Microbiologically active Mannich bases derived from 1,2,4-triazoles. The effect of C-5 substituent on antibacterial activity

Our research proved that chemical character of the C-5 substituent significantly determines the antibacterial activity of the Mannich bases derived from 4,5-disubstituted 1,2,4-triazole-3-thiones. This activity was considerably increased by an introduction of a chlorine atom to the phenyl ring. The obtained compounds were particularly active against opportunistic bacteria (Bacillus subtilis and Bacillus cereus). The antibacterial activity of some Mannich bases was similar or higher than the activity of commonly used antibiotics such as ampicillin and cefuroxime.


Introduction
Excessive and uncontrolled intake of antibiotics resulted in a selection of bacterial strains resistant to commonly used drugs. Recently, the world has been focused on the appearance of the so-called super resistant NDM-1 gene (Yong et al., 2009;Rolain et al., 2010) which spreads via DNA segments called plasmids. In the view of growing bacterial drug-resistance, the search of chemical substances which can efficiently treat infections caused by this type of bacteria seems to be necessary.
The Mannich reaction is known to be very useful for the synthesis of antibacterial compounds. This reaction makes it possible to introduce amine fragment into the different chemical scaffolds which can increase the affinity of the obtained molecule toward appropriate molecular target. 1,2,4-Triazole-3-thione derivatives known for their antibacterial activity (Turan-Zitouni et al., 2005;Eswaran et al., 2009;Shafiee et al., 2002) were used by many researchers as substrates for the Mannich reaction. The obtained aminomethyl derivatives included both compounds which acted stronger than their N2-unsubstituted predecessors (Isloor et al., 2009;Ashok et al., 2007;Bayrak et al., 2009a), as well as significantly less active compounds (Bayrak et al., 2009b;Almajan et al., 2009). In our previous studies we proved that the presence of the 4-bromophenyl moiety in the N-4 position benefited the antibacterial activity of 4,5-disubstituted 1,2,4-triazole-3-thione derivatives (Plech et al., 2011a, b). Further research also indicated that the activity of this type of Mannich bases decreases with the increased volume of substituent in the N2 position (Plech et al., 2011b). The goal of current research was to analyze the impact of the substituent in the C-5 position on the antibacterial activity of obtained compounds. First of all, it has been decided to examine if, and to what degree, the strength of the new derivatives' activity changes after introducing a chlorine atom to the phenyl ring. Also, the disparities in the activity of appropriate ortho-, meta-, and para-derivatives were analyzed.
Our earlier studies showed that the thione tautomer is energetically favored (Wujec et al., 2007). The IR spectra of compounds 7-9 showed the absorption bands at 3,437-3,411 cm -1 and 1,331-1,328 cm -1 , indicating the presence of NH and C=S groups, respectively. In the 1 H-NMR spectra, NH proton resonated as a singlet at *14 ppm. Crystallographic data (unpublished results) also confirm the existence of the mentioned compounds as the C=S tautomers.
The Mannich reaction was carried out in mild conditions; it was quick (30 min) and efficient (yields: 76-87 %). The structure and purity of the products (10-21) was confirmed using 1 H-NMR, 13 C-NMR (for compound 20), and IR spectra as well as elemental analysis. The 1 H-NMR spectra showed characteristic signals which indicated the presence of aminomethyl fragment. Two protons of the N2-CH 2group resonated as a singlet in the range of 5.22-5.34 ppm, while the signals of the amine residues were visible at 1.20-3.76 ppm. In addition to this, peaks characteristic for para-substituted phenyl rings were visible in the area typical for aromatic protons. The IR spectra also confirmed the suggested structure of the Mannich bases (10-21).

Antibacterial screening
The antibacterial activity of compounds 10-21 was determined for Gram-positive and Gram-negative bacteria. The growth of Gram-negative bacteria (Escherichia coli ATCC 25922, Klebsiella pneumoniae ATCC 13883, Proteus mirabilis ATCC 12453, and Pseudomonas aeruginosa ATCC 9027) was not inhibited by any of the compounds. Therefore, Table 1 4) activity. This may suggest a presence of more than one mechanism of action for these derivatives. In order to analyze the impact of the type of substituent in the C-5 position on the antibacterial activity, derivatives including phenyl (10-13), 2-chlorophenyl (14-17), and 4-chlorophenyl (18-21) rings were obtained. In order to ensure more comprehensive analysis, the discussion also considered the compounds with 3-chlorophenyl fragment (22-25) (Fig. 1)-synthesized and described by us recently (Plech et al., 2011a, b). Regardless of the type of aminomethyl substituent in the N2 position, none of the C5phenyl derivatives showed antibacterial activity which would be worth noticing. The activity of the obtained Mannich bases was significantly increased only after an electron-withdrawing chlorine atom had been introduced to the phenyl ring. Interesting conclusions can be drawn when comparing the activity of appropriate ortho-, meta-, and para-analogs. Balanced activity toward all analyzed Gram-positive bacteria was characteristic for compounds with 3-chlorophenyl fragment, regardless of the type of substituent in the N2 position. While the activity of orthoand para-analogs depended largely on the type of aminomethyl fragment. It is worth mentioning that compounds with pentatomic (apart from hydrogen atoms) amine substituent in the N2 position (pyrrolidine and diethylamine) were microbiologically more active than appropriate compounds with six-membered substituents (piperidine and morpholine). Also, the disparity between the activities of piperidinyl and morpholinyl derivatives shows that the oxygen atom in the morpholine molecule is important for the binding with a potential molecular target. This is probably caused by the fact that the oxygen atom can participate in the formation of hydrogen bonds in the drugtarget site.

Conclusions
Our research showed that chemical character of the C-5 substituent significantly determines the antibacterial activity of the N2-aminomethyl derivatives of the 1,2,4triazole. This activity can be considerably increased by an introduction of an electron-withdrawing chlorine atom to the phenyl ring in the C-5 position. In addition to this, the number of atoms which form the aminomethyl substituent seems to be important. The activity of the obtained Mannich bases was particularly strong toward opportunistic bacteria. The antibacterial activity of some compounds was similar or higher than the activity of commonly used antibiotics such as ampicillin and cefuroxime.

General comments
All reagents and solvents were purchased from Alfa Aesar (Ward Hill, USA) and Merck Co. (Darmstadt, Germany). Melting points were determined using Fisher-Johns apparatus (Fisher Scientific, Schwerte, Germany) and are uncorrected. The 1 H-NMR and 13 C-NMR spectra were recorded on a Bruker Avance spectrometer (Bruker Bio-Spin GmbH, Rheinstetten, Germany) using TMS as an internal standard. The IR spectra (KBr) were obtained on a Perkin-Elmer 1725X FTIR spectrophotometer. Elemental analyses were performed on an AMZ 851 CHX analyzer (PG, Gdańsk, Poland) and the results were within ±0.2 % of the theoretical value. All the compounds were purified by flash chromatography (PuriFlash 430evo, Interchim, USA).
Synthesis of 1,2,4-triazole derivatives (7-9) Appropriate thiosemicarbazides (4-6) were dissolved in 2 % solution of NaOH. Next, the resulting solution was heated under reflux for 2 h. After cooling, the reaction mixture was neutralized with HCl. The precipitated product was filtered off, washed with distilled water, and recrystallized from EtOH.