Synthesis and Antimicrobial Activity of 6-Thioxo-6,7-dihydro-2H-[1,2,4]triazino[2,3-c]-quinazolin-2-one Derivatives

Abstract Potassium 8-R1-9-R2-10-R3-3-R-2-oxo-2H-[1,2,4]triazino[2,3-c]quinazoline-6-thiolates 2.1–2.26 were synthesized via cyclocondensation of 6-R-3-(3-R1-4-R2-5-R3-aminophenyl)-1,2,4-triazin-5-ones 1.1–1.26 with carbon disulfide, potassium hydroxide, and ethanol or with potassium O-ethyl dithiocarbonate in 2-propanol. The corresponding thiones 3.1–3.26 were obtained by treatment of 2.1–2.26 with hydrochloric acid. It was found that the nature of the substituents in positions 3, 4, and 5 of the corresponding 6-R-3-(3-R1-4-R2-5-R3-aminophenyl)-1,2,4-triazin-5-ones were affected on the terms of the reaction. The structures of compounds were proven by a complex of physicochemical methods (1H, 13C NMR, LC–MS, and EI-MS). The results of the antibacterial and antifungal activity assay allowed the determination of the high sensitivity of Staphylococcus aureus ATCC 25923 (MIC 6.25–100 μg/mL, MBC 12.5–200 μg/mL) to the synthesized compounds.

Our attempt to record the NMR spectral data for potassium 8-R 1 -9-R 2 -10-R 3 -3-R-2-oxo-2Н- [1,2,4]triazino[2,3-с]quinazoline-6-thiolates in DMSO-d 6 , CDCl 3 , and D 2 O failed. We considered this as a consequence of the exchange processes, tautomeric transformations, and insufficient solubility. However, for the evaluation of the mentioned compound structure, we converted them into thiones 3.1-3.26 and recorded all of the necessary spectral data to prove their structures. So, as we considered, the confirmation of the structures 3.1-3.26 in combination with the IR and elemental analysis data, proved the structures of compounds 2.1-2.26.
The 1 H-NMR-spectra of compounds 3.1-3.26 are characterized by singlet signals of the thioamide group proton at 14.22-13.79 ppm and aromatic protons of the triazinoquinazoline system with corresponding chemical shifts [16]. The appearance at 171.05-168.79 ppm and 158.68-160.2 ppm of the characteristic signals of deshielded C-2 and C-6 carbons in the 13 С NMR-spectra of compounds 3.1, 3.3, 3.4, and 3.8 confirm the formation of the new heterocyclic system. Also in the 13 С NMR-spectra of the mentioned compounds, the signals of the corresponding aliphatic carbons are present. As we considered, the used physicochemical methods completely confirm the structure of the synthesized compounds.
The IR-spectra of compounds 3.1-3.26 are characterized by intensive absorption at 1767-1590 cm −1 , which correspond to vibrations of the С=S and С=О groups and substantially differentiate them from their initial compounds. At the same time in the IR-spectra of thiolates (2.1-2.26), the mentioned signals were subjected to the hypsochromic shift which may be explained by the formation of an ion bond. The vibrations of С=С bonds of the aromatic fragment at 1589-1468 cm −1 of the non-plate deforming vibrations of the =C-H bond at 850-666 cm −1 and intensive signals at 2960-2850 cm −1 , caused by the symmetric and asymmetric vibrations of CH 2 and CH 3 groups, are also present in the IR-spectra of the compounds 2.1-2.26 and 3.1-3.26. The IR-spectra of compounds, which contain halogen intensive signals of νС-F, νС-Cl, νС-Br, νС-I, are also present.
It is important to note that potassium thiolates 2.1-2.26, as more water-soluble in most cases, are less active (MIC 100 µg/mL, Table 2). The antimicrobial activity of compounds 2.1-2.26 and 3.1-3.26 towards the strain of Pseudomonas aeruginosa is also moderate (MIC 50-100 µg/mL) and substantially inferior to the inhibitory action of Nitrofural (MIC 6.25 µg/mL, tables 1 and 2).

General Methods
Melting points were determined in open capillary tubes and were uncorrected. The elemental analyses (C, H, N, S) were performed using the ELEMENTAR vario EL Cube analyzer (USA). Analyses were indicated by the symbols of the elements or functions within ±0.3% of the theoretical values. The IR spectra (4000-600 cm −1 ) were recorded on a Bruker ALPHA FT-IR spectrometer (Bruker Bioscience, Germany) using a module for measuring attenuated total reflection (ATR). The 1 H NMR spectra (400 MHz) and 13 C NMR spectra (100 MHz) were recorded on a Varian-Mercury 400 (Varian Inc., Palo Alto, CA, USA) spectrometer with TMS as the internal standard in DMSO-d 6 solution. The LC-MS were recorded using a chromato-mass spectrometric system which consisted of a highperformance liquid chromatograph «Agilent 1100 Series» (Agilent, Palo Alto, CA, USA) equipped with a diode-matrix and mass-selective detector «Agilent LC/MSD SL» (atmospheric pressure chemical ionization -APCI). The electron impact mass spectra (EI-MS) were recorded on a Varian 1200 L instrument at 70 eV (Varian, USA). The purity of all obtained compounds was checked by 1 H-NMR and LC-MS.
Substances 1.1-1.26 were synthesized according to the reported procedures [14]. Other starting materials and solvents were obtained from commercially available sources and used without additional purification.