Microwave Assisted Synthesis, Antifungal Activity, and DFT Study of Some Novel Triazolinone Derivatives

A series of some novel 1,2,4-triazol-5(4H)-one derivatives were designed and synthesized under microwave irradiation via multistep reaction. The structures of 1,2,4-triazoles were confirmed by 1H NMR, MS, FTIR, and elemental analysis. The antifungal activities of 1,2,4-triazoles were determined. The antifungal activity results indicated that the compounds 5c, 5f, and 5h exhibited good activity against Pythium ultimum, and the compounds 5b and 5c displayed good activity against Corynespora cassiicola. Theoretical calculation of the compound 5c was carried out with B3LYP/6-31G (d). The full geometry optimization was carried out using 6-31G(d) basis set, and the frontier orbital energy and electrostatic potential were discussed, and the structure-activity relationship was also studied.

Microwave-assisted technique is a green method in current organic synthesis [24][25][26][27][28]. It is attractive, offering reduced pollution, low cost, and high yields. The green technique can often shorten the reaction time.
In our previous work [29][30][31][32][33], some 1,2,4-triazole compounds were designed and synthesized. They showed good antifungal activities. In this paper, fifteen novel 1,2,4-triazole derivatives were designed and synthesized under microwave irradiation. Their chemical structures were confirmed by 1 H NMR, FTIR, MS, and elemental analysis. The antifungal activity of 1,2,4-triazoles was determined in vivo. be both methylated with (CH 3 ) 2 SO 4 . We found that the pH values of two NH groups are different. Therefore, pH value is controlled preferably about 8 to 9 which is given the intermediate 4. The target compounds 5a∼5o were synthesized using microwave irradiation method. The signal of NCH 2 proton appeared around 3.66-5.04 ppm. The infrared spectrum of the title compounds 5a∼5o showed absorption bands around 2930 cm −1 for CH 2 stretching. The characteristic stretching vibration ] (C=O) appears at 1720 cm −1 . The mass spectrum results showed that molecular ion is in accordance with its molecular formula. The elemental analysis results are in accordance with the calculated results.

Antifungal Activity.
The in vivo antifungal activity results of 1,2,4-triazol-5(4H)-ones against Phytophthora infestans, Botrytis cinerea, Corynespora cassiicola, Rhizoctonia solani, and Pythium ultimum were shown in Table 1; dimethomorph, fludioxonil, chlorothalonil, validamycin, and zhongshengmycin were used as controls. From Table 1, it is shown that compounds 5c, 5f, and 5h exhibited good control efficacy against Pythium ultimum at 500 ppm. Compounds 5a, 5g, 5i, 5m, and 5n showed moderate control efficacy against Pythium ultimum. The control zhongshengmycin had no control efficacy against Pythium ultimum. For the Rhizoctonia solani, most of the title compounds displayed no control efficacy, except compounds 5f and 5h. Surprisingly, all the compounds can not only inhibit the Botrytis cinerea, but also promote the Botrytis cinerea growth. Most of 1,2,4triazol-5(4H)-ones displayed weak control efficacy against Corynespora cassiicola; only compounds 5b and 5c showed good antifungal activity (about 70%) against Corynespora cassiicola, which is higher than that of control chlorothalonil. Unfortunately, the title compounds exhibited weak activity against Phytophthora infestans.
2.3. DFT Calculation. Molecular total energy and frontier orbital energy levels are listed in Table 2. 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 firstly [34][35][36]. Thus, study on the frontier orbital energy can provide useful information about the biological mechanism. From Figure 1, the HOMO of compound 5c is mainly located on the OCH 3 group and 1,2,4-triazol-5(4H)-one ring, while the LUMO of compound 5c is located on the OCH 3 group, 2,4-Cl 2 benzene ring, and 1,2,4-triazol-5(4H)-one ring. The fact that the compound 5c has strong affinity suggests the importance of the frontier molecular orbital in the -stacking or hydrophobic interactions. From Figure 1, the electron transfer process of the HOMO and LUMO implies that 2,4-Cl 2 phenyl ring had important impact on the antifungal activity.
The electrostatic potential of compound 5c was also calculated. From Figure 2, it is clear that the oxygen atom at the 1,2,4-triazole ring possessed the greatest negative charges and it is therefore possible that the oxygen atom had some interaction with the receptor or acceptor.
Furthermore, the combination of MO provided meaningful clues as to the structural features of these new family fungicides that will be helpful in the design of more potent compounds in the future.

Synthesis of Intermediate 4.
To a solution of 3 (65 mmol) and K 2 CO 3 (9.52 g) in CH 3 CN (100 mL) was added (CH 3 ) 2 SO 4 (68 mmol) at 55 ∘ C, and the mixture was stirred for another 2 h. The organic phase was extracted with CH 2 Cl 2 (3 × 10 mL). After drying over sodium sulphate and evaporation of the solvent, the crude was collected without being purified to give the corresponding intermediate 4.

General
Procedure for Thioether 5. DMF (5 mL), 4 (0.25 g, 1.00 mmol), RCH 2 Cl (1.10 mmol), and NaOH (0.05 g, 1.20 mmol) were charged into a CEM designed 10 mL pressure-rated vial. Then it was irradiated in a CEM Discover Focused Synthesizer (150 w, 90 ∘ C, 200 psi, 15 minutes). The mixture was cooled below 50 ∘ C. The mixture was poured into crushed ice and the title compound 1,2,4-triazole was collected after being recrystallized.       [37][38][39][40], 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 spray on the surface of the seed leaves on a fine morning at the standard concentration of 500 g/mL; dimethomorph, fludioxonil, chlorothalonil, validamycin, and zhongshengmycin were used as control. After 2 h, inoculation of Phytophthora infestans was carried out by spraying fungal suspension, inoculation of Rhizoctonia solani and Corynespora cassiicola was carried out by spraying mycelial suspension, and inoculation of Botrytis cinerea was carried out by radicle soaking. Pythium ultimum was found in the cucumber in nature. 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 activity was 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 that 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.

DFT Calculation.
DFT-B3LYP/6-31G (d) methods in Gaussian 03 package [41] were used to optimize the structure of 5c. 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 the DELL personal computer.

Conclusion
In summary, this paper reported some novel 1,2,4-triazol-5(4H)-one derivatives were successfully synthesized. The bioassay results showed that some of the title compounds exhibited considerable antifungal activity. The bioactivity of these novel compounds deserves further investigation.