Synthesis and Antibacterial Evaluation of N-phenylacetamide Derivatives Containing 4-Arylthiazole Moieties

A series of new N-phenylacetamide derivatives containing 4-arylthiazole moieties was designed and synthesized by introducing the thiazole moiety into the amide scaffold. The structures of the target compounds were confirmed by 1H-NMR, 13C-NMR and HRMS. Their in vitro antibacterial activities were evaluated against three kinds of bacteria—Xanthomonas oryzae pv. Oryzae (Xoo), Xanthomonas axonopodis pv. Citri (Xac) and X.oryzae pv. oryzicola (Xoc)—showing promising results. The minimum 50% effective concentration (EC50) value of N-(4-((4-(4-fluoro-phenyl)thiazol-2-yl)amino)phenyl)acetamide (A1) is 156.7 µM, which is superior to bismerthiazol (230.5 µM) and thiodiazole copper (545.2 µM). A scanning electron microscopy (SEM) investigation has confirmed that compound A1 could cause cell membrane rupture of Xoo. In addition, the nematicidal activity of the compounds against Meloidogyne incognita (M. incognita) was also tested, and compound A23 displayed excellent nematicidal activity, with mortality of 100% and 53.2% at 500 μg/mL and 100 μg/mL after 24 h of treatment, respectively. The preliminary structure-activity relationship (SAR) studies of these compounds are also briefly described. These results demonstrated that phenylacetamide derivatives may be considered as potential leads in the design of antibacterial agents.


Introduction
Xanthomonas oryzae pv. oryzae and Xanthomonas oryzae pv. oryzicola parasitizing rice can cause serious degradation of rice quality and yield [1]. Xanthomonas axonopodis pv. citri mainly harms citrus fruits while causing a plant bacterial disease, citrus canker [2]. In recent decades, the long-term use of traditional bactericides has resulted in their large accumulation in the soil and resistance of pathogenic bacteria, reducing control capability. For example, bismerthiazol, one of the most widely used bactericides, has indicated low efficiency of 25.5% at a high dosage of 200 µg/mL [3]. Therefore, it is necessary to develop highly efficient and environmentally friendly bactericides to protect crops. Thiazole-containing aromatic heterocycles are reported to bind to certain proteins and receptors of bacteria and exhibit a wide range of biological activities such as insecticidal [4,5], bactericidal [6][7][8], herbicidal [9], antiviral [10] and fungicidal ones [11,12]. In addition, compounds with an amide structure have revealed various activities as antimicrobial [13][14][15] and nematicidal [16,17] agents. Based on the reported good performance of derivatives containing thiazole or amide groups, splicing them to get new antibacterial structures was presumed to be a reasonable and promising approach.
Based on the reported good performance of derivatives containing thiazole or amide groups, splicing them to get new antibacterial structures was presumed to be a reasonable and promising approach.
Attracted by these facts, we have carried out the preparation of a series of novel Nphenylacetamide derivatives A1-A36 and the subsequent evaluation of their biological activities against Xoo, Xac and Xoc. Their preliminary structure-activity relationships are discussed. Additionally, the nematicidal activity of all target compounds against M. incognita were also checked.

Chemistry
As shown, compounds N-substituted-2-amino-4-arylthiazoles A1-A36 were synthesized following Scheme 1. Starting from p-phenylenediamine (PPD), the synthesis involves firstly aniline protection, amide formation and deprotection to afford the 4-amino-N-phenylacetamide intermediates 2 which were then turned into isothiocyanates 3 and converted to thioureas 4. Finally, 4 were condensed with different α-halocarbonyl compounds to give the target derivatives. The steps for each reaction described in Scheme 1 were elaborated as follows.

In Vitro Antibacterial Activity
The in vitro antibacterial activities of the target compounds against three phytopathogenic bacterial (Xoo, Xac and Xoc) were initially evaluated at the concentrations of 200 µg/mL and 100 µg/mL, respectively. As a comparison, the commercial bactericides bismerthiazol and thiodiazole copper served as positive controls (see preliminary screening results in the Supplementary Materials).
Compounds that performed well in the initial tests were further tested to determine their EC 50 values, with the results shown in Table 1. The EC 50 values of compounds A 1 , A 4 , A 6 against Xoo are 156.7, 179.2, 144.7 µM, which were significantly better than that of thiodiazole copper (545.2 µM). Moreover, compound A 4 had the best inhibitory effect on Xoc with an EC 50 value of 194.9 µM, which was slightly better than that of bismerthiazol (254.96 µM) and thiodiazole copper (607.5 µM). Compound A 4 (281.2 µM) showed the best antibacterial activity against Xac, which was better than that of the commercial bactericide thiodiazole copper (476.52 µM).

Structure-Activity Relationship Analyses
With the results indicated in Table 1, a preliminary structure-activity relationship of the target compounds can be discussed. It can be observed that the type and position of the substituent R on the benzene ring had an important effect on the bactericidal activity of the target compounds. First of all, the type and position of F, Cl, Br, and CF 3 at the 4-position of the benzene ring can increase the bactericidal activity of the compound, and the 3-position is not conducive to improving the bactericidal activity. For example, the order of the inhibitory effect of the target compound on Xoo bacteria follows the order A 1 > A 2 , A 4 > A 5 , confirming the above conclusion. In addition, the activity trend A 1 (4-F) > A 4 (4-Cl) > A 7 (4-Br) reveals that a 4-F substituted benzene ring is the most helpful for conferring antibacterial activity. Secondly, comparing different types of groups at the same position on the benzene ring, the corresponding compounds with 4-R electron-withdrawing substituents have a higher bactericidal activity against Xoo, Xac and Xoc than 4-R electron-donating substituents such as A 1 (4-F) > A 4 (4-Cl) > A 11  ). The type of amide-linked sidechain could also have a significant impact on the bactericidal activity of the corresponding target compounds.

Scanning Electron Microscopy Studies
Due to its outstanding activity shown in Table 1, compounds A 1 was further examined by SEM analysis to study the effect on Xoo. It can be observed that the cell membrane was damaged by the compound and the normal physiological functions of the cell would thus be affected. More importantly, this adverse effect becomes more severe with increasing compound concentration. For example, the surface of cells without compound treatment is smooth and the cell membrane is intact ( Figure 1A).
At the concentration of 100 µg/mL, a small part of the cell morphology appears abnormal ( Figure 1B). When the concentration was increased to 200 µg/mL, most of the cell surfaces were deformed, with few surviving cells ( Figure 1C). In summary, the inhibitory effect of compound A 1 on Xoo was further clarified by the SEM images.
Molecules 2020, 25, x FOR PEER REVIEW 4 of 14 example, the surface of cells without compound treatment is smooth and the cell membrane is intact ( Figure 1A). At the concentration of 100 µg/mL, a small part of the cell morphology appears abnormal ( Figure 1B). When the concentration was increased to 200 µg/mL, most of the cell surfaces were deformed, with few surviving cells ( Figure 1C). In summary, the inhibitory effect of compound A1 on Xoo was further clarified by the SEM images.

Nematicidal Biological Activities
The nematicidal activity results of the target compounds are summarized in Table 2. Unfortunately, most of the compounds showed poor activities against M. incognita, although compound A23, with its mortality rates of 100% at 500 µg/mL and 51.3% at 100 µg/mL which are comparable to the commercial nematicide avermectin (100% at 500 µg/mL and 71.8% at 100 µg/mL) in 24 h after treatment stuck out.

Nematicidal Biological Activities
The nematicidal activity results of the target compounds are summarized in Table 2. Unfortunately, most of the compounds showed poor activities against M. incognita, although compound A 23 , with its mortality rates of 100% at 500 µg/mL and 51.3% at 100 µg/mL which are comparable to the commercial nematicide avermectin (100% at 500 µg/mL and 71.8% at 100 µg/mL) in 24 h after treatment stuck out.

Chemicals and Instruments
All reagents used for reactions were purchased from a commercial source (Aladdin Chemistry Co., Shanghai, China) and were of analytical grade with no further purification. Basic alumina oxide (200 to 300 mesh, Aladdin Chemistry Co.) was applied for purification of target compounds by column chromatography. Melting points of the target compounds were recorded on an XT-4B binocular microscope (Beijing Tech Instrument Co., Beijing, China). NMR spectra were obtained on a 400 MHz spectrometer (Bruker BioSpin AG, Fällanden, Switzerland) using TMS as internal standard and DMSO-d 6 as solvent. HRMS data were recorded on a Thermo Scientific Q Exactive system (Thermo Fisher Scientific, Waltham, MA, USA). Single crystal structure data were collected using a single crystal diffractometer (Gemini E, Oxford Instruments, Oxford, UK). SEM analysis was carried out with a FEI Nova NanoSEM 450 (FEI Company, Hillsboro, OR, USA).

Synthesis of Intermediate 1
p-Phenylenediamine (PPD, 9.25 mmol, 1.0 equiv.) was dissolved in dry dichloromethane (50mL), degassed under N 2 (g) and cooled in an ice bath. BOC 2 O (4.62 mmol, 0.50 equiv.) was added dropwise to the p-phenylenediamine solution using a disposable syringe. After the addition, the mixture was reacted at room temperature (R.T.) for 5 h, monitored by thin layer chromatography (TLC). After the reaction was complete, the mixture was purified by flash column chromatography (petroleum ether:ethyl acetate, 3:1) to obtain yellow solid compound intermediate 1 in a yield of 63%.

Synthesis of Intermediates 2
A mixture of 1 (1.0 equiv.) and an acid chloride (1.10 equiv.) in DCM (40mL) was stirred at 0 • C for 1 h. Then, excess 4 M HCl was added and stirred at R.T. for 10 min. The reaction progress was monitored by TLC. After completion of the reaction, a saturated Na 2 CO 3 solution was added to adjust to 7.0 the pH of mixture which was then extracted with DCM (50 mL × 3). The organic phase was concentrated to give intermediate 2 as white solids in yields of 69-75%.

Synthesis of the Intermediates 4
The mixture of 3 (12.64 mmol, 1.0 equiv.) with excess ammonia solution in ethanol (30 mL) was stirred at R.T. The reaction was monitored by TLC using petroleum ether:ethyl acetate (1:1). The mixture was filtered through a Celite pad to afford the thioureas 4 in approximately 100% yield.

Synthesis of the α-Bromophenylethanone Intermediates
Br 2 (2.2 mmol, 1.10 equiv.) was added slowly to a DCM solution of substituted acetophenone (2.0 mmol, 1.0 equiv.) and the mixture was stirred at R.T. for 5 h, after which the reaction mixture was tpoured into distilled water and extracted with DCM three times. The organic phases were combined and the solvent removed under reduced pressure to obtain the α-bromophenylethanones in yields of 65-83%.

X-ray Diffraction Analysis
To further confirm the three-dimensional structure of the target compounds, crystals (1.9 mm × 0.5 mm × 1.6 mm) of A 4 ( Figure 2) were obtained by slow evaporation and analyzed by X-ray diffraction. Cell dimensions and intensities were measured at 298 K on a Bruker Smart Apex CCD diffractometer with MoKα radiation (λ = 0.71073 Å). The structure was solved by direct method with the SHELXS-97 program. The results show that crystals of A 4 is monoclinic system, which is characterized by no higher-order symmetry axis. A total of 2709 reflections were measured, of which 1714 were unique in the range of 2.19 < θ < 25.02 • (h, −14 to 19; k, −6 to 5; l, −22 to 21). The two benzene rings in the molecule are almost in the same plane. All of the non-H atoms were refined anisotropically by full-matrix least-squares to give the final R = 0.0732 and WR = 0.1820. The completeness of the crystal data is 99.9%. The crystal data of the compound A 4

X-ray Diffraction Analysis
To further confirm the three-dimensional structure of the target compounds, crystals (1.9 mm × 0.5 mm × 1.6 mm) of A4 ( Figure 2) were obtained by slow evaporation and analyzed by X-ray diffraction. Cell dimensions and intensities were measured at 298 K on a Bruker Smart Apex CCD diffractometer with MoKα radiation (λ = 0.71073 Å). The structure was solved by direct method with the SHELXS-97 program. The results show that crystals of A4 is monoclinic system, which is characterized by no higher-order symmetry axis. A total of 2709 reflections were measured, of which 1714 were unique in the range of 2.19 < θ < 25.02° (h, −14 to 19; k, −6 to 5; l, −22 to 21). The two benzene rings in the molecule are almost in the same plane. All of the non-H atoms were refined anisotropically by full-matrix least-squares to give the final R = 0.0732 and WR = 0.1820. The completeness of the crystal data is 99.9%. The crystal data of the compound A4 have been deposited at the Cambridge Crystallographic Data Center under code CCDC 1,975,217 that contains all the above data.

In Vitro Antibacterial Activity Bioassays
The in vitro antibacterial activity of target compounds against Xoo, Xac and Xoc was evaluated using a turbidimeter test [26,27]. For comparison, the commercial antibacterial agents bismerthiazol and thiodiazole copper was used as positive controls. DMSO in sterile water served as negative control. Nutrient broth medium (NB, 1 g of yeast powder, 3 g of beef extract, 10 g of glucose, 5 g of peptone and 1 L of distilled water, pH 7.0 to 7.2) in tubes was sterilized under high temperature and pressure. The tested compounds were dissolved in 120 µL DMSO then diluted with 0.1% Tween-20 solution, and finally working solution concentrations of 200 and 100 µg/mL were obtained. One mL of the solution containing compounds, bismerthiazol and thiadiazole copper was transferred into tubes (15 × 150 mm) containing 4 mL nutrient broth (NB) medium. Approximately 40 µL of activated bacteria was introduced into each tube. Finally, the test tubes were incubated at 28 ± 1 °C with continuous shaking at 180 rpm for 24-48h. The density value (OD595) of the solution in the tube was monitored on the microplates when the OD595 of the negative control group was 0.6 to 0.8. The inhibition rate was calculated using the following equation: Inhibitory Rate (%) = (CK -T) / CK × 100%. (1) where "CK" represents the density value (OD595) of negative control group, and "T" implies the density value (OD595) of the treated NB medium. The EC50 values against Xoo, Xac and Xoc of the target compounds were tested at five gradient concentrations and computed from analysis using the SPSS 17.0 software (IBM, New York, NY, USA). The experiments were repeated three times for each compound.

Scanning Electron Microscopy
The cell surface was observed as previously described [28]. Xoo culture (OD595 = 1.0) was centrifuged and washed with PBS (pH = 7.2), then resuspended in 1.0 mL of PBS buffer. Then, the

In Vitro Antibacterial Activity Bioassays
The in vitro antibacterial activity of target compounds against Xoo, Xac and Xoc was evaluated using a turbidimeter test [26,27]. For comparison, the commercial antibacterial agents bismerthiazol and thiodiazole copper was used as positive controls. DMSO in sterile water served as negative control. Nutrient broth medium (NB, 1 g of yeast powder, 3 g of beef extract, 10 g of glucose, 5 g of peptone and 1 L of distilled water, pH 7.0 to 7.2) in tubes was sterilized under high temperature and pressure. The tested compounds were dissolved in 120 µL DMSO then diluted with 0.1% Tween-20 solution, and finally working solution concentrations of 200 and 100 µg/mL were obtained. 1 mL of the solution containing compounds, bismerthiazol and thiadiazole copper was transferred into tubes (15 × 150 mm) containing 4 mL nutrient broth (NB) medium. Approximately 40 µL of activated bacteria was introduced into each tube. Finally, the test tubes were incubated at 28 ± 1 • C with continuous shaking at 180 rpm for 24-48h. The density value (OD 595 ) of the solution in the tube was monitored on the microplates when the OD 595 of the negative control group was 0.6 to 0.8. The inhibition rate was calculated using the following equation: where "CK" represents the density value (OD 595 ) of negative control group, and "T" implies the density value (OD 595 ) of the treated NB medium. The EC 50 values against Xoo, Xac and Xoc of the target compounds were tested at five gradient concentrations and computed from analysis using the SPSS 17.0 software (IBM, New York, NY, USA). The experiments were repeated three times for each compound.

Scanning Electron Microscopy
The cell surface was observed as previously described [28]. Xoo culture (OD 595 = 1.0) was centrifuged and washed with PBS (pH = 7.2), then resuspended in 1.0 mL of PBS buffer. Then, the cells were treated with compound A 1 for 10 h at 28 • C at concentrations of 50 µg/mL and 100 µg/mL. The untreated sample served as a negative control. Next, the compound solution was removed by washing three times with PBS buffer. The Xoo cells were fixed with 2.5% glutaraldehyde at 4 • C overnight and then dehydrated with 70%, and 90% ethanol, respectively.

Nematicidal Biological Activity In Vitro
The nematicidal activities of the compounds A 1 -A 36 against M. incognita were tested by a typical assay [29,30]. The tested compounds were dissolved with DMSO and then diluted with 1% Tween-80 solution to prepare 500 and 100 µg/mL solutions. Approximately 200 µL of test solution was added into a 48 well plate. Then a suspension that included approximately 200 living nematodes was added into the above solution. Avermectin was used as positive control. The solution without compound was used as a negative control. All experiments were repeated three times. The mortality of the nematodes was seen under a stereoscopic binocular microscope after 24 and 72 h. The corrected mortality of nematicide was calculated using the following equation:

Conclusions
In summary, a series of new N-phenylacetamide derivatives containing 4-arylthiazole moieties was designed and synthesized by introducing the thiazole moiety into the scaffolds of N-phenylacetamides. The target compounds were evaluated for their in vitro antibacterial activities against Xoo, Xac and Xoc. The bactericidal activity data show that most of the target compounds have moderate inhibitory activities against the above pathogens, among which compounds A 1 and A 3 have good inhibitory effects on Xoo and Xoc. The cell-rupturing effect of compound A 1 on Xoo was studied through SEM analysis. In addition their nematocidal activity was also determined and the compound A 23 exhibited strong toxicity against M. incognita, comparable to that of avermectin.