the lead compound 3,5,7-trisubstituted derivative (zgwatinib) and the quinoline 23 as novel anticancer agents with a potent and selective inhibition activity on c-Met (IC 50 of 0.93 and 0.95 nM, respectively) ( Figure 7a). In particular, compound 23 showed significant antiproliferative effects (IC 50 in the range 1-5 µM) especially on cancer cell lines characterized by c-Met overexpression (ICMKN45, SNU-5 and H1993), exhibiting a promising PK profile and a remarkable in vivo tumour growth inhibition in human glioblastoma xenograft models [55]. The crystal structure of zgwatinib in complex with the kinase domain of c-Met was determined in a further study, to elucidate the binding mode of this class of compounds. In detail, quinoline nitrogen forms an H-bond with Met 1160 in the hinge region of the kinase, the trifluoromethyl and the benzylamino groups occupy the hydrophobic pocket, and the nitro substituent forms H-bond with Asp 1222 (Figure 7b) [56].
Molecules 2020, 25, x FOR PEER REVIEW 12 of 42 growth inhibition in human glioblastoma xenograft models [55]. The crystal structure of zgwatinib in complex with the kinase domain of c-Met was determined in a further study, to elucidate the binding mode of this class of compounds. In detail, quinoline nitrogen forms an H-bond with Met 1160 in the hinge region of the kinase, the trifluoromethyl and the benzylamino groups occupy the hydrophobic pocket, and the nitro substituent forms H-bond with Asp 1222 (Figure 7b) [56]. Nevertheless, further toxicology studies of both derivatives demonstrated considerable cardiovascular safety risk in normal rats, arising from significant inhibition of hERG K + channels (IC50 in the range 37-463 nM). For this reason, in the attempt to reduce the off-target effect, the same researcher group developed a new set of quinoline molecules, among which the compounds 24 and 25 were identified as the most active, (Figure 7a). The modification was directed towards the C-3 piperazinyl portion with the aim of improve the PK profile and reduce the hERG activity without influencing the key interactions. In detail, bearing a Michael acceptor moiety to the N-4 piperazinyl ring, these new quinoline compounds showed c-Met IC50 values comparable to the parent molecules (2.3 and 2.6 nM respectively for 24 and 25), and an appreciable lower activity against hERG [57]. Nevertheless, further toxicology studies of both derivatives demonstrated considerable cardiovascular safety risk in normal rats, arising from significant inhibition of hERG K + channels (IC 50 in the range 37-463 nM). For this reason, in the attempt to reduce the off-target effect, the same researcher group developed a new set of quinoline molecules, among which the compounds 24 and 25 were identified as the most active, (Figure 7a). The modification was directed towards the C-3 piperazinyl portion with the aim of improve the PK profile and reduce the hERG activity without influencing the key interactions. In detail, bearing a Michael acceptor moiety to the N-4 piperazinyl ring, these new quinoline compounds showed c-Met IC 50 values comparable to the parent molecules (2.3 and 2.6 nM respectively for 24 and 25), and an appreciable lower activity against hERG [57].
Nishii and co-workers explored modifications at the C-3 and C-6 positions of quinoline scaffold. 3,6-disubstituted quinoline 26 (Figure 8), shows an inhibition effect selectivity against c-Met kinase among more than 20 kinases with IC 50 = 9.3 nM, and a potent antiproliferative activity against MKN45 cancer cell line (IC 50 = 0.093 µM), [58].  In 2019, focusing the attention on C-6 substitution, Lien and co-workers designed and evaluated several 4,6,7-substituted quinolines, analogues of cabozantinib ( Figure 8) [59]. In vitro antiproliferative assays highlight that derivatives 27 and 28 are more active than the parent compound on leukaemia, CNS, and breast cancer cell lines. The c-Met kinase inhibition assay denotes higher, or comparable activity than cabozantinib, with IC50 values of 19 and 64 nM respectively for 27 and 28 (IC50 cabozantinib = 40 nM). On this basis, from the point of view of structure-activity relationships, these derivatives demonstrated well fittings in the binding pocket of c-Met. Indeed, docking studies ( Figure 9) demonstrated that, besides the interactions with crucial amino acids residues, above observed, these compounds could engage, through the p-aminophenyl moiety at C-6, additional hydrogen bonding with Ala 1226 [59]. Different studies on hybrid derivatives, with a quinoline scaffold linked at the C-6 position to another heterocyclic system, as triazolo-pyrazine or imidazo-pyrazine, demonstrate inhibition activity on c-Met and antiproliferative effects unless the 4-phenoxy substitution typical of the cabozantinib/foretinib-like compounds. In detail, Cui and co-workers described anticancer properties (Figure 10), an extremely potent and selective c-Met inhibitor with good oral bioavailability and an acceptable safety profile in preclinical trials [60]. In 2019, focusing the attention on C-6 substitution, Lien and co-workers designed and evaluated several 4,6,7-substituted quinolines, analogues of cabozantinib ( Figure 8) [59]. In vitro antiproliferative assays highlight that derivatives 27 and 28 are more active than the parent compound on leukaemia, CNS, and breast cancer cell lines. The c-Met kinase inhibition assay denotes higher, or comparable activity than cabozantinib, with IC 50 values of 19 and 64 nM respectively for 27 and 28 (IC 50 cabozantinib = 40 nM). On this basis, from the point of view of structure-activity relationships, these derivatives demonstrated well fittings in the binding pocket of c-Met. Indeed, docking studies ( Figure 9) demonstrated that, besides the interactions with crucial amino acids residues, above observed, these compounds could engage, through the p-aminophenyl moiety at C-6, additional hydrogen bonding with Ala 1226 [59].  In 2019, focusing the attention on C-6 substitution, Lien and co-workers designed and evaluated several 4,6,7-substituted quinolines, analogues of cabozantinib ( Figure 8) [59]. In vitro antiproliferative assays highlight that derivatives 27 and 28 are more active than the parent compound on leukaemia, CNS, and breast cancer cell lines. The c-Met kinase inhibition assay denotes higher, or comparable activity than cabozantinib, with IC50 values of 19 and 64 nM respectively for 27 and 28 (IC50 cabozantinib = 40 nM). On this basis, from the point of view of structure-activity relationships, these derivatives demonstrated well fittings in the binding pocket of c-Met. Indeed, docking studies ( Figure 9) demonstrated that, besides the interactions with crucial amino acids residues, above observed, these compounds could engage, through the p-aminophenyl moiety at C-6, additional hydrogen bonding with Ala 1226 [59]. Different studies on hybrid derivatives, with a quinoline scaffold linked at the C-6 position to another heterocyclic system, as triazolo-pyrazine or imidazo-pyrazine, demonstrate inhibition activity on c-Met and antiproliferative effects unless the 4-phenoxy substitution typical of the cabozantinib/foretinib-like compounds. In detail, Cui and co-workers described anticancer properties of 2-(4-(1-(quinolin-6-ylmethyl)-1H-[1,2,3]triazolo [4-b]pyrazin-6-yl)-1H-pyrazol-1-yl)-ethanol methanesulfonate (PF-04217903), (Figure 10), an extremely potent and selective c-Met inhibitor with good oral bioavailability and an acceptable safety profile in preclinical trials [60]. Different studies on hybrid derivatives, with a quinoline scaffold linked at the C-6 position to another heterocyclic system, as triazolo-pyrazine or imidazo-pyrazine, demonstrate inhibition activity on c-Met and antiproliferative effects unless the 4-phenoxy substitution typical of the cabozantinib/foretinib-like compounds. In detail, Cui and co-workers described anticancer properties of 2-(4-(1-(quinolin-6-ylmethyl)-1H-[1,2,3]triazolo [4-b]pyrazin-6-yl)-1H-pyrazol-1-yl)-ethanol methanesulfonate (PF-04217903), (Figure 10), an extremely potent and selective c-Met inhibitor with good oral bioavailability and an acceptable safety profile in preclinical trials [60]. After SGX523 and JNJ-38877605 clinical failure [61][62][63], Zhang and co-authors synthetized 3-Nsubstituted quinoline triazolopyridine analogues to understand the electron-donating and steric hindrance effects on the metabolic activity of the aldehyde oxidase [64].

Quinoline as Inhibitor of PI3k/AkT/mTOR Pathway
A lot of quinoline derivatives, basically substituted or condensed with other ring systems, exhibit inhibitory activity on PI3K/AkT/mTOR pathway and several molecules are currently in clinical trials. For example, omipalisib (GSK2126458) is a quinoline derivative, substituted at C-6 with a 2-(methoxy)-3-benzenesulfonamide pyridinyl moiety, at present under investigation in the first-in-human phase I study. This molecule shows the high inhibition effect of both PI3K and mTOR with an acceptable oral bioavailability [67,68]. Several omipalisib-like derivatives, characterized by a N-{5-[6-quinolinyl]-3-pyridinyl}benzenesulfonamide scaffold, have been synthesized and biologically evaluated for their PI3K/mTOR inhibition capability (biological data of omipalisib and compounds 31-34 are reported in Table 2) [69][70][71][72].
Crystallography and in silico studies highlight some recurrent and crucial interactions between these ligands and the ATP-binding site of PI3Kγ: the backbone NH of Val 882 (Val 851 in PI3Kα) forms a hydrogen bond with quinoline nitrogen; Lys 883 interacts, through a charged bond, with the sulfonamide group; and the C-4 quinoline substituent frequently stabilizes the ligand-protein complex, accommodating itself in the ribose pocket physiologically occupied by ATP. In Figure 11 it is reported the co-crystal structure of omipalisib in complex with the catalytic subunit of PI3Kγ, [67,[69][70][71][72].
Ma and co-workers reported the synthesis and the biological activities of a series of quinoline derivatives with a substituted aniline at C-4 position, and linked to another quinoline moiety at the C-6 one (compound 35-37 in Table 3). These quinoline compounds showed interesting inhibitory activities on both mTORC1 and mTORC2, with an adequate stability in simulated gastric, intestinal fluids, and liver microsome. The docking studies for these derivatives into the mTOR catalytic cleft showed the importance of some recurrent interactions, that stabilize the complex: hydrogen bond between the nitrogen of central quinoline scaffold and the critical residue Val 2240 , π-π interaction between indole moiety of Trp 2239 and quinoline scaffold, and hydrogen bond between quinoline substituent at C-6 and Tyr 2225 [74,75]. and several molecules are currently in clinical trials. For example, omipalisib (GSK2126458) is a quinoline derivative, substituted at C-6 with a 2-(methoxy)-3-benzenesulfonamide pyridinyl moiety, at present under investigation in the first-in-human phase I study. This molecule shows the high inhibition effect of both PI3K and mTOR with an acceptable oral bioavailability [67,68]. Several omipalisib-like derivatives, characterized by a N-{5-[6-quinolinyl]-3-pyridinyl}benzenesulfonamide scaffold, have been synthesized and biologically evaluated for their PI3K/mTOR inhibition capability (biological data of omipalisib and compounds 31-34 are reported in Table 2) [69][70][71][72].   PI3Kα IC50 = 0.50 nM mTOR IC50 = 1.3 nM [72] Crystallography and in silico studies highlight some recurrent and crucial interactions between these ligands and the ATP-binding site of PI3Kγ: the backbone NH of Val 882 (Val 851 in PI3Kα) forms a hydrogen bond with quinoline nitrogen; Lys 883 interacts, through a charged bond, with the sulfonamide group; and the C-4 quinoline substituent frequently stabilizes the ligand-protein complex, accommodating itself in the ribose pocket physiologically occupied by ATP. In Figure 11 it is reported the co-crystal structure of omipalisib in complex with the catalytic subunit of PI3Kγ, [67,[69][70][71][72].  PI3Kα IC50 = 0.50 nM mTOR IC50 = 1.3 nM [72] Crystallography and in silico studies highlight some recurrent and crucial interactions between these ligands and the ATP-binding site of PI3Kγ: the backbone NH of Val 882 (Val 851 in PI3Kα) forms a hydrogen bond with quinoline nitrogen; Lys 883 interacts, through a charged bond, with the sulfonamide group; and the C-4 quinoline substituent frequently stabilizes the ligand-protein complex, accommodating itself in the ribose pocket physiologically occupied by ATP. In Figure 11 it is reported the co-crystal structure of omipalisib in complex with the catalytic subunit of PI3Kγ, [67,[69][70][71][72].  PI3Kα IC50 = 0.50 nM mTOR IC50 = 1.3 nM [72] Crystallography and in silico studies highlight some recurrent and crucial interactions between these ligands and the ATP-binding site of PI3Kγ: the backbone NH of Val 882 (Val 851 in PI3Kα) forms a hydrogen bond with quinoline nitrogen; Lys 883 interacts, through a charged bond, with the sulfonamide group; and the C-4 quinoline substituent frequently stabilizes the ligand-protein complex, accommodating itself in the ribose pocket physiologically occupied by ATP. In Figure 11 it is reported the co-crystal structure of omipalisib in complex with the catalytic subunit of PI3Kγ, [67,[69][70][71][72].  PI3Kα IC50 = 0.50 nM mTOR IC50 = 1.3 nM [72] Crystallography and in silico studies highlight some recurrent and crucial interactions between these ligands and the ATP-binding site of PI3Kγ: the backbone NH of Val 882 (Val 851 in PI3Kα) forms a hydrogen bond with quinoline nitrogen; Lys 883 interacts, through a charged bond, with the sulfonamide group; and the C-4 quinoline substituent frequently stabilizes the ligand-protein complex, accommodating itself in the ribose pocket physiologically occupied by ATP. In Figure 11 it is reported the co-crystal structure of omipalisib in complex with the catalytic subunit of PI3Kγ,  showed the importance of some recurrent interactions, that stabilize the complex: hydrogen bond between the nitrogen of central quinoline scaffold and the critical residue Val 2240 , π-π interaction between indole moiety of Trp 2239 and quinoline scaffold, and hydrogen bond between quinoline substituent at C-6 and Tyr 2225 [74,75].    14 [74] between the nitrogen of central quinoline scaffold and the critical residue Val , π-π interaction between indole moiety of Trp 2239 and quinoline scaffold, and hydrogen bond between quinoline substituent at C-6 and Tyr 2225 [74,75].   22 [75] between indole moiety of Trp and quinoline scaffold, and hydrogen bond between quinoline substituent at C-6 and Tyr 2225 [74,75].   30 [75] these ligands and the ATP-binding site of PI3Kγ: the backbone NH of Val 882 (Val 851 in PI3Kα) forms a hydrogen bond with quinoline nitrogen; Lys 883 interacts, through a charged bond, with the sulfonamide group; and the C-4 quinoline substituent frequently stabilizes the ligand-protein complex, accommodating itself in the ribose pocket physiologically occupied by ATP. In Figure 11 it is reported the co-crystal structure of omipalisib in complex with the catalytic subunit of PI3Kγ, [67,[69][70][71][72]. Ma and co-workers reported the synthesis and the biological activities of a series of quinoline derivatives with a substituted aniline at C-4 position, and linked to another quinoline moiety at the C-6 one (compound 35-37 in Table 3). These quinoline compounds showed interesting inhibitory activities on both mTORC1 and mTORC2, with an adequate stability in simulated gastric, intestinal The PI3K/mTOR inhibition activity is also conserved in numerous derivatives in which the quinoline scaffold is condensed with other heterocycles. Among these, imidazo [4,5-c]quinolines are the most important, such as investigational drugs like dactolisib (BEZ-235), panulisib (P-7170), LY-3023414, SHR8443, and BGT-226 ( Figure 13) [3].
LY-3023414, developed as a selective PI3K/mTOR dual inhibitor, was patented in 2012, with an absolute mTOR IC 50 and PI3Kα IC 50 values respectively of 0.165 µM and 0.00607 µM. In cancer cell human line panels, it showed broad antiproliferative activity [90,91]. In a first-in-human phase I study it demonstrated a tolerable safety profile and single-agent activity in patients with advanced tumours [92].
SHR8443 has been evaluated in vitro for the enzymatic inhibition assay of mTOR, showing IC 50 value of 1 nM [93]. Preclinical studies evidenced pharmaceutical properties favourable for the clinical use in antitumor treatments [94].
Remarkable interesting, are torin 1 and torin 2, two benzo[h][1,6]-naphthyridin-2(1H)-one compounds, containing a quinoline moiety and acting as selective mTOR inhibitors (Figure 16b). Developed by Liu and co-workers, these tricyclic pyridoquinolines derivatives are the result of two consecutive generations of quinoline-based compounds. They are active on the phosphorylation process of mTORC1/ mTORC2 substrates in the cell, exhibiting EC50 values in the range 0.25-7 nM. Furthermore, with respect to torin 1, second generation compound torin 2 demonstrates 800-fold This molecule demonstrated an mTOR IC 50 = 31 nM, but weak antiproliferative activity in vitro. Further structural optimization led to compound 43 (Figure 16), that showed a better mTOR inhibitory activity than the parent compound (IC 50 = 7 nM), good selectivity profile, potent antiproliferative activities against breast and cervical cancer lines, and significant tumor regression in the T-47D xenograft model after an oral once-daily dose [105].
Remarkable interesting, are torin 1 and torin 2, two benzo[h][1,6]-naphthyridin-2(1H)-one compounds, containing a quinoline moiety and acting as selective mTOR inhibitors (Figure 16b). Developed by Liu and co-workers, these tricyclic pyridoquinolines derivatives are the result of two consecutive generations of quinoline-based compounds. They are active on the phosphorylation process of mTORC1/ mTORC2 substrates in the cell, exhibiting EC 50 values in the range 0.25-7 nM. Furthermore, with respect to torin 1, second generation compound torin 2 demonstrates 800-fold selectivity over PI3K, improving the bioavailability (54%), metabolic stability, and plasma exposure [106,107].
inhibitory activity than the parent compound (IC50 = 7 nM), good selectivity profile, potent antiproliferative activities against breast and cervical cancer lines, and significant tumor regression in the T-47D xenograft model after an oral once-daily dose [105].
Remarkable interesting, are torin 1 and torin 2, two benzo[h][1,6]-naphthyridin-2(1H)-one compounds, containing a quinoline moiety and acting as selective mTOR inhibitors (Figure 16b). Developed by Liu and co-workers, these tricyclic pyridoquinolines derivatives are the result of two consecutive generations of quinoline-based compounds. They are active on the phosphorylation process of mTORC1/ mTORC2 substrates in the cell, exhibiting EC50 values in the range 0.25-7 nM. Furthermore, with respect to torin 1, second generation compound torin 2 demonstrates 800-fold selectivity over PI3K, improving the bioavailability (54%), metabolic stability, and plasma exposure [106,107].

Quinoline Derivatives as Inhibitors of Epidermal Growth Factor Receptors
Various quinoline derivatives, with EGFR inhibitory potential, have been studied and the 4-anilinoquinoline-3-carbonitrile class is one of the first group developed. These compounds have been designed by molecular modelling studies, considering the approved EGFR 4-anilinoquinazoline inhibitors (gefitinib, erlotinib, and afatinib) as lead compounds ( Figure 17). Various quinoline derivatives, with EGFR inhibitory potential, have been studied and the 4anilinoquinoline-3-carbonitrile class is one of the first group developed. These compounds have been designed by molecular modelling studies, considering the approved EGFR 4-anilinoquinazoline inhibitors (gefitinib, erlotinib, and afatinib) as lead compounds ( Figure 17).
Weissner et al. demonstrated that the N-3 of quinazoline ring system can be replaced by a C-X, where X is an electron withdrawing group (i.e., CN). It was found that 4-anilinoquinoline-3carbonitriles are effective inhibitors of EGFR kinase with activity comparable to the 4anilinoquinazoline-based inhibitors. Compound 44 (Figure 17) showed the interesting IC50 value of 7.5 nM [108]. The same authors developed a second generation of EGFR inhibitors, namely 4anilinoquinoline-3-carbonitrile derivatives bearing Michael acceptor groups (such as 4-(dimethylamino)crotonamide) at the position C-6 ( Figure 18). Weissner et al. demonstrated that the N-3 of quinazoline ring system can be replaced by a C-X, where X is an electron withdrawing group (i.e., CN). It was found that 4-anilinoquinoline-3-carbonitriles are effective inhibitors of EGFR kinase with activity comparable to the 4-anilinoquinazoline-based inhibitors. Compound 44 (Figure 17) showed the interesting IC 50 value of 7.5 nM [108].
Subsequently, Tsou et al., introducing a lipophilic substituent at the para position of the 4-arylamino ring, isolated the molecule HKI-272, more active than pelitinib, and approved in 2017 with the name neratinib ( Figure 18) for the extended adjuvant therapy against early-stage HER2/ErbB2-amplified/overexpressed breast cancer [3,4,109,111,112]. Crystal structure of EGFR/T290M mutant kinase domain in complex with neratinib showed the main interactions recurrent in the binding pocket ( Figure 19): non-covalent interactions (H-bond of quinoline nitrogen with residues of the hinge region; hydrophobic interactions between the 2-pyridinyl group and the Met 766 , Phe 856 and Met 790 ) and a covalent bond (Cys 797 at the edge of the active site cleft attacked the crotonamide group of the ligand) [113]. These molecules act as EGFR irreversible inhibitors, forming a covalent bond with a conserved cysteine residue located in the ATP binding pocket of EGFR (Cys 773 or Cys 797 ) and HER-2 (Cys 805 ) [109]. In particular, pelitinib (EKB-569; EGFR IC50 = 0.083 μM), currently under investigation in clinical trials, showed excellent oral in vivo activity [3,110].
Subsequently, Tsou et al., introducing a lipophilic substituent at the para position of the 4arylamino ring, isolated the molecule HKI-272, more active than pelitinib, and approved in 2017 with the name neratinib ( Figure 18) for the extended adjuvant therapy against early-stage HER2/ErbB2amplified/overexpressed breast cancer [3,4,109,111,112]. Crystal structure of EGFR/T290M mutant kinase domain in complex with neratinib showed the main interactions recurrent in the binding pocket ( Figure 19): non-covalent interactions (H-bond of quinoline nitrogen with residues of the hinge region; hydrophobic interactions between the 2-pyridinyl group and the Met 766 , Phe 856 and Met 790 ) and a covalent bond (Cys 797 at the edge of the active site cleft attacked the crotonamide group of the ligand) [113]. Starting from the lead compound neratinib, SHR1258 has been developed as irreversible EGFR/HER2 tyrosine kinase inhibitor, and afterwards named pyrotinib (Figure 18). Figure 20 shows a comparison between binding modes of neratinib and pyrotinib in the catalytic region of HER2 kinase. In this model the two molecules are completely superimposed (Figure 20b), except for the substituents on the Michael acceptor group. Both display the same stabilizing interactions: hydrogen Starting from the lead compound neratinib, SHR1258 has been developed as irreversible EGFR/HER2 tyrosine kinase inhibitor, and afterwards named pyrotinib ( Figure 18). Figure 20 shows a comparison between binding modes of neratinib and pyrotinib in the catalytic region of HER2 kinase. In this model the two molecules are completely superimposed (Figure 20b), except for the substituents on the Michael acceptor group. Both display the same stabilizing interactions: hydrogen bond between N-1 of the quinoline ring and the hinge region of Met 801 , a covalent bond between Cys 805 and Michael acceptor group (double bond) [114]. Starting from the lead compound neratinib, SHR1258 has been developed as irreversible EGFR/HER2 tyrosine kinase inhibitor, and afterwards named pyrotinib ( Figure 18). Figure 20 shows a comparison between binding modes of neratinib and pyrotinib in the catalytic region of HER2 kinase. In this model the two molecules are completely superimposed (Figure 20b), except for the substituents on the Michael acceptor group. Both display the same stabilizing interactions: hydrogen bond between N-1 of the quinoline ring and the hinge region of Met 801 , a covalent bond between Cys 805 and Michael acceptor group (double bond) [114].  In vitro preclinical studies show high potency of pyrotinib, comparable to neratinib, against HER2 dependent BT474 (breast cancer) and SK-OV-3 (ovarian cancer) cell lines and also in vivo efficacy in HER2-dependent mouse xenograft models. Moreover, pyrotinib displays high selectivity when tested against a panel of different kinases [114]. Clinical trials demonstrate a safety profiles, desirable pharmacokinetic properties, tolerability and promising antitumor activity in HER2-positive patients with metastatic breast cancer, especially in combination with capecitabine [115,116]. Currently, pyrotinib is also under trial evaluation for the use in HER2-positive gastric cancer and NSCLC [117]. Pannala et al. described the synthesis and the evaluation of EGFR inhibition of some 4-(2-arylcyclopropylamino)-quinoline-3-carbonitriles. These compounds possess an arylcyclopropylamino group at the C-4 position of the quinoline-3-carbonitrile core instead of 4-aniline one. Quinoline 45 ( Figure 21) shows an interesting inhibition effect on EGFR with IC 50 value of 5 nM [118]. In vitro preclinical studies show high potency of pyrotinib, comparable to neratinib, against HER2 dependent BT474 (breast cancer) and SK-OV-3 (ovarian cancer) cell lines and also in vivo efficacy in HER2-dependent mouse xenograft models. Moreover, pyrotinib displays high selectivity when tested against a panel of different kinases [114]. Clinical trials demonstrate a safety profiles, desirable pharmacokinetic properties, tolerability and promising antitumor activity in HER2-positive patients with metastatic breast cancer, especially in combination with capecitabine [115,116]. Currently, pyrotinib is also under trial evaluation for the use in HER2-positive gastric cancer and NSCLC [117]. Pannala et al. described the synthesis and the evaluation of EGFR inhibition of some 4-(2-arylcyclopropylamino)-quinoline-3-carbonitriles. These compounds possess an arylcyclopropylamino group at the C-4 position of the quinoline-3-carbonitrile core instead of 4-aniline one. Quinoline 45 ( Figure 21) shows an interesting inhibition effect on EGFR with IC50 value of 5 nM [118]. Aly et al. planned and synthesized a set of 4-anilino-3-carboxyamide derivatives. They based their studies on the high similarities between the 4-anilinoquinazoline, 4-anilinoquinoline-3carbonitrile, and 4-anilinoquinoline-3-carboxamide nuclei. In a previous work, quinoline 46 (EGFR IC50 = 5.283 μM) was selected as a lead structure for the design of other more active derivatives [119]. In particular, compound 47 with a substituted-thiophene moiety at C-6 ( Figure 22) exhibited selective activity on EGFR with IC50 value of 0.49 μM [120]. Aly et al. planned and synthesized a set of 4-anilino-3-carboxyamide derivatives. They based their studies on the high similarities between the 4-anilinoquinazoline, 4-anilinoquinoline-3-carbonitrile, and 4-anilinoquinoline-3-carboxamide nuclei. In a previous work, quinoline 46 (EGFR IC 50 = 5.283 µM) was selected as a lead structure for the design of other more active derivatives [119]. In particular, compound 47 with a substituted-thiophene moiety at C-6 ( Figure 22) exhibited selective activity on EGFR with IC 50 value of 0.49 µM [120]. Aly et al. planned and synthesized a set of 4-anilino-3-carboxyamide derivatives. They based their studies on the high similarities between the 4-anilinoquinazoline, 4-anilinoquinoline-3carbonitrile, and 4-anilinoquinoline-3-carboxamide nuclei. In a previous work, quinoline 46 (EGFR IC50 = 5.283 μM) was selected as a lead structure for the design of other more active derivatives [119]. In particular, compound 47 with a substituted-thiophene moiety at C-6 ( Figure 22) exhibited selective activity on EGFR with IC50 value of 0.49 μM [120].  In lasts years, several hybrids derivatives containing quinoline scaffold linked with other heterocyclic ring systems have been studied as EGFR inhibitors with interesting activity. Makawana et al. reported numerous Schiff's base derivatives bearing nitroimidazole and quinoline nuclei as potential EGFR tyrosine kinase inhibitors. Compound 50 ( Figure 24) showed an IC50 value on EGFR receptor of 0.12 ± 0.05 μM [122]. In lasts years, several hybrids derivatives containing quinoline scaffold linked with other heterocyclic ring systems have been studied as EGFR inhibitors with interesting activity. Makawana et al. reported numerous Schiff's base derivatives bearing nitroimidazole and quinoline nuclei as potential EGFR tyrosine kinase inhibitors. Compound 50 ( Figure 24) showed an IC 50 value on EGFR receptor of 0.12 ± 0.05 µM [122]. In lasts years, several hybrids derivatives containing quinoline scaffold linked with other heterocyclic ring systems have been studied as EGFR inhibitors with interesting activity. Makawana et al. reported numerous Schiff's base derivatives bearing nitroimidazole and quinoline nuclei as potential EGFR tyrosine kinase inhibitors. Compound 50 ( Figure 24) showed an IC50 value on EGFR receptor of 0.12 ± 0.05 μM [122].  The quinoline scaffold is frequently present in numerous polycondensed ring system compounds with proved selective inhibition activity on EGFR.
Abdelbaset et al., for example, designed several thieno[2,3-b]quinoline-2-carboxamide-chalcone derivatives, as molecule 54 (Figure 26a) that showed significant antiproliferative effects against tested cancer cell lines (IC 50 values in the range 0.9-1.2 µM) and an EGFR IC 50 = 0.5 µM. Molecular docking studies showed that the thienoquinoline moiety occupied the ATP-binding site of the receptor, whereas chalcone moiety was located in an allosteric pocket of the enzyme (Figure 26b) [124]. The quinoline scaffold is frequently present in numerous polycondensed ring system compounds with proved selective inhibition activity on EGFR.

Quinoline Derivatives as Inhibitors of Vascular Endothelial Growth Factor Receptor
VEGF-VEGFR abnormal signals play central roles in angiogenic processes in a variety of diseases, especially in cancer. Some studies demonstrated, that, although the VEGF binds more effectively the VEGFR-1, its major mitogenic and angiogenic effects seem to be mediated through the interaction with VEGFR of type 2. In recent years, a lot of selective angiogenesis and multi-receptor kinase inhibitors have been synthesized, and some of these have been approved for clinical use [24,25].
Lenvatinib, (Figure 28), approved in 2015 for the treatment of differentiated thyroid, hepatocellular cancers, and the second-line treatment of renal carcinoma, is a multikinase quinoline inhibitor with a selective effect against VEGFR [4,7].

Quinoline Derivatives as Inhibitors of Vascular Endothelial Growth Factor Receptor
VEGF-VEGFR abnormal signals play central roles in angiogenic processes in a variety of diseases, especially in cancer. Some studies demonstrated, that, although the VEGF binds more effectively the VEGFR-1, its major mitogenic and angiogenic effects seem to be mediated through the interaction with VEGFR of type 2. In recent years, a lot of selective angiogenesis and multi-receptor kinase inhibitors have been synthesized, and some of these have been approved for clinical use [24,25].
Lenvatinib, (Figure 28), approved in 2015 for the treatment of differentiated thyroid, hepatocellular cancers, and the second-line treatment of renal carcinoma, is a multikinase quinoline inhibitor with a selective effect against VEGFR [4,7].  (Figure 28), which exhibited the best inhibition effect on VEGFR with IC50 value of 0.9 nM [126].
Analysing the structural analogies between lenvatinib, Ki8751, and tivozanib, it is possible to underline the recurrent and important presence of the C-4 oxyphenyl urea substituted moieties and the C-6, C-7 substitutions on the quinoline scaffold. These structure features play a pivotal role for the stabilization of ligand-protein complex, as it is shown by the X-ray crystal structure of VEGFR-2 in complex with lenvatinib ( Figure 29, PDB id: 3WZD): the ligand binds the receptor kinase domain in its DFG-in conformation, occupying the ATP-binding site through its quinoline moiety and the neighboring region via the cyclopropane ring. In particular, the key interactions are three H-bonds (quinoline nitrogen and Cys 919 ; urea oxygen and Asp 1046 ; urea nitrogen and Glu 885 ), two hydrophilic interactions (the C-6 carboxamide with Asn 923 , bridged by water molecules) and several π-interactions (quinoline and C-4 phenoxy moiety with Leu 840 , Phe 818 and Lys 868 ) [130]. Quinoline core and urea moiety in tivozanib showed the same key interactions observed for the parent compound (PDB id: 4ASE) [131,132]. Yang et al. reported the synthesis of some quinoline amide derivatives with VEGFR-2 inhibition activity. Compound 56 ( Figure 30) resulted to be the most active to inhibit VEGFR kinase (IC50 = 3.8 nM) and proliferation of HUEVEC cancer cells (IC50 = 5.5 nM); the docking analysis confirmed that this compound is suitable bonded to VEGFR-2 [133]. Yang et al. reported the synthesis of some quinoline amide derivatives with VEGFR-2 inhibition activity. Compound 56 ( Figure 30) resulted to be the most active to inhibit VEGFR kinase (IC 50 = 3.8 nM) and proliferation of HUEVEC cancer cells (IC 50 = 5.5 nM); the docking analysis confirmed that this compound is suitable bonded to VEGFR-2 [133].
Through a combination of some structural elements present in molecules with antiproliferative activity, such as 4-piperazinoquinoline scaffold and aminoacyl chain, Aboul-Enein et al. planned and synthesized 7-Chloro-4-(piperazin-1-yl)quinoline derivatives as VEGFR-2 inhibitors. The most promising compound 57 (Figure 31), exhibited cytotoxicity higher than that of reference drug (doxorubicin) against MCF-7 cell line (6.502 µM vs. 6.774 µM) and a VEGFR-2 IC 50 = 1.38 µM, but less potent than the reference drug sorafenib (IC 50 of 0.33 µM). Docking analysis in the ATP-binding site of VEGFR-2 proved that molecule 57 shows a binding mode similar to that of other VEGFR-2 inhibitors as lenvatinib: both displayed H-bonds between quinoline nitrogen and Cys 919 , and between the carbonyl group (in the aminoacyl and in urea moieties in derivative 57 and tivozanib, respectively) and Asp 1046 . However, 57 showed some additional π-interaction with Ile 888 and Phe 918 [134]. Yang et al. reported the synthesis of some quinoline amide derivatives with VEGFR-2 inhibition activity. Compound 56 ( Figure 30) resulted to be the most active to inhibit VEGFR kinase (IC50 = 3.8 nM) and proliferation of HUEVEC cancer cells (IC50 = 5.5 nM); the docking analysis confirmed that this compound is suitable bonded to VEGFR-2 [133]. of VEGFR-2 proved that molecule 57 shows a binding mode similar to that of other VEGFR-2 inhibitors as lenvatinib: both displayed H-bonds between quinoline nitrogen and Cys 919 , and between the carbonyl group (in the aminoacyl and in urea moieties in derivative 57 and tivozanib, respectively) and Asp 1046 . However, 57 showed some additional π-interaction with Ile 888 and Phe 918 [134].   of VEGFR-2 proved that molecule 57 shows a binding mode similar to that of other VEGFR-2 inhibitors as lenvatinib: both displayed H-bonds between quinoline nitrogen and Cys 919 , and between the carbonyl group (in the aminoacyl and in urea moieties in derivative 57 and tivozanib, respectively) and Asp 1046 . However, 57 showed some additional π-interaction with Ile 888 and Phe 918 [134].    [136].

Quinoline Derivatives as Inhibitors of Ras/Raf/MEK Pathway
EGFR and VEGFR extracellular activations trigger the intracellular cascade of signalling connected to Ras/Raf/MEK/ERK. This deregulated pathway, in neoplastic cells, improve the tumour genesis, acting on the survival processes of cellular proliferation; such as differentiation, apoptosis, angiogenesis.
Several quinoline derivatives, tested as inhibitors of Ras/Raf/MEK cascade, demonstrate promising results both in antiproliferative and enzymatic inhibition assays. In detail, Feng and co-workers developed quinoline KAL-21404358 (Figure 34), the first ligand of the K-Ras P110 allosteric pocket able to disrupt downstream pathways (Raf/MEK/ERK and PI3K/AkT/mTOR). Through a combination of computational methods and with the validation of biochemical assays, the authors proposed a mechanism of action for KAL-21404358. The quinoline compound could interfere with the protein-protein interactions, binding and stabilizing K-Ras in its inactive GDP-bound state, then, it halts the nucleotide exchange process and the subsequent activation of Ras [137].

Quinoline Derivatives as Inhibitors of Ras/Raf/MEK Pathway
EGFR and VEGFR extracellular activations trigger the intracellular cascade of signalling connected to Ras/Raf/MEK/ERK. This deregulated pathway, in neoplastic cells, improve the tumour genesis, acting on the survival processes of cellular proliferation; such as differentiation, apoptosis, angiogenesis.
Several quinoline derivatives, tested as inhibitors of Ras/Raf/MEK cascade, demonstrate promising results both in antiproliferative and enzymatic inhibition assays. In detail, Feng and coworkers developed quinoline KAL-21404358 (Figure 34), the first ligand of the K-Ras P110 allosteric pocket able to disrupt downstream pathways (Raf/MEK/ERK and PI3K/AkT/mTOR). Through a combination of computational methods and with the validation of biochemical assays, the authors proposed a mechanism of action for KAL-21404358. The quinoline compound could interfere with the protein-protein interactions, binding and stabilizing K-Ras in its inactive GDP-bound state, then, it halts the nucleotide exchange process and the subsequent activation of Ras [137].
In 2016, Li et al. filled a patent of a new set of fused-tricyclic compounds containing quinoline scaffold with a promising inhibition activity on the mutated form of Gly12Cys K-Ras ( Figure 34). The analysis of the catalytic binding site, highlights that Cys 12 forms a covalent bond with the electrophilic acryloyl moiety of quinolines 62 and 63 [138]. El-Gamal et al. synthesized two class of Raf inhibitors possessing quinoline scaffold. In the first one, a diarylamide moiety, through a S or an O atom, was linked at the C-3 of a dimethoxy/dihydroxyquinoline scaffold. Biological assays realized by National Cancer Institute demonstrated that the most active compounds were dimethoxyquinolines with an oxygen as linker and with electronwithdrawing groups (-Cl or CF3) on the terminal ring. One of the most active compounds, 64 ( Figure  35a), exhibited GI50 values in the micromolar range against the full panel of NCI cancer cell lines and inhibited C-Raf kinase activity by 76.65% at 10 μM [139].
In the second class of quinoline compounds, the same authors substituted the amide linker with urea one to obtain diarylurea derivatives. In NCI five-dose screening protocol, molecule 65, with the insertion of 4-chloro-3-(trifluoromethyl)phenylurea moiety, was the most promising compound, with a relevant antiproliferative effect compared to the reference diarylurea drug sorafenib (Figure 35a). Inhibition assays of C-Raf kinase evidenced quinoline 65 as more active than the lead compound 64 (for 65 %inhibition at 10 μM = 99.67% and IC50 = 0.10 μM). Docking studies of the active site of C-Raf In 2016, Li et al. filled a patent of a new set of fused-tricyclic compounds containing quinoline scaffold with a promising inhibition activity on the mutated form of Gly12Cys K-Ras ( Figure 34). The analysis of the catalytic binding site, highlights that Cys 12 forms a covalent bond with the electrophilic acryloyl moiety of quinolines 62 and 63 [138].
El-Gamal et al. synthesized two class of Raf inhibitors possessing quinoline scaffold. In the first one, a diarylamide moiety, through a S or an O atom, was linked at the C-3 of a dimethoxy/dihydroxy-quinoline scaffold. Biological assays realized by National Cancer Institute demonstrated that the most active compounds were dimethoxyquinolines with an oxygen as linker and with electron-withdrawing groups (-Cl or CF 3 ) on the terminal ring. One of the most active compounds, 64 (Figure 35a), exhibited GI 50 values in the micromolar range against the full panel of NCI cancer cell lines and inhibited C-Raf kinase activity by 76.65% at 10 µM [139].  (Figure 35b,c) [140]. El-Damasy et al. designed and synthetized several quinoline analogues of sorafenib, substituting the central phenoxy nucleus with a quinoline one and inserting the two recurrent moieties of the lead compound, the arylurea/arylamide and the N-methyl picolinamide, respectively at the C-2 and C-5 positions of the quinoline ring system. NCI antiproliferative assays of urea derivatives with fluorinated phenyl ring, as 66 and 67 (Figure 36a), showed GI50 values in the low-submicromolar range for the majority of the tested cell lines. Furthermore, compound 67 obtained remarkable results in the kinase inhibition assays, with high selectivity against Raf family and IC50 of 316 nM and 61 nM against BRAF V600E and C-Raf, respectively (for the reference sorafenib BRAF V600E IC50 = 38 nM and C-Raf IC50 = 6 nM); instead of quinoline derivative 66, that exerted its inhibitory effect only against C-Raf. Docking studies showed a similar binding mode between 67 and sorafenib in the catalytic kinase domain of BRAF V600E , indeed both molecules formed interactions through the picolinamide, urea, and trifluoromethylphenyl moieties ( Figure 36b); on the other hand, the absence of activity of 66 against BRAF V600E was justified In the second class of quinoline compounds, the same authors substituted the amide linker with urea one to obtain diarylurea derivatives. In NCI five-dose screening protocol, molecule 65, with the insertion of 4-chloro-3-(trifluoromethyl)phenylurea moiety, was the most promising compound, with a relevant antiproliferative effect compared to the reference diarylurea drug sorafenib (Figure 35a). Inhibition assays of C-Raf kinase evidenced quinoline 65 as more active than the lead compound 64 (for 65 %inhibition at 10 µM = 99.67% and IC 50 = 0.10 µM). Docking studies of the active site of C-Raf kinase (PDB ID: 3OMV) further explained the high potency of 65 with respect to 64. Indeed, compound 65, due to the greater flexibility of the urea linker, formed additional H-bonds with Asp 486 and Lys 470 (Figure 35b,c) [140].
El-Damasy et al. designed and synthetized several quinoline analogues of sorafenib, substituting the central phenoxy nucleus with a quinoline one and inserting the two recurrent moieties of the lead compound, the arylurea/arylamide and the N-methyl picolinamide, respectively at the C-2 and C-5 positions of the quinoline ring system. NCI antiproliferative assays of urea derivatives with fluorinated phenyl ring, as 66 and 67 (Figure 36a), showed GI 50 values in the low-submicromolar range for the majority of the tested cell lines. Furthermore, compound 67 obtained remarkable results in the kinase inhibition assays, with high selectivity against Raf family and IC 50 of 316 nM and 61 nM against BRAF V600E and C-Raf, respectively (for the reference sorafenib BRAF V600E IC 50 = 38 nM and C-Raf IC 50 = 6 nM); instead of quinoline derivative 66, that exerted its inhibitory effect only against C-Raf. Docking studies showed a similar binding mode between 67 and sorafenib in the catalytic kinase domain of BRAF V600E , indeed both molecules formed interactions through the picolinamide, urea, and trifluoromethylphenyl moieties ( Figure 36b); on the other hand, the absence of activity of 66 against BRAF V600E was justified by the unfit orientation of the 2,4-difluorophenyl group, far from the hydrophobic pocket of the allosteric site (Figure 36c) [141]. by the unfit orientation of the 2,4-difluorophenyl group, far from the hydrophobic pocket of the allosteric site (Figure 36c) [141]. Considering 67 as lead compound, the same authors, by the substitution of the urea linker with an amine one, developed a new series of 2-anilinoquinolines bearing the N-methylpicolinamide group at the C-5 position of the quinoline scaffold. The derivative 4-chloro-3-(trifluoromethyl)aniline 68 (Figure 36a) manifested more remarkable antiproliferative effects than compound 67 and sorafenib, especially against melanoma and breast cancer panels. However, the replacement of the urea linker with an amine in compound 68 has a negative effect on Raf kinase inhibition, that resulted modestly. Docking studies in the catalytic kinase domain of BRAF V600E , in fact, underline the capability of quinoline 67 to form additional interactions right through its urea moiety; furthermore, the short amine spacer inhibited the insertion of the 4-chloro-3-(trifluoromethyl)phenyl group within the hydrophobic allosteric site adjacent the ATP binding site, precluding some stabilizing hydrophobic interactions [142].  Figure 37) with urea and a 4-fluoro-3-(trifluoromethyl)phenyl moieties, as sorafenib. It exhibited higher antiproliferative activity than the reference compound, against Hep G2, A549 and KCC-853 cancer cell lines and also a higher inhibitory activity against C-Raf (for 69 C-Raf IC50 = 8.7 nM; for sorafenib C-Raf IC50 = 28.5 nM) [143]. Considering 67 as lead compound, the same authors, by the substitution of the urea linker with an amine one, developed a new series of 2-anilinoquinolines bearing the N-methylpicolinamide group at the C-5 position of the quinoline scaffold. The derivative 4-chloro-3-(trifluoromethyl)aniline 68 (Figure 36a) manifested more remarkable antiproliferative effects than compound 67 and sorafenib, especially against melanoma and breast cancer panels. However, the replacement of the urea linker with an amine in compound 68 has a negative effect on Raf kinase inhibition, that resulted modestly. Docking studies in the catalytic kinase domain of BRAF V600E , in fact, underline the capability of quinoline 67 to form additional interactions right through its urea moiety; furthermore, the short amine spacer inhibited the insertion of the 4-chloro-3-(trifluoromethyl)phenyl group within the hydrophobic allosteric site adjacent the ATP binding site, precluding some stabilizing hydrophobic interactions [142].
Li et al. developed quinoline derivative 69 ( Figure 37) with urea and a 4-fluoro-3-(trifluoromethyl) phenyl moieties, as sorafenib. It exhibited higher antiproliferative activity than the reference compound, against Hep G2, A549 and KCC-853 cancer cell lines and also a higher inhibitory activity against C-Raf (for 69 C-Raf IC 50 = 8.7 nM; for sorafenib C-Raf IC 50 = 28.5 nM) [143]. In the Ras/Raf/MEK signalling cascade, MEK has been also identified as an interesting target for quinoline-based agents able to inhibit its catalytic activity.
In order to improve the solubility and the oral bioavailability, the same authors further developed molecule 74, with an alkenyl group at C-7 of the quinoline core; this compound demonstrated good in vitro potency (MEK1 IC50 = 12 nM) and high plasma levels after oral dosing in H358 xenograft models [147]. In the Ras/Raf/MEK signalling cascade, MEK has been also identified as an interesting target for quinoline-based agents able to inhibit its catalytic activity.
In order to improve the solubility and the oral bioavailability, the same authors further developed molecule 74, with an alkenyl group at C-7 of the quinoline core; this compound demonstrated good in vitro potency (MEK1 IC 50 = 12 nM) and high plasma levels after oral dosing in H358 xenograft models [147].
Polycondensed quinoline systems, as the 1H-imidazo[4,5-c]quinoline 75 ( Figure 39) show interesting results in MEK kinase inhibition assays, with IC50 values lower than 100 μM; it was demonstrated that the high selectivity for MEK over other kinases depends on the -F substituent at the C-7 position of the quinoline nucleus [148]. An interesting derivative of ursolic acid possessing a condensed chloro-substituted quinoline nucleus and a hydrazide moiety was developed recently by Jin et al. (76 in Figure 40). Compound 76 showed remarkable results as antiproliferative agent, capability to inhibit MEK1 kinase activity (MEK1 IC50 = 64 nM) and activation of Ras/Raf/MEK/ERK pathway. Molecular docking analysis into the MEK1 binding site proved that the ursolic acid skeleton ensured the correct orientation of the hydrazide side chain and of the quinoline ring into the pocket, while the latter were involved in stabilizing interactions with the amino acids [149]. An interesting derivative of ursolic acid possessing a condensed chloro-substituted quinoline nucleus and a hydrazide moiety was developed recently by Jin et al. (76 in Figure 40). Compound 76 showed remarkable results as antiproliferative agent, capability to inhibit MEK1 kinase activity (MEK1 IC 50 = 64 nM) and activation of Ras/Raf/MEK/ERK pathway. Molecular docking analysis into the MEK1 binding site proved that the ursolic acid skeleton ensured the correct orientation of the hydrazide side chain and of the quinoline ring into the pocket, while the latter were involved in stabilizing interactions with the amino acids [149].

Conclusions
The quinoline ring system is a scaffold highly frequent in druggable molecules. Currently, a great amount of quinoline derivatives exhibit pharmaceutical activity as antibacterial, antifungal, antimalarial, anthelmintic, local anaesthetic, antipsychotic, and anticancer drugs.
In the anticancer field, the quinoline nucleus has been observed as crucial molecular moiety

Conclusions
The quinoline ring system is a scaffold highly frequent in druggable molecules. Currently, a great amount of quinoline derivatives exhibit pharmaceutical activity as antibacterial, antifungal, antimalarial, anthelmintic, local anaesthetic, antipsychotic, and anticancer drugs.
In the anticancer field, the quinoline nucleus has been observed as crucial molecular moiety recurrent in several inhibitors of kinases, for the treatments of a wide range of tumours in the targeted therapeutic approach.
In particular, this review summarizes the biological data of several quinoline compounds, which are active on c-Met, VEGF, and EGF receptors, and on the related proteins involved in the intracellular signalling cascades. Overexpressed in tumour cells, these three protein kinases receptors trigger carcinogenic pathways closely connected with each other, regulating the survival processes in the cell, such as proliferation, apoptosis, differentiation, and angiogenesis.
The druggable attitude of the quinoline scaffold probably lies on its proved biocompatibility, versatility, and chemical accessibility, from which new derivatives can be easily designed and synthesized.
The quinoline nucleus is an electron-deficient ring system with tertiary base properties. The presence of nitrogen withdraws electrons by resonance, interfering with the equal distribution of the π-electron density and suggesting a chemical behaviour similar to the pyridine.
In view of an SAR study, the analysis of the interactions of the quinoline molecules in the protein binding sites, under investigation, highlights recurrent hydrogen bonds with the nitrogen of the quinoline ring and π-π stacking complexes with complementary amino acid residues. The presence of flexible moiety, especially at C-4, C-6, and C-7 positions, frequently consolidates the force of the ligand-protein complexes.
All the biological data of the quinoline compounds, analysed in the present review, are reported in detail, confirming the antiproliferative activity and the pivotal importance of this ring system in the efficacy of several approved drugs. In drug discovery, the information recovered in this manuscript could help in the design and rationale optimization studies for the development of new quinoline multitargets molecules.