Synthesis of Some Novel Fluorinated/Nonfluorinated α -Amino Acids, Bearing 3-Thioxo-5-oxo-1,2,4-triazin-6-yl and Steroidal Moieties, and Evaluation of Their Amylolytic Effects against Some Fungi, Part-II

Some new ﬂuorinated/nonﬂuorinated α -amino acids bearing 3-thioxo-5-oxo-1,2,4-triazin-6-yl and steroidal moieties have been obtained from condensation of the corresponding amino-triazinones with the steroid (Epiandrosterone). This was followed by the addition of HCN and, ﬁnally, acidic hydrolysis. The structure of the targets was established from their elemental analysis and spectral data. The amylolytic activity of the new products was evaluated against some fungi.


Introduction
α-Amino acids are one of the most important bioactive chemical substances (proteins and nucleoproteins) forming the basic constituents of living cells. Nine proteinogenic amino acids are considered as essential biochemicals for humans: valine, threonine, tryptophan, phenylalanine, leucine, isoleucine, methionine, lysine, and histidine. For instance, 5-fluorocytosine is an analogue of nucleotide, used as a chemotherapeutic antifungal when combined with amphotericin B [1]. Of equal importance, glycine is required for the biosynthesis of the heme group of haemoglobin; also, tryptophan is the precursor of a family of substances important in the biochemistry of the central nervous system (CNS), and tyrosine is the starting material for the biosynthesis of the skin pigment melanin [2]. e enzyme lactate dehydrogenase (LDH) illustrates isozymes very well ( Figure 1). 1,2,4-Triazine rings have been reported in the literature as having antifungal properties alongside their antitumor and antiviral activities. [3] Recently, Abdul-Rahman et al. [4][5][6][7][8][9] reported the synthesis, chemistry, and medicinal and biological activity, especially 6-(2-aminophenyl)3-thioxo-1,2,4-triazin-5-one [10][11][12][13][14]. Among all approved medicinal and pharmaceutical chemicals, nearly 20% have at least one fluorine atom existing which enhances phase II-III clinical trials [15].
us, the combination of fluorine with biomolecules such as fluorinated amino acids (FAAs), fluorinated steroids, and fluorinated nucleosides has increased, considerably, of the late years [16].
Incorporation of FAAs is one of the most utilized strategies in peptide and protein science. e effects of the combination of fluorinated α-amino acids into peptides and proteins on the primary and secondary structure have been widely reviewed by Koksch et al [17][18][19]. Furthermore, the fusion of unnatural/synthesized amino acids into peptides and proteins is generally closely accompanied with antimicrobial [20][21][22], antiviral [23], and metal chelating properties [24]such as thrombin, trypsin, and factor VIIa inhibitory activity.
Based on all these observations, this present work aims to find new synthetic fluorinated/nonfluorinated α-amino acids bearing both 3-thioxo 5-oxo-1,2,4-triazin-6-yl and steroidal moieties and evaluates their enzymatic effects toward some fungi activities (amylolytic activity) with an objective to obtain new highly bioactive substances.

Biological Evaluation.
e four synthesized a-amino acids, 4a, 4b, 5a, and 5b, were preliminarily tested toward some fungi such as Aspergillus flavus, Aspergillus fumigains, Aspergillus Niger, Aspergillus nidulaus, and Aspergillus terreus for their amylolytic activity according to the classical methods [27][28][29][30]. Using DMF as a solvent, about 0.01 g of each compound was dissolved in the presence of phosphate buffer saline (PBS) at PH 6.5 for 30 minutes. e amylase activity was assayed at the adjusted pH and temperature 38°C, according to the standard method. [28,29] e activity was estimated (in mg reducing sugar out of reaction mixture), and all the results obtained are reported in Table 1.
From these data, we can conclude that most of the tested compounds exhibited a good to moderate inhibition and/or acceleration activity. In specific, compound 4a, which contains both F and S atoms, showed a stronger effect towards all the tested fungi than other systems, and markably, fluorinated compounds 4 and 5 showed a complete control on the tested fungi as A. flavus, A. nedulaus, and A. niger. Finally, we report that the active new fluorinated α-amino acids are a promising candidate to be used as enzymatic catalysts in the vital biosynthesis process due to their activity.

Experimental
All reagents and solvents were purchased from commercial sources and used without further purification, unless otherwise stated. Melting points were measured using a Stuart SMB3 melting point apparatus and are uncorrected. IR spectra were recorded on a PerkinElmer Lambda 550 S spectrometer (KBr/cm −1 ). All the chemical shifts, 1 HNMR and 13 CNMR, were recorded relative to TMS and recorded on a varian-700 spectrometer (DMSO, d 6 ppm).

Conclusions
In a search for new α-amino acids, the present work reports a simple route to synthesize fluorinated and nonfluorinated α-amino acids derived from the corresponding 1,2,4-triazinone bearing an amino-group and a steroidal component. e new fluorinated synthetic skeletons exhibit an amylolytic activity greater than nonfluorinated systems against some fungi.

Data Availability
e data used to support the study can be made available upon request to the corresponding author.

Conflicts of Interest
e authors have no conflicts of interest to declare.