Design, Synthesis and Docking Studies of Novel Macrocyclic Pentapeptides as Anticancer Multi-Targeted Kinase Inhibitors

A series of macrocyclic pyrido-pentapeptide candidates 2–6 were synthesized by using N,N-bis-[1-carboxy-2-(benzyl)]-2,6-(diaminocarbonyl)pyridine 1a,b as starting material. Structures of the newly synthesized compounds were established by IR, 1H and 13C-NMR, and MS spectral data and elemental analysis. The in-vitro cytotoxicity activity was investigated for all compounds against MCF-7 and HepG-2 cell lines and the majority of the compounds showed potent anticancer activity against the tested cell lines in comparison with the reference drugs. Out of the macrocyclic pyrido-pentapeptide based compounds, 5c showed encouraging inhibitory activity on MCF-7 and HepG-2 cell lines with IC50 values 9.41 ± 1.25 and 7.53 ± 1.33 μM, respectively. Interestingly, 5c also demonstrated multitarget profile and excellent inhibitory activity towards VEGFR-2, CDK-2 and PDGFRβ kinases. Furthermore, molecular modeling studies of the compound 5c revealed its possible binding modes into the active sites of those kinases.


Introduction
Cancer is still considered one of the most serious diseases threatening human life. In the past three decades, there enormous efforts have been undertaken to confront cancer diseases. Within such efforts, many therapeutic agents have been developed to treat cancer patients in their early, as well as late cancer-developing stages [1]. These agents vary from antibiotics, chemically synthesized compounds, and natural products-based drugs.
Macrocyclic motifs are commonly defined as a ring system containing 12 or more atoms [2]. They are privileged scaffolds in the fields of chemistry, biology, and medicine [3][4][5]. There are different classes of macrocycles like peptidic and nonpeptidic natural products, non-natural (synthetic) peptides and non-natural (synthetic) macrocycles [6]. The advances in molecular biology and genetics help in identification of molecular targets that are related to cancer cells or overexpressed on them. The design of compounds affecting these targets improves the development of more selective anticancer drugs with less toxic side effects [24]. Macrocycles were reported to display antitumor properties, which may be attributed to inhibition of different enzymes involved in carcinogenesis cases. The prominent examples of macrocycles, illustrated in Figure 2, exhibited a potent inhibitory activity against various kinases, e.g., CDK-2, VEGFR-2, JAK-2, FLT-3, PDK-1 and EGFR [6,[25][26][27][28].
In view of these observations and as continuation of our previous works in peptide heterocyclic chemistry, we have herein synthesized some new linear and macrocyclic peptidopyridine derivatives, and tested their anticancer activity in comparison to Tamoxifen and 5-Fluorouracil ® as positive controls. Furthermore, they were screened for their inhibitory activity against VEGFR-2, EGFR, PDGFRβ and CDK-2 enzymes. Additionally, molecular modeling study was performed to explore the most appropriate binding modes of the most potent target compounds. The advances in molecular biology and genetics help in identification of molecular targets that are related to cancer cells or overexpressed on them. The design of compounds affecting these targets improves the development of more selective anticancer drugs with less toxic side effects [24]. Macrocycles were reported to display antitumor properties, which may be attributed to inhibition of different enzymes involved in carcinogenesis cases. The prominent examples of macrocycles, illustrated in Figure 2, exhibited a potent inhibitory activity against various kinases, e.g., CDK-2, VEGFR-2, JAK-2, FLT-3, PDK-1 and EGFR [6,[25][26][27][28]. In view of these observations and as continuation of our previous works in peptide heterocyclic chemistry, we have herein synthesized some new linear and macrocyclic peptidopyridine derivatives, and tested their anticancer activity in comparison to Tamoxifen and 5-Fluorouracil ® as positive controls. Furthermore, they were screened for their inhibitory activity against VEGFR-2, EGFR, PDGFRβ and CDK-2 enzymes. Additionally, molecular modeling study was performed to explore the most appropriate binding modes of the most potent target compounds.

Chemistry
In the previous work [9,29], L-amino acid methyl esters was initially coupled with dipicolinic acid via the conventional acid chloride method to give the corresponding 2,6-bis-N α -L-diamino acid pyridine methyl ester derivatives. In the present work, a series of linear and macrocyclic pyridopentapeptide derivatives 2-6 were synthesized based on N,N-bis-[1-carboxy-2-(benzyl)]-2,6-(diamino-carbonyl)pyridine (1a,b) and they are screened as anticancer agents. Treatment of 1 with L-amino acid methyl ester hydrochloride in the presence of ethyl chloroformate in dichloromethane afforded the corresponding tetrapeptide pyridine methyl ester derivatives 2a-c, respectively (Scheme 1). Hydrolysis of 2a-c with methanolic sodium hydroxide to afford the corresponding tetrapeptide pyridine derivatives 3a-c, which were cyclized with L-lysine methyl ester by different methods to afford the corresponding cyclic pentapeptide esters 4a-c, respectively. The cyclized pentapeptide esters 4a-c was hydrolyzed with methanolic sodium hydroxide to give the corresponding cyclic Hydrolysis of 2a-c with methanolic sodium hydroxide to afford the corresponding tetrapeptide pyridine derivatives 3a-c, which were cyclized with L-lysine methyl ester by different methods to afford the corresponding cyclic pentapeptide esters 4a-c, respectively. The cyclized pentapeptide esters 4a-c was hydrolyzed with methanolic sodium hydroxide to give the corresponding cyclic pentapeptide acids 5a-c, or by hydrazonolysis with hydrazine hydrate in methanol to give the corresponding cyclic pentapeptide acid hydrazides 6a-c, respectively (Scheme 2).

Anticancer Activity
The cytotoxic effects of all newly synthesized compounds were evaluated against MCF-7 and HepG-2 cell lines. Results obtained ( Figure 3) showed that all prepared compounds affected both cell lines in a dose-dependent manner, where increasing the applied concentration gradually decreased cell viability. All tested compounds exhibited moderate to excellent cytotoxic activities on both tested cell lines in comparison with control drugs. Compounds 4a, 4b, 5a, 5b, 6a and 6b were more potent at lower range of tested concentrations. However, compounds 2c, 3a, 4a and 6c were

Anticancer Activity
The cytotoxic effects of all newly synthesized compounds were evaluated against MCF-7 and HepG-2 cell lines. Results obtained ( Figure 3) showed that all prepared compounds affected both cell lines in a dose-dependent manner, where increasing the applied concentration gradually decreased cell viability. All tested compounds exhibited moderate to excellent cytotoxic activities on both tested cell lines in comparison with control drugs. Compounds 4a, 4b, 5a, 5b, 6a and 6b were more potent at lower range of tested concentrations. However, compounds 2c, 3a, 4a and 6c were considered inactive against MCF-7 cells, since they showed no practical IC 50 (>100 µM). Concerning IC 50 data (Table 1), it was noticed that only compounds 6a and 6b affected MCF-7 cells more than HepG-2 cells (IC 50 = 11.83 ± 1.62 and 10.87 ± 1.10 µM for MCF-7, and 12.44 ± 1.3 and 11.53 ± 1.70 µM for HepG-2, respectively). While, the rest of the prepared compounds showed more noticeable anticancer activities in case of HepG-2 cells rather than MCF-7 cells. Comparing the obtained IC 50 values for HepG-2 cells with those of the control drugs explored that compound 5c was the most potent in comparison with tamoxifen and 5-fluorouracil (IC 50 = 7.53 ± 1.33, 29.38 ± 1.15 and 43.84 ± 1.84 µM, respectively). Compounds 2a, 2b, 3b, 4a-c, 5a-c and 6a-c afforded higher cytotoxic activity, while compounds 3a and 3c gave approximately equipotent cytotoxic activity (IC 50 = 26.01 ± 2.35 and 26.64 ± 1.85 µM, respectively). For MCF-7 cell lines, 5c was also the most active derivative in comparison with tamoxifen (IC 50 = 9.41 ± 1. 25         By analysis of the previous results, the cytotoxic activities of the tested compounds, with the exception of compound 4c, were in the following order compound 5 > 6 > 4 > 3 > 2. Concerning the structure-activity relationships of the synthesized compounds, it was observed that the open chain derivatives 2 and 3 have lower activity than the cyclized derivatives 4-6. Furthermore, insertion of benzyl moiety at R 1 adjacent to the pyridine scaffold in 2c and 3c gave a marked decrease in the cytotoxic activity compared to their series 2a,b and 3a,b, respectively. This decrease may be due to formation of steric hindrance. The enhanced anticancer activities of compounds 4-6 may be contributed to the cyclization of these compounds. Contrary to what was found in the open chain derivatives 2c and 3c, the increased aromaticity of the cyclopeptide by the location of the phenyl group of phenylalanine neighboring the pyridine nucleus may be the cause of improved activity of compound 5c > 5b > 5a.

Molecular Modeling Studies
The kinase inhibitory assay revealed that compound 5c showed promising inhibitory activity against three kinases, namely VEGFR-2, CDK-2 and PDGFRβ. So, docking simulation was performed using Molecular Operating Environment (MOE ® ) 2008.10 [30,31] to predict the binding modes, affinities, and orientations of compound 5a at the active sites of them. The X-ray crystallographic structure of PDGFRβ was not fully resolved [32]. On the other hand, the X-ray crystallographic structures were reported for VEGFR-2 (pdb code: 4ASD) [33] with sorafenib and for CDK-2 (PDB ID: 2J9M) [34] with PY8. So the docking study was achieved for both VEGFR-2 and CDK-2 kinases The binding model, shown in Figure 5, was exemplified by the interaction of compound 5c with VEGFR-2. The carboxylic moiety of 5c shared in the binding pattern with three hydrogen bond donors; one H-bond was between CO group and the backbone of His1026 (distance: 2.99 Å), and the others were between OH group and the sidechain of Asp1046 (distance: 1.92 and 2.65 Å). Moreover, the residues Ser884, Arg1027 and Leu1049 were inserted nicely inside the centre of cyclic pentapeptide scaffold.

Molecular Modeling Studies
The kinase inhibitory assay revealed that compound 5c showed promising inhibitory activity against three kinases, namely VEGFR-2, CDK-2 and PDGFRβ. So, docking simulation was performed using Molecular Operating Environment (MOE ® ) 2008.10 [30,31] to predict the binding modes, affinities, and orientations of compound 5a at the active sites of them. The X-ray crystallographic structure of PDGFRβ was not fully resolved [32]. On the other hand, the X-ray crystallographic structures were reported for VEGFR-2 (pdb code: 4ASD) [33] with sorafenib and for CDK-2 (PDB ID: 2J9M) [34] with PY8. So the docking study was achieved for both VEGFR-2 and CDK-2 kinases The binding model, shown in Figure 5, was exemplified by the interaction of compound 5c with VEGFR-2. The carboxylic moiety of 5c shared in the binding pattern with three hydrogen bond donors; one H-bond was between CO group and the backbone of His1026 (distance: 2.99 Å), and the others were between OH group and the sidechain of Asp1046 (distance: 1.92 and 2.65 Å). Moreover, the residues Ser884, Arg1027 and Leu1049 were inserted nicely inside the centre of cyclic pentapeptide scaffold. VEGFR-2. The carboxylic moiety of 5c shared in the binding pattern with three hydrogen bond donors; one H-bond was between CO group and the backbone of His1026 (distance: 2.99 Å), and the others were between OH group and the sidechain of Asp1046 (distance: 1.92 and 2.65 Å). Moreover, the residues Ser884, Arg1027 and Leu1049 were inserted nicely inside the centre of cyclic pentapeptide scaffold.  Interaction of compound 5c with the binding site of CDK-2 kinase was illustrated in Figure 6. There was H-bond acceptor between CO of the carboxylic group and the backbone of Asp86 (distance: 2.89 Å). Another H-bond acceptor was established between the amide nitrogen and the backbone of Ile10 (distance: 1.80 Å). Furthermore, carbonyl group placed at p-2 of pyridine ring was linked to backbone of Glu12 (distance: 2.66 Å). In addition, Lys89 located in the centre of cyclic pentapeptide scaffold formed arene-cation interaction with centroid of benzyl ring. Interaction of compound 5c with the binding site of CDK-2 kinase was illustrated in Figure 6. There was H-bond acceptor between CO of the carboxylic group and the backbone of Asp86 (distance: 2.89 Å). Another H-bond acceptor was established between the amide nitrogen and the backbone of Ile10 (distance: 1.80 Å). Furthermore, carbonyl group placed at p-2 of pyridine ring was linked to backbone of Glu12 (distance: 2.66 Å). In addition, Lys89 located in the centre of cyclic pentapeptide scaffold formed arene-cation interaction with centroid of benzyl ring.
There was H-bond acceptor between CO of the carboxylic group and the backbone of Asp86 (distance: 2.89 Å). Another H-bond acceptor was established between the amide nitrogen and the backbone of Ile10 (distance: 1.80 Å). Furthermore, carbonyl group placed at p-2 of pyridine ring was linked to backbone of Glu12 (distance: 2.66 Å). In addition, Lys89 located in the centre of cyclic pentapeptide scaffold formed arene-cation interaction with centroid of benzyl ring.  Finally, it is deduced from molecular docking studies that the good fitting of compound 5c in the active sites of VEGFR-2 and CDK-2 enzymes via different types of interactions could be attributed to the presence of carboxylic group and cyclic pentapeptide scaffold.

Chemistry
Melting points were determined in an "Electro Thermal" Digital melting point apparatus (Shimadzu, Tokyo, Japan), (model: IA9100). Elemental analysis was found within the acceptable limits of the calculated values (Microanalytical Unit, NRC). Infrared spectra (KBr) were recorded on a Nexus 670 FTIR Nicolet, Fourier Transform infrared spectrometer (Perkin Elmer, Hopkinton, MA, USA). Proton nuclear magnetic resonance ( 1 H-NMR) spectra were run in [d6] DMSO on Jeol 270 MHz or 500 MHz instruments ((Tokyo, Japan). Chemical shifts d are given in ppm. Mass spectra Finally, it is deduced from molecular docking studies that the good fitting of compound 5c in the active sites of VEGFR-2 and CDK-2 enzymes via different types of interactions could be attributed to the presence of carboxylic group and cyclic pentapeptide scaffold.

Chemistry
Melting points were determined in an "Electro Thermal" Digital melting point apparatus It is generally known that basic reaction media enhance racemization. However, under the reaction conditions employed in this work, especially short reaction times and temperatures below (0 • C), only negligible racemization was observed.

Synthesis of N α -dipicolinoyl-bis[dipeptide methyl ester] Derivatives (2a-c)
Ethyl chloroformate (0:2 mL, 2 mmol) was added to a stirred and cold (−15 • C) dichloromethane solution (20 mL) of the corresponding N α -dipicolinoyl-bis[amino acid] (1a,b) (1 mmol), containing N-methylmorpholine (0:2 mL, 2 mmol). The reaction mixture was stirred for additional 10 min, then a cold dichloromethane solution (20 mL) of the free amino acid methyl esters, namely, L-isolucine-OMe or L-phenyalanine-OMe (2 mmol), was added. Stirring was maintained for 3 h at (−15 • C), then for 12 h at room temperature. The reaction mixture was washed with water, 1 N sodium bicarbonate, 1 N potassium hydrogen sulfate and water, and dried over anhydrous calcium chloride. The solvent was evaporated under reduced pressure to dryness, and the obtained oily residue was solidified by trituration with a dry ether-n-hexane mixture. The obtained solid was collected by filtration and crystallized from ethanol-n-hexane to give the corresponding 2,6-pyridine-bis-dipeptide ester derivatives (2a-c), respectively.

Synthesis of N α -Dipicolinoyl-bis[dipeptide]derivatives (3a-c)
To a cold (−15 • C) solution of the corresponding tetrapeptide ester (2a-c) (1 mmol) in methanol (20 mL) with stirring, sodium hydroxide (1 N, 25 mL) was gradually added. The reaction mixture was stirred for 2 h at the same temperature then for 3 h at room temperature. The solvent was distilled off under reduced pressure, and the remaining aqueous solution was cooled and acidified with 1 N hydrochloric acid to pH = 3. The obtained solid was filtered off, washed with water, dried and crystallized from ethanol/water to give the corresponding tetrapeptide acids (3a-c), respectively.  Stirring was maintained for 3 h, at (−15 • C), then for 12 h at room temperature. The reaction mixture was washed with water, 1 N sodium bicarbonate, 1 N potassium hydrogen sulfate and water, and then dried over anhydrous calcium chloride. The solvent was evaporated under reduced pressure to dryness, and the obtained oily residue was solidified by trituration with dry ether-n-hexane mixture. The crude product was purified by preparative thin layer chromatography using S3 as eluent to give the corresponding cyclic pentapeptide methyl esters (4a-c), respectively.   To a stirred and cold methanolic solution (−5 • C, 20 mL) of the corresponding cyclic pentapeptide methyl ester (4a-c) (1 mmol), sodium hydroxide (1 N, 25 mL) was gradually added. The reaction mixture was stirred for 2 h at the same temperature, then for 3 h at room temperature. The solvent was distilled off under reduced pressure, and the remaining aqueous solution was cooled and acidified with 1 N hydrochloric acid to pH = 3. The obtained solid was filtered off, washed with water, dried and crystallized from ethanol-water to give the corresponding cyclic pentapeptides (5a-c), respectively.