SYNTHESIS AND CHARACTERIZATION OF CYCLOHEXANE-1,3-DIONE DERIVATIVES AND THEIR IN SILICO AND IN VITRO STUDIES ON ANTIMICROBIAL AND BREAST CANCER ACTIVITY

Objective: The objective of this study was to evaluate in silico and in vitro anticancer activity for synthesized cyclohexane-1,3-dione derivatives. Methods: The new series of cyclohexane-1,3-dione derivatives were synthesized based on the Michael addition reaction. Further, the structures of the synthesized compounds were confirmed by Fourier-transform infrared spectroscopy, 1H nuclear magnetic resonance (NMR), and 13C NMR spectral data. Then, the in silico molecular docking studies were carried out using AutoDock tool version 1.5.6 and AutoDock version 4.2.5.1 docking program. The antimicrobial activity was carried out using the agar disk diffusion method, and the in vitro anticancer activity was performed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay for the synthesized compound. Results: In silico docking study, compound 5c showed good binding score and binding interactions with selected bacterial proteins and breast cancer protein. Further, compound (5a-5h) was tested for their antimicrobial activity and compound 5c was only tested for anticancer activity (human breast adenocarcinoma 3,4-methylenedioxyamphetamine-MB-231 cell line). Compound 5c was found to be the most active one of all the tested compounds. In the MTT assay compound, 5c showed the LC50 value of 10.31±0.003 μg/ml. In antimicrobial activity, the minimum inhibitory concentration of compound 5c is 2.5 mg/ml. Conclusion: An efficient synthesis of biologically active cyclohexane-1, 3-dione derivatives has been developed.


INTRODUCTION
Cancer is one of the most stressful and life aggressive diseases which implements cruel deaths in the world [1]. It exhibits at least 100 different disease conditions which shares several common symptoms. Cancer is the second leading cause of death in developed countries in spite of its prevention, early detection, and novel therapies. The World Health Organization has warned that nearly 13.1 million people may die of cancer in 2030. Among the women community, breast cancer is the most prevalent type of cancer [2]. The breast cells spread over the body by a process called cancer metastasis. By this process, the organs such as the liver, lungs, brain, and bones get affected and it becomes a major problem affecting the survival of cancer patients. Many therapies were introduced in the recent past to deal with the recurrence of cancer even though drugs and medicines have serious side effects. Hence, the development of drugs to serve as chemoprevention agents is warranted [3].
Heterocyclic compounds play a vital role in the development of pharmacologically active molecules and organic materials [4,5]. The hydroxyl carbonyl compounds prepared by the Claisen-Schmidt reaction between aldehydes and ketone also play an important role in synthetic organic chemistry [6]. Chalcone is a captivating moiety which consists of two aromatic rings linked by enone bridge and it belongs to the flavonoid family. Chalcones show various pharmacological activities such as anticancer, antiviral, antibacterial [7], and antifungal activity [8].
The C-C bond formation has been discovered in the past decades and its applications were reviewed [9][10][11][12][13][14]. Reactions such as Knoevenagel [15], Henry [16], Aldo [17], and Michael reaction [18] are base catalyzed transformations and are mostly used in organic synthesis. Further, heterocyclic compounds exhibited specific activity and are used in the treatment of many infectious diseases. Liu et al. developed successfully an efficient Michael addition between dimedone and cinnamons using unmodified chiral diphenylethylenediamine (DPEN) [19]. In the fight against the resistant bacterial strains, one of the strategies is the development of new antibacterial drugs affecting the integrity of the bacterial cell wall . According to that, many number of chalcones were evaluated for their anticancer and antibacterial activity. Studies on cyclohexane-1,3-dione remain scattered. Hence, this prompted as to undertake the present work. The antibacterial activity of cyclo-hexanone 1,3-dione compounds mechanism were done by in silico method.
Modern drug design process helps to identify and develop new ligands with a high binding affinity toward a target protein receptor. The molecular docking approaches help to reveal drug-receptor interaction to a greater detail. The study of receptor-ligand interaction is considered as one of the fundamental approaches for rational drug design and so the prediction of such interactions by molecular docking has been gaining importance [20].
General procedure for the synthesis of cyclohexane-1,3-dione derivatives (5a-5h) The dimedone (0.1 mol) and chalcones 3a-3h (0.1 mol) were taken in a round bottom flask containing 30 ml ethanol. After, 1 mole of sodium acetate was added. Then, this reaction mixture was stirred and refluxed for 42 h. After the reaction mixture was poured into 500 ml beaker containing crushed ice and it was kept in overnight at room temperature. Then, it was filtered, dried, and recrystallized from ethanol. The purity of the compound was checked by TLC using CHCl 3 as a solvent.

Preparation of the protein
The bacterial proteins and cancer protein were downloaded from Protein Data Bank (PDB) with id: 1UAG, 3UDI, 2X5O, and 2ZOQ.

Ligand preparation
Two-dimensional (2D) structure of the cyclohexane-1,3-dione derivatives is drawn using ChemDraw Ultra 8.0 (Chemoffice2002). After that, the chem 3D ultra were used to convert the 2D structure to 3D structure of the compounds, and the energy is minimized using the semi-empirical AM1 method. All the structures are saved as PDB file format for input to ADT. Finally, all the ligand structures are saved as PDB file format to carry out docking molecular docking in AutoDock Vina.

Grid formation
A grid box with a dimension of 40 × 40 × 40 A3 in 0.375 A spacing and centered on 30.473, 47.997, and 9.563 has created around the binding site of protein using ADT. The center of the box was set at ligand center and grid energy calculations have been carried out.

Docking protocol
Default parameters have been used for auto dock calculations. The energy calculation is done using genetic algorithms. The outputs are exported to Chimera 1.10 and Discovery Studio 4.5 for visual inspection of the binding modes and interaction of the compounds with amino acid residues in the active site [23].

Antimicrobial activity
The newly synthesized cyclohexane-1,3-dione compounds have been evaluated for their antimicrobial activity. The dimethyl sulfoxide was used as the solvent control of this activity. These studies were carried out by the agar disk diffusion method.

MTT assay
MDA-MB-231 (human breast adenocarcinoma) cell was initially procured from the National Center for Cell Science, Pune, India, and maintained Dulbecco's Modified Eagle's Medium (DMEM) (Sigma Aldrich, USA).
The viability of cells was evaluated by direct observation of cells by inverted phase contrast microscope and followed by MTT assay method.

Cell seeding in 96-well plate
Two-day-old confluent monolayer of cells trypsinized and the cells were suspended in 10% growth medium, and 100 µl cell suspension (5×10 4 cells/well) was seeded in 96-well tissue culture plate and incubated at 37°C in a humidified 5% CO 2 incubator.

Preparation of compound stock
Nearly 1 mg of sample was weighed and dissolved in 1 ml DMEM using a cyclomixer. The sample solution was filtered through 0.22 µm Millipore syringe filter to ensure the sterility.

Anticancer evaluation
After 24 h, the growth medium was removed, freshly prepared each compound in 5% DMEM was five times serially diluted by two-fold dilution (100 µg, 50 µg, 25 µg, 12.5 µg and, 6.25 µg) in 500 µl of 5% DMEM, and each concentration of 100 µl was added in triplicates to the respective wells and incubated at 37°C in a humidified 5% CO 2 incubator. Non-treated control cells were also maintained.

Anticancer assay by MTT method
About 15 mg of MTT (Sigma, M-5655) was reconstituted in 3 ml PBS until completely dissolved and sterilized by filter sterilization. After 24 h of the incubation period, the sample content in wells was removed and 30 µl of reconstituted MTT solution was added to all test and cell control wells; the plate was gently shaken well and then incubated at 37°C in a humidified 5% CO 2 incubator for 4 h. After the incubation period, the supernatant was removed and 100 µl of MTT solubilization solution (dimethyl sulfoxide, Sigma-Aldrich, USA) was added, and the wells were mixed gently by pipetting up and down to solubilize the formazan crystals. The absorbance values were measured using microplate reader at a wavelength of 540 nm [24].
The percentage of growth inhibition was calculated using the following formula: Mean OD 100 % of viability Mean OD of control group × =

Chemistry
In the present research work, the new series of cyclohexane-1,3-dione derivatives (5a-5h) were synthesized, which is shown in Scheme 1. The first step in Scheme 1 is the condensation reaction between substituted aldehydes and 4-acetyl biphenyl in the presence of a base to give compound (3a-3h). Finally, the compounds were recrystallized by using ethanol solvent. The chalcones (3a-3h) react with dimedone in the presence of sodium acetate by Michael addition reaction to give cyclohexane-1,3-dione derivatives. The cyclohexane-1,3-dione derivatives were purified by column chromatography using CHCl 3 as the solvent. The structure of the synthesized compounds (5a-5h) was confirmed by spectral techniques such as FT-IR, 1 H NMR, and 13 C NMR. The possible mechanism for this reaction is shown in Scheme 2.  Docking studies of cyclohexane-1, 3-dione derivatives (5a-5h) In the present study, in silico molecular docking studies of cyclohexane-1,3-dione derivatives (5a-5h) against bacterial proteins (PDB id: 1UAG, 3UDI, and 2X5O) and breast cancer protein (PDB id: 2ZOQ) have been carried out using ADT version 1.5.6 and AutoDock version 4.2.5.1 docking program. The molecular docking studies are reported in terms of binding affinity score and the compound which have a better interactions had a lower affinity score. In the ligand, cyclohexane-1,3-done derivatives (5a-5h) were individual, docked with bacterial proteins. Docking results are shown in Table 1. From the table, cyclohexane-1,3-dione derivatives (5a-5h) exhibit good docking scores compared to standard drug (amoxicillin). Based on the docking score, hydrophobic and hydrophilic interaction, cyclohexane-1,3-dione derivatives (5a-5h) show better binding affinity compared to the standard drug. The proteins IUAG, 3UDI, and 2X5O were involved in the cell wall synthesis mechanism. Molecular docking studies show that this cyclohexane-1,3-dione derivatives bind well in the active site pocket of bacterial proteins and interact with the active site of amino acid residues. The best compound of this cyclohexane-1,3-dione derivatives has been explained.

Binding affinity value
Finally, the binding affinity value, conventional hydrogen bond, hydrophobic interaction such as alkyl and pi-alkyl interactions, and other interactions were obtained. Cyclohexane-1, 3-dione derivatives showed good interaction with the studied proteins. Compound 5c showed good binding score with 1UAG (−9.3 kcal/mol), 3UDI (−9.6 kcal/mol), and 2X50 (−9.4 kcal/mol) compared with other synthesized compound of this series (5a-5h) and standard drug. Other synthesized compounds (5a-5h) binding affinity score are shown in Table 1.

Conventional hydrogen bond interaction
Based on high binding affinity value, compound 5c has two conventional hydrogen bond interactions (LEU A: 416 and SER A: 415) with carbonyl moiety of the biphenyl ring (1UAG); compound 5c has no hydrogen bond interaction with 3UDI protein; compound 5c has only one hydrogen bond interaction (ASN A: 211) with carbonyl moiety of the biphenyl ring (2X5O). Other synthesized compounds (5a-5h) conventional hydrogen bond interactions are shown in Table 1. Other synthesized compounds (5a-5h) hydrophobic interactions are shown in Table 1. 2D and 3D images of compound 5c are shown in Fig. 1.

Hydrophobic interaction
The new series of cyclohexane-1,3-dione derivatives (5a-5h) were subjected to molecular docking studies against breast cancer protein 2ZOQ. Compound 5c has high binding affinity score than other cyclohexane-1,3-dione derivatives of this series. For that reason, that compound was performed in vitro anticancer activity against  Table 2. 2D and 3D images of compound 5c are shown in Fig. 2. Compound 5c showed one conventional hydrogen bond interaction with the same amino acid (ARG A: 41) formed at carbonyl group of the biphenyl moiety and dimedone moiety. This compound has only one alkyl and pi-alkyl interaction with PRO A: 373 formed at the benzene ring of biphenyl moiety.     The synthesized compounds (5a-5h) were subjected to insilico analysis against different bacterial proteins (1UAG, 3UDI, 2X5O). Compound 5c showed binding scores as −9.4, −9.6, and −9.1 kcal/mol against 1UAG, 3UDI, and 2X5O proteins indicating a very good affinity. In a similar way when these compounds were docked against the human breast cancer protein 2ZOQ, compound 5c showed a binding score of −9.6 kcal/mol and thereby indicated that it is a good candidate for further studies. Hence, in vitro studies (MTT assay of antimicrobial studies) were carried out to confirm its activities.

Antimicrobial activity
The new series of cyclohexane-1,3-dione derivatives (5a-5h) were screened for antimicrobial activity. The results are shown in Table 3. From Table 3, compound 5c showed a good zone of inhibition against Staphylococcus aureus, Streptococcus pyogenes, and Pseudomonas sp. Compound 5a showed a good zone of inhibition against S. pyogenes, Escherichia coli, and Pseudomonas sp. Compound 5b showed a good zone of inhibition against S. pyogenes. Compound 5d showed a good zone of inhibition against E. coli and Pseudomonas sp. Compound 5e showed a good zone of inhibition against S. pyogenes. Compound 5f showed a good zone of inhibition against S. pyogenes and E. coli. Compound 5g showed a good zone of inhibition against S. pyogenes. Compound 5h showed a good zone of inhibition against S. pyogenes. These compound values are shown in Table 3.
The cyclohexane-1,3-dione derivatives were screened for antifungal activity at different concentrations such as 10, 5, and 2.5 mg/ml. The results are shown in Table 4. From Table 4, compound 5a shown the good zone of inhibition against Candida albicans (12 mm at 2.5 mg/ml).

CONCLUSION
The new series of cyclohexane-1,3-dione derivatives were synthesized by Michal addition reaction. The structures of the cyclohexane-1,3dione derivatives were confirmed by FT-IR, 1 H, and 13 C NMR spectral data. The docking study was carried out for cyclohexane-1,3-dione derivatives (5a-5h) using bacterial proteins (1UAG, 3UDI, and 2X5O) and cancer protein (2ZOQ). From this result, the receptor and the compound have greater potency of interactions in a binding site. Especially, the compound 5c showed good binding score with cancer protein. For that reason, anticancer (human breast adenocarcinoma) activity was studied only compound 5c. The LC 50 value of compound 5c is 10.3134 µg/ml. Furthermore, antimicrobial studies were carried out for cyclohexane 1,3-dione derivatives (5a-5h). From the antimicrobial and antifungal screening, it was observed that all compounds exhibited good activity against Gram-positive and Gram-negative strains. The present research work could be useful to develop the in vivo studies of the synthesized compounds.

AUTHORS' CONTRIBUTIONS
Designed the experiments: Raja Chinnamanayakar and Ezhilarasi M. R. Performed the reactions: Raja Chinnamanayakar.