Synthesis and anticancer activity of novel Eugenol derivatives against breast cancer cells

Abstract Eugenol chemically known as 4-allyl-2-methoxyphenol is a major phenolic component of Syzigium aromaticum and associated with significant biological activities. In the present work, new eugenol 1,2,3-triazole derivatives have been synthesized, characterized using NMR, mass spectrometry, IR, and elemental analysis and screened for their anticancer activity against breast cancer cells. Compound 9, namely 3-(4-((4-allyl-2-methoxyphenoxy)methyl)-1H-1,2,3-triazol-1-yl)-N'-(4-methylbenzoyl) benzohydrazide was found to be the most potent candidate and better than eugenol in exhibiting cytotoxicity with IC50 6.91 and 3.15 μM, comparable to Doxorubicin with IC50 6.58 and 3.21 μM against MDA-MB-231 and MCF-7 cells, respectively. Furthermore, compound 9 treated MCF-7 cells as observed by propidium iodide staining significantly increased cell population of S phase and G2 phase to 43.64% and 35.19%, respectively therefore arresting cell cycle at G2 and S phase. These results indicate that eugenol linked 1,2,3-triazole ring could be used as anticancer leads for the treatment of this deadly diseases. Graphical Abstract


Introduction
Structural modifications of biologically active natural products have been important strategies to overcome their side effects and enhance therapeutic effects, particularly in the area of cancer (Koehn and Carter 2005;Gordaliza 2007). Most of the anticancer drugs that reached to the markets like etoposide, teniposide, topotecan, irinotecan, etc or are under clinical trials were obtained through semi synthetic modifications of natural products (Abdel-Razek et al. 2020).
In continuation of our research involving structural modification of natural products Almalki et al. 2021) for lower toxicity and better efficacy, in the present work eugenol was selected due to its broad spectrum of pharmacological activities. On the other hand, previous work indicated that the incorporation of 1,2,3triazole ring in the structure of natural products have resulted in potential anticancer activity (Majeed et al. 2013;Alam 2022) due to its ability of non-covalent interactions and binding with biological targets (Alzahrani et al. 2022). Therefore, in this work, hydroxyl group of eugenol was explored by propargylation, followed by incorporation of 1,2,3-triazole ring by click chemistry approach and screened for anticancer activity which could be used as anticancer leads for the treatment of this deadly disease.

Chemistry and compound identification
Eugenol (1) was reacted with propargyl bromide in dry acetone using anhydrous potassium carbonate to yield o-propargylated eugenol 2 which upon click reaction with 3-azido-benzoic acid resulted in the formation of the key intermediate, 1,2,3-triazole incorporated eugenol derivative 3. Reaction of intermediate 3 with ethyl chloroformate and then with different amines and acid hydrazides, in the presence of triethylamine, afforded the new compounds 4-7 and 8-11 in moderate to good yield ( Figure 2).
The structure of all newly synthesized compounds was confirmed by FTIR, NMR, and mass spectrometry analyses. The absorption bands at 3400-2800 cm À1 (broad) and 1707 cm À1 in FT IR spectrum of 3 was due to OH and C ¼ O groups while in 1 H NMR, OH hydrogen appeared as singlet at 8.43 ppm and other singlet at d 5.40 ppm was assigned to oxy-methylene hydrogens bridging 1,2,3-triazole and eugenol fragments. Further confirmation was obtained from 13 C NMR displaying downfield signal at d 169.13 ppm for carbonyl carbon and mass spectrometry exhibiting molecular ion peak at 364.33 as [M-H] -.
Considering 4-7, FT IR displayed the carboxamide functional bands at 3157-3288 cm À1 and 1647-1683 cm À1 for -N-H and C ¼ O stretchings, respectively whereas in 1 H NMR, NH signal appeared as a singlet at 8.20-8.56 ppm. Besides the extra hydrogens arises due to introduction of new aromatic hydrogens (Figures S1-S4) and d 164.27-164.47 ppm for C ¼ O group of carboxamide in 13 C NMR (Figures S9-S11). However, in compound 7, two new signals as triplets in aliphatic region at d 1.19 and d 3.40 ppm were observed due to N-attached and O-attached morpholine ring hydrogens in 1 H NMR ( Figure S4) and C ¼ O peak at 168.79 ppm in 13 C NMR spectrum ( Figure S12). The structures of compounds 8-11 was also confirmed by FT IR showing similar absorption bands at 3170-3179 cm À1 and 1636-1682 cm À1 for -N-H stretching and -C ¼ O stretching of the carboxamide group. In 1 H NMR spectra of these compounds (Figures S5-S8), two new signals appeared at d 9.14-9.53 ppm and 9.89-10.28 ppm due to two -N-H hydrogens whereas two downfield signals at d163.64-164.05 ppm and d 165.85-166.65 ppm in 13 C NMR spectra (Figures S13-S16) for two carbonyl carbons confirmed the presence of two carboxamide groups in the structure. Further confirmatory observations were obtained from their mass spectra (Figures S17-S24).

MTT assay
The newly semisynthetic eugenol-1,2,3-triazole derivatives were screened for their cytotoxicity towards breast cancer cells, MDA-MB-231 and MCF-7 by MTT assay (Nazreen 2021). The results are presented in Table S1. These compounds showed significant to moderate cytotoxicity compared to eugenol and doxorubicin (standard drug). Compound 9 having tolyl group was found to be the most potent with IC 50 6.91 and 3.15 lM, comparable to doxorubicin with IC 50 6.58 and 3.21 lM whereas eugenol displayed IC 50 41.14 and 38.7 lM against MDA-MB-231 and MCF-7 cells, respectively. Compounds 4, 5, 6, 8 and 11 also exhibited better cytotoxicity than eugenol towards the tested breast cancerous cell lines. Compound 7 bearing morpholine ring and compound 8 bearing unsubstituted phenyl ring displayed weaker cytotoxicity. It was noted that most of the semisynthetic derivatives containing 1,2,3triazole moiety linked to eugenol possess better anticancer activity than eugenol towards breast cancer cells.

Cell cycle arrests
The most promising compound 9 was further investigated for cell cycle distribution in MCF-7 cells by flow cytometry after staining with propidium iodide. Compound 9 treated cells increased population of S phase significantly and G2 phase from 35.81% to 43.64% and 31.91% to 35.19%, respectively while decreased G1 phase to 21.17% from 32.28% ( Figure S25). This result demonstrated that compound 9 arrests cell cycle at G2 and S phase.

Semi synthesis of eugenol derivatives (4-11)
For intermediates 2 and 3, see supplementary material. In a 100 mL round bottom flask, 20 mL toluene were added and cooled to 0-5 C followed by addition of ethyl chloroformate (2.4 mmole) and triethylamine (2.4 mmole). To this reaction mixture, it was added compound 3 (2 mmole) at 0-5 C with stirring for 2-4 hrs, then added different amines or acid hydrazides (2 millimole) and further stirring was continued for 2-4 hrs. When the reaction was completed, 50 mL of water were added and toluene layer was separated out. The toluene layer was washed with water, evaporated and products 4-11 were recrystallized with isopropyl alcohol.

Biological activity
The experimental procedure can be found in supplementary material

Conclusion
The present work describes the synthesis of new eugenol derived 1,2,3-triazole derivatives and their anticancer activity against breast cancer cells. Compounds were successfully characterized using NMR, Mass, IR and elemental analysis. The anticancer activity showed that the compound 9 was found to be the most potent candidate and better than eugenol in exhibiting cytotoxicity with IC 50 6.91 and 3.15 lM, comparable to doxorubicin with IC 50 6.58 and 3.21 lM against MDA-MB-231 and MCF-7 cells, respectively. Furthermore, compound 9 treated MCF-7 cells as observed by propidium iodide staining significantly increased cell population of S phase and G2 phase to 43.64% and 35.19%, respectively therefore arresting cell cycle at G2 and S phase. These results indicated that eugenol linked 1,2,3-triazole ring could be used as anticancer leads for the treatment of this deadly disease

Disclosure statement
No potential conflict of interest was reported by the authors.