Synthesis and Bioactivities of Novel 1,3,4-Thiadiazole Derivatives of Glucosides

A series of novel 1,3,4-thiadiazole derivatives of glucosides were synthesized by the starting materials d-glucose and 5-amino-1,3,4-thiadiazole-2-thiol in good yields with employing a convergent synthetic route. The results of bioactivities showed that some of the target compounds exhibited good antifungal activities. Especially, compounds 4i showed higher bioactivities against Phytophthora infestans (P. infestans), with the EC50 values of 3.43, than that of Dimethomorph (5.52 μg/ml). In addition, the target compounds exhibited moderate to poor antibacterial activities against Xanthomonas oryzae pv. oryzae (Xoo), Xanthomonas campestris pv. citri (Xcc).


INTRODUCTION
Crop disease, caused by fungi, bacteria, viruses, and nematodes and parasitic seed plants, can effect on the biological or non-biological factors of plants causing the phenomenon of a series of morphological, physiological and biochemical pathologic changes, further blocking the normal growth and the development process and the human economic benefits (Zhan et al., 2015). Nowadays, some of the traditional fungicides and bactericides, such as Carbendazim, Kresoximmethyl, Streptomycin sulfate, Bismerthiazol, etc., have been widely used to prevent and control plant fungal and bacterial diseases. However, long-term using these pesticides could lead to drug resistance, serious ecological, and environmental problem (Aktar et al., 2009). Therefore, development of novel and promising fungicides and bactericides is still an urgent task.
Glycosides are secondary metabolites that widely exist in all organs of plants, such as flowers, fruits, leaves, skins, and roots, etc (Gruner et al., 2002), and previous studies found that glycosides had a wide range of pharmacological activities, such as antiviral (Chen W. et al., 2019;Khodair et al., 2019), antibacterial (Mohammed et al., 2019), anticancer (Gurung et al., 2018;Rahim et al., 2020), antioxidant (Jiang et al., 2018;Hawas et al., 2019), and anti-HIV (He et al., 2019) activities. Meanwhile, studies also found that glycoside derivatives showed exceeding inhibitory activities against plant pathogens. For example, Ningnanmycin, an important glycoside biological pesticide, is mainly used in rice seedling blight, soybean root rot, rice stripe disease, apple spot deciduous leaf disease and cucumber powdery mildew (Hu et al., 1997). In addition, it was also found that glycosylation is one of the effective ways to improve the functional activity of active lead compounds and develop new drugs. (Gurung et al., 2018;Wu et al., 2014).
In order to develop new lead compounds with highly bioactivity, in this study, we aimed to introduce a 1,3,4thiadiazole group into glucosides moiety to design a series of novel 1,3,4-thiadiazole derivatives of glucosides and then evaluate for their antifungal and antibacterial activities. Results indicated that some of the target compounds exhibited good antifungal activities. Especially, the compounds 4i showed higher bioactivities against Phytophthora infestans (P. infestans), with the EC 50 values of 3.43 μg/ml, respectively, than that of Dimethomorph (5.52 μg/ml). In addition, the target compounds showed moderate to poor antibacterial activities against Xoo and Xcc. As far as we know, this is the first report on the antifungal and antibacterial activities of 1,3,4-thiadiazole derivatives of glucosides.

Materials and Instruments
Melting points were determined on an XT-4 melting apparatus (Beijing Tech Instrument Co., China). 1 H NMR and 13 C NMR spectra were measured on a Bruker AVANCE III TM 400 and HD 600 MHz Digital NMR Spectrometer (Bruker Company, Billerica, MA, US.) in CDCl 3 as solvent and recorded in relative to internal standard tetramethylsilane. High-resolution mass spectrometry (HRMS) was carried out on an Agilent Technologies 6540 UHD Accurate-Mass Q-TOF LC/MS (Agilent Technologies, Palo Alto, CA, United States). The course of the reactions was monitored by thin-layer chromatography (TLC) analysis on silica gel GF254. All reagents and solvents meet the standards of analytical reagent before use.

Chemistry
Preparation of 2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl bromide (1). As shown in Figure 1, acetic anhydride (88 ml, 0.9 mol) was added to a solution of D-glucose (29.75 g, 0.15 mol) in glacial acetic acid (300 ml) and stirred at room temperature for 20 min. Then, perchloric acid (0.3 ml) was added to the above reaction mixture at room temperature. After TLC analysis showed complete disappearance of D-glucose, a solution of acetyl bromide (34 ml, 0.45 mol) in 50 ml CH 2 Cl 2 was added to the resultant reaction mixture and stirred at room temperature. After the completion of the reaction, the reaction mixture was poured into water and extracted with CH 2 Cl 2 . The organic layer was dried, filtered, and evaporated in vacuo to remove CH 2 Cl 2 . The crude product was recrystallized by a mixture of petroleum ether and diethyl ether (volume ratio 1:2) to afford intermediate 1. (Scattolin et al., 2020). 1  Preparation of (2R,3R,4S,5R,6R)-2-(acetoxymethyl)-6-((5amino-1,3,4-thiadiazol-2-yl)thio)tetra hydro-2H-pyran-3,4,5triyltriacetate (2). A mixture of 2-amino-5-mercapto-1,3,4thiadiazole (1.33 g, 10.0 mmol), acetone (50 ml), NaOH (0.4 g, 10.0 mmol), and water (10 ml) was stirred for 30 min at room temperature. Then, a solution of intermediate 1 (0.98 g, 10.0 mmol) in 5 ml acetone was added dropwise and continuously stirred at room temperature. After the reaction Frontiers in Chemistry | www.frontiersin.org March 2021 | Volume 9 | Article 645876 2 completed (monitored by TLC), acetone was evaporated in vacuo, the residues were diluted with water, extracted with CH 2 Cl 2 . The combined CH 2 Cl 2 extract was dried over anhydrous sodium sulfate, evaporated in vacuo and separated by silica gel column chromatography to afford intermediate 2 (Kamat et al., 2007). 1  General procedure for preparation of the target compounds 4a-4q. Substituted benzoic acid (1.2 mmol) was added in 2 ml SOCl 2 and refluxed for about 2 h SOCl 2 was distilled off in vacuo to obtain intermediates 3. And then, a solution of intermediate 3 in 2 ml CH 2 Cl 2 was added dropwise to a mixture of the intermediate 2 (1.0 mmol) and triethylamine (TEA, 1.2 mmol) in 10 ml CH 2 Cl 2 . After the reaction was completed (monitored by TLC), the mixture was diluted with water, the organic layer was dried over anhydrous sodium sulfate, filtered and distilled off in vacuo, and the crude products were recrystallized with isopropanol to afford title compounds 4a-4q.

Antifungal Activity In Vitro
The in vitro antifungal activities of the target compounds against G. zeae, Botryosphaeria dothidea (B. dothidea), Phomopsis sp., P. infestans, and Thanatephorus cucumeris (T. cucumeris) are evaluated by using the poison plate technique. All of the target compounds 4a-4q were dissolved in 1 ml DMSO before mixing with 90 ml potato dextrose agar (PDA) to prepare concentration of 50 μg/ml. Then, mycelia dishes of approximately 4 mm diameter were cut from the culture medium. A mycelium is obtained using a germ-free inoculation needle and inoculated in the middle of the PDA plate aseptically. The inoculated plates are incubated at 27 ± 1°C for 5 days. DMSO in sterile distilled water served as the negative control and Dimethomorph served as the positive control. Each treatment condition consisted of three replicates (Maddila et al., 2016). The relative inhibition rates I (%) were calculated as follows equation, where C was the diameter of fungal growth on untreated PDA, T was the diameter of fungi on treated PDA.

Antibacterial Activity In Vitro
The in vitro antibacterial activities of the target compounds 4a-4q against Xoo and Xcc were evaluated by using the turbidimeter test, the commercial agricultural antibacterial Thiodiazole-copper used as control. The test compounds were dissolved in 150 μL of dimethylformamide (DMF) and diluted with 0.1% (v/v) Tween-20 to prepare two concentrations of 200 and 100 μg/ml. One milliliter of the liquid sample was added to the 40 ml non-toxic nutrient broth medium (NB: 1.5 g of beef extract, 2.5 g of peptone, 0.5 g of yeast powder, 5.0 g of glucose, and 500 ml of distilled water, pH 7.0-7.2). Then, 40 μL of NB medium containing Xoo or Xcc was added to 5 ml of solvent NB containing the test compounds or Thiodiazole-copper. The inoculated test tubes were incubated at 30 ± 1°C under continuous shaking at 180 rpm for 48 h. The culture growth was monitored spectrophotometrically by measuring the optical density at 600 nm (OD 600 ) and expressed as corrected turbidity (Dalgaard et al., 1994). The relative inhibition rates I (%) were calculated as follows equation, where C tur was the corrected turbidity value of bacterial growth on untreated NB, T tur was the corrected turbidity value of bacterial growth on treated NB.
All the synthesized compounds were characterized by 1 H NMR, 13 C NMR, and HRMS. In the 1 H NMR spectra of the obtained amide, pyran and acetyl proton signals should be distinguished. For example, for compound 4i, the proton signals of NH group was observed as a singlet at 12.54 ppm, signals of benzene ring protons were registered at 8.36 and 7.85 ppm, respectively, and the proton signal of pyran was registered in the range of 5.18-3.82 ppm. Moreover, four singlets at 2.17, 2.09, 2.04, and 2.01 ppm indicated to CH 3 protons of acetyl.
The in vitro antifungal activities of the target compounds were evaluated against five different fungus including P. infestans, G. zeae, B. dothidea, Phomopsis sp., and T. cucumeris. Bioassay results, as shown in Table 1, revealed that the target compounds exhibited moderate to good antifungal activities against P. infestans, G. zeae, B. dothidea, Phomopsis sp., and T. cucumeris, with the inhibitory rates range of 19.8-83. 5%, 35.6-73.1%, 22.1-62.0%, 21.0-64.0%, and 17.1-65.1%, respectively. Meanwhile, it was found that the inhibitory rates of the target compounds against G. zeae in the range of 35.6-73.1% at the 50 μg/ml, which was higher than the previously reported  inhibitory activity of N-(2-chloro-4-phenyl-5-(trifluoromethyl) cyclopenta-1,4-dien-1-yl)-5-((4-nitrobenzyl)thio)-1,3,4-thiadiazol-2-amine against G. zeae (23.9%) at the 50 μg/ml (Xie et al., 2016). Especially, compound 4i and 4q showed higher antifungal activity against P. infestans, with the inhibition rates of 83.5%, 81.1%, respectively, than that of Dimethomorph (78.2%). Based on the preliminary antifungal bioassays, the EC 50 values of partial compounds against P. infestans were also tested and presented in Table 2. Table 2 showed that compounds 4i exhibited good bioactivities against P. infestans, with EC 50 values of 3.43 μg/ml, which were higher than that of Dimethomorph (5.52 μg/ml). While, the target compounds showed lower antibacterial activities (Table 3) against Xoo and Xcc at 200 and 100 μg/ml than those of Thiodiazolecopper as well as the amide derivatives containing 1,3,4thiadiazole of the previously reported by Chen . From the structure-activity relationships (SAR) analysis, it was found that there was clear SAR against P. infestans. Inspection of the chemical structures of the target compounds suggests that the group R in the target compounds significantly influence the antifungal activity against P. infestans. With a fluorinated or nitrificated substituent (4-F and 4-NO 2 ) on the phenyl ring, the compounds exhibited enhanced bioactivity against P. infestans (4i and 4q). Further, the position of substituent groups in the phenyl ring also plays an important role in the antifungal activity against P. infestans, with a four substituent (4-F or 4-NO 2 ) in the phenyl ring exhibited higher antifungal activity than other positions.

CONCLUSION
A series of novel 1,3,4-thiadiazole derivatives of glucosides were prepared via acetylation, bromination, thioetherification, chlorination, and condensation. Bioassay results showed that some of the target compounds revealed better inhibitory activity against P. infestans. In addition, SAR analysis found that the type and position of substituent groups in the phenyl ring of the target compounds plays an important role in increasing the antifungal activity against P. infestans.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material; further inquiries can be directed to the corresponding author/s.

AUTHOR CONTRIBUTIONS
MC and WW contributed to the synthesis, purification, characterization of all compounds, and prepared the original manuscript. XZ and DL performed the biological activity research. HL and ZZ analyzed the experimental results. GZ and XQ drafted the first and second version of the manuscript. All authors discussed, edited, and approved the final version.