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Microwave Assistant Synthesis, Antifungal Activity and DFT Theoretical Study of Some Novel 1,2,4-Triazole Derivatives Containing Pyridine Moiety

In order to investigate the biological activity of novel 1,2,4-triazole compounds, seventeen novel 1,2,4-triazole derivatives containing pyridine moiety were synthesized under microwave assistant condition by multi-step reactions. The structures were characterized by 1H NMR, MS and elemental analyses. The target compounds were evaluated for their fungicidal activities against Stemphylium lycopersici (Enjoji) Yamamoto, Fusarium oxysporum. sp. cucumebrium, and Botrytis cinerea in vivo, and the results indicated that some of the title compounds displayed excellent fungicidal activities. Theoretical calculation of the title compound was carried out with B3LYP/6-31G (d,p). The full geometry optimization was carried out using 6-31G (d,p) basis set, and the frontier orbital energy, atomic net charges were discussed, and the structure-activity relationship was also studied.

Furthermore, pyridine is an important class of heterocyclic ring, which have attracted attention in past few years. The pyridine nucleus exists in numerous natural products and is extremely important in the chemistry of biological systems, such as antioxidant activity [27], antimicrobial activity [28], acetylcholinesterase inhibitors [29], antibacterial activity [30,31], anticancer activity [32,33], anti-HBV activity [34], and so on. In the fungicidal activity area, some pyridine derivatives can prevent Ralstonia solanacearum [35], Cerospora beticola sacc. [36], Colletotrichum orbiculare [37], and so on. Currently, some pyridine compounds have been developed and commercialized, for example, Fluopicolid, Boscalid, Fluazinam, and Pyridinitril ( Figure 2). There are many reports about each of the two heterocycles, but the combination of the pyridine ring with the 1,2,4-triazole ring in one molecule is seldom reported both in chemistry and their biological activity studies. In view of the facts mentioned above, and also as a part of our work [38][39][40] on the synthesis of bioactive lead compounds, the title compounds were designed by introducing the pyridine ring pharmacophore into the 1,2,4-triazole scaffold. Twenty-one novel 1,2,4-triazole derivatives were synthesized and characterized by 1 H NMR, MS and elemental analysis. The antifungal activities of these compounds were tested in vivo.

Chemistry
The synthesis procedures for title compounds were shown in Scheme 1. with substituted benzyl chloride or alkyl chloride to afford compound 5. The microwave irradiation assistant synthesis and conventional method was also employed in this experiment. NaOH/DMF/H 2 O system was applied under microwave irradiation. The best reaction condition is at 90 °C for 15 min under microwave irradiation synthesis. The yield is higher than that of conventional methods, and, in addition, the reaction time is shorter ( Table 1).
The signal of CH 2 protons of thioether, neighboring the triazole ring, was observed at δ 2.23-4.67 ppm, respectively. The chemical shifts of pyridine are divided into two double peaks. The ESI (electrospray ionization)-MS spectrum showed that the m/z of molecular ion, in accordance with its molecular formula. The elemental analysis result is in accordance with the calculated results.

Molecular Total Energies and Frontier Orbital Energy Analysis
Molecular total energy and frontier orbital energy levels are listed in Table 3. The energy gap between HOMO and LUMO was calculated by B3LYP.
According to the frontier molecular orbital theory, HOMO and LUMO are the most important factors that affect the bioactivity. HOMO has the priority to provide electrons, while LUMO can accept electrons first [41]. Thus, study on the frontier orbital energy can provide useful information about the biological mechanism. Taking the DFT (density functional theory) result for example, the geometry of the frame of the compound 5h is hardly influenced by the introduction of, either the pyridine ring, 1,2,4-triazole ring, thioether group, or phenyl ring ( Figure 3). The HOMO of the title compound is mainly located on the 4-OCH 3 phenyl ring, 1,2,4-triazole ring, and thioether group. While, the LUMO of the title compound is located on the pyridine ring, 1,2,4-triazole ring, thioether group. The fact that the title compound has strong affinity suggests the importance of the frontier molecular orbital in the π-π stacking or hydrophobic interactions. This also implies that the orbital interaction between the title compound and the aromatic ring or some other side of residue chains of receptors is dominated by π-π or hydrophobic interaction among the frontier molecular orbitals.    Table 4 exhibits the calculated Mulliken atomic charges except for atoms H. Taking DFT, for example, again, four atoms S18, C3, C23, and C27 are the most positively charged ones, which can interact with the negative charged part of the receptor easily. The negative charges are mainly located on atoms N1, N2, N4, N14, and O29, so they can interact easily with the positive part of the receptor. Therefore, we supposed this compound can combine the amino-acid residue on its surface by the interaction of the pyridine, triazole sulfur ether group, and OCH 3 group, which may be responsible for the bioactivity.

Mulliken Atomic Charges and Electrostatic Potential Analyses
Electrostatic potential of title compound was also calculated. From Figure 4, both oxygen atoms and nitrogen atoms have more negative charges. Perhaps the oxygen atoms and nitrogen atoms had some interaction with the receptor or acceptor.

Materials and Reagents
All the reagents are of analytical grade or freshly prepared before use. The monitoring of the progress of all reactions and homogeneity of the synthesized compounds were carried out by thin layer chromatography (TLC), TLC analysis was performed on silica gel plate, which was obtained from Qingdao Ocean Chemicals (Qingdao, China). Melting points were determined using an X-4 apparatus (Taike, Beijing, China) and uncorrected (Taike, Beijing, China). 1 H NMR spectra were measured on a Bruker AV 400 or 500 (Bruker, Fallanden, Switzerland) instrument using TMS as an internal standard and CDCl 3 as the solvent. Mass spectra were recorded on a Thermo Finnigan LCQ Advantage LC/mass detector instrument (Thermo Finnigan, Waltham, MA, USA). Elemental analyses were performed on a Vario EL elemental analyzer (ELEMENTAR, Hanau, Germany). Microwave activation was carried out with CEM Discover™ focused microwave (2450 MHz, 300 W, CEM, Matthews, NC, USA).

Theoretical Calculations
On the basis of the above structure, an isolated molecule was selected as the initial structure, while DFT-B3LYP/6-31G (d,p) methods in Gaussian 03 package [42] were used to optimize the structure of the title compound. Vibration analysis showed that the optimized structures were in accordance with the minimum points on the potential energy surfaces. All the convergent precisions were the system default values, and all the calculations were carried out on a personal computer.

Synthesis of Intermediates
Ethyl Isonicotinate (1) A mixture of isonicotinic acid (1.23 g, 10 mmol), 20 mL of ethanol, and 0.5 mL of H 2 SO 4 were fluxed for 12 h in a 100 mL round-bottomed flask. After ethanol was evaporated under reduced pressure, about 20 mL of Na 2 CO 3 solution (1 M) was added into the mixture. Then the mixture was extracted with ether. After evaporation of the solvent, the product 1 was obtained. Colorless liquid, yield: 90%.
General Procedure for Thioether (5) A CEM designed 10 mL pressure-rated vial was charged with DMF (5 mL), 4 (0.25 g, 1 mmol), RCH 2 Cl (1.1 mmol), and NaOH (0.05 g, 1.2 mmol). The mixture was irradiated in a CEM Discover Focused Synthesizer (150 w, 90 °C, 200 psi, 15 min). The mixture was cooled to room temperature by passing compressed air through the microwave cavity for 2 min. It was poured into cold ice (40 mL) and the formed precipitate was filtered. The crude solid was recrystallized from EtOH to give the title compounds 5a. All the other compounds are synthesized according to the procedure.

Fungicidal Activities
Fungicidal activity of compounds 5a-5u against Stemphylium lycopersici (Enjoji) Yamamoto, Fusarium oxysporum. sp. cucumebrium, and Botrytis cinerea were evaluated according to reference, and a potted plant test method was adopted. Germination was conducted by soaking cucumber seeds in water for 2 h at 50 °C and then keeping the seeds moist for 24 h at 28 °C in an incubator. When the radicles were 0.5 cm, the seeds were grown in plastic pots containing a 1:1 (v/v) mixture of vermiculite and peat. Cucumber and tomato plants used for inoculations were at the stage of two seed leaves. Tested compounds and commercial fungicides were sprayed with a hand sprayer on the surface of the seed leaves on a fine morning, at the standard concentration of 500 μg/mL, zhongshengmycin, thiophanate-methyl and cyprodinil were used as a control. After 2 h, inoculations of Stemphylium lycopersici (Enjoji) Yamamoto was carried out by spraying fungal suspension, inoculation of Fusarium oxysporum. sp. cucumebrium was carried out by spraying mycelial suspension, inoculation of Botrytis cinerea was carried out by radicle soaking. The experiment was repeated 4 times. After inoculation, the plants were maintained at 18-30 °C (mean temperature of 24 °C and above 80% relative humidity (RH)). The fungicidal activities were evaluated when the nontreated cucumber plant (blank) fully developed symptoms. The area of inoculated treated leaves covered by disease symptoms was assessed and compared to those of nontreated ones to determine the average disease index. The relative control efficacy of compounds compared to the blank assay was calculated via the following equation: Relative control efficacy (%)= (CK − PT)/CK × 100% (1) where CK is the average disease index during the blank assay and PT is the average disease index after treatment during testing.

Conclusions
In summary, a series of 1,2,4-triazole derivatives were synthesized containing pyridine ring in good yields. The preliminary bioassays showed that some of the compounds had good fungicidal activity. The present findings provided a powerful complement to the SARs of fungicides, and warrant future investigation of the mechanism of action of these analogs.