Hydantoin Derivatives of L- and D-amino acids: Synthesis and Evaluation of Their Antiviral and Antitumoral Activity

3,5-Disubstituted hydantoin (1,3-imidazolidinedione) derivatives 5a-h were prepared by base induced cyclization of the corresponding N-(1-benzotriazolecarbonyl)-L- and D-amino acid amides 4a-h. Compounds 5a-h were evaluated for their cytostatic and antiviral activities. Among all the compounds evaluated, 3-benzhydryl-5-isopropyl hydantoin (5a) showed a weak but selective inhibitory effect against vaccinia virus (EC(50) = 16 microg/mL; selectivity index: 25). 3-Cyclohexyl-5-phenyl hydantoin (5g) showed inhibitory activity against cervical carcinoma (HeLa, IC(50) = 5.4 microM) and breast carcinoma (MCF-7, IC(50) = 2 microM), but also cytotoxic effects towards human normal fibroblasts (WI 38). On the contrary, the 3-benzhydryl-5-phenyl substituted hydantoin derivative 5h showed moderate inhibitory activity towards HeLa, MCF-7, pancreatic carcinoma (MiaPaCa-2), lung carcinoma (H 460) and colon carcinoma (SW 620) (IC(50) = 20-23 microM), but no effect on WI 38.


Results and Discussion
Chemistry A series of 3,5-disubstituted hydantoins 5a-h were prepared by intramolecular cyclization of N-[(1benzotriazolecarbonyl (Btc)]-amino acid amides 4a-h, following our previously described procedures [7−9] (Scheme 1). All these hydantoins are new compounds, except 5d and 5g, whose syntheses have been previously described [9−11], although their cytostatic and antiviral activities have not been reported until now. Synthesis of the N-Btc-amino acid amides 4a-h and their precursors N-Btc-amino acid derivatives 2a-d and the corresponding chloride derivatives 3a-d were also described in our previous reports [12]. Syntheses of the compounds 4a-h and 5a-h were carried out under mild reaction conditions and the products were obtained in high yields. In the preparation of the amides 4a-h, an equimolar ratio of the N-Btc-amino acid chloride derivatives 3a-d and the corresponding amine was crucial in order to avoid formation of ureido amides. Our previous research results had shown that 3cyclohexanemethyl-5-phenylhydantoin possessed marked antiviral activity (i.e. against cytomegalovirus) [7]. Based on that lead compound we have now prepared some novel hydantoins containing one or two lipophilic substituents at positions 3 and 5. Three amino acids of the L-series (L-valine, Lleucine and L-phenylalanine) and one D-amino acid (D-phenylglycine) and highly lipophilic amines (cyclopentyl-, cyclohexyl-, cyclohexanemethyl-or benzhydrylamines) were used as the starting reagents. As the chiral centre was not involved in chemical transformations of the L-and D-amino acid precursors (Scheme 1) these configurations should be retained in the target hydantoin molecules [7]. The validity of this assumption has been proven by X-ray crystallographic analysis of the stereostructure of the hydantoin derivative of L-valine [8]. Scheme 1. Synthesis of hydantoin derivatives of L-and D-amino acids, 5a-f and 5g,h, respectively The structures of the newly synthesized compounds were deduced from the analysis of their 1 Hand 13 C-NMR and IR spectra, and confirmed by elemental analysis. The IR spectra of the hydantoin derivatives (Table 1) showed characteristic bands at 3421−3227 (NH), 1775−1760 and 1718−1698 (CO) cm -1 . The chemical shifts in 1 H-and 13 C-NMR spectra (Tables 2 and 3) are in concordance with the proposed structures of the novel compounds and also consistent with previously described analogous hydantoin derivatives [7].       Table 3. 13 C-NMR spectral data of the novel N-Btc-L-valine benzhydrylamide (4a) and 3,5-disubstituted hydantoin derivatives 5a-h.

Conclusions
In summary, the hydantoin derivatives presented herein showed certain antiviral and antitumor activity. Derivatives 5f and 5h exhibited weak inhibitory effects against the evaluated viruses and hydantoin 5a showed a weak but selective inhibitory effect against vaccinia virus. With regards to their antitumor activity, hydantoin 5g showed rather marked inhibitory activity against HeLa and MCF-7 cell lines, while 5h showed inhibitory activity towards several tumor cell lines and no cytotoxic effects on normal cells. Further optimization of the hydantoin derivatives as potential antiviral and cytostatic agents is in progress.

General
Melting points were determined on a Boëtius Micro Heating Stage and are uncorrected. IR spectra were recorded on a FTIR Perkin Elmer Paragon 500 spectrometer. 1 H-and 13 C-NMR spectra were recorded on a Varian Gemini 300 spectrometer, operating at 300 and 75.5 MHz for the 1 H-and 13 Cnuclei, respectively. Samples were measured in DMSO-d 6 solutions at 20 ºC in 5 mm NMR tubes. Chemical shifts (δ) in ppm were referred to TMS. Precoated silica gel 60 F 254 plates (Merck) were used for thin-layer chromatography, with 1:1 cyclohexane/ethyl acetate as the eluent. Spots were visualized by short-wave UV light and iodine vapour.
N-Btc-L-valine benzhydrylamide (4a). To a cold solution of N-Btc-L-valine chloride 3a [9] (1.122 g, 4 mmol) in toluene (40 mL), a solution of benzhydrylamine (0.732 g, 4 mmol) and triethylamine (0.404 g, 4 mmol) in toluene (30 mL) was added dropwise. The reaction mixture was stirred at room temperature for 24 h. Product 4a, which precipitated together with TEA·HCl, was filtered off and dissolved in a dichloromethane/water mixture. The organic layers were extracted with diluted hydrochloride acid (w = 1%) and water, dried over sodium sulfate and evaporated in vacuo. The residue was triturated with ether to give the analytically pure 4a. The analytical, 1 H-and 13 C-NMR spectral data for 4a are given in Tables 1−3.

Antiviral Activity Assays
Antiviral activity against HSV-1, HSV-2, vaccinia virus, vesicular stomatitis virus, Coxsackie virus B4, respiratory syncytial virus, parainfluenza-3 virus, reovirus-1, Sindbis virus and Punta Toro virus was determined as described previously [13,14]. Antiviral activity was expressed as the EC 50 or concentration required to reduce virus-induced cytopathogenicy by 50%. EC 50 values were calculated from graphic plots of the percentage of cytopathogenicity as a function of concentration of the compounds.
The growth-inhibitory activity of HeLa, MCF-7, MiaPaCa-2, H 460, SW 620 and WI 38 cells was assessed according to a slightly modified procedure performed at the National Cancer Institute, Developmental Therapeutics Program [16]. The cells were seeded into a series of standard 96-well microtiter plates on day 0. Test agents were then added in five, 10-fold dilutions (10 -8 to 10 -4 M) and incubated for a further 72 hours. Working dilutions were freshly prepared on the day of testing. The solvent (DMSO) was also tested for eventual inhibitory activity by adjusting its concentration to be the same as for the tested concentrations. After 72 hours of incubation the cell growth rate was evaluated by MTT assay [17] which detects dehydrogenase activity in viable cells. The absorbance (OD, optical density) was measured by a microplate reader at 570 nm. The percentage of growth (PG) of the cell lines was calculated according to one or the other of the following two expressions: If (mean OD test -mean OD tzero ) ≥ 0, then: PG = 100 x (mean OD test -mean OD tzero ) / (mean OD ctrl -meanOD tzero ) If (mean OD test -mean OD tzero ) < 0, then: G = 100 x (mean OD test -mean OD tzero ) / OD tzero where mean OD tzero = the average of optical density measurements before exposure of cells to the test compound, mean OD test = the average of optical density measurements after the desired period of time and mean OD ctrl = the average of optical density measurements after the desired period of time with no exposure of cells to the test compound.
Each test point was measured in quadruplicate in three individual experiments. The results are expressed as IC 50 , a concentration necessary for 50% of inhibition. Each result is a mean value from three separate experiments. The IC 50 values for each compound were calculated from dose-response curves using linear regression analysis by fitting the test concentrations that gave PG values above and below the reference value (i.e. 50%).