Tetrahydro-2H-1,3,5- thiadiazin-t-thione Derivatives of The optical Isomers of Phenylalanine, Synthesis, Comparative Stability Study and Antifungal Activity

In order to investigate the effect of the optical properties of the 5-substituent on the stability and the antihngal activity of tetrahydro-2H-1,3,5-thiadizine-2-thione (THTT) moiety, optical isomers and racemic mixture of phenylalanine were incorporated in the 5th position of THTT to afford derivatives 2a-g. Chemical and enzymatic stability of these derivatives were studied in vitro in aqueous buffer solution of pH 7.4, physiological pH, and 80% human plasma at 37OC using HPLC. The chemical and enzy'matic degradation rates of the tested compounds revealed that the optical properties of the 5-substituent of THTT moiety have no role on their stability at the investigated media. However, the chemical nature of the 3-substiuents has a significant effect on their chemical and enzymatic liability as 3-aralkyl derivatives, 2f and 2g , were the least stable compounds under the investigation conditions. Solid state stability of these derivatives was studied using Differential Scanning Calorimetry (DSC). In spite of the distinguished DSC curves of the racemic cornpounds from that of the corresponding single optical isomers no constant pattern of their thermal stability was observed. The antifbngal activity of 2a-g, was investigated in vitro against Cnndida albicans, C. parasilosis and C. stellatoidea using tube dilution method. No role for the optical properties of the tested compounds on their antifungal activity was observed. The utmost antihngal activity revealed by compound 2g which has 3-phenethyl substituent. Moreover, 2g has the highest lipophilicity and the most susceptible compound for both chemical and enzymatic degradations.

Structure activity relationships which diverge from related compounds indicate that the polarity of the substituents at ring nitrogens determines the magnitude of antimicrobial action'5317. Optimum activities had been obtained in compounds bearing lipophilic groups at the 3ed and hydrophilic ones at the 5th position3.
As a part of studies in progress at our laboratories to develop various types of bioreversible derivatives of chemical entities occurring in amino acids with potential antimicrobial activity the present work outlines the incorporation of the optical isomers and racemic mixture of phenylalanine at the 5" position of THTT moiety. This aimed to investigate the effect of optical activity of the substituents at the 5" position of THTT moiety on the chemical and enzymatic degradation, and on the antimicrobial activity. Moreover, the carboxylic group of the phenylalanine moiety at the 5" position of THTT may reduces the toxicity for human cellslg. Additionally, investigation of the thermal (solid state) stability of these optical isomers seemed to be of interest.

Chemistry
The target compounds, 2a-g, were synthesized from appropriate amine, la-g, carbon disulfide, formaldehyde and proper optical isomer or racemic mixture of phenylalanine to provide the nitrogen atom at 5th position of thiazidiazine ring via cyclocondensation reaction, scheme 1  In IH-NMR spectra with the exception of the N~-substituents of the THTT moiety the resonance of the remaining sites of the protons of the synthesized derivatives is almost superimposable.

Lipophilicity
Lipophilicity of the syrlthesized derivatives, 2a-g, is expressed in the term of their log P values. These values,

Kinetic Measurements
It has been reported that the magnitude of activity of THTT derivatives may be attributed to the ease of hydrolysis of the ring system to f m i s h the i s o t h i~c~a n a t e s~~. Accordingly, the degradation kinetics of the synthesized derivatives, 2a-g, were studied in aqueous buffer solution of pH 7.4, physiological pH, at 37°C. At constant temperature disappearance of the tested compounds displayed strict first order kinetic reactions over several half-lives and all reactions proceeded to completion. The rate data obtained for the various derivatives, table 2, revealed that as a general pattern the rate of hydrolysis is unaffected by the optical activity. However, the chemical degradation rates of the synthesized derivatives affected by the variation of substituents on N-3 of the THTT moiety. THTT derivatives bearing phenethyl group at position 3 is the most susceptible for chemical hydrolysis.
The degradation rates of the tested compounds were also investigated in 80% human plasma at 37'C in order to get information about the susceptibility of the synthesized derivatives toward enzymatic metabolism. Strict first order kinetic reactions were also observed with the enzyme system used under the investigated conditions. The enzymatic degradation data, table 2, showed that the liability of the investigated THTT derivatives, 2a-g, toward human plasma enzymes is also unaffected by their optical activity and have the same pattern of the previously reported analogous14925126. Furthermore, the 3-aralkyl derivatives were more susceptible to the enzymatic degradation.

Solid State Stability Study:
Differential Scanning calorimetry (DSC) can be used for distinguishing optical isomers from the racemic mixtures in the solid state as well as investigation of solid state stability 27. Accordingly, Solid state stability of the optical isomers and the corresponding racemic mixtures of the derivatives
Kx lo3 min-' 2a,2c,2f and 2g was studied using DSC. Fig. 1 shows DSC curves as printouts from the instrument at heating rate 10°C/min for the tested compounds. As

Antifungal Activity
The antifungal activity of the synthesized compounds, 2a-g, were tested in vitro against Candida ulbicans, C. parasilosis and C. stellatoidea using tube dilution Results of the in vitro antifungal investigation, table 4, revealed that the antifu~~gal activity of the tested-compounds is independent on their optical properties. It is worthy notice that 2g is the most active compound  has the highest lipophilicity among the synthesized derivatives, and the most susceptible compound for both chemical and enzymatic degradation. This data is not only consistent with the structural requirements for the antimicrobial activity but also a strong evidence to that the activity attributed to the released isothiocyanates. 1% triethylarnine were used. The ratio of methanol : water was adjusted in order to give a retention time of 5-7 minutes for the synthesized derivatives.
The column effluent was monitored at 254 nm and the flow rate was 1 mllmin.
Quantification of the eluted compounds was done from peak area measurements in relation to those of standards chromatographed under the same conditions. DSC were determined using Shimadzu DSC-50 connected with a data station TA-501. The DSC apparatus was calibrated with indium (peak maximum at 156.6"C).
Antifungal activity was performed at the Department of Microbiology, Faculty of Medicine, Assiut University, Assiut, Egypt.

Calculation of log P values
The log P values of the synthesized derivatives were computed with a routine method called calculated log P (Clog P) contained in a PC-software package (MacLogP 2.0, BioByte Corp., CA, USA). A representation of the molecular structure where hydrogens are omitted, or 'suppressed7 (SMILES notation), is entered into the program, which computes the log P based on the fragment method developed by ~e o~' .

Kinetic Measurements
Degradation rates of the synthesized derivatives 2a-g in aqueous solution of isotonic phosphate buffer, pH 7.4, was determined at 37OC. The ionic strength of the prepared buffer solution was adjusted with KC1 to p = 0.5. Where a is the heating rate, T, is the temperature at peak maximum of the endothem, Ea is the activation energy, and R is the gas constant.
Based on equation 1, plotting ln(al~$) versus 1/Tm gives a linear relation, from its slope, the Ea can be calculated. Results are given in table 3.