Synthesis of ursolic acid arylidene-hydrazide hybrid compounds and investigation of their cytotoxic and antimicrobial effects

Abstract In this study, 13 new hybrid compounds (7a-m) were synthesised starting from ursolic acid, and their cytotoxic activities were investigated on the BEAS-2B and A549 cell lines. In addition, the synthesised compounds were tested against Staphylococcus aureus, Escherichia coli, and Candida albicans to determine their anti-microbial properties. The hybrid compounds that exhibited the lowest cytotoxicity against the BEAS-2B were 7k, 7b, and 7g. The cytotoxicity of the compounds against A549 was evaluated, the IC50 value of 7k, 7b, and 7g are found as 0.15 µM, 0.31 µM, and 0.26 µM, respectively. The results showed that the selectivity of 7k was 7 times higher than doxorubicin against the A549 cells. According to the antimicrobial activity studies 7c is found as the most effective compound against S. aureus. Almost all compounds showed a similar inhibition potential against E. coli and C. albicans. Graphical Abstract

Cancer is recognised as a major health problem worldwide. Indeed, it is one of the leading causes of death, being responsible for around 10 million deaths in 2019 alone. Globally, approximately one in six deaths are caused by cancer (Anonymous 2021; Ferlay et al. 2021). Based on the global cancer rates, the 10 most common types of cancers are lung, breast, colorectal, prostate, stomach, liver, esophagus, cervix uteri, thyroid, and bladder cancer (Ferlay et al. 2019).
Lung cancer has been determined to be the most common type of cancer in men worldwide, accounting for 15.5% of the total number of new cancer cases diagnosed in 2018. By contrast, breast cancer is the most common type of cancer in women worldwide, and it was responsible for 25.4% of all new cancer cases diagnosed in 2018. Yet, when all cancer-related deaths are taken into account, lung cancer is the most common cause of death among both genders worldwide, resulting in 18% of all cancer deaths (Ferlay et al. 2021;Merkouri et al. 2021).
Hybrid molecules are defined as chemical compounds that fulfill different biological functions and, further, that contain two or more structural domains that exhibit dual activity (Meunier 2008). Natural products and their semi-synthetic derivatives constitute a large percentage of the drugs used for the treatment of various diseases (Newman and Cragg 2007). However, due to the toxicity and poor bioavailability of anti-cancer drugs, it is considered necessary to improve the pharmacokinetic properties of new anti-cancer drugs. Although an increasing number of anticancer agents are being developed, their low selectivity and multi-drug resistance often prohibit successful cancer treatment (Kaya et al. 2017). Thus, medical professionals require new, potent, and selective anti-cancer agents capable of destroying tumor cells or, at the very least, stopping their proliferation (Pattnaik et al. 2017;Jin et al. 2019;Piccoli et al. 2020). The secondary metabolites of plants have traditionally been used in the treatment of cancer. Nowadays, the semi-synthetic derivatives of natural products comprise the majority of anti-cancer agents (Fu et al. 2014;Tuncay et al. 2018;Şenol et al. 2020).

Syntheses
Prior studies have found that both hydrazide and hydrazone compounds exhibit a wide variety of biological activities (Reddy and Kathale 2017;El-Sayed et al. 2018;Jin et al. 2019;Mohamed 2019;Settypalli et al. 2019). When these compounds are combined with natural products, potentially biologically active hybrid molecules with two or more effects are formed (Pawełczyk et al. 2016;Pertino et al. 2018;Chen et al. 2019;Sheng et al. 2019;Sun et al. 2019). In light of this, in the present study, 13 new hybrid compounds were synthesised from the natural product ursolic acid ( Figure 2). Moreover, the in vitro cytotoxic activities of these compounds were investigated in relation to BEAS-2B human non-tumorigenic lung epithelial cell lines and A549 adenocarcinomic human alveolar basal epithelial cell lines. In addition, the synthesised compounds were tested against Staphylococcus aureus, Escherichia coli, and Candida albicans to determine their anti-microbial properties. Initially, ursolic acid, a commercially available natural product, was converted into 3-methyl ursolic acid (4 b) using iodomethane in the presence of sodium hydride. During this reaction compound 4a was obtained as a minor product. Next, compound 4b was converted into acyl halide (5), which was then used to synthesise the hydrazide compound (6). Finally, the triterpene-arylidene-hydrazide hybrid compounds 7(am) were synthesised starting from hydrazide (6) and using 13 different aromatic aldehydes.

Cytotoxicity assay
The cytotoxic effects of compounds 6 and 7(a-m) were investigated with regard to the BEAS-2B and A549 cell lines. The cytotoxic effects and IC 50 values of the compounds in terms of the relative viability of the BEAS-2B and A549 cells at 0.1 mM, 0.5 mM, 1 mM, 5 mM, and 10 mM concentrations are given in Table S1 and S2, respectively. Furthermore, a relative cell viability graph showing the cytotoxic effects of the compounds on the BEAS-2B and A549 cells are presented in Figures S1 and S2, respectively.
The biological activity results of the synthesised compounds were compared with doxorubicin. The IC 50 value of the cytotoxic effect of doxorubicin on the BEAS- 1.67 mM) and 7d (IC 50 ¼ 1.46 mM). When the cytotoxic effects of compound 7k and doxorubicin were compared, it was determined that 7k had a toxic effect that was almost 15 times lower than the toxic effect of doxorubicin in the same healthy cells. In terms of compounds 7b, 7i, 7g, and 7d compounds, the cytotoxic effect was found to be reduced 14-, 11-, 10-, and 9-fold, respectively. The IC 50 value of the cytotoxic effect of doxorubicin against the A549 lung cancer cells was determined to be 0.07 mM (Kashkin et al. 2010), while the IC 50 values of the cytotoxic effects of the synthesised compounds against the same cell line ranged from 0.12 mM to 0.60 mM. When compared with the BEAS-2B cell line, the IC 50 values of, the cytotoxicity of compounds 7k, 7b, 7i, 7g, and 7d in relation to the A549 cells, were were 0.15 mM, 0.31 mM, 0.45 mM, 0.26 mM, and 0.60 mM, respectively. When the cytotoxic effects of compound 7k and doxorubicin were compared, 7k was found to exert half the inhibitory effect of doxorubicin on the same cancer cells. Moreover, the toxicity of compound 7k on healthy cells was 15 times lower than that of doxorubicin. Therefore, the selectivity (TD 50 /ED 50 ) of compound 7k was approximately seven times higher than the standard. In addition, the selectivity of compounds 7b, 7g, 7i, and 7d was 3, 3, 2, and 1.5 times higher, respectively, then that of doxorubicin.

Antimicrobial activity assay
Anti-microbial characteristics of synthesised compounds were tested against S. Aureus, E. Coli, and C. Albicans and the results were compared to standards such as amphotericin B, ciprofloxacin, and vancomycin. The tested compounds showed antibacterial activity against S. aureus and E. coli, at 62.5-250 mM and 125-250 mM concentrations, respectively. In addition, the tested compounds showed anti-fungal activity against C. Albicans at 125-250 mM concentration. The MIC values belonging to anti-microbial activity results were given in Table S3.

Structure activity relationship
According to the in vitro tests results, the structure-activity relationships were observed as follows. The selectivity for anticancer activity of compound 7a, which include an unsubstituted benzene ring, is 5.5. Compounds 7k, 7b, and 7g have higher selectivity than 7a, and these values are 15, 7 and 6 for 7k, 7b, and 7g, respectively. In compounds 7k, 7b, and 7g, the substituents are 2,4-dimethoxy, 4-methyl and 4nitro, respectively. Compounds 7e, 7c, 7f, and 7d have much lower selectivity than 7a and the substituents in these compounds are 4-fluoro, 4-chloro-, 3-nitro-and, 4bromo, respectively. It was observed that halogenated derivatives are unsuitable for the targeted anti-cancer activity, but electron-donating methoxy groups at the 2,4-position of the benzene ring are the optimum substituents. The lower selectivity of compound 7l and 7f explains why conjugation with the hydrazide group is important for activity. When the antimicrobial effects were evaluated, it was observed that the halogenated derivatives showed better activity than the others. The chloro-substituted 7c compound having the highest antimicrobial activity.

Experimental
The experimental procedure for syntheses and biological activities, as well as all corresponding spectra, are described in the Supplementary Information. The structures of all synthesised compounds were well characterised by 1 H NMR, 13 C NMR, HRMS, and HPLC analyses.

Conclusion
In this study, according to the in vitro test results, the hybrid molecule 7k may be a more selective potential anticancer drug when compared to doxorubicin. On the BEAS-2B and A549 cells, the hybrid compound 7k is seven times more selective and effective than doxorubicin. Furthermore, anti-microbial activity investigations revealed that compound 7c is the most efficient compound against S. aureus at 62.5 mM concentration when compared to standard antimicrobial agents. Almost all compounds showed a similar inhibition potential against E. coli and C. Albicans at 125 mM concentration. Finally, the hybrid compound 7k appears to be a viable option for further research as an anti-cancer drug.

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

Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Funding
This study financially supported by Bezmialem Vakif University, Scientific Research Project Number: BAP 20200207. Spectral data were recorded at Bezmialem Vakif University Drug Application and Research Centre (ILMER).