Identification of Novel Aryl Carboxamide Derivatives as Death-Associated Protein Kinase 1 (DAPK1) Inhibitors with Anti-Proliferative Activities: Design, Synthesis, In Vitro, and In Silico Biological Studies

Death-associated protein kinase 1 (DAPK1) is a serine/threonine protein kinase involved in diverse fundamental cellular processes such as apoptosis and autophagy. DAPK1 isoform plays an essential role as a tumor suppressor and inhibitor of metastasis. Consequently, DAPK1 became a promising target protein for developing new anti-cancer agents. In this work, we present the rational design and complete synthetic routes of a novel series of eighteen aryl carboxamide derivatives as potential DAPK1 inhibitors. Using a custom panel of forty-five kinases, a single dose of 10 µM of the picolinamide derivative 4a was able to selectively inhibit DAPK1 kinase by 44.19%. Further investigations revealed the isonicotinamide derivative 4q as a promising DAPK1 inhibitory lead compound with an IC50 value of 1.09 µM. In an in vitro anticancer activity assay using a library of 60 cancer cell lines including blood, lung, colon, CNS, skin, ovary, renal, prostate, and breast cancers, four compounds (4d, 4e, 4o, and 4p) demonstrated high anti-proliferative activity with mean % GI ~70%. Furthermore, the most potent DAPK1 inhibitor (4q) exhibited remarkable activity against leukemia (K-562) and breast cancer (MDA-MB-468) with % GI of 72% and 75%, respectively.


Introduction
Death-associated protein kinase 1 (DAPK1) (a Ca 2+ /calmodulin dependent Ser/Thr kinase) is an essential mediator in cell death and autophagy related signals [1,2]. It consists of 1430 residues and is the largest member in the DAPK protein family, including a Ca 2+ /CaM autoregulatory domain, a death domain, and a serine-rich C-terminal tail whose phosphorylation activity is known to be responsible for specific forms of apoptosis [3,4]. It coordinates cell-death signaling pathways in response to various stimuli such as death receptor activation, cytokines, matrix detachment, ceramide, ischemia, and glutamate toxicity [5]. As previously discussed in various studies, DAPK1 as a stress-responsive kinase is a crucial component that transmits ER stress signals into two distinct directions, caspase activation (via regulating type I apoptotic caspase-dependent cell death) and autophagy (by controlling type II autophagic caspase-independent cell death) [5][6][7][8][9][10][11][12][13].
Among novel effective approaches to hinder apoptosis pathways, some fusion proteins have been reported [14]. However, a peptide-based strategy has certain potential scientific and technical cautions, such as lack of cell selectivity, instability, as well as uncertainty of the effective therapeutic concentration, which influences the peptide cargo in addition to suffering from rapid degradation after administration orally [15]. Hence, the rational drug design of small molecule inhibitors could be a unique way to overcome such drawbacks. Although DAPK1 has gained a lot of interest regarding the comprehension of its functions, only a small number of chemical scaffolds, comprehensively discussed in our recent review [4], have been found in the literature with DAPK1 inhibitory activity, i.e., aminopyridazine [16,17], imidazo [1,2-b]pyridazine [18], pyridin-3-ylmethylene-1,3oxazol-5-one [19,20], pyrazolo [3,4-d]pyrimidinone [21,22], and 1H-pyrrolo [2,3-b]pyridine (7-azaindole) [23] (Figure 1). Inspired by the various unsolved issues of reported scaffolds, such as instability in biological systems, low potency, low selectivity profile, and/or insufficient toxicity studies, in addition to the absence of a current promising clinical candidate or an FDA-approved specific DAPK1 inhibitor, our institute has launched a project aimed at designing novel leads for DAPK1 activity modulation with potential anticancer activities. It coordinates cell-death signaling pathways in response to various stimuli such as death receptor activation, cytokines, matrix detachment, ceramide, ischemia, and glutamate toxicity [5]. As previously discussed in various studies, DAPK1 as a stress-responsive kinase is a crucial component that transmits ER stress signals into two distinct directions, caspase activation (via regulating type I apoptotic caspase-dependent cell death) and autophagy (by controlling type II autophagic caspase-independent cell death) [5][6][7][8][9][10][11][12][13]. Among novel effective approaches to hinder apoptosis pathways, some fusion proteins have been reported [14]. However, a peptide-based strategy has certain potential scientific and technical cautions, such as lack of cell selectivity, instability, as well as uncertainty of the effective therapeutic concentration, which influences the peptide cargo in addition to suffering from rapid degradation after administration orally [15]. Hence, the rational drug design of small molecule inhibitors could be a unique way to overcome such drawbacks. Although DAPK1 has gained a lot of interest regarding the comprehension of its functions, only a small number of chemical scaffolds, comprehensively discussed in our recent review [4], have been found in the literature with DAPK1 inhibitory activity, i.e., aminopyridazine [16,17], imidazo [1,2-b]pyridazine [18], pyridin-3-ylmethylene-1,3oxazol-5-one [19,20], pyrazolo [3,4-d]pyrimidinone [21,22], and 1H-pyrrolo [2,3-b]pyridine (7-azaindole) [23] (Figure 1). Inspired by the various unsolved issues of reported scaffolds, such as instability in biological systems, low potency, low selectivity profile, and/or insufficient toxicity studies, in addition to the absence of a current promising clinical candidate or an FDA-approved specific DAPK1 inhibitor, our institute has launched a project aimed at designing novel leads for DAPK1 activity modulation with potential anticancer activities. In previous studies, a structure-based virtual screening strategy was followed to develop an oxazol-5-one derivative III and its related analogues as DAPK inhibitors. Further in silico studies were conducted to define the structure-activity relationship (SAR) of the developed inhibitors [19,24]. The SAR studies stated that a nitrogen containing group, such as pyridinyl moiety, is essential for DAPK activity due to the role of the N-atom in H-bond formation with the backbone NH of Val96 in the hinge region at the ATP binding site. In addition, substitution on the terminal phenyl ring was found to improve potency against DAPK compared to the unsubstituted analogues, which might be explained by In previous studies, a structure-based virtual screening strategy was followed to develop an oxazol-5-one derivative III and its related analogues as DAPK inhibitors. Further in silico studies were conducted to define the structure-activity relationship (SAR) of the developed inhibitors [19,24]. The SAR studies stated that a nitrogen containing group, such as pyridinyl moiety, is essential for DAPK activity due to the role of the N-atom in H-bond formation with the backbone NH of Val96 in the hinge region at the ATP binding site. In addition, substitution on the terminal phenyl ring was found to improve potency against DAPK compared to the unsubstituted analogues, which might be explained by the increase in electron density of the phenyl ring which allows for ring contribution in hydrophobic interactions at the ATP binding site. Regarding phenyl ring substitution, the studies reported that meta-substitution seems to be more appropriate for binding at the ATP binding site than para-substitution because of electronic effects. Additionally, substitution with electron withdrawing groups at meta-positions is more effective for activity than Pharmaceuticals 2022, 15, 1050 3 of 23 electron donating ones. It was also observed that the phenyl ring is located near Asp161 in a very tight area of the binding pocket, so bulky substitution would badly affect inhibitor affinity to the binding site and reduce their stability and activity, as well. Finally, docking of compound III in DAPK binding sites illustrated an observed vacancy around the phenyl group, which is considered a point of optimization for developing new DAPK inhibitors with extended substitution on the phenyl ring.
Relying on the aforementioned SAR studies, structural optimizations of the reported inhibitor III were conducted, while keeping the essential binding interactions, with the aim of producing a novel series of DAPK inhibitors (4a-r) ( Figure 2). In the current work, the designed carboxamide derivatives (4a-r) were designed to retain the hinge binding interaction with the Val96 backbone by introducing different nitrogen containing groups (pyridine, pyridazine, and pyrazine). However, the principal modification was the ring opening of the central oxazole into carboxamide moiety, which has been reported in many DAPK inhibitors. The lateral phenyl group of the designed series was substituted by an electron withdrawing meta-chloro group that was reported for better hydrophobic interaction. The new designed compounds possessed a unique extension in the hydrophobic area via additional phenoxy substitutions on the terminal phenyl group in an attempt to boost molecular interaction with the enzyme pocket.
the increase in electron density of the phenyl ring which allows for ring contribution in hydrophobic interactions at the ATP binding site. Regarding phenyl ring substitution, the studies reported that meta-substitution seems to be more appropriate for binding at the ATP binding site than para-substitution because of electronic effects. Additionally, substitution with electron withdrawing groups at meta-positions is more effective for activity than electron donating ones. It was also observed that the phenyl ring is located near Asp161 in a very tight area of the binding pocket, so bulky substitution would badly affect inhibitor affinity to the binding site and reduce their stability and activity, as well. Finally, docking of compound III in DAPK binding sites illustrated an observed vacancy around the phenyl group, which is considered a point of optimization for developing new DAPK inhibitors with extended substitution on the phenyl ring.
Relying on the aforementioned SAR studies, structural optimizations of the reported inhibitor III were conducted, while keeping the essential binding interactions, with the aim of producing a novel series of DAPK inhibitors (4a-r) ( Figure 2). In the current work, the designed carboxamide derivatives (4a-r) were designed to retain the hinge binding interaction with the Val96 backbone by introducing different nitrogen containing groups (pyridine, pyridazine, and pyrazine). However, the principal modification was the ring opening of the central oxazole into carboxamide moiety, which has been reported in many DAPK inhibitors. The lateral phenyl group of the designed series was substituted by an electron withdrawing meta-chloro group that was reported for better hydrophobic interaction. The new designed compounds possessed a unique extension in the hydrophobic area via additional phenoxy substitutions on the terminal phenyl group in an attempt to boost molecular interaction with the enzyme pocket.

Chemistry
The chemical synthesis of the target compounds 4a-r was carried out as illustrated in Scheme 1. A nucleophilic aromatic substitution reaction of the commercially available starting material 2-chloro-1-fluoro-4-nitrobenzene (1) was performed via adding the appropriate phenolic derivative and a catalytic amount of potassium carbonate to generate 2-chloro-4-nitrophenoxybenzene derivatives (2a-c). Catalytic hydrogenation of compounds 2a-c

Chemistry
The chemical synthesis of the target compounds 4a-r was carried out as illustrated in Scheme 1. A nucleophilic aromatic substitution reaction of the commercially available starting material 2-chloro-1-fluoro-4-nitrobenzene (1) was performed via adding the appropriate phenolic derivative and a catalytic amount of potassium carbonate to generate 2-chloro-4-nitrophenoxybenzene derivatives (2a-c). Catalytic hydrogenation of compounds 2a-c was performed using Pt/C catalyst under hydrogen atmosphere to yield compounds 3a-c. Introduction of amide functionality to intermediate 3a-c was achieved by coupling with the appropriate benzoic acid derivative using HATU to afford the final target compounds 4a-r (Table 1)

Chemistry
The chemical synthesis of the target compounds 4a-r was carried out as illustrated in Scheme 1. A nucleophilic aromatic substitution reaction of the commercially available starting material 2-chloro-1-fluoro-4-nitrobenzene (1) was performed via adding the appropriate phenolic derivative and a catalytic amount of potassium carbonate to generate 2-chloro-4-nitrophenoxybenzene derivatives (2a-c). Catalytic hydrogenation of compounds 2a-c was performed using Pt/C catalyst under hydrogen atmosphere to yield compounds 3a-c. Introduction of amide functionality to intermediate 3a-c was achieved by coupling with the appropriate benzoic acid derivative using HATU to afford the final target compounds 4a-r (Table 1)

Chemistry
The chemical synthesis of the target compounds 4a-r was carried out as illustrated in Scheme 1. A nucleophilic aromatic substitution reaction of the commercially available starting material 2-chloro-1-fluoro-4-nitrobenzene (1) was performed via adding the appropriate phenolic derivative and a catalytic amount of potassium carbonate to generate 2-chloro-4-nitrophenoxybenzene derivatives (2a-c). Catalytic hydrogenation of compounds 2a-c was performed using Pt/C catalyst under hydrogen atmosphere to yield compounds 3a-c. Introduction of amide functionality to intermediate 3a-c was achieved by coupling with the appropriate benzoic acid derivative using HATU to afford the final target compounds 4a-r (Table 1)

Chemistry
The chemical synthesis of the target compounds 4a-r was carried out as illustrated in Scheme 1. A nucleophilic aromatic substitution reaction of the commercially available starting material 2-chloro-1-fluoro-4-nitrobenzene (1) was performed via adding the appropriate phenolic derivative and a catalytic amount of potassium carbonate to generate 2-chloro-4-nitrophenoxybenzene derivatives (2a-c). Catalytic hydrogenation of compounds 2a-c was performed using Pt/C catalyst under hydrogen atmosphere to yield compounds 3a-c. Introduction of amide functionality to intermediate 3a-c was achieved by coupling with the appropriate benzoic acid derivative using HATU to afford the final target compounds 4a-r (Table 1).

Chemistry
The chemical synthesis of the target compounds 4a-r was carried out as illustrated in Scheme 1. A nucleophilic aromatic substitution reaction of the commercially available starting material 2-chloro-1-fluoro-4-nitrobenzene (1) was performed via adding the appropriate phenolic derivative and a catalytic amount of potassium carbonate to generate 2-chloro-4-nitrophenoxybenzene derivatives (2a-c). Catalytic hydrogenation of compounds 2a-c was performed using Pt/C catalyst under hydrogen atmosphere to yield compounds 3a-c. Introduction of amide functionality to intermediate 3a-c was achieved by coupling with the appropriate benzoic acid derivative using HATU to afford the final target compounds 4a-r (Table 1).

Chemistry
The chemical synthesis of the target compounds 4a-r was carried out as illustrated in Scheme 1. A nucleophilic aromatic substitution reaction of the commercially available starting material 2-chloro-1-fluoro-4-nitrobenzene (1) was performed via adding the appropriate phenolic derivative and a catalytic amount of potassium carbonate to generate 2-chloro-4-nitrophenoxybenzene derivatives (2a-c). Catalytic hydrogenation of compounds 2a-c was performed using Pt/C catalyst under hydrogen atmosphere to yield compounds 3a-c. Introduction of amide functionality to intermediate 3a-c was achieved by coupling with the appropriate benzoic acid derivative using HATU to afford the final target compounds 4a-r (Table 1).

Chemistry
The chemical synthesis of the target compounds 4a-r was carried out as illustrated in Scheme 1. A nucleophilic aromatic substitution reaction of the commercially available starting material 2-chloro-1-fluoro-4-nitrobenzene (1) was performed via adding the appropriate phenolic derivative and a catalytic amount of potassium carbonate to generate 2-chloro-4-nitrophenoxybenzene derivatives (2a-c). Catalytic hydrogenation of compounds 2a-c was performed using Pt/C catalyst under hydrogen atmosphere to yield compounds 3a-c. Introduction of amide functionality to intermediate 3a-c was achieved by coupling with the appropriate benzoic acid derivative using HATU to afford the final target compounds 4a-r (Table 1).

Chemistry
The chemical synthesis of the target compounds 4a-r was carried out as illustrated in Scheme 1. A nucleophilic aromatic substitution reaction of the commercially available starting material 2-chloro-1-fluoro-4-nitrobenzene (1) was performed via adding the appropriate phenolic derivative and a catalytic amount of potassium carbonate to generate 2-chloro-4-nitrophenoxybenzene derivatives (2a-c). Catalytic hydrogenation of compounds 2a-c was performed using Pt/C catalyst under hydrogen atmosphere to yield compounds 3a-c. Introduction of amide functionality to intermediate 3a-c was achieved by coupling with the appropriate benzoic acid derivative using HATU to afford the final target compounds 4a-r (Table 1).

Chemistry
The chemical synthesis of the target compounds 4a-r was carried out as illustrated in Scheme 1. A nucleophilic aromatic substitution reaction of the commercially available starting material 2-chloro-1-fluoro-4-nitrobenzene (1) was performed via adding the appropriate phenolic derivative and a catalytic amount of potassium carbonate to generate 2-chloro-4-nitrophenoxybenzene derivatives (2a-c). Catalytic hydrogenation of compounds 2a-c was performed using Pt/C catalyst under hydrogen atmosphere to yield compounds 3a-c. Introduction of amide functionality to intermediate 3a-c was achieved by coupling with the appropriate benzoic acid derivative using HATU to afford the final target compounds 4a-r (Table 1).

Chemistry
The chemical synthesis of the target compounds 4a-r was carried out as illustrated in Scheme 1. A nucleophilic aromatic substitution reaction of the commercially available starting material 2-chloro-1-fluoro-4-nitrobenzene (1) was performed via adding the appropriate phenolic derivative and a catalytic amount of potassium carbonate to generate 2-chloro-4-nitrophenoxybenzene derivatives (2a-c). Catalytic hydrogenation of compounds 2a-c was performed using Pt/C catalyst under hydrogen atmosphere to yield compounds 3a-c. Introduction of amide functionality to intermediate 3a-c was achieved by coupling with the appropriate benzoic acid derivative using HATU to afford the final target compounds 4a-r (Table 1).

Chemistry
The chemical synthesis of the target compounds 4a-r was carried out as illustrated in Scheme 1. A nucleophilic aromatic substitution reaction of the commercially available starting material 2-chloro-1-fluoro-4-nitrobenzene (1) was performed via adding the appropriate phenolic derivative and a catalytic amount of potassium carbonate to generate 2-chloro-4-nitrophenoxybenzene derivatives (2a-c). Catalytic hydrogenation of compounds 2a-c was performed using Pt/C catalyst under hydrogen atmosphere to yield compounds 3a-c. Introduction of amide functionality to intermediate 3a-c was achieved by coupling with the appropriate benzoic acid derivative using HATU to afford the final target compounds 4a-r (Table 1).

Chemistry
The chemical synthesis of the target compounds 4a-r was carried out as illustrated in Scheme 1. A nucleophilic aromatic substitution reaction of the commercially available starting material 2-chloro-1-fluoro-4-nitrobenzene (1) was performed via adding the appropriate phenolic derivative and a catalytic amount of potassium carbonate to generate 2-chloro-4-nitrophenoxybenzene derivatives (2a-c). Catalytic hydrogenation of compounds 2a-c was performed using Pt/C catalyst under hydrogen atmosphere to yield compounds 3a-c. Introduction of amide functionality to intermediate 3a-c was achieved by coupling with the appropriate benzoic acid derivative using HATU to afford the final target compounds 4a-r (Table 1).

Chemistry
The chemical synthesis of the target compounds 4a-r was carried out as illustrated in Scheme 1. A nucleophilic aromatic substitution reaction of the commercially available starting material 2-chloro-1-fluoro-4-nitrobenzene (1) was performed via adding the appropriate phenolic derivative and a catalytic amount of potassium carbonate to generate 2-chloro-4-nitrophenoxybenzene derivatives (2a-c). Catalytic hydrogenation of compounds 2a-c was performed using Pt/C catalyst under hydrogen atmosphere to yield compounds 3a-c. Introduction of amide functionality to intermediate 3a-c was achieved by coupling with the appropriate benzoic acid derivative using HATU to afford the final target compounds 4a-r (Table 1).

Chemistry
The chemical synthesis of the target compounds 4a-r was carried out as illustrated in Scheme 1. A nucleophilic aromatic substitution reaction of the commercially available starting material 2-chloro-1-fluoro-4-nitrobenzene (1) was performed via adding the appropriate phenolic derivative and a catalytic amount of potassium carbonate to generate 2-chloro-4-nitrophenoxybenzene derivatives (2a-c). Catalytic hydrogenation of compounds 2a-c was performed using Pt/C catalyst under hydrogen atmosphere to yield compounds 3a-c. Introduction of amide functionality to intermediate 3a-c was achieved by coupling with the appropriate benzoic acid derivative using HATU to afford the final target compounds 4a-r (Table 1).

Chemistry
The chemical synthesis of the target compounds 4a-r was carried out as illustrated in Scheme 1. A nucleophilic aromatic substitution reaction of the commercially available starting material 2-chloro-1-fluoro-4-nitrobenzene (1) was performed via adding the appropriate phenolic derivative and a catalytic amount of potassium carbonate to generate 2-chloro-4-nitrophenoxybenzene derivatives (2a-c). Catalytic hydrogenation of compounds 2a-c was performed using Pt/C catalyst under hydrogen atmosphere to yield compounds 3a-c. Introduction of amide functionality to intermediate 3a-c was achieved by coupling with the appropriate benzoic acid derivative using HATU to afford the final target compounds 4a-r (Table 1).

Chemistry
The chemical synthesis of the target compounds 4a-r was carried out as illustrated in Scheme 1. A nucleophilic aromatic substitution reaction of the commercially available starting material 2-chloro-1-fluoro-4-nitrobenzene (1) was performed via adding the appropriate phenolic derivative and a catalytic amount of potassium carbonate to generate 2-chloro-4-nitrophenoxybenzene derivatives (2a-c). Catalytic hydrogenation of compounds 2a-c was performed using Pt/C catalyst under hydrogen atmosphere to yield compounds 3a-c. Introduction of amide functionality to intermediate 3a-c was achieved by coupling with the appropriate benzoic acid derivative using HATU to afford the final target compounds 4a-r (Table 1).

Chemistry
The chemical synthesis of the target compounds 4a-r was carried out as illustrated in Scheme 1. A nucleophilic aromatic substitution reaction of the commercially available starting material 2-chloro-1-fluoro-4-nitrobenzene (1) was performed via adding the appropriate phenolic derivative and a catalytic amount of potassium carbonate to generate 2-chloro-4-nitrophenoxybenzene derivatives (2a-c). Catalytic hydrogenation of compounds 2a-c was performed using Pt/C catalyst under hydrogen atmosphere to yield compounds 3a-c. Introduction of amide functionality to intermediate 3a-c was achieved by coupling with the appropriate benzoic acid derivative using HATU to afford the final target compounds 4a-r (Table 1).

Chemistry
The chemical synthesis of the target compounds 4a-r was carried out as illustrated in Scheme 1. A nucleophilic aromatic substitution reaction of the commercially available starting material 2-chloro-1-fluoro-4-nitrobenzene (1) was performed via adding the appropriate phenolic derivative and a catalytic amount of potassium carbonate to generate 2-chloro-4-nitrophenoxybenzene derivatives (2a-c). Catalytic hydrogenation of compounds 2a-c was performed using Pt/C catalyst under hydrogen atmosphere to yield compounds 3a-c. Introduction of amide functionality to intermediate 3a-c was achieved by coupling with the appropriate benzoic acid derivative using HATU to afford the final target compounds 4a-r (Table 1).

Chemistry
The chemical synthesis of the target compounds 4a-r was carried out as illustrated in Scheme 1. A nucleophilic aromatic substitution reaction of the commercially available starting material 2-chloro-1-fluoro-4-nitrobenzene (1) was performed via adding the appropriate phenolic derivative and a catalytic amount of potassium carbonate to generate 2-chloro-4-nitrophenoxybenzene derivatives (2a-c). Catalytic hydrogenation of compounds 2a-c was performed using Pt/C catalyst under hydrogen atmosphere to yield compounds 3a-c. Introduction of amide functionality to intermediate 3a-c was achieved by coupling with the appropriate benzoic acid derivative using HATU to afford the final target compounds 4a-r (Table 1).  In an attempt to test our design hypothesis and discover if DAPK1 is the potential conceivable kinase target of the designed candidates, a kinase selectivity assay was carried out to the first synthesized derivative (4a) against a group of forty-five different kinases. Compound 4a was evaluated at a single dose concentration of 10 µM and % inhibition was determined against each corresponding kinase ( Table 2). Interestingly, the preliminary data revealed a remarkable selectivity of compound 4a towards DAPK1, with mean % inhibition of 44.19%. On the other hand, the results for the other 44 kinases showed no activity for most of the kinases and low activity (less than 25%) for a few of the tested kinases ( Figure 3). An additional inspection of compound 4a selectivity was carried out by evaluating its activity against other DAPK isoforms (DAPK2 and DAPK3). The obtained results showed no activity for compound 4a with DAPK2 and DAPK3 isoforms (data not shown).

In Vitro DAPK1 Kinase Assay and Optimization towards Lead Development
Based on the previous panel of kinase data, a series of aryl carboxamide derivatives (4b-r) were synthesized and evaluated in vitro for their inhibitory activity against DAPK1 using the ELISA technique (enzyme-linked immune sorbent assay). The enzyme inhibition assay was conducted with a 10 µM dose of the tested compounds and the mean % inhibition was measured. As illustrated in Table 3, derivatives possessing the pyridinyl carboxamide moiety (4c, 4e, 4f, 4h, 4j, 4k, 4m, 4p, and 4q) exhibited the highest inhibitory activity (59-81%) in comparison to the pyridazine or pyrazine possessing derivatives (38-61%). In addition, the attachment point of these nitrogen heterocycles (pyridine, pyridazine, and pyrazine) with the amide group did not show a remarkable effect in terms of activity. In regard to the substitution on the terminal phenoxy moiety, the overall results showed that para-substitution with the fluoro group in compounds 4a and 4h-l has a better effect on activity than the meta-substituted derivatives 4b-g, except for the pyridazine bearing derivatives 4a and 4b. Moreover, replacing the m-fluoro substitution with m-chloro in compounds 4m-r showed a slight improvement in activity, except in compounds 4d and 4o where the chloro substitution dramatically reduced activity to 37.99%. Among the tested series, compounds 4h, 4j, 4k, and 4q revealed the highest activity of 81%, 79%, 80%, and 72%, respectively.

In Vitro DAPK1 Kinase Assay and Optimization towards Lead Development
Based on the previous panel of kinase data, a series of aryl carboxamide derivatives (4b-r) were synthesized and evaluated in vitro for their inhibitory activity against DAPK1 using the ELISA technique (enzyme-linked immune sorbent assay). The enzyme inhibition assay was conducted with a 10 µM dose of the tested compounds and the mean % inhibition was measured. As illustrated in Table 3, derivatives possessing the pyridinyl carboxamide moiety (4c, 4e, 4f, 4h, 4j, 4k, 4m, 4p, and 4q) exhibited the highest inhibitory activity (59-81%) in comparison to the pyridazine or pyrazine possessing derivatives (38-61%). In addition, the attachment point of these nitrogen heterocycles (pyridine, pyridazine, and pyrazine) with the amide group did not show a remarkable effect in terms of activity. In regard to the substitution on the terminal phenoxy moiety, the overall results showed that para-substitution with the fluoro group in compounds 4a and 4h-l has a better effect on activity than the meta-substituted derivatives 4b-g, except for the pyridazine bearing derivatives 4a and 4b. Moreover, replacing the m-fluoro substitution with mchloro in compounds 4m-r showed a slight improvement in activity, except in compounds 4d and 4o where the chloro substitution dramatically reduced activity to 37.99%. Among the tested series, compounds 4h, 4j, 4k, and 4q revealed the highest activity of 81%, 79%, 80%, and 72%, respectively.   For more clarification of the structural activity relationship of the synthesized compounds, the most active derivatives (4h, 4j, 4k ,and 4q) were subjected to a dose-dependent IC 50 kinase assay. The selected candidates showed low micromolar IC 50 values against DAPK1 kinase with a range of 1.09-7.26 µM. The data obtained in Table 4 revealed the impact of the attachment point of the pyridine moiety with the amide linker and, for the 4-F phenoxy derivatives, nicotinamide owing compound 4j exhibited the highest activity (IC 50 = 1.7 µM). In contrast, substitution with picolinamide (4h) and isonicotinamide (4k) declined activity to 6.81 µM and 7.26 µM, respectively. Interestingly, the isonicotinamide derivative with the terminal m-chlorophenoxy moiety (4q) emerged to be the most potent inhibitor with an IC 50 value of 1.09 µM. In order to determine its selectivity against DAPK1, compound 4q was evaluated at a single dose concentration (10 µM) against the other two isoforms (DAPK2/DAPK3). The two isoforms exhibited no inhibition at all by compound 4q (data not shown), indicating the high potential of compound 4q to be a promising selective DAPK1 inhibitory lead compound.
 Anti-proliferative activity on colon cancers: The target compounds were assayed against a panel of seven colon adenocarcinoma cell lines (COLO-205, HCC-2998, HCT-116, HCT-15, HT29, KM12, and SW-620) and the results are summarized in Table 7. The tested compounds revealed a wide range of inhibitory activity against the seven cell lines, with average % GI from 0% to 78%. The overall data indicated that compounds 4d, 4e, 4o, and 4p had the highest growth inhibitory activity and showed average % GI of 71.84%, 69.73%, 78.16%, and 71.11%, respectively (Figure 7). Compounds 4d and 4e significantly inhibited the growth of HOP92 adenocarcinoma with % GI of 90.87% and 87.13%, respectively. The two compounds also exhibited outstanding activity towards the COLO 205 cell line (% GI = 84.52% and 77.29%, respectively).   Anti-proliferative activity on CNS cancers: The anti-proliferative activities of the target derivatives were determined against six CNS cancer cell lines (SF-268, SF-295, SF-539, SNB-19, SNB-75, and U251) and the resulting % GIs were tabulated in Table 8. As illustrated, compounds 4d, 4e, 4o, and 4p kept their ranking among the tested derivatives as the most potent anti-proliferative agents. It was observed that SF-539 and SNB-75 were the most sensitive cell lines that showed growth inhibition around 100% upon treatment with 10 µM of compounds 4d, 4o, 4p, 4e (Figure  8). In addition, the mentioned derivatives broadly inhibited the growth of the remaining four cell lines (44.50-83.53%). Compound 4q moderately inhibited the growth of SF-295 KM12, and SW-620 and exhibited % GI of 52.26%, 76.34%, 72.49%, and 63.04%, respectively (Figure 7).

Docking Study
In an attempt to introduce a reasonable explanation for the observed DAPK1 kinase activity with respect to the inhibitors' binding affinity, the potent inhibitor 4j was subjected to a molecular docking study as a representative example of the designed inhibitors. In this study, Molecular Operating Environment (MOE, 2014) software was used to operate the docking protocol. The X-ray crystallographic structures of DAPK1 in complex with Genistein (PDB ID: 5AUZ) were downloaded from the protein data bank (PDB). Validation of the docking protocol was achieved by re-docking of the co-crystalized ligand, Genistein, in the binding site of DAPK1. The re-docked ligand retained the same binding manner in the DAPK1 active site (docking score= −6.1511 kcal/mol) with an RMSD value

Docking Study
In an attempt to introduce a reasonable explanation for the observed DAPK1 kinase activity with respect to the inhibitors' binding affinity, the potent inhibitor 4j was subjected to a molecular docking study as a representative example of the designed inhibitors. In this study, Molecular Operating Environment (MOE, 2014) software was used to operate the docking protocol. The X-ray crystallographic structures of DAPK1 in complex with Genistein (PDB ID: 5AUZ) were downloaded from the protein data bank (PDB). Validation of the docking protocol was achieved by re-docking of the co-crystalized ligand, Genistein, in the binding site of DAPK1. The re-docked ligand retained the same binding manner in the DAPK1 active site (docking score= −6.1511 kcal/mol) with an RMSD value of 0.8894 Å. As illustrated in Figure 14A, the iso-flavone moiety of the native ligand, Genistein, is binding in the adenine pocket via H-bonding between the hydroxyl group and Glu100 residue while the pyran ring is additionally embedded in the pocket by AreneH interaction with Val27. Furthermore, the DAPK1 hydrophobic pocket is occupied by the lateral hydroxy phenyl moiety, which exhibits H-bonding between its para-hydroxyl group and the Glu94 residue. Accordingly, the target compounds should conserve the binding mode of the native ligand, which in turn would also be an indication of their binding affinity and enzymatic activity. In Figure 14B, docking of compound 4j in the active site of DAPK1 (docking score= -5.5545 kcal/mol) illustrates that the NH of the amide linker is introduced to the adenine pocket and forms H-bonding with Glu100, while the phenoxy moiety is inserted deeply in the pocket via Arene-H interaction with Val27. On the other hand, the pyridine ring, which is substituted on the amide linker, exhibits additional H-bonding with Arg150. of 0.8894 Å. As illustrated in Figure 14A, the iso-flavone moiety of the native ligand, Genistein, is binding in the adenine pocket via H-bonding between the hydroxyl group and Glu100 residue while the pyran ring is additionally embedded in the pocket by AreneH interaction with Val27. Furthermore, the DAPK1 hydrophobic pocket is occupied by the lateral hydroxy phenyl moiety, which exhibits H-bonding between its para-hydroxyl group and the Glu94 residue. Accordingly, the target compounds should conserve the binding mode of the native ligand, which in turn would also be an indication of their binding affinity and enzymatic activity. In Figure 14B, docking of compound 4j in the active site of DAPK1 (docking score= -5.5545 kcal/mol) illustrates that the NH of the amide linker is introduced to the adenine pocket and forms H-bonding with Glu100, while the phenoxy moiety is inserted deeply in the pocket via Arene-H interaction with Val27. On the other hand, the pyridine ring, which is substituted on the amide linker, exhibits additional H-bonding with Arg150.

Chemistry
General: All reactions and manipulations were performed in a nitrogen atmosphere using standard Schlenk techniques. All reaction solvents and reagents were purchased from commercial suppliers and used without further purification. The NMR spectra were obtained with a Bruker Avance 400 (400 MHz 1 H and 100.6 MHz 13 C NMR). 1 H NMR spectra were referenced to tetramethylsilane (δ = 0.00 ppm) as an internal standard and were reported as follows: chemical shift, multiplicity (b = broad, s = singlet, d = doublet, t = triplet, dd = doublet of doublet, m = multiplet). Column chromatography was performed on Merck Silica Gel 60 (230-400 mesh) and eluting solvents for all of these chromatographic methods were noted as appropriated-mixed solvent with given volume-to-volume ratios. TLC was carried out using glass sheets pre-coated with silica gel 60 F254 purchased by Merk.
3.1.1. General Procedure of 2-Chloro-4-nitrophenoxybenzene Derivatives (2a-c) Potassium carbonate (1.25 mmol) and the appropriate phenol (1 mmol) were added to a solution of commercially available 2-chloro-1-fluoro-4-nitrobenzene (1 mmol) in MeCN (12 mL). The reaction mixture was heated at 85 °C for 6 h. The mixture was extracted with EtOAc and water. The organic layer was dried over Na2SO4 and concentrated under reduced pressure to give the target compound.

Chemistry
General: All reactions and manipulations were performed in a nitrogen atmosphere using standard Schlenk techniques. All reaction solvents and reagents were purchased from commercial suppliers and used without further purification. The NMR spectra were obtained with a Bruker Avance 400 (400 MHz 1 H and 100.6 MHz 13 C NMR). 1 H NMR spectra were referenced to tetramethylsilane (δ = 0.00 ppm) as an internal standard and were reported as follows: chemical shift, multiplicity (b = broad, s = singlet, d = doublet, t = triplet, dd = doublet of doublet, m = multiplet). Column chromatography was performed on Merck Silica Gel 60 (230-400 mesh) and eluting solvents for all of these chromatographic methods were noted as appropriated-mixed solvent with given volume-to-volume ratios. TLC was carried out using glass sheets pre-coated with silica gel 60 F 254 purchased by Merk. Potassium carbonate (1.25 mmol) and the appropriate phenol (1 mmol) were added to a solution of commercially available 2-chloro-1-fluoro-4-nitrobenzene (1 mmol) in MeCN (12 mL). The reaction mixture was heated at 85 • C for 6 h. The mixture was extracted with EtOAc and water. The organic layer was dried over Na 2 SO 4 and concentrated under reduced pressure to give the target compound.