Novel N-Acyl Hydrazone Compounds as Promising Anticancer Agents: Synthesis and Molecular Docking Studies

In this study, a new series of N-acyl hydrazones 7a-e, 8a-e, and 9a-e, starting from methyl δ-oxo pentanoate with different substituted groups 1a-e, were synthesized as anticancer agents. The structures of obtained target molecules were identified by spectrometric analysis methods (FT-IR, 11H NMR, 13C NMR, and LC–MS). The antiproliferative activity of the novel N-acyl hydrazones was evaluated on the breast (MCF-7) and prostate (PC-3) cancer cell lines by an MTT assay. Additionally, breast epithelial cells (ME-16C) were used as reference normal cells. All newly synthesized compounds 7a-e, 8a-e, and 9a-e exhibited selective antiproliferative activity with high toxicity to both cancer cells simultaneously without any toxicity to normal cells. Among these novel N-acyl hydrazones, 7a-e showed the most potent anticancer activities with IC50 values at 7.52 ± 0.32–25.41 ± 0.82 and 10.19 ± 0.52–57.33 ± 0.92 μM against MCF-7 and PC-3 cells, respectively. Also, molecular docking studies were applied to comprehend potential molecular interactions between compounds and target proteins. It was seen that the docking calculations and the experimental data are in good agreement.


INTRODUCTION
Today, one of the main causes of death worldwide is cancer, which develops when one or more cells from a certain tissue in the body deviate from their usual properties and multiply uncontrollably. 1,2 According to the findings of recent studies, female breast cancer is the most commonly diagnosed cancer, with 11.7% of cases and 6.9% of all cancer deaths, followed by lung (11.4%), colorectal (10.0%), prostate (7.3%), and stomach (5.6%) cancers. 3,4 In recent years, intensive studies have been carried out around the world for the development of new drugs that can act against cancer. Today, many factors, such as the rapidly increasing number of patients, serious side effects caused by drugs in use, toxicity, and the development of drug resistance by tumors, increase the importance of these studies. Chemotherapy, one of the anticancer treatments, is widely used because of its effects on tumor cells. However, it is known that many anticancer drugs have serious adverse effects and toxicity. Therefore, it is of great importance to develop new anticancer agents that can stop the growth of cancer cells or kill them while at the same time not harming healthy cells.
Small organic compounds serve as bioactive scaffolds, which are a crucial component of drug design. N-Acyl hydrazones, which are an important member of the class of organic compounds and are represented by the general formula R 1 − NHN�CH−R 2 , are small organic molecules in which R 1 and R 2 represent different functional groups. In recent years, the interest of researchers has focused on N-acyl hydrazones, which have very important pharmacological properties, thus a good option for the development of new biologically active drug molecules. 5−13 In the research carried out to find more effective and, at the same time, low-toxic anticancer drugs, it was determined that N-acyl hydrazone derivatives have anticancer activity, and this result increased the importance of this substance group in cancer treatment. In the literature survey, it is observed that new derivatives of N-acyl hydrazones are prepared and their anticancer activities are measured, and these drugs are modified by preparing their analogues with commercially available anticancer drugs. 14−23 For example, in a study by Popiołek et al., N-acyl hydrazones derived from 3-hydroxy-2naphthoic acid were found to show significant antitumor activities against HepG2 and 769-P cell lines. 24 In another study, a series of N-acyl hydrazone derivatives synthesized from ethyl paraben were reported to have potential activity against liver cancer (HepG2). 25 The synthesized dipyrromethane N-acyl hydrazone derivatives exhibited moderate to extremely strong cytotoxic effects against HL-60 (leukemia) and HCT-116 (colon) cancer cells according to a different study by Gautam et al. 26 Novel (R,S)-etodolac derivatives containing the N-acyl hydrazone moiety synthesized by Koçet al. have been found to have good cytotoxic effects against PC-3, DU-145, and LNCaP cell lines (prostate cancer). 27 Further, N-acyl hydrazones are well-known to exhibit a wide spectrum of biological properties as antioxidant, 28−31 analgesic, 32 anti-inflammatory, 33−35 antimicrobial, 36−41 antiviral, 42,43 anticonvulsant, 44 antiprotozoal, 45 antimalarial, 46 larvicidal, 47 antituberculosis, 48 and antifungal 49 activity. Some compounds with known biological activities containing the N-acyl hydrazone structure are shown in Figure 1.
In the previous study of our group, we synthesized γand δimino esters from γand δ-oxo methyl ester derivatives and found that they exhibited high antioxidant activity. 50 In another study, we obtained γ-oxime esters and determined that they showed elastase inhibition activity. 51 Based on our previous studies and literature data, in this work, we focused to designing and synthesizing novel N-acyl hydrazones from methyl δ-oxo pentanoate with different substituted groups with promising considerable anticancer properties. For this purpose, we synthesized fifteen novel N-acyl hydrazone derivatives 7a-e, 8a-e, and 9a-e from their corresponding methyl δ-oxo pentanoates with aryl, substituted aryl, and heteroaryl groups (Scheme 1). The structures of the compounds 7a-e, 8a-e, and 9a-e were elucidated by FT-IR, 1 H NMR, 13 C NMR, and LC− MS analysis methods, and their purity was confirmed by HPLC. Compounds 7a-e, 8a-e, and 9a-e were screened for their in vitro anticancer activity against breast (MCF-7) and prostate (PC-3) cancer cell lines using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. In addition, molecular docking studies were applied to investigate the antiproliferative effects of these novel compounds.
First, we obtained the methyl δ-oxo pentanoate derivatives with substituted aryl and heteroaryl groups 1b-e used as starting compounds according to the Friedel−Crafts acylation reaction in 85−90% yield. 52,53 Compound 1a was obtained from the reaction of δ-oxo-δ-phenyl-pentanoic acid with methanol in the presence of concentrated sulfuric acid in 100% yield. Next, compounds 1a-e were reacted with hydrazine monohydrate in ethanol in order to obtain δ-oxo pentane hydrazide derivatives as the key intermediate. When we examined the reaction of similar molecules with hydrazine in the literature, we saw that the reaction was carried out without protecting the carbonyl group. 54,55 However, in our experiments, we observed that both C�O groups in the molecule reacted with hydrazine hydrate, thus reducing the reaction efficiency (45−50%). Thus, we decided to protect the carbonyl group in the δ-position in order to increase the reaction yield and prevent the formation of byproducts. A model reaction was used to determine optimum reaction conditions; we reacted compound 1a with varying molar ratios of ethylene glycol (EG), triethyl orthoformate (TMOF), and p-toluenesulfonic acid monohydrate (p-TsOH). The results are given in Table 1. In Table 1, entry 2 shows that the reaction was performed with molar ratios of 1:3:3:0.01 1a/EG/TMOF/p-TsOH with a 96% yield. Therefore, compounds 2a-e were successfully obtained in 90−96% isolated yields. Product formation was determined by the GC−MS chromatographic method. Then, ketal the intermediates 3a-e were prepared by refluxing compounds 2a-e with hydrazine monohydrate in ethanol in 95−98% yields. The formation of the intermediates 3a-e was confirmed by the molecular ion peak observed by the GC−MS spectrum. Finally, compounds 3a-e were reacted with various aldehydes (p-anisaldehyde, 2-furaldehyde, and isovaleraldehyde) in dimethylformamide (DMF) in the refluxing condition to generate δ-ketal N-acyl hydrazones 4a-e, 5a-e, and 6a-e in 51−95% isolated yields. 56,57 Then, the hydrolysis reaction of 4a was investigated in the presence of several acid catalysts and solvents in order to optimize reaction conditions. The results are shown in Table 2. In Table 2, entry 4 shows that the reaction was carried out in the presence of Bi(NO 3 ) 3 · 5H 2 O in CH 2 Cl 2 at room temperature with a 98% yield. According to these reaction conditions, N-acyl hydrazone derivatives 7a-e, 8a-e, and 9a-e were obtained with 95−98% isolated yields. All the target molecules 7a-e, 8a-e, and 9a-e are shown in Figure 2.
In the FT-IR spectra, weak −NH bands were observed at 3279−3172 cm −1 and N−N bands at 1178−1015 cm −1 in all target compounds. Carbonyl absorption (C�O), belonging to both the azomethine group and the aliphatic chain, was seen between 1515−1477 and 1684−1556 cm −1 , respectively. The peaks around 1596−1488 cm −1 are evidence of the presence of the C�N group. The peaks of aromatic ring vibrations were observed between 3090−3026 and 1608−1538 cm −1 . The literature suggests that the hydrazones could exist as cis/trans amide conformers and E/Z geometrical isomers centered on C−N double bonds. 25,58 In the present study, the peaks observed in the 1 H and 13 C NMR spectra of the N-acyl hydrazone compounds showed that 7a-e and 8a-e were obtained as a mixture of E and Z isomers. However, compounds 9a-e were determined to be in single isomer form. Therefore, we evaluated the signals of the 1 H NMR spectra of the compounds 7a-e and 8a-e as set I and set II. 58 In each compound's 1 H NMR spectrum for 7a-e, 8a-e, and 9a-e, the characteristic signals of the azomethine �CH protons found at δ 11.23−8.98 ppm were observed as broad singlets and those of −NH amide protons (−CONHNCH−) as singlets and triplets at δ 7.89−7.09 ppm. Aromatic protons are seen as a doublet, triplet, and multiplet between δ 7.98 and 6.56 ppm. The chemical shift and integral values of other protons are in agreement with their compound structures. In the 13 C NMR spectra, the azomethine group (−N�CH−) signals were detected at δ 168.7−146.3 ppm. The signals corresponding to C�O of ester and amide were observed at δ 200.2−192.7 and δ 175.2−174.2 ppm, respectively. Other aliphatic and aromatic carbon signals of N-acyl hydrazones 7ae, 8a-e, and 9a-e were seen at the expected values of chemical shift.

Cytotoxicity Assay.
In this work, cells were treated with the compounds at concentrations ranging from 1 to 1000 M for 48 h in order to evaluate the in vitro cytotoxic effects on cancer (MCF-7, PC-3) and normal (ME-16C) cell lines. Doxorubicin, the most commonly used chemotherapy drug to treat various types of cancer cells, was chosen as the positive   (Table 3). Among selected cancer cell lines, MCF-7 cells were more sensitive to the antiproliferative effects of newly synthesized Nacyl hydrazones. Doxorubicin demonstrated significantly low IC 50 values at 0.83 ± 0.07, 0.75 ± 0.04, and 0.80 ± 0.09 for MCF-7, PC-3, and ME-16C cell lines, respectively (Table 3, entry 16). Although doxorubicin seems to be very effective in killing cancer cells at low concentrations, it is also highly toxic to normal cells at the same concentration.
Compounds 7a-e exhibited more potent cytotoxic activity in cancer cells than compounds 8a-e and 9a-e. Therefore, the selectivity index (SI) of compounds 7a-e was calculated and is presented in Figure 3. SI is a ratio of the IC 50 value in cancer cell lines (MCF-7, PC3) to the IC 50 value in non-cancer cell lines (ME16C). SI values greater than 1.0 demonstrate higher anticancer activity. 59 The data revealed that compound 7d was the most cytotoxic compound with IC 50 values at 7.52 ± 0.32 and 10.19 ± 0.52 μM against MCF-7 and PC-3 cells, respectively (Table 3,    The cytotoxic activity of synthesized N-acyl hydrazones in cancer cell lines differed among cells. 7d, 7c, and 7b exhibited the highest antiproliferative effects in MCF-7 cells, whereas 8c, 8e, and 8d were the lowest toxic compounds, respectively. On the other hand, the cytotoxicity order differs in PC-3 cells as 7d, 7e, and 7c were characterized as the most toxic compounds, whereas 8d, 8e, and 8c were the least toxic compounds. The methoxy group in the phenyl ring attached to the azomethine group at the R 2 position activates the ring due to its electron donor effect. As a result of this effect, it can be assumed that compounds 7a-e carrying the p-methoxy phenyl group in the R 2 position have higher antiproliferative activity than other compounds (8a-e and 9a-e). Among synthesized Nacyl hydrazones, 8b, 8d, 8e, and 9a-e did not cause any cytotoxic effect in ME-16C cells in the tested range of concentrations. As a result, it was revealed that the p-methoxy phenyl group at the R 2 position (R 1 −NHN�CH−R 2 ) is crucial for inducing cytotoxicity as well as selectivity against the MCF-7 cancer cell line. By replacing the p-methoxy phenyl group with furyl or isovaleryl groups, cytotoxic activities were reduced.
2.3. Molecular Docking Studies. By molecular docking experiments, these novel compounds' antiproliferative effects have been examined, and comprehensive evaluations of the molecules' ideal poses have been conducted. By evaluating the best binding affinity and receptor-ligand interaction of each compound, the good interactions of the compounds within the receptor active pocket of the target receptor proteins are shown in Tables 4 and 5. Considering prior studies of identical structures that contain hydrazone subunits, we made the decision to look for potential binding motifs for MCF-7 and PC-3 in order to examine their anticancer properties for breast cancer (MCF-7) and prostate cancer (PC-3). 60,61 Thus, the proteins with the highest affinity for the target molecules were determined as 1Z5M for PC-3 and 1X7B for MCF-7. Doxorubicin, an often-used anticancer medicine, was selected to compare our results.
After the docking interactions, the conformation with relatively low docking energy scores is selected because the ligand's strongest binding potential inside the target is indicated by the conformation with the lowest negative binding energy values.
Nearly all of the compounds demonstrated adequate binding free energies for MCF-7 and PC-3 that ranged between −9.67 and −11.74 and −7.28 and −9.37 Kcal/mol, respectively. Figures 4−7 show that the compounds bind to the active position and overlap with the reference compounds. According to our preliminary findings, these substances have a respectable level of ligand-receptor binding interactions.
Compounds 7b-d and 8b have the smallest RMSD scores with the lowest binding energy scores in each target. As shown in the tables, some of them also exhibited powerful hydrogen bonding with related amino acid residues. The figures show that the active conformations of each compound bind the active site and overlap with each other. The results established that these compounds forecasted the best ligand-receptor binding interactions. Additionally, we evaluated the interactions involved between the target proteins (1Z5M and 1X7B) and compound 7d which exhibited high affinity toward 1Z5M and 1X7B and has proven to possess good in vitro anticancer activity. Figures 8 and 9 show the interactions between ligands and proteins. Compound 7d interacts with nine residues to be complexed with 1Z5M as seen in Figure 8. It implicated five alkyl interactions with VAL A: 96, ALA A: 109, VAL A: 143, LEU A: 159, and ALA A: 162; one Pi-sigma interaction with LEU A: 212; attractive charges with GLU A:166 and hydrogen bonds with GLY A:165 and LEU A:88 residues which interact with compound 7d were identified to be relevant in the complexation process between 7d and 1Z5M. In Figure 9, compound 7d interacts with eight residues to be complexed with 1X7B. It implicated five alkyl interactions with LEU A: 298, LEU A: 301, LEU A: 339, LEU A: 343, and LEU A: 476; one carbon-hydrogen bond with PHE A: 356; one pi-sulfur bond with MET A:340; and a hydrogen bond with HIS A:475.

Drug-like Properties.
The Swiss ADME Calculation program has been used to determine drug-likeness rankings in order to better understand the structure−activity correlations of compounds. Table 6 lists the molecular weight, logP, TPSA, blood−brain barrier (BBB) crossing, GI absorption characteristics, and kind of CYPP450 inhibition of drugs. Almost all substances have been shown to have quite low levels that will cross lipid barriers. Compounds have been identified to have lipophilicity values less than 4.

CONCLUSIONS
In summary, we designed, synthesized, and identified novel Nacyl hydrazone derivatives 7a-e, 8a-e, and 9a-e and tested them for in vitro cytotoxicity. Overall, this study presents evidence that N-acyl hydrazone derivatives exhibit significant and  Molecular docking experiments were used to explore the antiproliferative properties of these new compounds, and appropriate binding free energies of almost all compounds for MCF-7 and PC-3 were determined between −9.67 and −11.74 and −7.28 and −9.37 Kcal/mol, respectively. According to the   results obtained from molecular docking studies, it was determined that all synthesized N-acyl hydrazone derivatives exhibited very good ligand-receptor binding interactions. It has also been discovered that all compounds have relatively favorable values for crossing lipid barriers (lipophilicity value <4).
Since conventional chemotherapy drugs are highly toxic to normal cells, newly synthesized N-acyl hydrazone derivatives could be promising candidates for anticancer drug development through their selective cytotoxic activity. Therefore, the potential anticancer efficacy and mechanism of action of N-acyl hydrazone derivatives must be further investigated in vivo.

Materials and Apparatuses.
All of the chemicals and solvents utilized in the syntheses and in vitro tests were obtained from commercial suppliers (Merck, Sigma-Aldrich, Acros Organics, and Thermo Fisher Scientific) and used without additional purification. Solvents used for chromatography were of technical grade and distilled before use. Thinlayer chromatography (TLC) was used to monitor chemical reactions under 254 nm UV light. The synthesized starting compounds and N-acyl hydrazones were purified using column chromatography on silica gel (0.063−0.200 mm) with hexaneethylacetate. Melting points were measured with a Buchi melting point apparatus B-540. Gas chromatography−mass spectrometry (GC−MS) data were recorded on a Shimadzu QP2010 Plus. The purity of the N-acyl hydrazone derivatives was determined on the Shimadzu/DGU-20A5 HPLC apparatus. FT-IR spectra were recorded on Bruker Vertex. 1 H NMR and 13 C NMR spectra were recorded at 500 and 126 MHz, respectively. DMSO d 6 was used as a solvent, and Me 4 Si was used as the internal standard. LC−MS data were recorded on Shimadzu 8040. Pentanoate Derivatives (2a-e).

Synthesis. 4.2.1. General Procedure for the Protection of Methyl δ-Oxo
To a solution of δ-oxo methyl ester 1a-e (1 mmol), ethylene glycol (3 mmol), and triethyl orthoformate (3 mmol) in toluene (5 mL) was added p-toluenesulfonic acid monohydrate (0.01 mmol). The reaction mixture was heated to reflux for 24 h until all starting material was consumed in the TLC analysis. Next, the reaction mixture was cooled and quenched with a saturated NaHCO 3 solution. The mixture was extracted with petroleum ether (40−60°C) three times. The combined organic phases were washed with saturated NaCl solution and then dried over anhydrous Na 2 SO 4 and concentrated under reduced pressure. The products 2a-e were obtained as yellow oil in a 90−96% yield. Hydrazides (3a-e). The 80% hydrazine monohydrate (3 mL) was added to a solution of protected δ-oxo methyl ester 2a-e (5 mmol) in absolute ethanol (15 mL) and was stirred at reflux for 6 h. The reaction mixture was monitored by TLC (eluent: hexanes/EtOAc 1:1) and visualized using UV light. After the reaction is complete, the ethanol was removed under reduced pressure. The mixture was extracted with ethyl acetate three times. The organic phase was washed with distilled water, dried over anhydrous Na 2 SO 4 , and concentrated under reduced pressure. The ketal hydrazides 3a-e were obtained in a 95− 98% isolated yield. (4a-e, 5a-e, 6a-e). 56,57 The δ-ketal hydrazide 3a-e (1 mmol) and aldehyde (anisaldehyde, furfural, or isovaleraldehyde) (2 mmol) in DMF (2 mL) were added to a reaction flask, and then the mixture was heated to reflux. The reaction progress was monitored by TLC (eluent: hexane:E-tOAc (1:1). After finishing the reaction, the solvent was removed under reduced pressure. The crude product was purified by column chromatography on silica gel (eluent: hexanes/EtOAc 3:1). The pure products 4a-e, 5a-e, and 6a-e were obtained in 51−95% isolated yields. Hydrazones (7a-e, 8a-e, 9a-e). A solution of δ-ketal N-acyl hydrazones 4a-e, 5a-e, 6a-e (1 mmol) and Bi(NO 3 ) 3 ·5H 2 O (0.25 mmol) in CH 2 Cl 2 (5 mL) was stirred at room temperature for 2−4 h. Next, the mixture was filtered, and the filtrate was washed first with a 10% aqueous NaHCO 3 solution and then with saturated NaCl, dried over anhydrous Na 2 SO 4 , and concentrated under reduced pressure. The pure products 7a-e, 8a-e, and 9a-e were obtained in a 98% isolated yield.

HPLC Analysis.
The purity of the target compounds was defined by normal phase high-performance liquid chromatography (HPLC) using a 250 × 4,6 mm, 5 μm AD-H chiral column using Hekzan/isopropanol (70:30). The   : LGC Standards, Wessel, Germany) to be used in the cytotoxic activity assays. Cells were detached with a 0.25% Trypsin/EDTA solution upon 70−80% confluency and seeded into a new culture flask including DMEM-LG with 10% FBS and 0.1 mg/mL primocin, incubated in a 37°C, 5% CO 2 incubator. The medium was refreshed every 48 h, and the cells were passaged every 5−6 days.

MTT Assay.
Newly synthesized compounds were tested for their cytotoxic activity on cancer (MCF-7, PC-3) and normal (ME-16C) cell lines using the MTT assay. MTT is used to assess cell viability by determining the mitochondrial activity of living cells according to their ability to reduce the yellow tetrazolium salt MTT into purple formazan crystals. The MTT assay (Thermo) was carried out according to the manufacturer's instructions. Briefly, cells were seeded on 96well plates at a density of 1 × 10 4 cells/well and incubated in a 37°C, 5% CO 2 incubator. Cells were then treated with different concentrations (1−1000 μM) of compounds for 48 h. Following incubation, 10 μL of MTT solution was added to the cells and incubated for 4 h, and then 100 μL of solubilization solution was added and incubated overnight. Absorbance was measured at 570 nm using the microplate reader (Synergy H1, Biotek). For all experiments, doxorubicin was used as the positive control, and untreated cells were used as the negative control. DMSO was used as the solvent of the compounds, and the final concentration did not exceed 0.5% v/v. All the experiments were performed three times, and each was carried out in triplicate.  To provide a theoretical perspective on probable molecular interactions between compounds from the 7a-e, 8a-e, and 9a-e series and target proteins, molecular docking experiments were conducted. Energy minimization was used to calculate theoretical binding affinities based on the results of docking calculations. The computation of molecular docking, energy reduction, and molecular visualization of docking data was carried out using the Autodock Vina software suite. The Chem Draw drawing program was used to prepare model inhibitor compounds for molecular docking in the 7a-e, 8a-e, and 9a-e series. Before the docking procedure, the special 7a-e, 8a-e, and 9a-e series compounds were drawn and edited in the SD File format using the Chem 3D suite program. Protonation, charging, and conformation minimization utilizing the root-mean-square gradient have all been applied to these molecular structures.
Three-dimensional coordinates of X-ray crystal structures of target proteins were obtained from the Structural Bioinformatics Research Collaboration (RCSB) Protein Data Bank. 62 For use in docking calculations, a structure with the PDB IDs 1X7B for MCF-7 and 1Z5M for PC-3 was selected as the crystal structure model matching these target proteins. The software tool Autodock Vina was used to correct structural defects in these target proteins. As docking calculations are being performed, default parameters are being used (temperature 300 Kelvin, pH 7, solvent 0.1 M, electrostatic energy cutoff 15 A). The final molecular docking score values were calculated using the average score of the top 10 final docking postures determined by the binding minimum energy (kcal/ mol) for each molecule. 62