Study of the Anticancer and Antimicrobial Biological Activity of a New Series of Thiohydantoin Derivatives

: Recently, some prostate cancer patients have acquired resistance to the second -generation drugs (anzalutamide and apalutamide) prescribed for the treatment of this disease due to the emergence of the F876L mutation, which represents a challenge to modern medicine. In this study, a new series of 2-thiohydantoin derivatives were prepared through the reaction of different derivatives of maleimide (1c-4c) with isothiocyanate derivatives. The prepared compounds were diagnosed using FT-IR, 1 H-NMR , 13 C-NMR, Mass spectra. The prepared series compounds has been studied against prostate cancer cells. The MTT assay was used to determine the activity of the prepared compounds against prostate cancer cells. The data indicated, depending on the IC50 values, that some of the prepared compounds have anti-prostate cancer activity. The results indicate that compounds 1d and 2d have good anti-prostate cancer activity compared to the rest of the compounds. The prepared series of compounds were also studied against selected types of bacteria and fungi, as the results showed that some of the compounds in the series had anti-bacterial and anti-fungal activity.


Introduction:
Hydantoin was first isolated by Bayer in 1861 in the course of her research on uric acid 1 . 2thiohydantoin was first reported in 1913, and by evaluating the evidence, it was found that the first compound prepared for this type of compound was by Peter Klason in 1890 2 . 2-thiohydantoin is one of the biologically active molecules and has diverse activities to supply the medical and pharmaceutical industries in facing the challenges faced by modern medicine after the spread of drug-resistant diseases, which are increasingly spreading all over the world, where it has been used as anti-cancer [3][4][5][6][7][8] , antileishmaniasis 9 , an antiviral 10 , protect PC12 cells 11 , antibacterial [12][13][14] , also showed anti-TMV activity 15 , anti-neuroinflammatory 16 . Perhaps one of the most important things that drew the attention of researchers to the thiohydantoin nucleus in the last decade is that it is the main nucleus in the secondgeneration of drugs (enzalutamide and apalutamide) intended for the treatment of prostate cancer. Enzalutamide and apalutamide were discovered in a series of 2-thiohydantoin derivatives prepared in 2006 by Sawyers and Jung in a series of UCLA patent applications and subsequently published in a journal in 2010 [17][18] . In 2012, enzalutamide was officially approved as a drug for the treatment of prostate cancer by the US Food and Drug Administration, and it was approved in the European Union in 2013 and in Japan in 2014. As for apalutamide, it was approved in the United States of America as a drug for the treatment of prostate cancer in 2018 and in the European Union in 2019 19 . Prostate cancer is one of the most common diseases in men. In 1989, flutamide was approved as a drug for the treatment of prostate cancer by the US Food and Drug Administration, while bicalutamide was approved in 1995, and these drugs became ineffective as cancer progressed to the stage of hormone resistance. The ineffectiveness of the firstgeneration anti-prostate cancer drugs led to the design of the second-generation drugs (enzalutamide and apalutamide), but unfortunately, as the disease progressed to the stage of resistance to this generation of drugs, the second-generation drugs have become ineffective at the present time .On the contrary, in some cases, their use leads to strengthening The disease instead of being cured, and the reason is due to the emergence of the F876L mutation [20][21] . In this study, we prepared a new series of 2-thiohydantoin derivatives and they were studied against prostate cancer using the MTT assay 22 . In comparison to the other compounds, the data show that compound 1d has good anti-prostate cancer activity. The compounds were also studied against selected types of bacteria and fungi, and it was clear from the results that some compounds had antibacterial and anti-fungal activity under study.

Materials and Methods:
Infrared spectra of the prepared compounds were recorded using a Japanese-made Shimadzu 8400 FT.IR device and in the form of potassium bromide tablets in the (400-4000) region at room temperature. At room temperature, 1 H and 13 C-NMR spectra were recorded using BRUCKER-500MHz and 125MHz instruments, DMSO-d6 as a solvent, and TMS as an internal reference. All chemical displacements were measured in ppm. Mass spectra were recorded using a quadruple device analyzer spectrometer 5975 Agilent with electron collision technology at electronic energy of 70 eV.1-(4bromophenyl)-1H-pyrrole-2,5-dione(1a), 1-(p-tolyl) -1H-pyrrole-2,5-dione(2a),1-(4-methyl-3nitrophenyl) -1H-pyrrole-2,5-dione(3a), 1-(4acetylphenyl) -1H-pyrrole-2,5-dione(4a), acetone, hexane, absolute ethanol, acetonitrile from( Sigma-Aldrich). Phenyl isothiocyanate, glacial acetic acid, Isoniazid, TLC, cyclohexyl isothiocyanate from (Merck). Synthesis of compounds (1c-4) 23,24 The first step: 0.01mol of different maleimides (1a-4) was dissolved with 0.01mol of Isoniazid in 30ml of absolute ethanol and the mixture was left under reflux and stirred for 6-12 hrs. The reaction was monitored by TLC (thin layer chromatography). After the reaction was completed, it was cooled, filtrated, and washed in absolute ethanol. Recorded spectra data of the prepared compounds:     23,24 In this experiment, 0.01mol of maleimide derivatives (1c-4) was dissolved in 30ml of acetonitrile as a solvent and few drops of glacial acetic acid as a catalyst.. Then was added 0.01mol of cyclohexyl isothiocyanate or phenyl isothiocyanate. The mixture was left under reflux with stirring for 16-70 hrs. The reaction was monitored by TLC (thin layer chromatography). After the reaction was completed, the solvent was evaporated at room temperature and then recrystallized by a mixture of acetone and hexane. Recorded spectra data of the prepared compounds:

Results and Discussion:
In this study, the authors prepared a new series of thiohydantoin derivatives. In the first step, a new series of succinimide derivatives 1c-4 were prepared by reacting different maleimides 1a-4 with isoniazid in absolute ethanol, and the time required for the reaction to occur was 6-12 hrs. In the second step, and to obtain the final product, different succinimide derivatives (1c-4) were reacted with isothiocyanate derivatives in acetonitrile as a solvent and few drops of glacial acetic acid as a catalyst. The period required for the reaction to occur was 16-70 hrs. Scheme 1. The reaction mechanism for the synthesis of compounds 1d-4 and 1e-4 is illustrated in Scheme. 2, as the synthesis process goes through an intermediate compound that suffers a rearrangement process to give the final product. The compounds of the prepared series were diagnosed by FT-IR, 1 HNMR, 13 C-NMR and mass spectra. The chemical structures of all the resulting succinimide derivatives 1c, 3c and 4c were confirmed by FT-IR, 1 H-NMR, and 13 C-NMR. The IR spectrum was used to identify the functional groups of these compounds. The NH amide and NH groups were observed in the range of 3564-3317 and 3483-3217 cm -1 , respectively. The absorption bands in the 1788-1637 cm −1 area are linked to C=O. The band in the range 1546-1409 cm -1 was assigned to the C=C aromatic stretching.
The succinimide derivatives 1c, 3c and 4c were used to generate 1 H-NMR spectra. The compounds were also distinguished by the appearance of signal doublet of doublets at around δ 3.13-3.09 and 2.83-2.80, which belong to Hi and Hk protons, respectively. Because the hydrogen atoms of the methylene group are adjacent to the chiral center. The Hr signal is at δ 4. 32-4.27. The signals at around δ 6.20-6.15 were assigned to the protons of Hm. Signals of singlet at around δ 10.57-10.11 were due to He. signals (doublet and signal) for protons Hn in the arrange at δ 10.51-10.47. Aromatic protons were given the signals (multiplet, doublet and singlet) at roughly δ 8.77-7.23. The methyl groups responsible for the singlet are at δ 2.57.
The 13 C-NMR of the compounds 1c, 3c and 4c that showed signals at around δ 197-164 were attributed to carbonyl groups. The signals of the carbon aromatic ring appeared in the range of δ 150-121. The aliphatic carbons are present in the range δ 58. 27-21.45.
The obtained spectra data indicates the correctness of the structures of the prepared compounds 1d-4d and 1e-4e. The most important bands recorded by the FT-IR spectra, 3479-3300 (NH amide group), 3300-3200 (NH), 3186-3000 (CH -aromatic ring), 1449-1400 (thiocarbonyl group), 1350-1300 (C-N). The recorded NMR spectra of the prepared compounds were characterized by the fact that the signals appeared in almost identical chemical shifts. The 1 HNMR spectrum revealed a triple signal for Hc at 4.99 to 4.63 and a multiple for Ha and Hb at 3.2 to 2.7. The protons of aromatic rings showed multiple signals in the region of 8.83-7.09. The two singlets at δ 11.83 to 11.63 and δ 10.57 to 9.98 correspond to protons Hd and He, respectively. Signals of a multiplet were observed at δ 4.52-4-44 attributed to the Hf, while protons of the cyclohexyl group showed a multiple signals in the region at δ 2.25-1.14. 13 (1d-4) and (1e-4).
The anti-prostate cancer activity: 22 The discovery of anti-prostate cancer drugs is one of the most important challenges facing modern medicine, and the reason is the large number of mutations that occur in AR (androgen receptor). In this study, we examined a new series of thiohydantoin derivatives as anti-prostate cancer agents. Depending on the IC50 value of the studied compounds, we note that there is a noticeable variation in the activity of the compounds against prostate cancer cells Fig.1.The difference in activity is attributed to the different Ar-substitutions in the prepared series. Among the compounds studied, compounds 1d, 2d, and 3d showed anti-prostate cancer activity Fig.2. The results showed that compound 1d has good activity compared to the rest of the series compounds, and the reason is due to the presence of the bromine atom. Also, the good activity of compound 2d compared to compound 3d is due to the presence of the methyl group, as shown in Table. 7.   Evaluation of prepared compounds against bacteria and fungi: 25,26 Most of the heterocyclic compounds are characterized by their anti-bacterial and anti-fungal activity 27,28 . The biological activity of thiohydantoin derivatives is found in a wide range of heterocyclic compounds, and the biological activity of this type of compound usually depends on the nature of the substituent groups on the thiohydantoin ring. Previous studies confirm that compounds containing in their structures the thiohydantoin nucleus have anti-bacterial and anti-fungal properties [29][30][31][32][33] . In this study, we evaluated the prepared thiohydantoin series against selected strains of bacteria and fungi.The prepared compounds were studied as antibacterial E. coli and Staph compared with Spiromycin as a positive control. Also, the prepared series of compounds were evaluated as anti-fungal Candida albicans and Aspargilus. The growth inhibition of bacteria and fungi under study was measured in mm, as one concentration of 100 mg/ml was prepared for each compound.50 μL of each concentration was taken and added to each well (7 mm diameter holes cut in the agar gel, 20 mm apart from one another). Plates were incubated for 24 hours at 36ºC ± 1ºC, under aerobic conditions. The obtained data indicate that some compounds of the prepared series have anti-bacterial and anti-fungal activity. The two compounds 1c and 3c showed antifungal activity against Aspargilus, while the rest of the compounds under study did not show any activity. The data showed that all the compounds under study had anti-Candida albicans activity. All the compounds under study showed antibacterial activity against staph except the compounds 2d, 3e and 4e, which showed no activity against this particular type of bacteria. The compounds 1c, 3c, and 1d showed anti-bacterial activity against E. coli, while the rest of the compounds under study did not have anti-bacterial activity against this type of bacteria, Table 8.

Conclusion:
The aim of this study was to prepare compounds that have anti-prostate cancer activity. The relevant dose-response curve data confirm that the substituent groups in the aromatic rings of the prepared series of compounds have a significant effect on their antiprostate cancer activity. The two compounds 1d and 2d showed moderate activity against prostate cancer cells, and further study is needed to increase their activity. When replacing the phenyl group in the compounds 1e, 2e and 3e with a cyclohexyl group 1d, 2d, 3d, it is noted that the activity of the compounds against prostate cancer increases. Hence, we recommend keeping the cyclohexyl group in the event that further study is conducted on this type of compound for its role in increasing activity.