Design, Synthesis, and Bioactivity Evaluation of New Thiochromanone Derivatives Containing a Carboxamide Moiety

In this study, using the botanical active component thiochromanone as the lead compound, a total of 32 new thiochromanone derivatives containing a carboxamide moiety were designed and synthesized and their in vitro antibacterial activities against Xanthomonas oryzae pv. oryzae (Xoo), Xanthomonas oryzae pv. oryzicolaby (Xoc), and Xanthomonas axonopodis pv. citri (Xac) were determined, as well as their in vitro antifungal activities against Botryosphaeria dothidea (B. dothidea), Phomopsis sp., and Botrytis cinerea (B. cinerea). Bioassay results demonstrated that some of the target compounds exhibited moderate to good in vitro antibacterial and antifungal activities. In particular, compound 4e revealed excellent in vitro antibacterial activity against Xoo, Xoc, and Xac, and its EC50 values of 15, 19, and 23 μg/mL, respectively, were superior to those of Bismerthiazol and Thiodiazole copper. Meanwhile, compound 3b revealed moderate in vitro antifungal activity against B. dothidea at 50 μg/mL, and the inhibition rate reached 88%, which was even better than that of Pyrimethanil, however, lower than that of Carbendazim. To the best of our knowledge, this is the first report on the antibacterial and antifungal activities of this series of novel thiochromanone derivatives containing a carboxamide moiety.


Introduction
Plant bacterial and fungal diseases have posed serious threats in agricultural production and in spite of the best control efforts of plant pathologists, continue to contribute to heavy crop losses worldwide each year [1,2]. In recent years, the irrational use of traditional pesticides for plant bacterial and fungal disease control have posed a danger to living systems, killing not only target bacteria and fungi, but also affecting beneficial living systems [3]. Therefore, the resistance of plant bacterial and fungal diseases against pesticides is rapidly becoming a serious problem, and in pesticide research the development of novel antibacterial and antifungal agents is still a major challenge to be tackled [4].
Chromone, a kind of botanical active component with extensive biological activities, is widely found in the secondary metabolites of flowers, roots, stems, and pericarp of many plants [5,6]. Thiochromanone, a kind of chromone compound, is an important botanical active component with extensive biological activities, including antiviral [7], antibacterial [8,9], antifungal [8,[10][11][12], herbicidal [13,14], and insecticidal [15] activity. Therefore, using thiochromanone as the leading compound to develop promising agrochemical candidates will become a reality. In our previous study, we reported a series of novel thiochromanone derivatives containing a sulfonyl hydrazone moiety ( Figure 1) with moderate to good antibacterial activities against Xanthomonas oryzae pv. oryzae (Xoo), Xanthomonas oryzae pv. oryzicolaby (Xoc), and Xanthomonas axonopodis pv. citri (Xac) [16]. Meanwhile, carboxamides, as important nitrogen-containing compounds in organic synthesis, have attracted considerable attention due to their broad range of biological activities, including antiviral [17], antibacterial [18,19], antifungal [20][21][22], herbicidal [23], and insecticidal [24,25] activity. Therefore, carboxamide could reasonably be considered as a potential active group in the design of new lead compounds. In this study, using the botanical active component thiochromanone as the lead compound, a series of new thiochromanone derivatives containing a carboxamide moiety were designed and synthesized. We then determined the in vitro antibacterial activities of the derivatives against Xoo, Xoc, and Xac as well as their in vitro antifungal activities against Botryosphaeria dothidea (B. dothidea), Phomopsis sp., and Botrytis cinerea (B. cinerea).

Chemistry
The synthetic route to the target compounds 3a-3h and 4a-4x was carried out in three consecutive steps as shown in Scheme 1. Using a 4-substituted thiophenol as the starting material, the target compounds 3a-3h and 4a-4x were prepared with yields of 68-88% and their structures were determined by 1 H NMR, 13 C NMR, and HRMS. The 1 H NMR, 13 C NMR, and HRMS spectra for all the target compounds are shown in Supplementary Materials.
In the 1 H NMR spectra for compound 4d, two singlets at δ 11.73 and 10.36 ppm indicated the presence of -OH and -NH-groups, respectively; a chemical shift at 7.85-7.14 ppm indicated the presence of hydrogen atoms of the benzene ring in the thiochromanone group; two doublet-doublets at 3.32-3.12 ppm indicated the presence of CH 2 in the thiochromanone group. Meanwhile, in the 13 C NMR spectra for compound 4d, a singlet at 168.30 ppm indicated the presence of C=O in the thiochromanone group; a doublet at 159.79 and 157.41 ppm indicated the presence of C=O; a singlet at 149.88 ppm indicated the presence of C=N in the thiochromanone group. Scheme 1. Synthetic route of the target compounds 3a-3h and 4a-4x.

Biological Evaluations
The in vitro antibacterial activities of the racemic target compounds 3a-3h and 4a-4x against Xoo, Xoc, and Xac were determined by turbidimeter tests [26,27] and the bioassay results are listed in Tables 1 and 2. As shown in Table 1, at 200 and 100 µg/mL, some of the target compounds exhibited moderate to good antibacterial activities against Xoo, Xoc, and Xac. Among of them, compound 4e at 200 µg/m, exhibited excellent in vitro antibacterial activity (100%) against Xoo, which was even better that that of Bismerthiazol and Thiodiazole copper. Meanwhile, as shown in Table 2, compounds 4d, 4e, 4f, 4h, and 4i displayed in vitro antibacterial activities against Xoo, Xoc, and Xac, with EC 50 values in the range of 15-29, 19-34, and 23-41 µg/mL, respectively, and their antibacterial activities were better than those of Bismerthiazol and Thiodiazole copper. In particular, compound 4d revealed the best in vitro antibacterial activity against Xoo, Xoc, and Xac, and its EC 50 values of 15, 19, and 23 µg/mL, respectively, were even better than those of Bismerthiazol and Thiodiazole copper as well as the other target compounds; however, lower than those of compound methyl 6-chloro-4-(2-((4-fluorophenyl)sulfonyl)hydrazineylidene)thiochromane-2carboxylate [16]. Meanwhile, the in vitro antifungal activities of the racemic target compounds 3a-3h and 4a-4x against B. dothidea, Phomopsis sp., and B. cinerea were tested at 50 µg/mL by the mycelial growth rate method [28] and the results are listed in Table 3. As shown in Table 3, the target compounds revealed certain antifungal activities against B. dothidea, Phomopsis sp., and B. cinerea at 50 µg/mL with inhibition rate ranges of 0-22%, 0-60%, and 2-88%, respectively. In particular, compound 3b revealed moderate antifungal activity against B. dothidea at 50 µg/mL, and the inhibition rate reached 88%, which was even better than that of Pyrimethanil, however, lower than that of Carbendazim.
a Average of three replicates (mean ± SD).

Structure-Activity Relationship Analysis
The structure-activity relationship (SAR) analysis was deduced on the basis of the antibacterial and antifungal activity values listed in Tables 1-3. First, the introduction of an oxime ether or oxime fragment to the 4-position of thiochromanone can increase the antibacterial activity against Xoo, Xoc, and Xac (4a > 3a and 4d > 3b); to the contrary, it can decrease the antifungal activity against B. dothidea, Phomopsis sp., and B. cinerea (3a > 4a and 3b > 4d). Second, on comparing the same substituent at the R 2 and R 3 substituent groups, with the presence of a −Cl group at the R 1 substituent group, the corresponding compounds presented better in vitro antibacterial and antifungal activities which followed the order 3a (R 1 = −Cl) > 3e (R 1 = −CH 3 ) and 4a (R 1 = −Cl) > 4m (R 1 = −CH 3 ). Third, compared with the same substituent at the R 1 and R 3 substituent groups, a smaller electron drawing group at the R 2 substituent group could cause an increase in the antibacterial and antifungal activities which followed the order 3b Forth, compared with the same substituent at the R 1 and R 2 substituent groups, a -CH 3 at the R 3 substituent group could cause an increase in the antibacterial and antifungal activities which followed the order 4b (R 3

General Information
The melting points were determined by an uncorrected WRX-4 binocular microscope (Shanghai Yice Tech. Instrument Co., Shanghai, China). 1 H NMR and 13 C NMR spectral analyses were performed on a Bruker DRX-400 NMR spectrometer (Bruker, Rheinstetten, Germany). HRMS data were measured on a Waters Xevo G2-S QTOF mass spectrometer (Waters, Milford, MA, USA).

Preparation Procedure of Intermediate 2
As shown in Scheme 1, intermediate 2 was prepared according to our previously reported method [16].

Preparation Procedure for the Target Compounds 3a-3h
To a 50 mL round bottom flask equipped with a magnetic stirrer, intermediate 2 (0.02 mol) was dissolved in DMF (10 mL), and then substituted phenylamine (0.02 mol), dimethylaminopyridine (DMAP, 0.0002 mol), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI, 0.03 mol) were added. The reactions were performed overnight at room temperature. Upon completion of the reaction (determined by TLC), the mixture was quenched with distilled water (50 mL) and the precipitated residues were filtered, dried under vacuum, and recrystallized from methanol to give the pure racemic target compounds 3a-3h. The physical characteristics, 1 H NMR, 13 C NMR, and HRMS data for the target compounds 3a-3h are shown below. The 1 H NMR, 13

In Vitro Antibacterial Activity Test
Each target compound (7.5 mg) was dissolved in 150 µL DMSO and then 80 and 40 µL of the solution, respectively, was poured into two 15 mL centrifuge tubes each containing 4 mL 0.1% Twain aqueous solution. The solutions (1 mL) were then added into glass test tubes each containing 4 mL nutrient broth (NB) medium to prepare 5 mL test solutions with concentrations of 200 and 100 µg/mL, respectively. Then, 40 µL of the NB mediums containing Xoo, Xoc, and Xac, respectively, were added to the test tubes mentioned above. The inoculated test tubes were incubated at 30 • C and 180 rpm for 24-48 h until the OD 595 values of the negative control reached 0.6-0.8 (the logarithmic growth phase). DMSO served as the negative control, whereas Thiodiazole copper and Bismerthiazol served as positive controls. Three replicates were conducted for each treatment. The OD 595 values of the cultures were monitored on a Multiskan Sky 1530 spectrophotometer (Thermo Scientific, Poland). The inhibition rate I (%) was calculated by the following formula (1), where C is the corrected turbidity value of the untreated NB medium, and T is the corrected turbidity value of the treated NB medium.
Inhibition rate I (%) = (C-T)/C × 100 (1) On the basis of the preliminary bioassay results, the antibacterial activities (expressed by EC 50 ) of some of the target compounds against Xoo, Xoc and Xac were evaluated and calculated using SPSS 17.0 software. Three replicates were conducted for each treatment.

In Vitro Antifungal Activity Test
Each target compound (5 mg) was dissolved in 1 mL DMSO and mixed with 90 mL potato dextrose agar (PDA) medium. The mixed PDA mediums were then poured into 6 dishes and cooled to room temperature to prepare the PDA plates with the test solution concentration of 50 µg/mL. Mycelia dishes of approximately 0.4 cm diameter were then cut from the culture medium and picked up with a germfree inoculation needle and placed into the middle of PDA plates aseptically. The inoculated PDA plates were fostered in an incubator at 28 • C for 3-4 days until the colony diameter of the negative control reached 5-6 cm. DMSO served as the negative control, whereas Pyrimethanil and Carbendazim acted as positive controls. Three replicates were conducted for each treatment. The inhibition rate I (%) was calculated by the following formula (2), where C (cm) represents the diameter of fungi growth on the untreated PDA plate, and T (cm) represents the diameter of fungi on the treated PDA plate.

Conclusions
In this study, a total of 32 new thiochromanone derivatives containing a carboxamide moiety were designed and synthesized. The bioassay results demonstrated that compound 4e exhibited excellent in vitro antibacterial activity against Xoo, Xoc, and Xac which was superior to those of Bismerthiazol and Thiodiazole copper. Meanwhile, compound 3b revealed moderate in vitro antifungal activity against B. dothidea at 50 µg/mL which was even better than that of Pyrimethanil, nevertheless, lower than that of Carbendazim. For controlling plant bacterial and fungal diseases, this study provided a practical tool for guiding the design and synthesis of novel and more promising active small molecules of thiochromanone derivatives containing a carboxamide moiety.