INTRODUCTION:

Lung cancer is one of the most common cancers in the world, where it has caused approximately 1.8 million deaths across the globe¹. This type of cancer can be classified into two categories according to its histology, namely small cell lung cancer and non-small cell lung cancer. The latter is the more dominant type with prevalence of 85% of lung cancer². Overall survival rates of this cancer remain low, despite the current development of therapy³. Bis(arylidene) cycloalkanone based compound has been commonly found in various fields, namely polymer sciences⁴, agrochemicals, perfume industry, and pharmaceuticals⁵.

This compound could also potentially be utilized as starting point for developing novel drug, since this scaffold possess various biological activity^6,7. This class of compound possess almost identical scaffold to curcumin, one of the well-known constituents in Curcuma genus plant⁸. Several studies of structure modifications of curcumin have suggested the replacement of diketo moiety with cycloalkanone ring, namely^9-12. One of the reasons for this modification is to eliminate tautomeric functional group, which could affect bioactivity¹³ and stability¹⁴ of curcumin-based compound (figure 1).

Figure 1: (above) tatutomeric keto-enol form of curcumin. (below) bis(benzylidene) cyclohexanone class of compound

Various research has been performed to confirm the bioactivity of this class of compound. It has been shown that cyclohexanone-curcumin analogs with methoxy group yielded anti-cancer activity against various cancer cell line such as carcinoma in gastric, esophagus¹⁰, prostate¹¹, and colon¹². Nakhjiri et al. have designed various asymmetric bis(nitro-benzylidene) cyclohexanone and tested their bioactivity against various cancer cell-line. The result showed that most of the analogs possessed better anti-cancer activity⁹. This class of compound can be synthesized in a facile manner using aldol condensation from cyclohexanone and benzaldehyde derivates. Normally this reaction is performed under strong acid or strong base condition (eg. HCl and NaOH)^7,15. Many other catalysts have also been utilized to further improve the outcome of this condensation process, such as p-toluenesulfonic acid¹⁶, sulfamic acid¹⁷, magnesium bisulfate¹⁸, boron trifluoride-diethyl etherate complex¹⁹, ionic liquid²⁰, and NMSDSA⁵. Interestingly, some of the reactions explained previously were performed under microwave irradiation. This method has significant advantage over conventional reaction, especially increasing product yield and minimizing use of chemical reagents²¹.

In this study, we aimed to synthesize 2,6-bis-(4-nitrobenzylidene) cyclohexanone and evaluated its anticancer property. This compound belongs to symmetric bis(nitrobenzylidene) class and has been previously evaluated its bioactivity as antioxidant and anti-inflammatory agent⁷. Synthesis process was performed using microwave-assisted aldol condensation in basic condition by using 10% NaOH. Afterwards, the product was tested against lung cancer cell A549. In addition, molecular docking study was performed to verify the possible mechanism of interaction between designed compound and its molecular target²².

MATERIALS AND METHODS:

Instruments and Chemicals:

Synthesis procedure was performed in the Laboratory of Organic Chemistry, Faculty of Pharmacy, University of Surabaya. All chemicals and reagents used in the synthesis process was purchased from Merck Millipore (Singapore). Commercial microwave (LG MH6548FR) was used for synthesis process. Thin Layer Chromatography was used to monitor the completion of reaction (Type 60 F254, Merck). Detection of the spots was aided by UV lamp in 254nm. Melting point determination of the product was performed using Sybron-Thermolyne-MP12615. Infrared and Ultraviolet spectra measurement was done using Agilent and Shimadzu, respectively. These data were obtained from Research Laboratory, Faculty of Pharmacy, University of Surabaya. NMR spectra measurements were conducted using JEOL ECS-400 spectrophotometer (400 MHz) in Institute of Tropical Disease, Airlangga University. HRMS-TOF spectra was recorded in Padjadjaran University. Anticancer activity against A549 and Vero cell line was evaluated in Gadjah Mada University. Ultimately, molecular docking was performed in standard personal computer specification (Intel Core i7-9700F 3.00 GHz, RAM 16 GB) using Vina 1.2.3²³.

Synthesis and structure identification:

4.82mmol cyclohexanone (0.5mL) was put to Erlenmeyer flask over an ice bath, then added by 8mL of 10% NaOH solution. Afterwards, 9.66mmol 4-nitrobenzaldehyde (1.46g) was weighed and dissolved in ethanol 96%, separately. The flask containing cyclohexanone was then set on an ice bath while slowly 4-nitrobenzaldehyde solution was added dropwise. The mixture was then irradiated with 200W microwave for twenty seconds. This irradiation process was replicated five times, to ensure the completion of reaction (figure 2). The reaction was monitored using TLC with n-hexane/chloroform system of 2:5. The product was then acidified in HCl 2M to yield solid product. After washing the crystal with water and ethanol, it was recrystallized using chloroform-ethanol solvent and dried in oven. The final product was then characterized using various instrument mentioned previously (melting point apparatus, UV spectrophotometer, infrared spectrophotometer, NMR, and HRMS-TOF).

Figure 2: Synthesis of 2,6-bis-(4-nitrobenzylidene) cyclohexanone

Anticancer bioassay:

Anticancer activity was evaluated in vitro against A549 and Vero cell lines. Stock solution of 50.000ppm was made by dissolving the test compound, positive control (erlotinib), and negative control with DMSO. This solution was then diluted in several concentration using RPMI media to make working solution for normal cell. A549 and Vero cell lines were seeded into 96-well plates and then incubated overnight in 5% CO₂ incubators. Various concentration of test compound, positive, and negative compound were added into the well plates in triplicate. At the end of incubation, each well was added with 100μL of 0.5mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), followed by incubation for 3 h. The reaction was quenched by adding 100μL of 10% sodium lauryl sulfate solution in HCl 0.1M into each well. Afterwards, the plates were reincubated at 37°C overnight. Enzyme-linked immunosorbent assay (ELISA) reader was used to quantify the formation of formazan blue color from MTT at 595nm. The IC₅₀ values were obtained using probit analysis and compared to positive control²⁴.

Molecular docking study:

Prediction of molecular interaction between test compound and protein target was done using molecular docking approach. EGFR was selected as macromolecule target (PDB ID: 1M17)²⁵. It is argued that EGFR can be found in non-small cell lung cancer, such as A549 cell line²⁶. This protein was prepared by stripping of its native ligand (erlotinib) followed by adding hydrogen and Kollman charges. Meanwhile, the native ligand and designed compound was also prepared by ensuring the correct three-dimensional structure with addition of Gasteiger charge. The first step performed was to validate the method by redocking the native ligand back to the original binding site in the protein. Grid box size of 16x16x16 Å³ was built centered on the binding site of erlotinib. Vina use Monte-Carlo stochastic search algorithm in combination with BFGS optimization to produce the most optimal docking pose ²³. RMSD value was utilized to evaluate the process, where less than 2Å was considered acceptable. Afterwards the test compound was docked to the designated binding site and then evaluated its binding energy with Vina-based scoring function. In addition, ligand interaction with amino acid residues within the binding site using Discovery Studio Visualizer.

RESULT AND DISCUSSION:

Synthesis and structure identification:

To the best of our knowledge, 2,6-bis-(4-nitrobenzylidene) cyclohexanone has been previously synthesized^5,7 and utilized as a starting material for synthesis potential anticancer agent²⁷. In this study, the target compound was synthesized via Claisen-Schmidt aldol condensation with the assistance of microwave irradiation, in contrast to the previous studies which utilized reflux conditions. This method has helped in increasing the reaction rate significantly, where the product can be obtained in just less than 2 minutes. The compound (2,6-bis-(4-nitrobenzylidene) cyclohexanone) obtained from this reaction has a physical characteristic of yellow crystal without any observable by-product, as indicated by TLC and NMR results.

Figure 3: Reaction mechanism of synthesis of 2,6-bis-(4-nitrobenzylidene) cyclohexanone

2,6-bis-(4-nitrobenzylidene) cyclohexanone:

Yield: 85%, yellow crystal (Rf = 0.40); m.p. = 204°C; UV (λ max) = 333nm; IR(ATR) (cm^-1-ν: 3060 (C-H aromatic), 2924(C-H cycloalkane), 1690(C=O), 1662 (C=C alkene), 1584 (C=C aromatic), 1511(-NO₂), 1446 (C=C aromatic), 1336(-NO₂), 829(p-disubstituted benzene); ¹H-NMR(400 MHz,CDCl₃): δ (in Hz) 1.85 (q,J = 2.4 Hz, 2H), 2.93 (t,J = 10.3 Hz, 4H), 7.59 (d,J = 8.7 Hz, 4H), 7.79 (s, 2H), 8.26 (d,J = 8.7 Hz, 4H); ¹³C-NMR (100MHz,CDCl3): δ (in Hz) 22.92, 28.46, 123.78, 130.91, 134.99, 138.74, 142.20, 147.44, 189.32; HRMS m/z ESI+ (M+H⁺): 365.1135 (figure 4-8)

Figure 4: UV spectrum of 2,6-bis-(4-nitrobenzylidene) cyclohexanone

Figure 5: IR spectrum of 2,6-bis-(4-nitrobenzylidene) cyclohexanone

Anticancer bioassay:

Several analogs of benzylidene-cyclohexanone have exhibited promising anti-cancer properties^9-12. However, this compound possesses anticancer activity only on millimolar scale against A549 cell line. Similar result was observed for erlotinib as well which also yielded millimolar inhibitory activity. The findings indicated that both the tested compound and erlotinib are considered not adequate to inhibit A549 cell line. Although erlotinib is known to possess potent inhibitory activity against EGFR receptor and has been approved for usage against non-small-cell lung cancer by FDA²⁸, its potency against A549 cell line was found to be minimal in other study²⁹. On the other hand, the results from Vero cell line bioassay showed that both compounds indicating the non-toxic characteristic against normal cell line.

Table 1: IC₅₀of 2,6-bis-(4-nitrobenzylidene) cyclohexanone and erlotinib

Compound Name	A549 cell line (mM)	Vero cell line (mM)
2,6-bis-(4-nitrobenzylidene) cyclohexanone	0.48 ± 0.05	> 1
Erlotinib	0.28 ± 0.03	> 0.2

Figure 6: 1 H-NMR spectrum of 2,6-bis-(4-nitrobenzylidene) cyclohexanone

Figure 7: 13C-NMR spectrum of 2,6-bis-(4-nitrobenzylidene) cyclohexanone

Figure 8: Mass spectrum of 2,6-bis-(4-nitrobenzylidene) cyclohexanone

Molecular docking:

This study aims to predict the binding interaction between designed compounds and evaluate its binding energy compared to erlotinib. Prior to the docking process, method validation was performed by redocking its native ligand (erlotinib). This process has yielded 10 best docking poses ranked according to their binding energy. However, RMSD value obtained from all of the best scoring poses was found to be higher than the commonly acceptable value of 2Å. It is possible that this result was due to the flexibility of erlotinib which has 10 rotatable bonds, mainly on the methoxyethoxy group, which caused difficulty for docking algorithm in reproducing original docking pose³⁰. In this case, we decided to select docking pose which is closer to the original conformation rather than choosing the first ranked pose³¹. The selected pose has an RMSD value of 2.21 Å with binding energy value of -6.80. Meanwhile, the docking result of designed compound also yielded 10 docking poses. After evaluating every conformation based on its interaction with amino acid residues, it was decided to choose the best scoring pose with binding energy value of -8.25. It is also found that although 2,6-bis-(4-nitrobenzylidene) cyclohexanone cannot form hydrogen bond with any amino acid, the ligand possesses π-alkyl interactions with VAL702, ALA719, LYS721, and CYS773 (Table 2). Despite the similar result with previous study of another curcumin-cyclohexanone analogue²² it did not correlate well with in vitro cell based assay against A549 cell.

Table 2: Molecular docking result of 2,6-bis-(4-nitrobenzylidene) cyclohexanone and erlotinib against EGFR receptor (PDBID:1M17)

Ligand

Docking Score

Hydrogen Bond Interaction

π-Alkyl Interaction

2,6-bis-(4-nitrobenzylidene) cyclohexanone

-8.25

VAL702

ALA719

LYS721

CYS773

Erlotinib

-6.80

MET769

CYS773

VAL702

LYS721

CONCLUSION:

Compound 2,6-bis-(4-nitrobenzylidene) cyclohexanone can be synthesized using microwave assisted aldol condensation in a time-efficient manner. Anticancer activity of this compound was found to be nominal. Further structure modifications are needed in order to improve its bioactivity as anticancer agent.

CONFLICT OF INTEREST:

All authors declare no conflict of interest.

ACKNOWLEDGMENTS:

This research was supported by Lembaga Penelitian dan Pengabdian Masyarakat University of Surabaya

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Received on 12.09.2023 Modified on 06.12.2023

Research J. Pharm. and Tech 2024; 17(7):3246-3250.

DOI: 10.52711/0974-360X.2024.00508