L-proline catalyzed green synthesis and anticancer evaluation of novel bioactive benzil bis-hydrazones under grinding technique

ABSTRACT L-Proline organocatalyst was investigated as an efficient and environmentally benign catalyst for synthesis of some novel bioactive benzil bis(2-cyano-3-aryl-2-propenoylhydrazone) derivatives from reaction of the benzil bis(cyanoacetylhydrazone) with the respective aldehydes. The reusability of the L-proline was tested and the results revealed that the recovered catalyst can be reused at least three additional times in subsequent reactions without significant decrease in product yield. The current protocol is very efficient as it provides mild reaction, cleaner reaction profiles, effortless work phase with outstanding purity, and with short reaction time, high performance of the desired products. IR, 1H-NMR and MS and alternative methods, whenever available, have verified the chemical structures of the newly prepared compounds. Compared to the Doxorubicin reference drug using the MTT assay, the in vitro growth inhibitory activity of ten new compounds was investigated against the liver carcinoma cell line (HepG-2) and the findings revealed promising activities of six derivatives. GRAPHICAL ABSTRACT


Introduction
Green chemistry concentrates on research aimed at developing safe and environmentally sustainable chemical procedures for the design, in synthetic organic chemistry, of biologily active and industryleading molecules (1-3). Green chemistry provides various alternatives, including the use of fewer solvents, high nuclear economies and selectivity, removal of hazardous wastes, the use of climate, simple methods for product separation and purification, as well as use of alternative energy sources, in order to carry out organic transformations on environmentally sustainable terms (4)(5)(6)(7)(8)(9)(10)(11). 'Mechanosynthesis' using 'ball milling' or simple grinding methods (12,13) is one of the protocols belonging to this sustainable and 'greener' definition. Due to its simple experimental setup, manual grinding using a mortar and pestle is a very useful technique on the laboratory scale. This method does not involve external heating, so organic synthesis is an energy efficient and more economical and ecologically advantageous process (14). The work carried out by Toda and his colleagues reported many of the organic exothermic reactions can be done using mortar and pestle grinding at good yields (15). This approach is often used for condensation reactions, including the formation of Schiff's base and oxime (16). Recently, the construction of heteroaromatic compounds of biological interest has been found to be equally efficient (17)(18)(19)(20).
In recent years, organocatalysis has been a subject of great interest due to the ease of obtaining, easily stored, stable, non-toxic and inexpensive. In addition, organocatalysis is a type of catalysis that can be done without the need for inert atmosphere or anhydrous conditions in mild conditions (21,22). Organocatalysis has now seen an exponential growth in many publications, the interest in new catalysts and reactions in which asymmetric products can be produced in classical and valuable reactions using organocatalysts, such as Diels-Alder, Mannich, Michael reactions and others (23,24). Among organocatalysts, L-proline is a readily obtainable naturally occurring amino acid and is easy to obtain in high enantiomeric purity it has been reported as an ecofriendly catalyst for the synthesis of several heterocycles with high efficiency (25)(26)(27)(28)(29)(30). In particular, L-proline has received a lot of attention because of its dual position as a ligand and catalyst (31)(32)(33).
Cyanoacetic acid hydrazide is a powerful and convenient precursor for the synthesis of a wide range of heterocyclic compounds. The molecule's β-functional nitrile (34-37) is a favorable unit for addition, accompanied by cyclization or cycloaddition, of various reagents supplying one or more heteroatoms with heterocyclic compounds of different ring sizes that are of interest as antibacterial agents, herbicides, pharmaceuticals (38)(39)(40) and as dyes (41).
In view of these facts, we reported here the synthesis of novel bioactive benzil bis(2-cyano-3-aryl-2-propenoylhydrazone) derivatives via solvent free mechanical grinding of the benzil bis(cyanoacetylhydrazone) with the respective aldehydes using L-proline as an efficient organocatalyst, in short reaction time, high efficiency, and low costs high percent yields.
Under the grinding process with moist L-proline (5 mol percent) at room temperature, Knoevengel condensation of benzil bis(cyanoacetylhydrazone) 3 with various benzaldehyde derivatives 4a-j was achieved to provide an excellent yield of the corresponding compounds 5a-j. Structures of bis-hydrazones 5a-j were compatible with spectroscopic data (Scheme 1). 1 H-NMR of compound 5a, taken as an example of the prepared series, showed two singlet signals at δ = 8.64 and 11.06 ppm attributable to -CH = N and = N-NH, respectively. The theoretical measurement was also in line with molecular ion peaks and microanalysis findings for the synthesized products.
To test the completeness of reactions after grinding for 10 min, we repeated the synthesis of compounds 5a, 5e and 5g after 10 min and the results revealed that the yield% for compounds 5a and 5g have decreased from 94% to 83% and 90% to 89%., respectively while the yield% for compound 5e haven't changed. We can conclude that the electron donating and the electron withdrawing groups enhance the rate of reaction so the yield% isn't significantly decreased. While in case of the unsubstituted derivative 5a was sharply decreased (from 94% to 83%).
Next, we checked the reusability of the catalyst for the synthesis of 5a in the presence of a catalytic amount of L-proline (5 mol%) in water. After completion of the reaction, the reaction mixture was diluted with water (10 mL) and the crude solid product was filtered and washed with H 2 O (2-5 mL). The L-proline was removed from the reaction media by washing with H 2 O. Since the catalyst is soluble in water, the catalyst was recovered by evaporation of the water and washed with diethyl ether and reused for the subsequent catalytic runs. The reusability of the L-proline was tested for four runs, providing 94-73% of the desired product 5a yield in a similar reaction time. The results of recycling experiments are given in Table 1. The recovered catalyst can be reused at least three additional times in subsequent reactions without significant decrease in product yield (Table 1).
The electronic absorption spectral data of compounds 5a,e,g in dioxan exhibited in each case two characteristic absorption bands at ∼ 341 and 269 nm ( Table 2). Such an absorption pattern is similar to that of the typical hydrazone chromophore (43).
Scheme 2 demonstrates a possible mechanism for the synthesis of benzil bis-hydrazones 5a-j derivatives. The mechanism based on the reaction of benzil bis(cyanoacetylhydrazone) 3 in the presence of L-proline with two benzaldehyde derivatives, producing a Knoevengel adduct (II) through the condensation reaction of Knoevengel followed by the removal of 2 water molecules to supply the final desired products 5a-j.
As can be shown in the experimental section three singlet signals at δ 5.21 (s, 2O-CH 2 -O), 8.73 (s, 2CH = N) and 10.99 (s, D 2 O exchangeable, 2NH) ppm were shown by the 1 H-NMR spectra of 7, in addition to an aromatic multiplet in the 7.10-7.43 (m, 16H, ArH) ppm region. The mass spectrum of reaction products 7 and 9 displayed molecular ion peaks at m/z = 636 and 560, respectively, in accordance with the planned structures (see Experimental).
In addition, in the presence of a moist L-proline organocatalyst (5 mol percent) under solvent-free conditions (Scheme 4), bis-coumarin derivatives 13a-c were generated at excellent yields when compound 3 was ground by mortar and pestle with two equivalents of salicyaldehydes 10a-c.
In order to take account of the formation of product 13, it was proposed that the reaction of compound 3 with salicyaldehyde 10 initially results in benzil bishydrazone intermediate 11 followed by intramolecular cycloaddition of the OH group to an activated cyano group forming a non-isolable bis-iminointermediate 12 followed by hydrolysis of the imino groups to the carbonyl groups forming the respective bis-coumarins.
In addition to the absorption bands of the NH function in the region v ∼3426 cm −1 , the IR spectra of the latter products showed carbonyl absorption bands in the region v ∼1667 and 1705 cm −1 . In addition to the coumarin-H4 singlet signal at δ = 9.01 ppm, their 1 H- NMR of bis-coumarin derivative 13a showed one D 2 O exchangeable NH group signal at δ = 11.14 ppm. The assigned structures 13a-c (Scheme 3) confirmed the analysis of the above products. The structure of 13 was chemically demonstrated by an alternate approach (Scheme 3). Thus the reaction of benzil with 2-oxo-2H-chromene-3-carbohydrazide derivatives 14a-c (2 moles) (40) with one drop of acetic acid under the grinding process resulted in the formation of products which are identical in all respects (mp, mixed mp and IR) with 13a-c compounds, respectively.

Cytotoxic activity
Discovering new heterocyclic compounds used for production of new, active, selective and less toxic anticancer agents remains a major challenge for researchers in medicinal chemistry. Holding this in mind, and in the continuation of our earlier work on the discovery of different anticancer agents (44)(45)(46)(47)(48)(49)(50)(51)(52)(53), we are presenting an effective synthesis of a new series of benzil bis-hydrazone derivatives 5a-j for the evaluation of their antitumor activity against human hepatocellular carcinoma (HepG-2).
The antitumor activity of the newly synthesized bishydrazones 5a-j was investigated against the liver carcinoma cell line HepG-2. As a reference standard, Doxorubicin was used and showed IC 50 = 0.72 μM against the cell line of liver carcinoma. The obtained results were used to graph a dose-response curve that was calculated by the concentration (μM) of test compounds needed to kill 50 percent of the cell population (IC 50 ). Cytotoxic activity was presented as the mean IC 50 of the three independent studies.
The findings showed a high variable behavior relative to the reference drug as seen in Table 3, Figures 1 and 2 which was mainly evaluated. The activities of the synthesized compounds depend on structural skeleton and electronic environment of the molecules. The following was the descending order of activity of the newly synthesized compounds: 5i > 5j > 5d > 5e > 5b > 5f > 5a > 5c > 5g > 5h. Examination of the SAR leads to the following conclusions.

Chemistry
Melting points were determined on a digital melting point apparatus of the Electrothermal IA 9000 series. Elemental analyses were measured using a German made Elementarvario LIII CHNS analyzer. Shimadzu TGA-50 H Thermal Analyzer was utilized to study the thermal degradation behavior of the samples from room temperature to 500°C with a heating rate of 10°C min −1 . Mass spectra were recorded at 70 eV on a mass spectrometer of the Shimadzu GCMS-QP1000 EX. NMR spectra were recorded on a Varian Mercury VX-300 NMR spectrometer operating at 300 MHz ( 1 H-NMR) and run in deuterated dimethylsulfoxide (DMSO-d6). Chemical shifts were related to that of the solvent. 13 C-NMR was recorded on a BRUKER spectrometer at 75 MHz. IR spectra were recorded in potassium bromide discs on Pye Unicam SP 3300 and Shimadzu FTIR 8101 PC infrared spectrophotometers.

Synthesis of benzil bis (cyanoacetylhydrazone) (3)
A mixture of benzil (2.1 g, 10 mmol), 2-cyanoacetohydrazide (1.98 g, 20 mmol) and 50 μL of HCl was ground with a pestle in a mortar at 25°C. The initial syrupy reaction  3.3. General procedure for the synthesis of compounds 5a-j, 7, 9 and 13a-c In the presence of L-proline (0.006 g, 5 mol percent) and 50 μL of water, a mixture of compound 3 (0.372 g, 1 mmol) and the respective aldehyde 4a-j, 7, 9 or 13a-c (2 mmol) was ground with a pestle in a mortar at 25°C . The initial syrupy reaction mixture solidified within 10-20 min. The TLC (EtOAc: n-hexane 1:1) tracked reaction progress. The solid was washed with cold water and recrystallized from the appropriate solvent to give products 5a-j, 7, 9 or 13a-c, respectively. The physical constants and spectral data of the obtained products are listed below: Benzil bis   Using a mortar and pestle at 25°C, a mixture of benzil (0.210 g, 1 mmol) and the required 2-oxo-2H-chromene-3-carbohydrazide derivative 14a-c (2 mmol) was ground together for 10-15 min. TLC tracked the progress of the reaction. The reaction mixture was recrystallized from DMF to give the respective 13a-c compounds that were similar in all respects (m.p., mixed m.p. and IR spectra) with those obtained from the reaction of 3 with 10a-c.

Biological evaluation
Evaluation of the antitumor activity using Viability assay: Human hepatocellular carcinoma (HepG2) cell line was obtained from the American Type Culture Collection (ATCC, Rockville, MD). The cells were grown on RPMI-1640 medium supplemented with 10% inactivated fetal calf serum and 50 µg/mL gentamycin. The cells were maintained at 37°C in a humidified atmosphere with 5% CO 2 and were subcultured two to three times a week. Potential cytotoxicity of the compounds was evaluated on tumor cells using the method of Gangadevi and Muthumary. The cells were grown as monolayers in growth RPMI-1640. The monolayers of 10 4 cells adhered at the bottom of the wells in a 96-well microtiter plate incubated for 24 h at 37°C in a humidified incubator with 5% CO 2 . The monolayers were then washed with sterile phosphate buffered saline (0.01 M pH 7.2) and simultaneously the cells were treated with 100 µL from different dilutions of tested sample in fresh maintenance medium and incubated at 37°C. A control of untreated cells was made in the absence of tested sample. Positive controls containing doxroubcin drug was also tested as reference drug for comparison. Six wells were used for each concentration of the test sample. Every 24 h the observation under the inverted microscope was made. The number of the surviving cells was determined by staining the cells with crystal violet followed by cell lysing using 33% glacial acetic acid and read the absorbance at 590 nm using microplate reader (SunRise, TECAN, Inc, USA) after well mixing. The absorbance values from untreated cells were considered as 100% proliferation. The number of viable cells was determined using microplate reader as previously mentioned before and the percentage of viability was calculated as [1-(ODt/ODc)] x 100% where ODt is the mean optical density of wells treated with the tested sample and ODc is the mean optical density of untreated cells. The relation between surviving cells and drug concentration is plotted to get the survival curve of each tumor cell line after treatment with the specified compound. The 50% inhibitory concentration (IC 50 ), the concentration required to cause toxic effects in 50% of intact cells, was estimated from graphic plots (54).

Conclusion
In conclusion, this research highlights a simple, efficient and novel green protocol for the preparation, under solvent-free conditions of bezil bis-acylhydrazones using L-proline as a reusable catalyst. The main aspects of this process include: quick operation; high product yield, commercially viable, safe handling, no toxicity and catalyst reusability. Structures of all newly prepared products been established on the basis of both elemental analysis and spectroscopic data and wherever possible, by alternative methods. In addition, the mechanisms of formation of the compounds of the title were discussed. In comparison with a well-known standard anticancer drug (Doxorubicin), ten new compounds were assessed for their anti-cancer efficacy against the human hepatocellular carcinoma (HepG-2) cell line. Their structure activity (SAR) has also been studied. Promising activities for six compounds (5i, 5j, 5d, 5e, 5b and 5f) were revealed in the results (IC 50 = 0.93, 1.18, 2.15, 5.27, 6.12 and 8.01 µM, respectively).