Study on the Synthesis, Characterization and Bioactivities of 3-Methyl-9’-fluorenespiro-5-hydantoin

This work describes a method for synthesis, as well as in vitro antiproliferative and antibacterial investigation of 3-methyl-9’-fluorenespiro-5-hydantoin. The structure of the substituted fluorenylspirohydantoin derivative was verified by UV-Vis, FT-IR, Raman, H NMR and C NMR spectroscopy, and by using a combination of 2D NMR experiments, which included H-H COSY, HMQC and HMBC sequences. The geometry of the compound was optimized by the B3LYP density functional with 6-31G(d) basis set and the H and C NMR spectra were predicted with the HF/631G(d) calculations at the optimized geometry. The anticancer activity of the 3-methyl-9’-fluorenespiro-5-hydantoin was determined in suspension cell lines originating from tumors in humans (WERI-Rb-1). The cytotoxic effect was evaluated by WST-assay (Roche Applied Science). The antimicrobial effect of the compound against Gram-negative, Gram-positive bacteria and the yeast Candida albicans was investigated.


Introduction
Hydantoins, or 2,4-imidazolidinediones are compounds of considerable interest both from a chemical and biological point of view. 1 Several compounds of this class have shown a pharmaceutically useful activity that led in some cases to clinical applications. In particular, 5-substi-tuted and 5,5-disubstituted hydantoins are important medicinal compounds: phenytoin, or 5,5-diphenylhydantoin, is widely used as an anticonvulsant agent, for the treatment of epilepsy, and as a cardiac antiarrhytmic agents. 2,3 Among the medicinally useful properties exhibited by other 5-substituted hydantoins, at least their antidepressant and antiviral activities, the inhibition of platelet aggrega-tion as well as human aldose reductase and human leukocyte elastase inhibition are worth mentioning. A number of other biological activities of hydantoin derivatives are known, including possible uses as herbicides, 4-7 fungicides 8,9 and insecticides. [10][11][12][13][14][15] Lee et al. presented the molecular modeling of six structurally diverse ARIs (aldose reductase inhibitors), being carried out at the active site of aldose reductase to probe the charge interactions between the ionizable group (e.g. carboxylate or hydantoin) of the ARIs and the positively charged His 110. 16 An attempt was also made to correlate the binding mode of these structurally diverse inhibitors to observed inhibitory activity. Palm et al. investigated the influence of diabetes-induced changes in oxygen tension and consumption in relation to regional renal metabolism in rats. 17 In the second set of experiments, the putative role of the polyol pathway for hyperglycaemiainduced alterations in renal metabolism was studied. Sugiyama et al. reported a method for the in vitro isolation of a non-covalent complex formed in solution by the interaction of human muscle or rat lens aldose reductase with either NADP + or NADPH and the aldose reductase inhibitors tolrestat, AL1576 (2,7-difluorospirofluorene-9,5'-imidazolidine-2',4'-dione), or ponalrestat. 18 Kato et al. investigated the effects of novel aldose reductase inhibitors, , on neuropathy in streptozotocin-induced (STZ) diabetic rats. 19 The effect of a single oral administration of M16209, a novel aldose reductase inhibitor, on serum glucose was investigated by Nakayama et al. 20 The group of Nakayama investigated the stimulatory effects of M16209 on insulin secretion using isolated, perfused pancreases in rats. 21 M16209 showed no appreciable effect on ATP-sensitive K + -channels in pancreatic β-cells. Two potent aldose reductase inhibitors, l-[(2,5dichlorophenyl)sulfonyl]hydantoin (Di-ClPSH) and 1-[β-naphthyl)sulfonyl]hydantoin (β-NSH), were tested for usefulness in the treatment of diabetic and galactosemic complications in animal experiments. 22 Sorbitol formation from glucose, catalyzed by the enzyme aldose reductase, is believed to play a role in the development of certain chronic complications of diabetes mellitus. Spirohydantoins derived from five-and six-membered ketones fused to an aromatic ring or ring system inhibit aldose reductase isolated from calf lens. In vivo these compounds are potent inhibitors of sorbitol formation in sciatic nerves of streptozotocinized rats. Optimum in vivo activity is reached in spirohydantoins derived from 6-halogenated 2,3-dihydro-4H-l-benzopyran-4-ones (4-chromanones). In 2,4-dihydro-6-fluorospiro[4H-l-benzopyran-4,4'-imidazolidine]-2',5'-dione, the activity resides exclusively in the 4S isomer, compound 115 (CP-45,634, USAN: sorbinil). This compound is currently being used to test, in humans, the value of aldose reductase inhibitors in the therapy of diabetic complications. 23 A series of 27 hydantoins was prepared and tested as antitumor agents. These were variously substituted at the 5 position but with special emphasis on the substituents (chloro, acetyl, chloroacetyl, and methyl) at the 1 and/or 3 positions. The most active compound was 5,5-bis(4-chlorophenyl)-l,3-dichlorohydantoin with a T/C value of 190% against P-388 lymphocytic leukemia in mice. 24 Hydantoinases are valuable enzymes for the production of optically pure D-and L-amino acids. They catalyze the reversible hydrolytic ring cleavage of hydantoin or 5-monosubstituted hydantoins and therefore are classified in the EC-nomenclature as cyclic amidases C 3.5.2 group. 25 Hydantoinases have been classified into D-, L-, unselective or ATP-requiring enzymes due to their substrate specificity, stereoselectivity and cofactor dependency. From recent findings based on protein sequence data all hydantoin cleaving enzymes, with the exception of the ATP-dependent N-methylhydantoinases, belong to a protein superfamily of »amidohydrolases related to urease« 26 and seem to have evolved from a common ancestor in a divergent evolution. 27 A D-specific hydantoinase has been purified to homogeneity from Arthrobacter crystallopoietes DSM 20117 with a yield of 5% related to the crude extract. 28 The group of Yamada was the first to study intensively the D-selective cleavage of 5-monosubstituted hydantoins in microorganisms. 29 They postulated the identity of microbial D-hydantoinases with dihydropyrimidinases and proved this hypothesis for the enzyme from Pseudomonas striata. 30 In the meantime, several publications described various similar D-selective microbial hydantoinases from microorganisms, such as Pseudomonas fluorescens DSM 84, 31 Pseudomonas sp. AJ11220, 32,33 Agrobacterium sp. IP-I 671, 34,35,40 several Bacillus spp. [36][37][38] and even from anaerobic microorganisms. 39 However, recently a hydantoinase from Agrobacterium was identified which exhibits no dihydropyrimididase activity. 34 DL-5-Monosubstituted hydantoins are converted to D-amino acids via N-carbamoyl-D-amino acids by some bacteria. 32,41,42 Takahashi et al. 30 revealed that in Pseudomonas putida (P. striatu) IFO 12996, D-hydantoinase is identical with dihydropyrimidinase, which catalyzes the cyclic ureide-hydrolyzing step of the reductive degradation of pyrimidine bases. The same results were obtained for other Pseudomonas species, 43,44 Comamonas species, 44 Bacillus species, 45 Arthrobacter species, 41 Agrobacterium species, 43 and rat liver. 46 Various 5-chloroarylidene-2-amino substituted derivatives of imidazoline-4-one were synthesized and evaluated for their activity in vitro against Mycobacterium tuberculosis and other type strains of bacteria and fungi. 2-Chloro-and 2,4-dichlorobenzylidene substituted hydantoins exhibited antimycobacterial effect. 47 The antimitotic effect of the investigated hydantoins was also examined. In the course of structure-activity relationship (SAR) studies and to explore the antiproliferative effect associated with the hydantoin framework, several diversely substituted diazaspiro hydantoins were synthesized. 48 Variation in the functional group at N-terminal of the hydan-toin ring and coupling of different substituted aromatic acids in 4-aminocyclohexanone ring led to three sets of compounds. The antiproliferative effect of the compounds was evaluated in vitro using the MTT test against one normal cell line (NDF-103 skin fibroblast cells) and four human cancer cell lines (MCF-7 breast carcinoma cell line, HepG-2 hepatocellular carcinoma cell line, HeLa cervix carcinoma cell line and HT-29 colon carcinoma cell line) for the time period of 24 h. Among the series, some compounds exhibited interesting growth inhibitory effects against all four cell lines. The SAR studies revealed that the substitution at N-terminal in hydantoin ring played a key role in the antiproliferative activity.
For this reason, the goal of the present paper is to describe a method for synthesis of 3-methyl-9'-fluorenespiro-5-hydantoin, its structural elucidation and biological properties (its cytotoxic and antimicrobial effects).

1. Instrumentation and Methods
All chemicals used were purchased from Merck and Sigma-Aldrich. UV/Vis spectrum was measured on a Lambda 9 Perkin-Elmer UV/Vis/NIR Spectrophotometer from 200 nm to 1000 nm. The IR spectrum of 3-methyl-9'-fluorenespiro-5-hydantoin was obtained as KBr pellet on a Bruker FT-IR VERTEX 70 Spectrometer from 4000 cm -1 to 400 cm -1 at resolution 2 cm -1 with 25 scans. The Raman spectrum of the obtained product (the stirred crystals placed in aluminium disc) was measured on a RAM II (Bruker Optics) with a focused laser beam of 200 mW power of Nd:YAG laser (1064 nm) from 4000 cm -1 to 400 cm -1 at resolution 2 cm -1 with 25 scans. The NMR spectra were taken on a Bruker Avance II+ 600 MHz NMR spectrometer operating at 600.130 and 150.903 MHz for 1 H and 13 C, respectively, using the standard Bruker software. Chemical shifts were referenced to tetramethylsilane (TMS). Measurements were carried out at ambient temperature.

3. WST-1 Cell Proliferation Assay
The cytotoxic effect of compound 2 was assessed on a suspension cell line using WST-1 assay (Cat. No11 644 807 001, Roche). The suspension retinoblastoma cells (WERI-Rb1, ATCC-HTB-169) were cultured in RPMI 1640 medium, containing 10% FCS, 100 μg/mL streptomycin and 100 units/mL penicillin. Cells were cultured at 37 °C in a humidified atmosphere containing 5% CO 2 . The compound was first dissolved in DMSO and then diluted in the respective culture medium. The concentration of DMSO in the wells did not exceed 1%. Cells were seeded in triplicates in 96-well flat-bottom plates at a density of 6.5 × 10 4 cells/well (WERI-Rb1). After a cultivation period of 24 h, the compound was added at a concentration 100 μM on WERI-Rb1 cells and incubated for 24, 48 and 72 h, respectively. WST-1 was added to the cells at these time points and incubated for 4 h. After the incubation period the absorbance was measured using a microplate ELISA SUNRISE reader at a wavelength of 450 nm with a reference filter at 620 nm. The percentage of viable cells was calculated as a ratio of the OD value of the sample to the OD value of the control. The data are presented as mean ± standard deviation of the mean.

4. Antimicrobial Assay
The antimicrobial activity of 2 against Gram-positive bacteria -Staphylococcus aureus ATCC 25923 and Bacillus subtilis, Gram-negative bacteria -Escherichia coli ATCC 25922, Salmonella enterica subsp enterica ATCC BAA-2162, Pseudomonas aeruginosa ATCC 9027 and the yeasts Candida albicans ATCC 10231 was investigated using the agar diffusion method. Melted PCA (Scharlau) nutrient medium was inoculated through the addition of 1 mL of microbial suspension (1 × 10 10 CFU/mL for the bacteria and 1 × 10 9 CFU/mL for the yeast) and was poured in Petri dishes, 20 mL in each dish. Wells with 7 mm diameter were made in the solidified and cooled agar medium. 50 μL of the tested substance solution (5.38 mg/mL in 15% DMSO) was pipetted into the wells. The current concentrations of the test microorganisms in the suspensions were as follow: S. aureus 3.4 × 10 9 ; Bacillus subtilis 2.21 × 10 9 ; E.coli 1.02 × 10 9 ; S. enterica subsp enterica 1.12 × 10 9 ; P. aeruginosa 1.07 × 10 9 ; C. albicans 4 × 10 9 cfu/mL. The Petri dishes were incubated at 37 °C for 24-48 h. The inhibition zone was measured. Zones with diameter more than 7 mm were considered zones of inhibition.

5. Computational Details
To additionally verify the proposed assignments, quantum chemistry calculations were performed by using the Gaussian 98, Revision A.7. 65 For the geometry optimization the B3LYP density functional with 6-31G(d) basis set was used and for the 1 H and 13 C NMR spectra prediction the HF/6-31G(d) calculations were carried out at the optimized geometry.

Results and Discussion
A synthesis procedure for 9'-fluorenespiro-5hydantoin methylation with diazomethane has already been described. 66 Here we present a new method for 3-methyl-9'-fluorenespiro-5-hydantoin (2) preparation. The method discussed here is based on the reaction of 9'-fluorenespiro-5-hydantoin with dimethyl sulfate. The target product was obtained with high yield (78%) and showed m.p. 227-228 °C. The synthesis of 2 was carried as shown in Scheme 1. The structure of 2 was determined by UV-Vis, FT-IR, Raman, 1 H NMR and 13 C NMR spectroscopy. Maxima in the UV/Vis spectrum of the 2 were observed at 306, 271, 235, 228, 210 nm. The IR band at 3232 cm -1 of 2 that was observed may refer to the stretching vibration of the N-H group of the hydantoin ring. The vibrational (N 1 -H) stretching mode did not appear in the Raman spectrum. In the IR spectrum of the 2 the bands at 1775 cm -1 and 1717 cm -1 can be attributed to stretching vibrations of the two C=O groups of the hydantoin ring. In the Raman spectrum of 2 the one of the two C=O groups appeared at 1771 cm -1 . The other vibrational (C=O) stretching mode did not appear in the Raman spectrum. Several bands in the IR spectrum (3058, 3041 cm -1 ) and in the Raman spectrum (3058, 3003 cm -1 ) were for stretching vibrations of CH in fluorene moiety. In the IR spectrum of the 2 the bands at 2946 cm -1 and 2814 cm -1 can be attributed to stretching vibrations of the CH 3 group. In Raman spectrum of 2 the former vibration appeared at 2949 cm -1 .
The 1 H-broadband-decoupled 13 C NMR spectrum of 2 showed 10 signals: 6 pairs of atoms were magnetically equivalent. The two signals with the highest chemical shift in 13 C NMR spectrum, 173.06 and 157.58 ppm, were for the carbonyl groups (C 4 =O) and (C 2 =O). The signals at 71.54 and 25.49 ppm were for the spiro-carbon and methyl group. The structure of multiplets and coupling constants in 1 H NMR spectrum were consistent with the structure of 2. The assignment of signal at 71.54 to the spiro carbon, C-9', was supported also by an HMBC correlation of HN with it (δ H 8.87-δ C 71.54). There was also an HMBC correlation δ H 7.47-δ C 71.54 which points out that this δ H is for H-1'/8'. This inference and the COSY correlations allow to unambiguously assign all proton signals. As only the meta (vicinal) coupling ( 3 J CH ) in benzene rings is usually resolved, 67 the assignments of the quaternary carbons, C-1a', C-4a', C-5a' and C-8a', can be made ( Table 1).
The effect of the compound 2 on the proliferation of WERI-Rb1 cells after 24 and 72 h of treatment is presented in Fig. 1. The results from the cytotoxicity assay on the human WERI-Rb-1 cell line showed that the product 2 reduced the number of tumor cells by around 2% after 24 h. It showed a significant cytotoxic effect after 72 h of treatment when cell vitality decreased by 80%.
The results for the antimicrobial activity of 2 are presented in Table 2. Compound 2 showed strong antimi-crobial effect only against Bacillus subtilis (inhibition zone 15 mm) and moderate antimicrobial activity against S. aureus and P. aeruginosa (inhibition zones 9 mm). The   presence of single cell colonies in the inhibition zone for Bacillus subtilis shows that there are cell with different sensitivity towards this substance within the strain.

Conclusions
The method for synthesis of 3-methyl-9'-fluorenespiro-5-hydantoin (2) was presented. The structure of the obtained product 2 was determined by UV-Vis, FT-IR, 1 H, 13 C NMR and Raman spectroscopy, as well as by means of one-and two-dimensional NMR techniques, including HMQC, 1 H-1 H COSY, and HMBC spectra. The preliminary results of our study showed that the compound could serve as a potential anticancer agent. Further investigations are needed to elucidate the exact mechanisms of this action and to exclude any cytotoxic effect on normal cells. The results for the compound 2 showed that it has potential as antimicrobial agent against Gram positive bacteria.
The numbered structure of 2, complete spectral data, with enlarged detailed sections for multiplets and cross peaks in NMR spectra, as well as the archive Gaussian job results are included in Supporting Information.