Synthesis and biological activity of novel zingerone–thiohydantoin hybrids J. Serb. Chem. Soc. 87 (12) 1349–1358 (2022) Original scientific paper JSCS–5598 Published 1 August 2022 1349 Synthesis and biological activity of novel zingerone–thiohydantoin hybrids KATARINA D. VIRIJEVIĆ1, PETAR B. STANIĆ1, JOVANA M. MUŠKINJA1, JELENA S. KATANIĆ STANKOVIĆ1, NIKOLA SREĆKOVIĆ2, MARKO N. ŽIVANOVIĆ1 and BILJANA M. ŠMIT1* 1University of Kragujevac, Institute for Information Technologies, Department of Science, Jovana Cvijića bb, 34000 Kragujevac, Serbia and 2University of Kragujevac, Faculty of Science, Department of Chemistry, Radoja Domanovića 12, 34000 Kragujevac, Serbia (Received 4 April, revised 28 May, accepted 9 June 2022) Abstract: A series of zingerone–thiohydantoin hybrids were synthesized from O-alkyl zingerone derivatives by cyclocondensation with thiosemicarbazide in a two-step reaction. The obtained new potentially bioactive compounds were structurally characterized by IR and NMR spectroscopy, as well as by elemen- tal and HRMS analysis. In addition, their antimicrobial and in vitro anticancer activities were tested. The tested compounds showed low to moderate antimic- robial activity. Zingerone–thiohydantoin hybrid with an O-butyl substituent exerted the significant cytotoxic activity on colon HCT-116 cancer cells, with- out toxicity on healthy MRC-5 cells. Keywords: molecular hybrids; antimicrobial activity; cytotoxic activity. INTRODUCTION The development of novel synthetic molecular hybrids is one of the main challenges in the drug discovery field. Hybrid drugs represent a combination of specific agents aimed to be more efficient than classic single synthesized compounds. In that way, the hybrid approach allows the connection of two distinct compounds in one molecule, increasing the biological potential of at least one of the compounds.1 Many natural products play an important role in this field. For example, zingerone also called vanillylacetone, obtained from a ginger extract, is a natural compound that belongs to the methoxyphenol class, along with its derivatives. Both natural and synthetic zingerone derivatives exhibit dif- ferent biological and pharmacological activities such as anti-inflammatory, anti- -microbial, anti-cancer and hepatoprotective.2 Furthermore, zingerone appears to be a potential agent for inhibiting colon cancer progression, as the number of * Corresponding author. E-mail: biljana.smit@uni.kg.ac.rs https://doi.org/10.2298/JSC220404047V ________________________________________________________________________________________________________________________ (CC) 2022 SCS. Available on line at www.shd.org.rs/JSCS/ 1350 VIRIJEVIĆ et al. larger foci was found to be significantly lower after zingerone treatment, com- pared to dimethyl hydrazine-induced colon cancer cells.2 On the other hand, 2-thiohydantoin (2-thioxoimidazoline-4-one) is a non- -aromatic five-membered heterocyclic compound with a cyclic ureide core.3 Many synthesized thiohydantoin derivatives with various substituents attached to their nucleus exhibit a wide range of biological and pharmacological potentials, such as antimicrobial,4 anti-convulsive,5 anti-diabetes6 and anti-HIV.7 However, novel studies showed that thiohydantoins and their synthesized derivatives could be used as promising anti-proliferative and anti-metastatic agents.8 Taking into account that colon cancer is one of the most prominent tumors in the world and less sensitive to cytostatics, the search for new effective therapeutic drugs is cru- cial. In this study, different zingerone derivatives were prepared as starting mat- erials for obtaining a short series of new zingerone-thiohydantoin hybrids for the evaluation of their potential biological activity. EXPERIMENTAL General methods All reagents and chemicals were commercially available and used without additional purification. Solvents were distilled before use. Anhydrous methanol was prepared by stan- dard drying methods. Zingerone, starting material for a preparation of O-alkyl zingerone deri- vatives, was obtained by condensation reaction of vanillin and acetone and subsequent reduct- ion of yielded dehydrozingerone, according to well-known procedure.9 IR spectra were rec- orded through KBr pellets on a Perkin–Elmer FT-IR spectrometer model Spectrum One in the 4000 to 450 cm-1 range. 1H- and 13C-NMR spectra were recorded on a Varian Gemini 2000 NMR spectrometer using CDCl3 as the solvent and TMS as the internal standard. Elemental analysis was done on an Elemental Vario ELIII CHNSO analyzer. HRMS were measured on an Agilent 6550 iFunnel Q-TOF LC/MS system. For biological assays microtiter plates and Multiskan SkyHigh Microplate spectrophotometer by Thermo Scientific were used. General procedure for the preparation of O-alkyl zingerone derivatives 1a and b Zingerone derivatives 1a and b were synthesized according to a procedure that uses dimethyl and diethyl sulfate, respectively.10 A mixture of zingerone (0.971 g, 5 mmol) and 50 mL of boiling water is heated on a steam bath. A 2 mL portion of 20 % NaOH solution is heated to about 100 °C and added in one lot to the hot mixture of zingerone and water. Heat- ing is continued and 6.25 mmol of methyl/ethyl sulphate is slowly added in portions. After the addition of all methyl/ethyl sulphate, which requires about 1.5 h, the reaction mixture is heated for 45 min longer and an additional portion of 1.1 mmol of methyl/ethyl sulphate is added at the same rate as the first portion. At the end of this addition the reaction mixture should show an acid reaction. The reaction mixture is rendered slightly alkaline with NaOH solution, and the addition of Me/Et sulfate and NaOH solution is done two more times until the total amount of Me/Et sulfate (11.25 mmol) is added. The mixture is then made strongly alkaline by the addition of 1 mL NaOH solution and is heated another 20 min. The reaction mixture is rapidly cooled at ambient temperature with continued stirring and the product is ________________________________________________________________________________________________________________________ (CC) 2022 SCS. Available on line at www.shd.org.rs/JSCS/ ZINGERONE–THIOHYDANTOIN HYBRIDS 1351 extracted with diethyl ether (3×10 mL). The combined ether extracts are dried over anhydrous MgSO4 and the ether is evaporated, giving yellow oil that soon solidifies. General procedure for the preparation of O-alkyl zingerone derivatives 1c–g Zingerone derivatives 1c–g were synthesized according to a known procedure that uses alkyl halides with potassium carbonate in acetone.11 The mixture of zingerone (0.971 g, 5 mmol), alkyl halide (12.5 mmol) and anhydrous K2CO3 (2.25 g, 16.3 mmol) in acetone (25 mL) were heated to reflux for 3 h. The mixture was cooled at ambient temperature and then poured into cold water. The products were extracted from the mixture with ethyl acetate (3×10 mL). Combined extracts were rinsed with water and then dried over anhydrous Na2SO4. The solvent was removed by vacuum distillation and the product was separated from the residue by column chromatography with hexane/EtOAc (from 3:1 to 6:1). Zingerone derivatives 1a, 1b and 1f are known and commercially available chemicals, while the others are novel. The structure and purity of all new products were confirmed by IR and NMR spectroscopy. Synthesis of zingerone–thiohydantoin derivatives 2a–g The zingerone–thiohydantoin derivatives were synthesized according to a previously published protocol for the synthesis of arylidene thiohydantoin derivatives.12 A mixture of O-alkyl zingerone derivative 1a–g (2 mmol) and thiosemicarbazide (0.182 g, 2 mmol) in 30 mL of methanol were heated to reflux for 3 h and then cooled to ambient temperature, result- ing in the corresponding intermediate thiosemicarbazone without isolation. Ethyl chloroacet- ate (0.245 g, 2 mmol) and anhydrous sodium acetate (0.492 g, 6 mmol) were added in situ and the mixture was refluxed for another 6 h. The reaction mixture was cooled to room tempera- ture at first and then poured into cold water. The resulting precipitate was filtered off, rinsed with hot water and re-crystallized from hot methanol, giving white amorphous powder in all cases. The structure of the synthesized compounds was confirmed by IR and NMR spectro- scopy, as well as elemental analysis and HRMS (Supplementary material to this paper). All compounds are obtained as an inseparable mixture of Z and E stereoisomers, as can be seen through the duplication of most signals in the 1H-NMR spectra. NMR spectral data are given for the major stereoisomer. Antimicrobial activity determination In this preliminary testing, five microbial strains were used, four of which were bacteria, Salmonella enteritidis ATCC 13076, Pseudomonas aeruginosa ATCC 10145, Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, and the yeast Candida albicans ATCC 10259. The microorganisms were acquired from the Institute of Public Health Kragujevac, University of Kragujevac, Serbia, kept at 4 °C with subcultivation once a month. The broth used for bacteria cultivation was nutrient agar and yeast was cultured on Sabouraud dextrose agar. For antimicrobial evaluation of synthesized compounds, a standard microdilution method by Sarker et al. was used.13,14 Cytotoxic activity determination All synthesized derivatives were dissolved in dimethyl sulfoxide (DMSO) in order to obtain stock solutions of 5 mМ concentration, followed by further dilution in Dulbecco’s Modified Eagle Medium (DMEM) to obtain working concentrations (0.1, 1, 10, 50, 100 and 250 µM). At the highest applied concentration of derivatives, the concentration of DMSO in the solution was lower than 0.05 %, which was previously confirmed as non-toxic to cancer cells.15 ________________________________________________________________________________________________________________________ (CC) 2022 SCS. Available on line at www.shd.org.rs/JSCS/ 1352 VIRIJEVIĆ et al. The healthy human lung fibroblasts (MRC-5) and colorectal carcinoma cell lines (HCT- -116) were obtained from the European Collection of Authenticated Cell Cultures. Cells were cultured in a complete medium in humidified conditions, at 37 °C and 5 % CO2. When cells reached 70–80 % of confluence, the detachment was done using 0.25 % trypsin–EDTA, fol- lowed by seeding (1×104 cells/well) in 96-well flat-bottomed microtitre plates. The treatment with 100 µL of synthesized compounds solution was done 24 h after cell seeding. The effects of synthesized compounds on the viability of tested cell lines were assessed after 24 and 72 h using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay according to the previously described protocol.16 At the end of the incubation period, 25 μL of MTT solution (from a 5 mg mL-1 stock) was added to each well, followed by incubation at 37 °C for 2 h, after which 100 μL of DMSO was added. The evaluation of cytotoxic activity was done by measuring the absorbances at 550 nm wavelength. The obtained results are pre- sented as mean ± standard error (SE), expressed as a percent of cell viability (%). IC50 values (minimal inhibitory treatment concentration that induces the death of 50 % of treated cells) were calculated from dose curves obtained by the MTT test. 5-Fluorouracil (5-FU) was used as positive control. The magnitude of the correlation between variables was calculated using statistical soft- ware SPSS (SPSS for Windows, ver. 20, 2008, Chicago, IL, USA) whereat the ANOVA test was applied, and for all comparisons p < 0.05 was considered as a statistically significant dif- ference between the control and the tested compounds. The IC50 values were calculated by using the CalcuSyn software program. RESULTS AND DISCUSSION Synthesis of the zingerone–thiohydanoin molecular hybrids The zingerone–thiohydantoin derivatives 2a–g were obtained through a con- densation reaction with thiosemicarbazide, utilizing a previously published two- -step protocol (Scheme 1).12 In the first step, synthesized O-alkyl zingerone deri- vatives 1a–g reacted with thiosemicarbazone, giving corresponding thiosemicar- bazides. The thiosemicarbazides then undergo intramolecular cyclocondenzation with ethyl chloroacetate in the presence of anhydrous sodium acetate, yielding the final thiohydantoin products 2a–g. The structure and purity of the novel zin- gerone–thiohydantoin derivatives were confirmed by IR and NMR spectroscopy, as well as by elemental and HRMS analysis. The compounds were obtained in medium to high yields, with the exception of 2d, which was obtained in a modest yield. Naturally occurring zingerone itself did not react in this manner and the corresponding thiohydantoin derivative was not obtained. Similar to some Schiff- -base derivatives,17 all newly synthesized zingerone–thiohydantoin derivatives are obtained as a mixture of Z and E stereoisomers, which could be found in cor- responding NMR spectra. Most signals in the 1H-NMR spectra are duplicated, which indicates the presence of isomers. This is best seen through the singlets of CH2-5, CH3-6 and CH3-16, which have the most pronounced difference of the chemical shifts and thus, less overlap. The ratios of Z/E isomers are obtained from relative integration of the most suitable and distinct pairs of singlets. Their ratios range from 1:1.06 to 1:9.82 (Scheme 1). In all cases the E isomer is favoured. ________________________________________________________________________________________________________________________ (CC) 2022 SCS. Available on line at www.shd.org.rs/JSCS/ ZINGERONE–THIOHYDANTOIN HYBRIDS 1353 Scheme 1. Synthesis of the zingerone–thiohydantoin hybrids 2a–g. Furthermore, the computational data for both possible configurations of 2a were also calculated using the DFT method in order to determine the more stable isomer as seen in Fig. 1. When any internal interconnection between molecules for both isomers is neglected the less internal steric repulsion factor plays an important role in fixing the E isomer over Z isomer, as it is with less internal repulsion. The gaseous state DFT calculations also showed that the energy dif- ference value ΔE = 4.0 kJ mol–1 between E and Z isomer was very small. Fig. 1. The optimized geometries of E and Z isomers of 2a, with relative free energy values indicated in kJ mol-1. Antimicrobial activity The results of antimicrobial effects of the zingerone–thiohydantoin hybrids are presented in Table I. The compounds were tested against two Gram-negative (S. enteritidis and P. aeruginosa) and two Gram-positive (S. aureus and E. coli) bacterial species, as well as against the yeast C. albicans. S. aureus and C. albi- cans were the most resistant to the action of 2a–g, wherein the compounds had not shown any activity even at the highest applied concentration (MIC > 4 mg mL–1). Moreover, compound 2c was completely inactive towards all used micro- organisms at the same concentration, while in the cases of other compounds MIC values were quite high, mostly in range 2–4 mg mL–1. 2b was able to inhibit the ________________________________________________________________________________________________________________________ (CC) 2022 SCS. Available on line at www.shd.org.rs/JSCS/ 1354 VIRIJEVIĆ et al. growth of S. enteritidis in moderate concentration (MIC 0.5 mg mL–1). Only E. coli was more susceptible to the action of the compounds, particularly 2a and g with MIC 0.25 mg mL–1. Nevertheless, the quite high concentrations at which the compounds exhibit their activity cannot be easily compared with the activity of the reference standards, erythromycin and nystatin, where MICs were expressed in mg/mL. This is the first study regarding the antimicrobial potential of zinger- one–thiohydantoin hybrids. The literature data about similar compounds are scarce. There are some recently published results regarding the influence of some thiohydantoin derivatives on several bacterial species.4 These compounds were the most active against Gram-positive bacteria such as S. epidermidis, S. pyo- genes, S. agalactiae, E. faecium and S. aureus with MICs below 1 mg mL–1, but were less effective against Gram-negative bacteria like K. pneumonia, P. mirabilis and E. coli. TABLE I. Antimicrobial activity (MIC / mg mL-1) of the synthesized zingerone–thiohyd- antoin derivatives 2a–g; –: not tested Compound Microorganism S. enteritidis P. aeruginosa S. aureus E. coli C. albicans 2a 4 4 ˃ 4 0.25 ˃ 4 2b 0.5 4 ˃ 4 2 ˃ 4 2c ˃ 4 ˃ 4 ˃ 4 ˃ 4 ˃ 4 2d – – – – – 2e 4 4 ˃ 4 2 ˃ 4 2f ˃ 4 4 ˃ 4 4 ˃ 4 2g 4 4 ˃ 4 0.25 ˃ 4 Erythromycin 20 20 1.25 2.5 – Nystatin – – – – 1.25 Cytotoxic activity Thiohydantoin analogs have been already confirmed as potent anti-tumor agents. Furthermore, apoptosis-inducing activity of some thiohydantoin derivat- ives has been demonstrated.18 Previous research has identified zingerone as a potential anti-cancer agent, an inhibitor of colon cancer progression,2,19 with sig- nificant cytotoxicity (IC50 = 11.49 mM) when applied to mesothelioma cells. Su et al. showed that the zingerone-induced cytotoxic effect on colon cancer cells (HCT-116) was achieved through the mechanism of ROS-mediated apoptosis.20 Besides that, the cytotoxic potential of thiohydantoin derivatives on HCT-116 was noticed when applied to colon cancer cells (with IC50 > 50 µM).21 In this study, the cytotoxic activity of newly synthesized zingerone–thiohyd- antoin derivatives was evaluated on healthy lung (MRC-5) and human colorectal carcinoma (HCT-116) cell lines by MTT assay. As positive control, the commer- cial chemotherapeutic drug, 5-fluorouracil (5-FU) was used. 5-FU is widely used ________________________________________________________________________________________________________________________ (CC) 2022 SCS. Available on line at www.shd.org.rs/JSCS/ ZINGERONE–THIOHYDANTOIN HYBRIDS 1355 in the treatment of different types of cancer, such as gastric, pancreatic, breast, and ovarian cancers.22 In regard to the influence on the viability of the healthy MRC-5 cells, a moderate cytotoxic effect was observed only for compound 2a after an extended time of exposure with IC5072 h = 184.15 µM (Fig. S-44 of the Supplementary material) and control with IC5072 h = 181.71 µM (Fig. S-46 of the Supplementary material). The tested compounds manifest their cytotoxicity potentials in a time and dose-dependent manner. The reduction of HCT-116 cells viability was obtained mainly after 72 h and at the highest applied concentration of the inves- tigated compounds (Fig. S-45 of the Supplementary material). The effects of the synthesized compounds were expressed by dose curves (Figs. S-44 and S-45) and IC50 values (Fig. 2). Based on the results, 2e exhibited the most prominent anti- proliferative activity on the HCT-116 cell line with an IC5024 h = 209.08 µM and IC5072 h = 160.93 µM. 5-FU exerted a weaker cytotoxic effect than 2e on HCT- -116 after 24 and 72 h with IC5024 h > 250 µM and IC5072 h = 181.71 µM, respectively. (Fig. 2). Fig. 2. The cytotoxic effects of zingerone-thiohydantoin derivatives and 5-FU after 24 and 72 h exposure, represented as IC50 values. Unlike other tested compounds, 2e contains a butyl group. The result of its activity is probably related to the lipophilicity of the substituents in the structure of the tested molecule. The lipophilicity of molecules plays a major role in the transport of molecules across biological membranes.23 As the length of hydro- carbon chain increases, the polarity of compounds decreases resulting in inc- reased molecule permeability. Cell membranes are relatively impermeable to hydrophilic compounds, hence the permeability of molecules depends on the hydrophobic characteristics, i.e., lipophilicity of the compound.24 In addition, a previous study reported that the lipophilicity of the substituents (Ph > Allyl > Me) had a significant impact on the cytotoxic effect.25 Compound 2f, with a benzyl group, did not exhibit a cytotoxic effect (IC50 > 250 µM). However, based on the obtained dose curves, it can be concluded that ________________________________________________________________________________________________________________________ (CC) 2022 SCS. Available on line at www.shd.org.rs/JSCS/ 1356 VIRIJEVIĆ et al. the wider range of concentrations could provide promising effects. Increasing the concentration range of the tested compounds would probably lead to a rise of the cytotoxic activity of some of them, and also would allow the determination of the selectivity index on tested cell lines. The presented results indicate the potential of tested compounds as antican- cer agents with no significant toxicity on healthy cell lines. Compound 2e exhi- bited the most promising bioactivity and is the leading candidate in the synthe- sized series. The increased cytotoxicity of 2e, compared to zingerone itself, could be attributed to the introduction of thiohydantoin moiety. CONCLUSION This study presents the synthesis, characterization, and biological evaluation of new zingerone–thiohydantoin molecular hybrids as potential anticancer agents. In addition to the zingerone-induced cytotoxic effect, it is known that the biological activity of thiohydantoin compounds depends largely on the nature of the substitution of the thiohydantoin ring. The difference in the cytotoxic activity of the tested compounds depends on the nature of the O-alkyl substituent of the benzene ring. Among the tested compounds, 2e exerted significant cytotoxic pot- ential without toxicity to healthy MRC-5 cells. However, all tested compounds showed low to moderate antimicrobial activity. Further toxicological testing is required to assess its therapeutic potential. SUPPLEMENTARY MATERIAL Additional data and information are available electronically at the pages of journal website: https://www.shd-pub.org.rs/index.php/JSCS/article/view/11747, or from the corres- ponding author on request. Acknowledgement. The authors are grateful for financial support from the Ministry of Education, Science and Technological Development of the Republic of Serbia (Agreements No. 451-03-68/2022-14/200378 and 451-03-68/2022-14/200122). И З В О Д СИНТЕЗА И БИОЛОШКА АКТИВНОСТ НОВИХ ЗИНГЕРОН–ТИОХИДАНТОИНСКИХ ХИБРИДА КАТАРИНА Д. ВИРИЈЕВИЋ1, ПЕТАР Б. СТАНИЋ1, ЈОВАНА М. МУШКИЊА1, ЈЕЛЕНА С. КАТАНИЋ СТАНКОВИЋ1, НИКОЛА СРЕЋКОВИЋ2, МАРКО Н. ЖИВАНОВИЋ1 и БИЉАНА М. ШМИТ1 1Универзитет у Крагујевцу, Институт за информационе технологије, Сектор за природно–мате- матичке науке, Јована Цвијића бб, 34000 Крагујевац и 2Универзитет у Крагујевцу, Природно– –математички факултет, Институт за хемију, Радоја Домановића 12, 34000 Крагујевац Серија зингерон–тиохидантоинских хибрида је синтетисана из деривата О-алкил зингерона циклокондензацијом са тиосемикарбазидом у двостепеној реакцији. Добијена нова потенцијално биоактивна једињења структурно су окарактерисана IC и NMR спек- троскопијом, као и елементалном анализом. Поред тога, тестиране су њихове антимик- робне и in vitro антиканцерогене активности. Испитана једињења су показала ниску до умерену антимикробну активност. Зингерон–тиохидантоински хибрид са О-бутил суп- ________________________________________________________________________________________________________________________ (CC) 2022 SCS. 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Available on line at www.shd.org.rs/JSCS/ @Article{Virijevic2022, author = {Virijevi{\'{c}}, Katarina D and Stani{\'{c}}, Petar B and Mu{\v{s}}kinja, Jovana M and {Katani{\'{c}} Stankovi{\'{c}}}, Jelena S and Sre{\'{c}}kovi{\'{c}}, Nikola and {\v{Z}}ivanovi{\'{c}}, Marko N and {\v{S}}mit, Biljana M}, journal = {Journal of the Serbian Chemical Society}, title = {{Synthesis and biological activity of novel zingerone–thiohydantoin hybrids}}, year = {2022}, issn = {1820-7421}, month = {aug}, number = {12}, pages = {1349--1358}, volume = {87}, abstract = {A series of zingerone–thiohydantoin hybrids were synthesized from O-alkyl zingerone derivatives by cyclocondensation with thiosemicarbazide in a two-step reaction. The obtained new potentially bioactive compounds were structurally characterized by IR and NMR spectroscopy, as well as by elemen­tal and HRMS analysis. In addition, their antimicrobial and in vitro anticancer activities were tested. The tested compounds showed low to moderate antimic­robial activity. Zingerone–thiohydantoin hybrid with an O-butyl substituent exerted the significant cytotoxic activity on colon HCT-116 cancer cells, with­out toxicity on healthy MRC-5 cells.}, doi = {10.2298/JSC220404047V}, file = {:D\:/OneDrive/Mendeley Desktop/Virijevi{'{c}} et al. - 2022 - Synthesis and biological activity of novel zingerone–thiohydantoin hybrids.pdf:pdf;:01_11747_5598.pdf:PDF}, keywords = {antimicrobial activity, cytotoxic activity}, publisher = {Serbian Chemical Society}, url = {https://www.shd-pub.org.rs/index.php/JSCS/article/view/11747}, }