Introduction

Cancer can be definitely situated by means of the hysterical production of irregular cells in every portion of the body. These atypical cells are mentioned as malignancy cells, tumor cells, or cancer cells. The process of sarcoma cells departure one physical location and rising in another location which called metastasis [1].

Natural plant products have sparked a lot of awareness in the avoidance and cure of prolonged illnesses and normal founded compounds have been for a long time utilized equally medications or drug indications, whether on an experimental or rational basis [2].

Ferulic acid has anti-cancer properties, mostly in vitro and in vivo research condition. It scavenges free radicals, controls cell growing, and spread. Cytoprotective enzymes are promoted and cytotoxic systems are inhibited [3].

Triazole products own a great importance in therapeutic attraction and many heterocyclic triazole chain compounds consuming several biological processes. Triazole is a five-membered basic chemical with the molecular formula C₂H₃N₃ that contains two carbon and three nitrogen atoms. It forms a pair of isomers [4, 5].

The 1-2-3 triazole remains measured to be the most constant compound in relationship to other complexes having three neighboring N atoms. Triazole derivatives have an extensive variety of medicinal actions; for example, antifungal, antibacterial, antiviral, antitumor, anti-convulsant, anti-neuropathic, and others [6]. Therefore, new synthesized compound uses ferulic acid as a starting material with heterocyclic moiety and evaluate the cytotoxicity by MTT assay method.

Materials and Methods

2-Chloroacetaphenone, Benzoyl chloride, Propagyl chloride, Ferulic acid, Hyperchem China, Benzyl bromide, Thionyl chloride, CDH India, Phenylisothiocyanate Sigma Aldrich Germany, Sodium hydroxide pellets              Spectrum USA. The equipment used in the work are electrical melting point apparatus Stuart UK, FT-IR spectrophotometer 8400s Shimadzu-Bruker Japan, ¹H-NMR Jeol-Bruker USA, and ¹³C-NMR Jeol-Bruker USA.

Chemical synthesis

Scheme 1 shows the Synthesis of ferulic acid derivatives.

General synthesis of 4 new ferulic acid derivatives (4a-d) in the same procedure

The solution of benzyl/benzoyl/phenacyl/propagyl chloride (0.005 mol) was stirred in acetone (10 mL), and then it was added to the solution of (E)-4-(2-(5-mercapto-4-phenyl-4H-1,2,4-triazol-3-yl)vinyl)-2-methylphenol (0.96 g, 0.005 mol) in 40 mL acetone containing triethylamine (1.39 mL, and 0.010 mol). The mixture was stirred for 30 hours at 25 °C. After that, it was vaporized under vacuum, and water was added to the precipitate, and then the product compound (4a-d) was obtained by filtration [7].

(E)-4-(2-(5-(benzylthio)-4-phenyl-4H-1,2,4-triazol-3-yl)vinyl)-2-methylphenol (4a)

Yellow green, mp 160-165 °C, yield 50%, IR (KBr) (ν_max/ cm^-1): 3301 (O-H phenol), 3024 (C-H aromatic ring), 1631 (C=N), 1516-1593 (C=C aromatic ring), 1213 (C-O-CH₃), 1070 (C-S). ¹H-NMR (600 MHz, DMSO): δ 3.70 (3H, CH₃ methoxy), 4.39 (2H-Ar ring), 6.35 (1H, CH=CH α, β unsaturated carbonyl), 6.53-755 (13H-Ar ring), 6.82 (1H, CH=CH unsaturated next to aromatic ring), 9.40 (1H, OH phenolic). ¹³C-NMR (150 MHz, DMSO): δ 39.9 (1C, Ar ring), 56.2 (3C, CH₃ of methoxy group), 108 (1C, CH=CH α, β unsaturated carbonyl), 121-130 (6C, Ar ring), 133.5 1C, CH=CH α, β unsaturated carbonyl), 128-137, (6C-Ar ring), 148.4 (1C ,C-triazole), 111-148.7, (6C, Ar ring), 154.1 (1C, C-S).

(E)-2-((5-(4-hydroxy-3-methoxystyryl)-4H-1,2,4-triazol-3-yl)thio)-1-phnylethan-1-one (4b(

Dark brown, mp 180-182 °C, yield 40%, IR (KBr) (ν_max/ cm^-1): 3273 (O-H phenol), 3059 (C-H aromatic ring), 1676 (C=O ketone), 1635 (C=N), 1524 (C = C aromatic ring), 1128 (C-O-CH₃), 1199 cm^-1 (C-S). ¹H-NMR (600 MHz, DMSO): δ 3.76 (3H, CH₃ methoxy), 4.36 (2H-adjucent to carbonyl), 6.31 (1H, CH=CH α, β unsaturated carbonyl), 6.54-8.30 (13H, Ar ring), 7.07 (1H, CH=CH unsaturated next to aromatic ring), 9.15 (1H, OH phenolic); ¹³C-NMR (150 MHz, DMSO): δ 39 (1C adjacent to carbonyl), 56.1 (3C, CH₃ of methoxy group), 116 (1C, CH=CH α, β unsaturated carbonyl), 111-128 (6C, Ar ring), 128.7 (1C, CH=CH α, β unsaturated carbonyl), 121-128.9 (6C, Ar ring), 141 (1C ,C- triazole), 129-156 (6C, Ar ring), 148.6 (1C, C-S), 193 (1C carbonyl group).

(E)-S-(-5-(4-hydroxy-3-methoxystyryl)-4H-1,2,4-triazol-3-yl) benzothioate (4c)

Light brown, mp 175-178 °C, yield 30%, IR (KBr) (ν_max/ cm^-1): 3254 (O-H phenol), 3115 (C-H aromatic ring), 1703 (C=O ketone), 1595 (C=N), 1545 (C = C aromatic ring), 1220 (C-O-CH₃), 1120 cm^-1 (C-S). ¹H-NMR (600 MHz, DMSO): δ 3.33 (3H, CH₃ methoxy), 6.88-7.97 (13H, Ar ring), 7.39 (1H, CH=CH unsaturated next to the aromatic ring), 7.47 (1H, CH=CH α, β unsaturated carbonyl), 10.15 (1H, OH phenolic). ¹³C-NMR (150 MHz, DMSO): δ 56 (3C, CH₃ of methoxy group), 112 (1C, CH=CH α, β unsaturated carbonyl), 133 1C, (CH=CH α, β unsaturated carbonyl), 126-131(6C, Ar ring), 128-136 (6C, Ar ring), 144.5 (1C ,C – triazole), 116-149 (6C, Ar ring),162.5 (1C, C-S), 210 (1C carbonyl group).

Scheme 1: Synthesis of ferulic acid derivatives

(E)-4-(2-(4-cyclohexyl-5-(prop-2-yn-1-ylthio)-4H-1,2,4-triazol-3-yl)vinyl)-2-methoxyphenol (4d)

Black, mp 105-110 °C, yield 15%, R (KBr) (ν_max/ cm^-1): 3270 (H attach to C triple bond), 3046 (O-H phenol), 2339 (C≡ C), 1703 (C=N), 1515-1596 (C = C aromatic ring), 1234 (C-S), 1157 (C-O-CH₃), ¹H-NMR (600 MHz, DMSO): δ 3.38 (2H adjacent to thio), 3.76 (3H, CH₃ methoxy), 4.38 (1H-adjacent to triple bond), 6.31 (1H, CH=CH α, β unsaturated next to aromatic ring), 6.34 (1H, CH=CH  unsaturated carbonyl), 6.74-7.32 (8H-Ar ring), 9.16 (1H, OH phenolic). ¹³C-NMR (150 MHz, DMSO): δ 21 (1C-carbon between triple and thiol), 56.1 (3C, CH₃ of methoxy group), 75-76 (C≡C), 112 (1C, CH=CH α, β unsaturated carbonyl), 133 (1C, CH=CH α, β unsaturated carbonyl), 126-138 (6C-Ar ring), 144.5 (1C, C-triazole), 148.7 (1C, C-S), 111-149 (6C, Ar ring).

Molecular docking studies for the synthesized compounds

The docking research for the produced compounds were carried out by using the Genetic Optimization for Ligand Docking (GOLD) (v 5.7.1). Tool program was obtained from the Cambridge Crystallographic Data Center (CCDC). The Hermes visualizer software (v.1.10.1) was used to show receptors, ligands, active positions, interface modes (hydrogen bonds or brief contacts), posture estimate, bond length assessment, and fold images [8].

Interpretation of the results of ADME studies

The Swiss ADME server examined the physicochemical and ADME properties of the new synthesized compounds to examine which synthesized molecules are subjected to organism given orally, to make known the unharmed, a medication view, and to exclude compounds that inadequate in the phases of drug development deponding on ADME properties [9].

Cytotoxicity assay

It was possible to get the human A459 lung cancer cell line and the human MCF-7 breast cancer cell line. They were in Tissue Culture Research Database Center, Cell Bank in Al-Qahera University, College of Science.

Cell viability by MTT colorimetric assay

The cytotoxic effects of newly developed and manufactured compounds on lung cancer cells and breast cancer cells were determined by using the MTT colorimetric test.

Cell viability was measured by using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cells of lung and the breast cancers were seeded in 96-well plates when growth was reaching 70% confluence. Serial indicated concentration of tamoxifen, erlotinib, and newly developed and manufactured compounds were added to culture medium. After 24 hours, twenty microliters of MTT (5 mg/mL) was added to each well, and then the 96-well plates were incubated at 37 °C. After 4 hours, 150 µl of DMSO was added to adherent cells of each well to dissolve crystal. Then, the luminescence of each well was measured by using a microplate reader at a wavelength (570 nm) [10].

Determination of the half-maximal inhibitory concentration (IC₅₀)

The IC₅₀ of the studied substances may be calculated by using the dose-response curve. The IC₅₀ in the MTT experiment (in vitro) may be defined as the concentration of produced compounds (4a, 4b, 4c, and 4d) required to achieve 50% cell inhibition.

Results and Discussion

Interpretation of ADME result

TPSA or topological polar surface area was considered, this is a useful characteristic for forecasting different ADME features, including drug brain access and bioavailability [11]. The compound with a TPSA > 140 Ao stays likely to be situated weakly absorbed from the GIT.

Bioavailability of all ligands was 0.55 and TPSA for all synthesized compounds was less than 140, indicating that they entered the systemic circulation, as demonstrated in Table 1.

Interpretation of the docking result

Docking study results in the determination of binding energies and specification of the designed compounds to proteins, e.g., EGFR, and ER-α via investigating the molecular interaction between the active binding sites of proteins and designed chemical compounds. The EGFR and ER-α inhibitory effect of developed compounds, erlotinib, and tamoxifen were classified depending on their PLP fitness associated in the complex formation at the active site.

The outcomes of docking analysis of newly created compounds are displayed in Figures 1 to 8 and Tables 2 and 3.

Table 1: ADME result of the final compound

BBB: Blood Brain Barriar, TPSA: Topological Polar Surface Area            

Table 2: New compound and tamoxifen, a predictable drug, docked with ER-α. Binding energies

PLP: Pull Ups, Lunges, and Push Ups

Table 3: The binding energies of new compound and erlotinib, a predictable drug, docked with EGFR

PLP: Pull Ups, Lunges, and Push Ups, EGFR: Epidermal Growth Factor Receptor

All of the proposed ligands showed binding energies in the active pocket of the receptor, according to docking data, and a positive interaction with EGFR, H-bonds, and other brief contacts was anticipated that strengthen the binding are used by the ER-protein and other proteins to attach to the amino acid residue of the active site.

Results of compounds' cytotoxicity tests on a breast cancer cell line (MCF-7)

By using the MTT assay, the synthesized compounds' IC₅₀ values were calculated. The experiment was conducted by using 96-well flat plates with various concentrations of the produced compounds (10000-78.12 µg/mL) of the synthesized compound (4a, 4b, 4c, and 4d) compared with the standard anticancer cell, Tamoxifen, the IC₅₀ values were obtained after 72 hours of chemical treatment of the cancer cells.

Prism Pad 8.1's nonlinear regression analysis was used to create the dose-response curves for the synthetic substances in MCF-7 cells (12) are demonstrated in Figures 9, 10, 11, and 12. The IC50 of the new synthesized compound was indicated in the Table 4.

From the mentioned result above, we noticed that the new synthesized compounds 4a, 4b, and 4c, respectively are the more active anticancer than standard Tamoxifen as IC₅₀ value, which have IC₅₀ (89.17, 108.69, and 118.24) lower than standard tamoxifen IC₅₀ (392.3).

Serafim et al. found that ferulic acid derivatives increase cytotoxicity effect against breast cancer MCF-7 cell line [13]. In the same line, Elkhazendar reported the promising therapeutic effect of ferulic acid in breast cancer treatment [14].

Table 4: IC₅₀ values for the active chemicals (4a, 4b, 4c, and 4d) and Tamoxifen as a standard against breast cancer MCF-7 cell

Results of compounds' cytotoxicity tests on a lung cancer cell line (A549)

By using the MTT assay, the synthesized compounds IC₅₀ values were calculated. The experiment was conducted by using 96-well flat plates with various concentrations of the produced compounds (10000-78.12 µg/mL) of the synthesized compounds 4a, 4b, 4c, and 4d compared with standard anticancer cell, erlotinib, the IC₅₀ values were obtained after 72 hours of chemical treatment of the cancer cells.

Prism Pad 8.1's nonlinear regression analysis was used to create the dose-response curves for the synthetic substances in A549 cells [15] are indicated in Figures 13, 14, 15, and 16. The IC50 of the new synthesized compound was shown in Table 5.

Table 5: IC₅₀ values for the active chemicals (4a, 4b, 4c, and 4d) and erlotinib as a standard drug against lung cancer A 549 cell

Although erlotinib is important for first line treatment of patients with non-small lung cancer that EGFR mutated, the result of our study noticed the new synthesized compounds 4a and 4b, respectively are more active anticancer than standard erlotinib as IC₅₀ value, which have IC₅₀ (478.32 and 561.12) lower than standard erlotinib IC₅₀ (582.73). This finding agrees with Fong et al. study indicated inhibitory effect of ferulic acid dervitive against lung cancer by suppressing proliferation and migration of cancer cells [16].

Conclusion

Successful synthesis of a new family of ferulic acid derivatives was accomplished and the anticancer activities of the synthesized compounds were evaluated against the lung cancer cell line A549. Among the examined substances, the most promising compounds 4a and 4b which are more active than reference anticancer drug erlotinib. While the others synthesized compound 4d and 4c have higher IC₅₀ values than standard anticancer drug erlotinib, the anticancer activities of the synthesized compounds were evaluated against the MCF-7 (breast) cancer cell line. We noticed the new synthesized compound 4a, 4b, and 4c, respectively is the more active anticancer than standard tamoxifen as IC₅₀ value, which have IC₅₀ lower than standard tamoxifen, the results of docking experiments for ligand interactions with the EGFR protein were in agreement with one compound and disagreement with those obtained in vitro, the results of docking studies for ligand interactions with the ER-protein were not consistent with those from in vitro experiments.

Acknowledgment

The authors would like to thank and chairman for their support and help, I would want to thank Assistant Professor, Dr. Ayad M.R. Raauf, for his scientific guidance, assistance, and support during the research period.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Authors' contributions

All authors contributed to data analysis, drafting, and revising of the paper and agreed to be responsible for all the aspects of this work.

Conflict of Interest

We have no conflicts of interest to disclose.

ORCID:

Samir A. Malik

https://orcid.org/0000-0002-2110-2386

HOW TO CITE THIS ARTICLE

Samir A. Malik, Karima F. Al, Ashour H. Dawood. Synthesis, Characterization, and Preliminary Evaluation of Ferulic Acid Derivatives Containing Heterocyclic Moiety. J. Med. Chem. Sci., 2023, 6(6) 1444-1456

https://doi.org/10.26655/JMCHEMSCI.2023.6.24

URL: http://www.jmchemsci.com/article_160865.html

 Karima F. Al

https://orcid.org/0000-0003-3496-6471