Anticancer Activities of Newly Synthesized Chiral Macrocyclic Heptapeptide Candidates

As important cancer therapeutic agents, macrocyclic peptides have recently drawn great attention, mainly because they are synthetically accessible and have lower toxicity towards normal cells. In the present work, we synthesized newly macrocyclic pyridoheptapeptide derivatives. The synthesized derivatives were characterized using standard chemical and spectroscopic analytical techniques, and their anticancer activities against human breast and hepatocellular cancer cells were investigated. Results showed that compounds 1a and 1b were the most effective against hepatocellular (HepG2) and breast (MCF-7) cancer cell lines, respectively.


Introduction
Cancer is the second most common causative disease threatening human life. Recently, researchers have focused their works on developing successful therapeutic drugs capable of treating different cancer cells. Most research has been focused on developing early-stage cancer-treating drugs, which have received better attention in comparison to drugs used to treat late cancer phases [1]. Besides naturally obtained preparations; i.e., plant-derived extracts and microbially produced antibiotics, chemical synthesis is still used as a traditional method for obtaining potential anticancer drugs.
Additionally, peptides comprise a major group of pharmaceutical drugs with potential anticancer effects [7]. Previous reports showed that chemical synthesis of peptides proved successful in obtaining new derivatives with potent antimicrobial, anti-inflammatory [8][9][10][11][12][13][14][15][16], and anticancer properties [17][18][19][20][21]. In our previous works, we were able to synthesize, chemically characterize, and biologically evaluate different bis-amino acid and peptide conjugates of dipicolinic acid [22]. Our previous work with compound (A) (Figure 1) showed potential anti-proliferative effects, mainly due to DNA intercalation, and metal sensor properties, particularly for pollutant lead (Pb2+) cations [23]. Moreover, human cells react towards toxicants; i.e., injury and infection, by developing a natural inflammatory response, which finally results in damage to the concerned tissues [24]. Inflammatory reactions include the initiation of various biological pathways such as production and secretion of pro-inflammatory mediators [25]. There has been a well-established connection between inflammatory response and cancer development. Tumor growth and development is dramatically increased by the presence of inflammatory cells; inflammatory mediators are considered a tumor microenvironment [26,27]. Recently, cancer treatments based on the application of synthesized peptides as anti-inflammatory agents have been used [28].
Based on previous investigations and our continuous work in the field of peptide synthesis [17][18][19][20][21][22][23], we have prepared different new macrocyclic heptapeptidopyridine candidates, and evaluated their anticancer potential in relation to standard used anticancer drugs.

Anticancer Activity
The newly synthesized derivatives were evaluated for their anticancer potential towards breast (MCF-7) and hepatocellular (HepG2) cancer cell lines cell lines. Compounds 6a-c did not show any cytotoxic activity against both tested cell lines. Generally, all other derivatives, with the exception of compound 5c, showed concentration-dependent effects on both cell lines ( Figure 2). Moreover, potential positive anticancer compounds showed varying effects ranging from potent to moderate effects. Increasing the applied concentration gradually increased the cytotoxic effects and correspondingly decreased cell viability. On the other hand, compound 5c did not show any activity against MCF-7 cells, and was only active against HepG2 cells. Additionally, compounds 2a and 5a-c were considered practically inactive against MCF-7 cells, since they exhibited higher compound concentrations required to inhibit cell viability by 50% (IC 50 ; > 100 µM) at the investigated concentration ranges. Furthermore, all compounds which exhibited anticancer activities were found to be more effective (lower IC 50 values, Table 1) on HepG2 cells than MCF-7 cells. This can be attributed to the fact that different biological systems react differently toward same affecting compounds due to inherent morphological and membrane-structural differences among different cell lines [30][31][32]. For HepG2 cells, compounds 1a,b, 3a,b, 4a-c, and 5a-c were more potent than compounds 1c, 2a-c, and 3c, which were the least effective against HepG2 cells. According to the IC 50 values, the order of activity of the most potent compounds can be arranged as 1a > 5b > 5a > 4b > 4a > 3a > 1b > 5c > 4c > 3b (IC 50 : 6.62 ± 0. 35 From the aforementioned results, it can be concluded that most of the prepared compounds with potent promising anticancer activities against HepG2 cells showed better activities than the positive control tamoxifen, whereas the most potent compounds against MCF-7 cells showed cytotoxic effects comparable to those of tamoxifen. Finally, it can be concluded that compounds 1a and 1b were the most potent synthesized derivatives against HepG2 and MCF-7 cells, respectively. The structure activity relationship (SAR) was outlined in order to explain the activity of the prepared derivatives. The order of activity against cancer cell lines can be explained due to presence of free carboxylic groups, which increase the acidity, and thus increase the potential anticancer activity. Moreover, the difference in cytotoxic effects can be correlated with the substituted amino acid residues.

Chemistry
Melting points were determined in open glass capillary tubes with an "Electro Thermal" Digital melting point apparatus, (model: IA9100) and are uncorrected. Elemental micro-analyses results for carbon, hydrogen, and nitrogen (Microanalytical Unit, NRC) were found within the acceptable limits of the calculated values. Infrared spectra (KBr) were recorded on a Nexus 670 FTIR Nicolet, Fourier Transform infrared spectrometer. Proton nuclear magnetic resonance ( 1 H NMR) spectra were run in [D 6 ] DMSO on Jeol 270 MHz or 500 MHz instruments. Chemical shifts d are given in ppm. Mass spectra were run on a MAT Finnigan SSQ 7000 spectrometer, using the electron impact technique (EI). Analytical thin layer chromatography (TLC) was performed on silica gel aluminum sheets, 60 F254 (E. Merck). Specific optical rotations were measured with a A. Krawss, Optronic, P8000a polarimeter, in a 1-dm length observation tube at the indicated conditions, and according to the equation: [a]T D = 100. a = (c l), where: a = observed rotation angle, D = sodium line (l = 589 nm), c = concentration (g = 100 mL), l = path length in dm, and T = temperature ( • C). The following solvent systems (by volume) were used as eluents for the development of the plates: S: chloroform-methanol-acetic acid (85: 10: 5); S 1 : S-petroleum ether (40-60 • C) (1:1); S 2 : S-petroleum ether (40-60 • C) (3: 2); S 3 : S-petroleum ether (40-60 • C) (1:2), and S 4 : butanol-water-acetic acid-pyridine (120:48:12:40). It is generally known that basic reaction media enhance racemization. However, under the reaction conditions employed in this work, especially with short reaction times and temperatures below 0 • C, only negligible racemization was observed. [29] (1 mmol) and N-methylmorpholine (0.2 mL, 2 mmol) in dichloromethane (25 mL), ethyl chloroformate (0.2 mL, 2 mmol) was added with stirring at the same temperature (−15 • C). The reaction mixture was stirred for 20 min. Then, a cold dichloromethane solution (20 mL) of the free amino acid methyl ester of L-Phe and/or L-ILeu (2 mmol) was added, with stirring for 3 h at −15 • C and then for 12 h at room temperature. The reaction mixture was washed with water, 1 N sodium bicarbonate, and 1 N hydrochloric acid and water, and dried over anhydrous calcium chloride. The solvent was evaporated under reduced pressure; the obtained residue was triturated with dry ether/n-hexane mixture. The obtained solid was filtered off and crystallized from ethanol/n-hexane to give the corresponding bis-esters (2a-c), respectively.   To a stirred and cold methanolic solution (−15 • C, 20 mL) of the corresponding tripeptide ester (2a-c) (1 mmol), sodium hydroxide (1N, 25 mL) was gradually added. The reaction mixture was stirred for 2 h at the same temperature and 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 acids (3a-c).  The reaction mixture was washed with water, 1 N sodium bicarbonate, and 1 N potassium hydrogen sulfate and water, and then dried over anhydrous sodium sulfate. 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 heptapeptide methyl esters (4a-c). To a stirred and cold methanolic solution (−5 • C, 20 mL) of cyclic heptapeptide 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, and 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 heptapeptide methyl acids (5a-c). To a stirred methanolic solution (20 mL) of the corresponding cyclic pentapeptide methyl ester (4a-c) (1 mmol), anhydrous hydrazine hydrate (0:35 mL, 10 mmol) was added with refluxing for 3 h. The solvent was evaporated and the obtained residue was triturated with ether, filtered off, and crystallized from methanol/ether to afford the hydrazides (6a-c).