Microwave Assisted Synthesis, Antifungal Activity and DFT Theoretical Study of Some Novel 1,2,4-Triazole Derivatives Containing the 1,2,3-Thiadiazole Moiety

In order to investigate the biological activity of 1,2,4-triazole compounds, seventeen novel 1,2,4-triazole derivatives containing 1,2,3-thiadiazole moieties were synthesized by multi-step reactions under microwave assisted conditions. The structures were characterized by 1H-NMR, 13C-NMR, MS and elemental analyses. The target compounds were evaluated for their in vivo fungicidal activities against Corynespora cassiicola, Pseudomonas syringae pv. Lachrymans, and Pseudoperonospora cubensis, and the results indicated that some of the title compounds displayed good fungicidal activities. Theoretical calculations on the title compounds were carried out at the B3LYP/6-31G (d,p). level. The full geometry optimization was carried out using the 6-31G(d,p) basis set, and the frontier orbital energy, atomic net charges were discussed, and the structure-activity relationships were also studied.


Chemistry
The synthesis procedures for title compounds are shown in Scheme 1. The intermediates 1~6 were synthesized according our previous method [25]  The signal of the CH 2 protons of the thioether neighboring the triazole ring was observed at δ 4.43~4.63 ppm, respectively. The chemical shifts around 3.0 ppm are from the methyl of the 1,2,3-thiadiazole ring. The ESI-MS spectra showed the m/z of the molecular ions, according with their molecular formulas. The elemental analysis results were in accord with the calculated results.

Fungicidal Activities and Structure-Activity Relationship (SAR)
The in vivo fungicidal results of the title compounds against Corynespora cassiicola (CC), Pseudomonas syringae pv. Lachrymans (PS) and Pseudoperonospora cubensis (PC) are listed in Table 2, where iprodione, validamycin and topsin-M were used as controls. As shown in Table 2, lots of the title compounds showed good control efficacy against Corynespora cassiicola at a concentration of 500 μg/mL. When the R 1 is a phenyl group, only compound 7e exhibited moderate activity (46.9%) against Corynespora cassiicola. Notably, compounds 7b, 7c, 7d and 7e displayed excellent activity (>80%) against Corynespora cassiicola, especially compound 7b which exhibited higher activity than the control. Also the activity of benzene substituted derivatives is higher than that of cyclopropyl substituted ones. Compounds 7b, 7c, 7d, 7j, 7k, 7l, 7m exhibited fair inhibition effects against Pseudomonas syringae pv. Lachrymans, and the fungicidal activities (control efficacy of 52%-79%) were higher than those of iprodione, validamycin and topsin-M. All of them were less effective than the control validamycin. Compound 7e (71.03%) and 7g (68.79%) showed about 70% control effect against Pseudoperonospora cubensis, at the same level as iprodione, validamycin and topsin-M. In general, the in vivo fungicidal activity of compounds 7 indicated that the substituent changes affect the activity.
In order to further research the structure-activity relationships of the title compounds, some compounds (7o, 7p, 7q) containing cyclopropyl groups synthesized in our other paper [29] were used for comparison. From the data in Table 2, for Corynespora cassiicola, the compounds containing phenyl groups exhibited higher activity than those with cyclopropyl groups, but for Pseudoperonospora cubensis, the results are the opposite, and the compounds containing cyclopropyl groups displayed higher activity than that of phenyl group-containing compounds. For Pseudomonas syringae pv. Lachrymans, the structure-activity relationships are not obvious. Further biological evaluation of all compounds is in progress. The mode of action of title compounds will be explored by molecular modeling.

Molecular Total Energies and Frontier Orbital Energy Analysis
Molecular total energy and frontier orbital energy levels are listed in Table 3. 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 [30]. Thus study on the frontier orbital energy can provide useful information about the biological mechanism. Taking the DFT result for example, the geometry of the frame of the title compound is hardly influenced by the introduction of either a 1,2,3-thiadiazole ring, 1,2,4-triazole ring, thioether group or phenyl ring ( Figure 3). The HOMO of the title compound is mainly located on the 1,2,3-thiadiazole ring, 1,2,4-triazole ring and thioether group, while, the LUMO of the title compound is located on the 1,2,3-thiadiazole ring, 1,2,4-triazole ring, thioether group and phenyl ring. 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 interactions between the title compound and the aromatic ring or some other side of residue chains of receptors is dominated by π-π or hydrophobic interactions among the frontier molecular orbitals.  Table 4 lists the calculated Mulliken atomic charges except for the H atoms. Taking DFT for example again, three atoms C1, C2, C17, S1 and S2 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, N3, N4 and N5, so they can interact easily with the positive part of the receptor. Therefore, we supposed that this compound can combine the amino-acid residues on its surface by the interaction of S2-N4-N5 and S1-C1-N3, which may account in part for the bioactivity. Table 4. Mulliken atomic charges except for H atoms (e).

Atom
Charge

Materials and Reagents
All the reagents were 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 chromatography (TLC), performed on silica gel plates obtained from Qingdao Ocean Chemicals (Qing Dao, China). Melting points were determined using an X-4 apparatus and are uncorrected. 1 H-NMR and 13 C-NMR spectra were measured on a Bruker AV 400 or 500 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. Elemental analyses were performed on a Vario EL elemental analyzer.
Microwave activation was carried out with a CEM Discover TM focused microwave (2,450 MHz, 300 W).

Therotical Calculations
On the basis of the above structure shown in Scheme 1, compound 7b was selected as the initial structure, and the DFT-B3LYP/6-31G (d,p) [31] methods in the Gaussian 03 package [32] 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. The synthetic route is shown in Scheme 1. The intermediate 4-methyl-1,2,3-thiadiazole-5carboxylic acid hydrazide (2) was synthesized according to the literature [25]. Carbonic acid diethyl ester (11.8 g, 0.1 mol) and hydrazine hydrate (5.6 mL, 0.095 mol, 85%) were added into a 250 mL round-bottom flask equipped with a condenser. The reaction mixture was heated to 50 °C and stirred for 20 min, and then cooled down to room temperature and further stirred for 30 h. Water, ethanol, and excess carbonic acid diethyl ester were distilled off under reduced pressure. After drying, compound (2) (9.88 g, 95% yield) was obtained as white crystals. To a stirred solution of compound 2 (6.36 g, 0.06 mol) in ethanol (16.7 mL), a solution of ethyl acetoacetate (7.8 g, 0.06 mol) in ethanol (3.7 mL) was added at room temperature. Stirring was continued for 6 h. Then, the solvent was removed in vacuo and the crude 3-ethoxycarbonyl hydrazonoacetic acid ethyl ester was directly used in the next step without further purification. 3-Ethoxycarbonyl hydrazonoacetic acid ethyl ester (12.8 g, 0.06 mol) was dissolved in dry dichloromethane (25 mL), and thionyl chloride (20 mL) was added to the stirred reaction mixture dropwise at 0 °C for 1 h. Next, the reaction mixture was allowed to stand for 20 h at room temperature. The excess thionyl chloride and dichloromethane were distilled off, and the remaining residue was subjected to fractional distillation under reduced pressure. A slightly yellowish oil of 3 (7.95 g, 77% yield) was obtained. A mixture of 4-methyl-1,2,3-thiadiazole-5-carboxylic acid ethyl ester (1.72 g, 10 mmol) and hydrazine hydrate (12 mmol) in 10 mL of methanol was stirred vigorously for 0.5 h at room temperature, the resulting mixture was then filtered, and the solid was washed with cold methanol. After drying, the solid was recrystallized from methanol to give intermediate (4).

Fungicidal Activities
Fungicidal activity of compounds 7a-q against Pseudoperonospora cubensis(PC), Corynespora cassiicola(CC), Pseudomonas syringae pv. Lachrymans(PS) were evaluated according to reference [11], 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 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 μg/mL, iprodione, validamycin and topsin-M were used as a control. After 2 h, inoculations of Pseudoperonospora cubensis, Corynespora cassiicola were carried out by spraying a conidial suspension, inoculation of Pseudomonas syringae pv. Lachrymans was carried out by spraying fungal suspension. 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 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 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% 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 containing 1,2,3-thiadiazole rings were synthesized 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 analogues.