Optimization of Aminoimidazole Derivatives as Src Family Kinase Inhibitors

Protein kinases have emerged as crucial targets for cancer therapy over the last decades. Since 2001, 40 and 39 kinase inhibitors have been approved by FDA and EMA, respectively, and the majority are antineoplastic drugs. Morevoer, many candidates are currently in clinical trials. We previously reported a small library of 4-aminoimidazole and 2-aminothiazole derivatives active as Src family kinase (SFK) inhibitors. Starting from these results, we decided to perform an optimization study applying a mix and match strategy to identify a more potent generation of 4-aminoimidazoles. Firstly, a computational study has been performed, then compounds showing the best predicted docking scores were synthesized and screened in a cell-free assay for their SFK inhibitory activity. All the new chemical entities showed IC50s in the nanomolar range, with 2–130 fold increased activities compared to the previously reported inhibitors. Finally, the most active compounds have been tested on three cancer cell lines characterized by Src hyperactivation. Compounds 4k and 4l showed an interesting antiproliferative activity on SH-SY5Y neuroblastoma (NB) cell line. In this assay, the compounds resulted more potent than dasatinib, a tyrosine kinase inhibitor approved for the treatment of leukemias and in clinical trials for NB.


Introduction
Protein kinases are a large class of enzymes (to date 518 members have been identified) which are involved in different phases of the cell life [1]. These proteins are overexpressed and/or hyperactivated in several diseases, including cancer, neurodegenerative disorders and inflammation [2]. For this reason, protein kinases have become a thoroughly studied target in medicinal chemistry and, to date, 40 and 39 kinase inhibitors have been approved by the FDA and EMA, respectively, and the majority are antineoplastic drugs [3].
TKs are further divided in two main families: receptor tyrosine kinases and non-receptor tyrosine kinases. Into the latter, Src family kinases (SFKs) are the biggest subfamily of enzymes. This class includes nine highly homologous members, i.e., Src, Fyn, Yes, Blk, Yrk, Fgr, Hck, Lck, and Lyn. All SFKs present a common structure characterized by a N-terminal Src homology domain (SH4), a 'unique' region-different among SFK members, two Src homology domains (SH2 and SH3), a catalytic domain (SH1), and a short C-terminal tail. The catalytic domain constitutes the core of the enzyme since it possesses the kinase activity. SH1 presents a bilobal structure, with a small Nterminal lobe and a large C-terminal lobe, linked by a flexible chain, named hinge region [5]. At the interlobe cleft there are the ATP-and substrate-binding sites. Under basal conditions, SFKs present a close and inactive conformation that prevents ATP and substrate binding. After phosphorylation of a specific tyrosine residue (Tyr419 in human Src) in the C-terminal lobe by upstream kinases, a structural rearrangement occurs and results in a flip to an open and active conformation. At this point, the enzyme is able to bind ATP and the opportune peptide substrate, that will be phosphorylated [6,7]. SFKs are involved in the regulation of different signal transduction pathways such as growth, proliferation, differentiation, migration, metabolism, and apoptosis and, as reported for kinases in general, their overexpression and/or hyperactivation have been shown in many types of tumors [8,9]. Interestingly, despite the high homology among SFK members, different pathological implications have been detected inside the family. For instance, Fyn plays a key role in brain pathologies, such as Alzheimer's disease [10], whereas Hck, Fgr, and Lyn are the main SFK members involved in inflammation [11].
To date, three molecules active as SFK inhibitors have been approved for clinical use, i.e., dasatinib, bosutinib, and ponatinib ( Figure 1) [3,12,13]. Anyway, these compounds are not selective SFK inhibitors, but also inhibit other kinases. Dasatinib (Sprycel ® , Bristol-Myers Squibb, approved in 2006), and bosutinib (Bosulif ® , Wyeth, approved in 2012), are two dual Src/Bcr-Abl (another nonreceptor TK) inhibitors which bind the enzymes in their active conformation. Dasatinib potently inhibits all nine members of SFKs [14] and, in detail, a KINOMEscan analysis published by Davis et al. in 2011 [15] showed that it possesses Kd values of 0.21, 0.30, 0.53, and 0.79 nM on c-Src, Yes, Lyn, and Fyn, respectively. The compound also inhibits other TKs, including Kit, PDGFR, ephrin A receptor kinase, and the Tec kinase Btk [16]. Dasatinib has been approved for the treatment of chronic myeloid leukemia (CML) and acute lymphoblastic leukemia Philadelphia chromosome-positive (ALL Ph+), and is currently in clinical trials for NB and other solid tumors [17]. NB is an extracranial solid tumor of childhood often characterized by poor prognosis and for which an effective treatment still lacks. Bosutinib has been approved for the treatment of CML Ph+ with resistance or intolerance to prior therapy, and in December 2017, the FDA granted accelerated approval as first line therapy [18]. Ponatinib (Iclusig ® , Ariad Pharmaceuticals, approved in 2012) is a so-called "pan-kinase" inhibitor, since it is active against many kinases, including SFKs, Bcr-Abl, VEGFR, PDGFR, and Ret. Ponatinib binds Src and Bcr-Abl in their inactive conformation. It has been approved for CML or ALL Ph+, in particular for resistant T315I-positive CML or T315I-positive, Ph+ ALL [19]. Although the majority of protein kinase inhibitors occupy the ATP binding site, few compounds are able to bind pockets that are far from the catalytic cleft and act as allosteric inhibitors [20]. The complexity in regulating kinases' activity offers many potential routes for pursuing their inhibition Although the majority of protein kinase inhibitors occupy the ATP binding site, few compounds are able to bind pockets that are far from the catalytic cleft and act as allosteric inhibitors [20]. The complexity in regulating kinases' activity offers many potential routes for pursuing their inhibition and, despite the initial concerns about the possibility to identify potent and selective kinases inhibitors, significant advances have been made over the past two decades [21][22][23].
As widely reported in the literature, kinase inhibitors usually have heteroaromatic scaffolds capable of interacting with the hinge region [8]. Therefore, the synthesis of new hinge interacting moieties is crucial to obtain new molecules that may be selective for one or a few kinases and endowed with a high inhibitory potency. Zhang et al. described in detail the binding site of different kinase inhibitors and showed many heterocyclic ring systems that occupy the purine binding site [24]. The strength of the binding between these heterocyclic moieties and the target kinase is due to electrostatic interactions; in particular, some hinge binders were designed to establish one to three hydrogen bonds to the hinge region.
From an extensive literature study, the 4-aminoimidazole and the 2-aminothiazole rings emerged as interesting starting entities for the development of new ATP pocket binders. The 4-aminoimidazole moiety has been first explored by AstraZeneca [25], who highlighted the ability of this scaffold to bind the Janus kinase hinge region. The authors compared their clinical candidate AZD1480 (a Jak2 inhibitor with an IC 50 of 58 nM, Figure 2), containing the 3-aminopyrazole moiety, with the related 4-aminoimidazole derivative (IC 50 value of 120 nM towards Jak2), and found that this bioisosteric substitution could be an effective replacement for the 3-aminopyrazole ring. The subsequent modulation of this first hit led to the discovery of the potent and orally bioavailable Jak2 inhibitor 1 (IC 50 < 3 nM, Figure 2) [25].
Molecules 2018, 23, x FOR PEER REVIEW  3 of 17 and, despite the initial concerns about the possibility to identify potent and selective kinases inhibitors, significant advances have been made over the past two decades [21][22][23].
As widely reported in the literature, kinase inhibitors usually have heteroaromatic scaffolds capable of interacting with the hinge region [8]. Therefore, the synthesis of new hinge interacting moieties is crucial to obtain new molecules that may be selective for one or a few kinases and endowed with a high inhibitory potency. Zhang et al. described in detail the binding site of different kinase inhibitors and showed many heterocyclic ring systems that occupy the purine binding site [24]. The strength of the binding between these heterocyclic moieties and the target kinase is due to electrostatic interactions; in particular, some hinge binders were designed to establish one to three hydrogen bonds to the hinge region.
From an extensive literature study, the 4-aminoimidazole and the 2-aminothiazole rings emerged as interesting starting entities for the development of new ATP pocket binders. The 4aminoimidazole moiety has been first explored by AstraZeneca [25], who highlighted the ability of this scaffold to bind the Janus kinase hinge region. The authors compared their clinical candidate AZD1480 (a Jak2 inhibitor with an IC50 of 58 nM, Figure 2), containing the 3-aminopyrazole moiety, with the related 4-aminoimidazole derivative (IC50 value of 120 nM towards Jak2), and found that this bioisosteric substitution could be an effective replacement for the 3-aminopyrazole ring. The subsequent modulation of this first hit led to the discovery of the potent and orally bioavailable Jak2 inhibitor 1 (IC50 < 3 nM, Figure 2) [25]. On the other hand, the 2-aminothiazole represents the hinge binder moiety of dasatinib, the potent SFK inhibitor already reported.
In a previous work, we described the synthesis and the biological evaluation of a set of 4aminoimidazole and 2-aminothiazole derivatives as SFK inhibitors [26]. The 4-aminoimidazole ring was demonstrated to be an effective hinge binder for c-Src kinase, suggesting that this moiety, properly functionalized, is a good replacement for the aminothiazole ring [26]. Indeed, the most active compound 2 ( Figure 3) showed IC50 values of 220, 689, 1300, and 167 nM for the isolated enzymes Src, Fyn, Lyn, and Yes, respectively (Table 1), and resulted active on SH-SY5Y neuroblastoma (NB) cell line with an IC50 of 25 μM. On the other hand, 2 showed a weak activity on K562 CML cell line, possessing an IC50 > 25 μM. As a continuation of our work, herein we present an optimization study aimed at obtaining a new generation of (1H-imidazol-4-yl)-pyrimidin-4-ylamines endowed with a higher affinity towards SFKs and a stronger activity on cells compared to compounds previously reported by us. On the other hand, the 2-aminothiazole represents the hinge binder moiety of dasatinib, the potent SFK inhibitor already reported.
In a previous work, we described the synthesis and the biological evaluation of a set of 4-aminoimidazole and 2-aminothiazole derivatives as SFK inhibitors [26]. The 4-aminoimidazole ring was demonstrated to be an effective hinge binder for c-Src kinase, suggesting that this moiety, properly functionalized, is a good replacement for the aminothiazole ring [26]. Indeed, the most active compound 2 ( Figure 3) showed IC 50 values of 220, 689, 1300, and 167 nM for the isolated enzymes Src, Fyn, Lyn, and Yes, respectively (Table 1), and resulted active on SH-SY5Y neuroblastoma (NB) cell line with an IC 50 of 25 µM. On the other hand, 2 showed a weak activity on K562 CML cell line, possessing an IC 50 > 25 µM. As a continuation of our work, herein we present an optimization study aimed at obtaining a new generation of (1H-imidazol-4-yl)-pyrimidin-4-yl-amines endowed with a higher affinity towards SFKs and a stronger activity on cells compared to compounds previously reported by us.

Docking Studies
Starting from our hit 2 [26] and 3 (a potent Src inhibitor) [27], a mix and match strategy combined with the use of computational tools has been applied for the identification of new chemical entities (NCEs) acting as ATP pocket binders ( Figure 3). As a first step towards a better understanding of the molecular determinants for the inhibitory activity of this class of compounds against Src, a molecular docking simulation has been performed on NCEs and the upcoming results were compared with the best aminoimidazole hit 2 reported in our previous work [26]. In particular, more than one hundred NCEs were designed and docked into the ATP binding site of Src by using the 3G5D X-ray structure [28]. The mix and match strategy has been applied in order to (i) introduce different alkyl groups as linkers to investigate the steric hindrance allowed around the hinge region; (ii) introduce hydrophobic and hydrophilic moieties interacting with the hydrophobic region I (HRI) to improve the primary activity by achieving electrostatic interactions that would be missing with an unsubstituted phenyl group; (iii) replace the solvent exposed substituent with different types of heterocycles.

Docking Studies
Starting from our hit 2 [26] and 3 (a potent Src inhibitor) [27], a mix and match strategy combined with the use of computational tools has been applied for the identification of new chemical entities (NCEs) acting as ATP pocket binders ( Figure 3). As a first step towards a better understanding of the molecular determinants for the inhibitory activity of this class of compounds against Src, a molecular docking simulation has been performed on NCEs and the upcoming results were compared with the best aminoimidazole hit 2 reported in our previous work [26]. In particular, more than one hundred NCEs were designed and docked into the ATP binding site of Src by using the 3G5D X-ray structure [28]. The mix and match strategy has been applied in order to (i) introduce different alkyl groups as linkers to investigate the steric hindrance allowed around the hinge region; (ii) introduce hydrophobic and hydrophilic moieties interacting with the hydrophobic region I (HRI) to improve the primary activity by achieving electrostatic interactions that would be missing with an unsubstituted phenyl group; (iii) replace the solvent exposed substituent with different types of heterocycles. Docking studies were performed by means of Glide [29] software and the reliability of the applied protocol was first assessed by reproducing the experimental binding mode of two known inhibitors of Src: dasatinib (PDB code: 3G5D) [28] and CGP77675 (PDB code: 1YOL [30], Figure 2). Compounds were drawn, minimized and finally docked into the ATP-binding site of Src (3G5D) [28].  Docking studies were performed by means of Glide [29] software and the reliability of the applied protocol was first assessed by reproducing the experimental binding mode of two known inhibitors of Src: dasatinib (PDB code: 3G5D) [28] and CGP77675 (PDB code: 1YOL [30], Figure 2). Compounds were drawn, minimized and finally docked into the ATP-binding site of Src (3G5D) [28].
As a result, the program was able to reproduce the experimental poses of the two compounds with a RMSD of 0.55 Å suggesting that the docking procedure could be reliable to predict the binding mode of our NCEs ( Figure 4).

Docking Studies
Starting from our hit 2 [26] and 3 (a potent Src inhibitor) [27], a mix and match strategy combined with the use of computational tools has been applied for the identification of new chemical entities (NCEs) acting as ATP pocket binders ( Figure 3). As a first step towards a better understanding of the molecular determinants for the inhibitory activity of this class of compounds against Src, a molecular docking simulation has been performed on NCEs and the upcoming results were compared with the best aminoimidazole hit 2 reported in our previous work [26]. In particular, more than one hundred NCEs were designed and docked into the ATP binding site of Src by using the 3G5D X-ray structure [28]. The mix and match strategy has been applied in order to (i) introduce different alkyl groups as linkers to investigate the steric hindrance allowed around the hinge region; (ii) introduce hydrophobic and hydrophilic moieties interacting with the hydrophobic region I (HRI) to improve the primary activity by achieving electrostatic interactions that would be missing with an unsubstituted phenyl group; (iii) replace the solvent exposed substituent with different types of heterocycles. Docking studies were performed by means of Glide [29] software and the reliability of the applied protocol was first assessed by reproducing the experimental binding mode of two known inhibitors of Src: dasatinib (PDB code: 3G5D) [28] and CGP77675 (PDB code: 1YOL [30], Figure 2). Compounds were drawn, minimized and finally docked into the ATP-binding site of Src (3G5D) [28].  Compounds 4a-g (Table 1), showing the best predicted docking scores, are characterized by a series of polar moieties in the solvent exposed region, and hydroxyl or methoxyl groups on the phenyl ring of the N-1-(2-phenylethyl)-1H-imidazol-4-yl side chain. These substitution patterns have been selected with the aim of improving the water solubility and getting further insights into this class of inhibitors. All compounds showed a similar interaction pattern characterized by two hydrogen bonds involving the imidazole nucleus and the hinge region: one between the N3 and the NH backbone of Met341, and one between the 4-NH and the CO backbone of Met341. The phenyl ring was located into the HR1, forming Van der Waals interactions with hydrophobic amino acids of this region. Moreover, the new N-[1-(2-phenylethyl)-1H-imidazol-4-yl]pyrimidinamines interacted with two different residues in HR1 and in solvent exposed area. The best compounds in terms of docking scores show a meta or ortho hydroxyl group on the phenyl ring and an amide, ester, or carbamate group in N4 position of the piperazine chain. In detail, the pose example of compound 4j (GB = −11.33 kcal/mol) has been reported in Figure 5: the meta hydroxyl group acts as both H-bond donor and acceptor in the interactions with Glu310 and Asp464 respectively. The ortho substituted derivative 4g (GB = −11.23 kcal/mol) establishes a hydrogen bond interaction with Asp464 belonging to the DFG motif.
with a RMSD of 0.55 Å suggesting that the docking procedure could be reliable to predict the binding mode of our NCEs ( Figure 4).
Compounds 4a-g (Table 1), showing the best predicted docking scores, are characterized by a series of polar moieties in the solvent exposed region, and hydroxyl or methoxyl groups on the phenyl ring of the N-1-(2-phenylethyl)-1H-imidazol-4-yl side chain. These substitution patterns have been selected with the aim of improving the water solubility and getting further insights into this class of inhibitors. All compounds showed a similar interaction pattern characterized by two hydrogen bonds involving the imidazole nucleus and the hinge region: one between the N3 and the NH backbone of Met341, and one between the 4-NH and the CO backbone of Met341. The phenyl ring was located into the HR1, forming Van der Waals interactions with hydrophobic amino acids of this region. Moreover, the new N-[1-(2-phenylethyl)-1H-imidazol-4-yl]pyrimidinamines interacted with two different residues in HR1 and in solvent exposed area. The best compounds in terms of docking scores show a meta or ortho hydroxyl group on the phenyl ring and an amide, ester, or carbamate group in N4 position of the piperazine chain. In detail, the pose example of compound 4j (GB = −11.33 kcal/mol) has been reported in Figure 5: the meta hydroxyl group acts as both H-bond donor and acceptor in the interactions with Glu310 and Asp464 respectively. The ortho substituted derivative 4g (GB = −11.23 kcal/mol) establishes a hydrogen bond interaction with Asp464 belonging to the DFG motif.    phenyl ring of the N-1-(2-phenylethyl)-1H-imidazol-4-yl side chain. These substitution patterns have been selected with the aim of improving the water solubility and getting further insights into this class of inhibitors. All compounds showed a similar interaction pattern characterized by two hydrogen bonds involving the imidazole nucleus and the hinge region: one between the N3 and the NH backbone of Met341, and one between the 4-NH and the CO backbone of Met341. The phenyl ring was located into the HR1, forming Van der Waals interactions with hydrophobic amino acids of this region. Moreover,

the new N-[1-(2-phenylethyl)-1H-imidazol-4-yl]pyrimidinamines interacted
with two different residues in HR1 and in solvent exposed area. The best compounds in terms of docking scores show a meta or ortho hydroxyl group on the phenyl ring and an amide, ester, or carbamate group in N4 position of the piperazine chain. In detail, the pose example of compound 4j (GB = −11.33 kcal/mol) has been reported in Figure 5: the meta hydroxyl group acts as both H-bond donor and acceptor in the interactions with Glu310 and Asp464 respectively. The ortho substituted derivative 4g (GB = −11.23 kcal/mol) establishes a hydrogen bond interaction with Asp464 belonging to the DFG motif.  -3OH -COOtert-butyl −10.99 50 ± 3 14 ± 1.5 26 ± 3 7 ± 0.35 a The compound was tested in two independent experiments, and IC 50 values are the mean ± SD. b Dasatinb was used as reference; the IC 50 values of dasatinib were less than that of enzyme concentrations, which were 4, 9, 0.9, and 3 nm for Src, Fyn, Lyn, and Yes, respectively.
As shown in Table 1, compounds 4g and 4j, having the highest values of docking score, are predicted to be the most active compounds on the selected kinase, while compounds 4b and 4d (−7.266 and −8.336 kcal/mol respectively) resulted as the least active ones.

Chemistry
The best predicted derivatives, in respect to our previous reported hit 2, were selected to be synthesized and tested (Table 1). 4b and 4d were also prepared as negative controls in enzymatic assays. Compounds 4a-d, bearing a hydrophobic group -OMe exposed to HR1, were first synthesized (Scheme 1) [26]. The commercially available 4-nitro-1H-imidazole 5 was regioselectively functionalized using opportune alkylating agents to give intermediates 6a,b in high purity and yield [26,31]. Subsequent palladium mediated hydrogenation of 6a,b afforded the corresponding amino derivatives as free bases, which were immediately converted to hydrochloride salts, since the compounds are unstable as free bases, to yield derivatives 7a,b. Compounds 7a,b were regioselectively coupled with the commercially available 4,6-dichloro-2-methylpyrimidine at 50 • C in the presence of N,N-diisopropylethylamine (DIPEA) to afford intermediates 8a,b.
Dasatinb was used as reference; the IC50 values of dasatinib were less than that of enzyme concentrations, which were 4, 9, 0.9, and 3 nm for Src, Fyn, Lyn, and Yes, respectively.
As shown in Table 1, compounds 4g and 4j, having the highest values of docking score, are predicted to be the most active compounds on the selected kinase, while compounds 4b and 4d (−7.266 and −8.336 kcal/mol respectively) resulted as the least active ones.

Chemistry
The best predicted derivatives, in respect to our previous reported hit 2, were selected to be synthesized and tested (Table 1). 4b and 4d were also prepared as negative controls in enzymatic assays. Compounds 4a-d, bearing a hydrophobic group -OMe exposed to HR1, were first synthesized (Scheme 1) [26]. The commercially available 4-nitro-1H-imidazole 5 was regioselectively functionalized using opportune alkylating agents to give intermediates 6a,b in high purity and yield [26,31]. Subsequent palladium mediated hydrogenation of 6a,b afforded the corresponding amino derivatives as free bases, which were immediately converted to hydrochloride salts, since the compounds are unstable as free bases, to yield derivatives 7a,b. Compounds 7a,b were regioselectively coupled with the commercially available 4,6-dichloro-2-methylpyrimidine at 50 °C in the presence of N,N-diisopropylethylamine (DIPEA) to afford intermediates 8a,b. The intermediates 8a,b were reacted with the appropriate piperazines at 110 • C under microwave irradiation in the presence of DIPEA to afford the final compounds 4a,c and the intermediates 9a-c, used for the synthesis of phenol derivatives 4f,i,l described in the Scheme 2. Furthermore, 9a,b were reacted with methyl bromoacetate to obtain intermediates 10a,b, that were treated with ammonia 7N in MeOH to yield the final compounds 4b,d, according to the procedure used by Novartis (Scheme 1) [27].
Furthermore, 9a,b were reacted with methyl bromoacetate to obtain intermediates 10a,b, that were treated with ammonia 7N in MeOH to yield the final compounds 4b,d, according to the procedure used by Novartis (Scheme 1) [27].

Enzymatic Assays
Compounds 4a-l have been tested against the isolated Src enzyme and showed IC50 values in the nanomolar range. Derivatives 4e-l were found more active on Src than the previous reported hit 2, showing IC50 values from 93 nM to 40 nM. In particular, compounds 4g, 4j and 4k, bearing a hydroxyl group in the ortho or meta positions of the phenyl ring and an amide or methylester substituent as side chain, resulted to have the highest inhibitory activity (IC50 values of 40 nM). On the other hand, the methoxy derivatives 4a-d are less potent on Src (IC50 values 225-1533 nM) compared with the phenolic derivatives, confirming the importance of the hydroxyl group, as predicted by modeling studies. In addition, all new compounds were tested for their activity against other members of SFKs. As expected, the most promising compounds were also potent inhibitors of Yes, Lyn, and Fyn with IC50 values in the range 3-73 nM. These results confirmed the hypothesis that the 4-aminoimidazole template, properly decorated, is an effective hinge binder for SFKs and has a good/high in vitro potency on these enzymes.

Cellular Assays
Starting from these promising results in enzymatic assays, we decided to test NCEs 4 on K562 CML and SH-SY5Y NB cell lines, to evaluate if they are endowed with an increased antiproliferative activity compared with the hit compound 2. A hyperactivation of SFKs has been detected in both K562 and SH-SY5Y cell lines [9,32,33]. Cells were treated with increasing concentrations of compounds and cell proliferation was measured by counting viable cells after 72 h of incubation. Dasatinib and 2 were used as reference compounds. In Figure 6 we show the activity of 4k and 4l that demonstrated, in comparison with the other NCEs (see Supplementary Materials, Figure S1), the best antiproliferative activity on SH-SY5Y cells. In detail, 4k and 4l possess IC50 values of 8.6 and 7.8 μM, respectively, and show a more than 2-fold increased activity compared to the hit compound 2. Importantly, in NB cells, 4k and 4l exerted an antiproliferative effect similar or higher than dasatinib. The activity of these compounds could be due not only to Src inhibition, but also to their effect on Fyn and Lyn, both involved in NB development [34]. Furthermore, both the compounds showed a similar activity on K562 cells, with an antiproliferative effect comparable with the one observed on NB cells. In fact, compounds 4k and 4l show IC50 values of 11.7 and 18.9 μM, respectively, and are more active than 2, but less active than dasatinib ( Figure 6).

Enzymatic Assays
Compounds 4a-l have been tested against the isolated Src enzyme and showed IC 50 values in the nanomolar range. Derivatives 4e-l were found more active on Src than the previous reported hit 2, showing IC 50 values from 93 nM to 40 nM. In particular, compounds 4g, 4j and 4k, bearing a hydroxyl group in the ortho or meta positions of the phenyl ring and an amide or methylester substituent as side chain, resulted to have the highest inhibitory activity (IC 50 values of 40 nM). On the other hand, the methoxy derivatives 4a-d are less potent on Src (IC 50 values 225-1533 nM) compared with the phenolic derivatives, confirming the importance of the hydroxyl group, as predicted by modeling studies. In addition, all new compounds were tested for their activity against other members of SFKs. As expected, the most promising compounds were also potent inhibitors of Yes, Lyn, and Fyn with IC 50 values in the range 3-73 nM. These results confirmed the hypothesis that the 4-aminoimidazole template, properly decorated, is an effective hinge binder for SFKs and has a good/high in vitro potency on these enzymes.

Cellular Assays
Starting from these promising results in enzymatic assays, we decided to test NCEs 4 on K562 CML and SH-SY5Y NB cell lines, to evaluate if they are endowed with an increased antiproliferative activity compared with the hit compound 2. A hyperactivation of SFKs has been detected in both K562 and SH-SY5Y cell lines [9,32,33]. Cells were treated with increasing concentrations of compounds and cell proliferation was measured by counting viable cells after 72 h of incubation. Dasatinib and 2 were used as reference compounds. In Figure 6 we show the activity of 4k and 4l that demonstrated, in comparison with the other NCEs (see Supplementary Materials, Figure S1), the best antiproliferative activity on SH-SY5Y cells. In detail, 4k and 4l possess IC 50 values of 8.6 and 7.8 µM, respectively, and show a more than 2-fold increased activity compared to the hit compound 2. Importantly, in NB cells, 4k and 4l exerted an antiproliferative effect similar or higher than dasatinib. The activity of these compounds could be due not only to Src inhibition, but also to their effect on Fyn and Lyn, both involved in NB development [34]. Furthermore, both the compounds showed a similar activity on K562 cells, with an antiproliferative effect comparable with the one observed on NB cells. In fact, compounds 4k and 4l show IC 50 values of 11.7 and 18.9 µM, respectively, and are more active than 2, but less active than dasatinib ( Figure 6).
On the basis of the exciting activity of 4k and 4l on NB cell lines in comparison with dasatinib, we decided to test NCEs also on U87 glioblastoma multiforme (GBM), another tumor characterized by Src hyperactivation (Figure 6) [35]. Compounds 4k and 4l showed IC 50 s of 12.6 and 13.3, respectively, but, unfortunately, resulted less active than dasatinib. A possible explanation could be the multidrug resistance mechanisms that GBM cells usually carry: an example is the overexpression of membrane channels (ABCB1) that are able to pump different kind of drugs out of the cells [36]. In Supplementary Materials, Figure S1 On the basis of the exciting activity of 4k and 4l on NB cell lines in comparison with dasatinib, we decided to test NCEs also on U87 glioblastoma multiforme (GBM), another tumor characterized by Src hyperactivation (Figure 6) [35]. Compounds 4k and 4l showed IC50s of 12.6 and 13.3, respectively, but, unfortunately, resulted less active than dasatinib. A possible explanation could be the multidrug resistance mechanisms that GBM cells usually carry: an example is the overexpression of membrane channels (ABCB1) that are able to pump different kind of drugs out of the cells [36]. In Supplementary Materials, Figure S1, the activity of other NCEs on U87 GBM cell line are reported.
In conclusion, a small library of aminoimidazole derivatives was synthesized and screened in a cell-free assay for their SFK inhibitory activity. Enzymatic assays showed an increase in potency against isolated Src (from a micromolar range of our previously reported compounds [13] to nanomolar of the new molecules), with an exceptional increase in potency also against other SFK members. Furthermore, the most active inhibitors have been tested on three different cancer cell lines, i.e., NB, GBM, and CML cell lines. Interestingly, compounds 4k and 4l showed good antiproliferative activity in the SH-SY5Y NB cell line. In this assay the compounds resulted more potent than dasatinib, a TKI inhibitor which is currently in clinical trials for NB [17].
Further studies on this class of compounds will be focused on the improvement of the ADME properties, with the aim of obtaining more potent compounds in cell assays.

Protein Preparation
Crystal structures of c-Src in complex with dasatinib and CGP77675 (PDB IDs:3G5D [28] and 1YOL [30]), were retrieved from the RCSB Protein Data Bank. After removal of bound ligands, the proteins were prepared by using the Protein Preparation Wizard [37] workflow (Schrodinger Suite). In particular, all water molecules were deleted, hydrogen atoms were added, and partial charges assigned. In addition, the ionization and tautomeric states of His, Asp, Glu, Arg, and Lys were adjusted to match pH 7.4. Next, optimization of the hydrogen bonding network was obtained by reorienting hydroxyl and thiol groups, amide groups of Asn and Gln, and the His imidazole ring. Finally, the systems were refined by running a restrained minimization (OPLS3 force field) which was stopped when the RMSD of heavy atoms reached 0.30 Å , the default limit. In conclusion, a small library of aminoimidazole derivatives was synthesized and screened in a cell-free assay for their SFK inhibitory activity. Enzymatic assays showed an increase in potency against isolated Src (from a micromolar range of our previously reported compounds [13] to nanomolar of the new molecules), with an exceptional increase in potency also against other SFK members. Furthermore, the most active inhibitors have been tested on three different cancer cell lines, i.e., NB, GBM, and CML cell lines. Interestingly, compounds 4k and 4l showed good antiproliferative activity in the SH-SY5Y NB cell line. In this assay the compounds resulted more potent than dasatinib, a TKI inhibitor which is currently in clinical trials for NB [17].

Ligands Preparation
Further studies on this class of compounds will be focused on the improvement of the ADME properties, with the aim of obtaining more potent compounds in cell assays.

Protein Preparation
Crystal structures of c-Src in complex with dasatinib and CGP77675 (PDB IDs:3G5D [28] and 1YOL [30]), were retrieved from the RCSB Protein Data Bank. After removal of bound ligands, the proteins were prepared by using the Protein Preparation Wizard [37] workflow (Schrodinger Suite). In particular, all water molecules were deleted, hydrogen atoms were added, and partial charges assigned. In addition, the ionization and tautomeric states of His, Asp, Glu, Arg, and Lys were adjusted to match pH 7.4. Next, optimization of the hydrogen bonding network was obtained by reorienting hydroxyl and thiol groups, amide groups of Asn and Gln, and the His imidazole ring. Finally, the systems were refined by running a restrained minimization (OPLS3 force field) which was stopped when the RMSD of heavy atoms reached 0.30 Å, the default limit.

Molecular Docking
Docking simulations were performed using the Glide program (Schrödinger, LLC, New York, NY, USA) [38,39] within the ATP binding site of Src (3G5D). A grid box of default size was centered on the X-ray ligand. No constraints were included during grid generation while rotation of the hydroxyl groups was allowed only for those residues closer to the ligand. After grid preparation, NCEs were flexibly docked and scored using the Glide standard-precision (SP) mode, treating the proteins as rigid. Docking experiments were performed using 0.80 factor to scale vdW radii of the nonpolar ligand atoms with a partial atomic charge of <0.15. Flash silica gel chromatography was performed on Biotage automatic flash chromatography systems (Uppsala, Sweden) (Isolera or SP1) using Biotage SNAP HP silica cartridges or Biotage SNAP KP-NH cartridges. Reverse phase chromatography was performed on a Biotage automatic flash chromatography system (Isolera) using Redisep Gold C-18Aq cartridges. Purification was also done by SPE with SCX cartridges. Reactions were monitored by thin-layer chromatography on 0.25 mm E. Merck silica gel plates (60F-254), visualized with UV light. Melting points (Mp) were determined with a Büchi B-540 apparatus.

Chemistry
Microwave irradiation experiments were conducted on a Biotage Initiator microwave reactor.
To obtain compounds 4a,c and 9c the column was eluted with 2CV of water + 0.1% AcOH and then the eluent was gradually changed to acetonitrile + 0.1% AcOH over 13CV. The fractions were combined and evaporated. The orange-brown residue was dissolved in DCM and treated with a saturated solution of sodium bicarbonate to avoid the acetic acid salt formation. Finally, the organic phase was dried, filtered and evaporated under vacuum affording the desired compounds.
To obtain compounds 9a,b, the column was eluted with 3CV of water + 0.1% AcOH and then the eluent was gradually changed to acetonitrile + 0.1% AcOH over 13CV. The fractions containing the desired product were combined and evaporated to obtain a brown residue. It was dissolved in MeOH and loaded onto a SPE-SCX (5 g) eluted first with methanol and then with ammonia in methanol (2N). Basic fractions were collected and evaporated affording the desired compounds. and Yes1, all human recombinant full-length proteins) according to a procedure already reported. Final results have been expressed as percent inhibition, and IC 50 values were calculated by non-linear curve fitting using GraphPad Prism software (version 6 for Windows). The inter-experimental variability of IC 50 values resulted within accepted limits of ±0.5 log units.

Cellular Assays
In vitro experiments were carried out using human neuroblastoma cell line SH-SY5Y, human glioblastoma cell line U-87, and human erythroleukemia cell line K-562. Cell lines were obtained from American Tissue Culture Collection (ATCC, SH-SY5Y CRL-2266; U-87 HTB-14; K-562 CCL-243). K-562 cells were cultured in RPMI medium with 10% FCS. SH-SY5Y and U-87 cells were cultured in DMEM medium with 10% FCS. In order to determine antiproliferative effect of NCE compounds cells were seeded at density of 5 × 10 4 cells/mL (K-562) or 10 × 10 4 cells/cm 2 (SH-SY5Y, U-87) and treated with increasing concentrations of NCE compounds. Control cells were treated with the vehicle of the experimental point containing the highest percentage of DMSO. Cell cultures were maintained at 37 • C in 5% v/v CO 2 for 72 h. Cell number and vitality were evaluated on cell suspension using the automatic cell counter NucleoCounter ® (Chemometec, Denmark). Results from the NucleoCounter represented either total or non-viable cell concentration, depending on the sample preparation indicated by the manufacturer. Each experiment was performed at least three times and results were expressed as mean and standard deviation.

Conflicts of Interest:
The authors declare no conflict of interest.