Novel Pyridyl–Oxazole Carboxamides: Toxicity Assay Determination in Fungi and Zebrafish Embryos

Eight novel pyridyl–oxazole carboxamides were evaluated against fungi and displayed good fungicidal activities against Botrytis cinereal and Rhizoctonia solani. Preliminary bioassay results indicated that at 100 mg/L, compounds 6a–6e, 6g and 6h exhibited 100% fungicidal activities against Botrytis cinerea, and the compound 6b to Rhizoctonia solani at 100%. Then, the zebrafish embryo acute toxicity test was performed to assess the toxicity of 6b and 6c. A series of malformations appeared, when the zebrafish embryos were exposed to 6b and 6c, such as delayed yolk sac resorption, significant shortening of body length, pericardial edema, bending spine, lack of melanin, heart hemorrhage, head hemorrhage, delayed swim sac development, yolk malformation and head malformation. In addition, the acute toxicity of 6b to zebrafish embryo is 4.878 mg/L, and 6c is 6.257 mg/L.


Introduction
In the early 20th century, pesticide, as a helpful partner for agriculture, came into our lives. Lots of people realized the fact that every coin has two sides: the effective pesticides may be hazardous to human health and the environment. In recent decades, the mushrooming human population has come with a booming economy, followed by the unexpected impact from the accumulation of environmental pollution caused by abusing pesticides. The residues of pesticides slipped into the food chain quietly, through bioconcentration and biomagnification, invisibly and continuously, affecting all natural creatures.
Since quality of life has improved, people have been concerned with the pesticide residues and setting higher demands on pesticides, including insecticides, herbicides, and fungicides. Currently, it is hard to imagine the world without pesticides, which have already permeated so many aspects of our lives. Thus, toxicological study of pesticides, by helping people directly and visually recognize the toxicity of pesticides, is absolutely significant. In recent years, because of certain inherent features, such as the comparable genetic and physiological make-up of mammals, zebrafish are widely used in toxicity research as an alternative model. Since zebrafish can absorb small molecules in their environment through skin and gills [1], their embryos and larvae were commonly used in developmental toxicity research to assess toxicity, such as cardiovascular toxicity, neurotoxicity and ototoxicity [2][3][4].
Along with social progress and the awakening of people's awareness of human health and environmental protection, many highly toxic pesticides were gradually eliminated by market forces, which urged researchers to search for ideal alternatives to those pesticides. Amide compounds with ideal biological activity and metabolic properties were widely used in insecticide and fungicide [5,6]. Additionally, in the process of creating green pesticides, heterocyclic pesticides have the characteristics of high activity and good selectivity, thus becoming one of the hot spots in the development of pesticide lead compounds [7][8][9][10][11]. Since the 20th century, many highly active amide-containing fungicidal drugs containing Along with social progress and the awakening of people's awareness of human health and environmental protection, many highly toxic pesticides were gradually eliminated by market forces, which urged researchers to search for ideal alternatives to those pesticides. Amide compounds with ideal biological activity and metabolic properties were widely used in insecticide and fungicide [5,6]. Additionally, in the process of creating green pesticides, heterocyclic pesticides have the characteristics of high activity and good selectivity, thus becoming one of the hot spots in the development of pesticide lead compounds [7][8][9][10][11]. Since the 20th century, many highly active amide-containing fungicidal drugs containing heterocyclic structures [12][13][14][15] have been developed (Figure 1). Nitrogen-containing heterocyclic compounds have various structural forms and good biological activity. Thus, the compound is widely used in the field of pesticides. At the same time, pyridine derivatives [16][17][18][19][20][21] and oxazole derivatives [22][23][24][25][26][27] (Figure 2) have also attracted attention as important nitrogen-containing heterocyclic compounds.  Chlorantraniliprole was used as the lead compound, relating to the phenylcyclobioxazole structure of oxazole derivatives, while the principle of bioelectronic isostery was used to obtain the pyridixazole structure, introduce the benzamide structure and, finally, design and synthesize several pyridines bioxazole amide compounds ( Figure 3). The synthetic route is shown in Scheme 1. Furthermore, their fungicidal activities and toxicity test on zebrafish embryos were studied.  Along with social progress and the awakening of people's awareness of human health and environmental protection, many highly toxic pesticides were gradually eliminated by market forces, which urged researchers to search for ideal alternatives to those pesticides. Amide compounds with ideal biological activity and metabolic properties were widely used in insecticide and fungicide [5,6]. Additionally, in the process of creating green pesticides, heterocyclic pesticides have the characteristics of high activity and good selectivity, thus becoming one of the hot spots in the development of pesticide lead compounds [7][8][9][10][11]. Since the 20th century, many highly active amide-containing fungicidal drugs containing heterocyclic structures [12][13][14][15] have been developed ( Figure 1). Nitrogen-containing heterocyclic compounds have various structural forms and good biological activity. Thus, the compound is widely used in the field of pesticides. At the same time, pyridine derivatives [16][17][18][19][20][21] and oxazole derivatives [22][23][24][25][26][27] (Figure 2) have also attracted attention as important nitrogen-containing heterocyclic compounds.  Chlorantraniliprole was used as the lead compound, relating to the phenylcyclobioxazole structure of oxazole derivatives, while the principle of bioelectronic isostery was used to obtain the pyridixazole structure, introduce the benzamide structure and, finally, design and synthesize several pyridines bioxazole amide compounds ( Figure 3). The synthetic route is shown in Scheme 1. Furthermore, their fungicidal activities and toxicity test on zebrafish embryos were studied.  Chlorantraniliprole was used as the lead compound, relating to the phenylcyclobioxazole structure of oxazole derivatives, while the principle of bioelectronic isostery was used to obtain the pyridixazole structure, introduce the benzamide structure and, finally, design and synthesize several pyridines bioxazole amide compounds ( Figure 3). The synthetic route is shown in Scheme 1. Furthermore, their fungicidal activities and toxicity test on zebrafish embryos were studied.
Along with social progress and the awakening of people's awareness of human health and environmental protection, many highly toxic pesticides were gradually eliminated by market forces, which urged researchers to search for ideal alternatives to those pesticides. Amide compounds with ideal biological activity and metabolic properties were widely used in insecticide and fungicide [5,6]. Additionally, in the process of creating green pesticides, heterocyclic pesticides have the characteristics of high activity and good selectivity, thus becoming one of the hot spots in the development of pesticide lead compounds [7][8][9][10][11]. Since the 20th century, many highly active amide-containing fungicidal drugs containing heterocyclic structures [12][13][14][15] have been developed ( Figure 1). Nitrogen-containing heterocyclic compounds have various structural forms and good biological activity. Thus, the compound is widely used in the field of pesticides. At the same time, pyridine derivatives [16][17][18][19][20][21] and oxazole derivatives [22][23][24][25][26][27] (Figure 2) have also attracted attention as important nitrogen-containing heterocyclic compounds.  Chlorantraniliprole was used as the lead compound, relating to the phenylcyclobioxazole structure of oxazole derivatives, while the principle of bioelectronic isostery was used to obtain the pyridixazole structure, introduce the benzamide structure and, finally, design and synthesize several pyridines bioxazole amide compounds ( Figure 3). The synthetic route is shown in Scheme 1. Furthermore, their fungicidal activities and toxicity test on zebrafish embryos were studied.

Biological Activities of Target Compounds
The fungicidal activity test results of the compounds 6a-6h against Botrytis cinereal and Rhizoctonia solani are shown in Tables 1 and 2. It can be seen that the target compounds had a high inhibitory effect on Botrytis cinereal, of which at a concentration of 100 mg/L, 6f had a 90% inhibitory rate, and 6a-6e, 6g and 6h were 100%. Additionally, at a concentration of 100 mg/L, the inhibition rates of 6a, 6e, and 6h against Rhizoctonia solani were 90%, and 6b was 100%. According to the data analysis in Tables 1 and 2, the results indicated that the inhibition rate of fluorine substitution was higher than that of chlorine substitution and methyl substitution when the benzene ring was substituted.
It can be seen from Tables 1 and 2 that at a concentration of 50 mg/L, the inhibition rates of 6c (79.12%) and 6f (66.77%) on Rhizoctonia solani were better than azoxystrobin, and 6c-6f (62.06-66.47%) and 6h (64.61%) on Botrytis cinereal were better than azoxystrobin. According to the data in Table 2, it can be seen that at a concentration of 50 mg/L, when the benzene ring was substituted, the inhibition rate of di-fluorine substitution was higher than mono-fluorine substitution against Rhizoctonia solani. On Botrytis cinereal, the inhibition rate of its di-substitution was higher than that of mono-substitution when the substituents on the benzene ring were fluorine and methyl.

Biological Activities of Target Compounds
The fungicidal activity test results of the compounds 6a-6h against Botrytis cinereal and Rhizoctonia solani are shown in Tables 1 and 2. It can be seen that the target compounds had a high inhibitory effect on Botrytis cinereal, of which at a concentration of 100 mg/L, 6f had a 90% inhibitory rate, and 6a-6e, 6g and 6h were 100%. Additionally, at a concentration of 100 mg/L, the inhibition rates of 6a, 6e, and 6h against Rhizoctonia solani were 90%, and 6b was 100%. According to the data analysis in Tables 1 and 2, the results indicated that the inhibition rate of fluorine substitution was higher than that of chlorine substitution and methyl substitution when the benzene ring was substituted. Note: nt = not tested. All the data were determined three times. It can be seen from Tables 1 and 2 that at a concentration of 50 mg/L, the inhibition rates of 6c (79.12%) and 6f (66.77%) on Rhizoctonia solani were better than azoxystrobin, and 6c-6f (62.06-66.47%) and 6h (64.61%) on Botrytis cinereal were better than azoxystrobin. According to the data in Table 2, it can be seen that at a concentration of 50 mg/L, when the benzene ring was substituted, the inhibition rate of di-fluorine substitution was higher than mono-fluorine substitution against Rhizoctonia solani. On Botrytis cinereal, the inhibition rate of its di-substitution was higher than that of mono-substitution when the substituents on the benzene ring were fluorine and methyl.

Toxicity to Zebrafish Embryos
According to the data mentioned above, comparing their fungicidal activities, we chose compound 6b and 6c to study the lethal and teratogenic effects exposure on zebrafish embryos from 6 to 96 hpf.
At 24 hpf of 6b ( Figure 4A), the autonomous movement of zebrafish embryo within 1 min was obviously inhibited, and there was a concentration-dependent effect. Among them, the 3 and 4 mg/L groups had a decrease in the autonomous movement of the embryo (p < 0.01), and in the 6 and 8 mg/L treatment groups, the number of autonomous movements of embryos was even less (p < 0.001). At 48 hpf ( Figure 4B), compared to the control group, the hatching rate of the 6b drug-exposed group was significantly suppressed. At 72 hpf ( Figure 4B), under 6b concentration of 6 mg/L, the hatching rate was about 5%, and under 8 mg/L exposure, the hatching rate was 0%. At 96 hpf of 6b ( Figure 4C), the zebrafish malformation rate gradually increased with a concentration-dependency (p < 0.01 and p < 0.001). The main phenotypic changes of zebrafish under 6b acute exposure ( Figure 4E) were shortened body length, delayed tail development, delayed eye development, yolk cyst and even deformity, and there was a concentration-dependent effect. At the 48-96 hpf, a series of malformations appeared, such as delayed yolk sac resorption, significant shortening of body length, pericardial edema, bending spine, lack of melanin, heart hemorrhage, head hemorrhage, delayed swim sac development, yolk malformation and head malformation. The LC 50 concentration of 6b ( Figure 4D) was 4.878 mg/L.
The main manifestations of acute violent poisoning of 6c ( Figure 4I) were yolk cyst, venous sinus hemorrhage, developmental delay, bending spine, hypopigmentation, pericardial cyst, and delayed swim sac development (represented by the black arrow). The pericardial edema was the most obvious. The hatching rate at 72 hpf was shown in Figure 4F. At 8 mg/L and 16 mg/L, the hatching rate was significantly reduced (p < 0.05, p <0.01, p < 0.001). The malformation rate at 96 hpf was in Figure 4G. In the high-concentration exposure group, the malformation rate was significantly increased and showed a concentrationdependent (p < 0.001). Due to the impact of incubation and stillbirth, the mortality rate at 96 hpf was counted ( Figure 4H), and the LC 50 concentration was 6.257 mg/L.
Comparing the lethal and teratogenic effects of 6b and 6c, we found that the toxicity of 6b to zebrafish embryos was higher than that of 6c. Therefore, we speculated that the structure of di-substitution was more beneficial than mono-substitution to reduce the toxicity to zebrafish embryos.    The hatchability of zebrafish embryos exposed to 6b from 24 to 96 hpf; (C) The developmental abnormalities of zebrafish embryos exposed to 6b at 96 hpf; (D) The mortality of zebrafish embryos exposed to 6b at 96 hpf; (E) The main phenotypic changes of zebrafish embryos and larvae under 6b exposure from 24 to 96 hpf; (F) The hatching rate of zebrafish embryos exposed to 6c at 72 hpf; (G) The malformation rate of zebrafish embryos and larvae under 6c exposure at 96 hpf; (H) The mortality of zebrafish embryos exposed to 6c at 96 hpf; (I) The main phenotypic changes of zebrafish embryos and larvae under 6c exposure from 24 to 72 hpf. "*" represents significant differences at * p ≤ 0.05, ** p ≤ 0.01 and *** p ≤ 0.001 by one-way ANOVA followed by a Dunnet test. The hatchability of zebrafish embryos exposed to 6b from 24 to 96 hpf; (C) The developmental abnormalities of zebrafish embryos exposed to 6b at 96 hpf; (D) The mortality of zebrafish embryos exposed to 6b at 96 hpf; (E) The main phenotypic changes of zebrafish embryos and larvae under 6b exposure from 24 to 96 hpf; (F) The hatching rate of zebrafish embryos exposed to 6c at 72 hpf; (G) The malformation rate of zebrafish embryos and larvae under 6c exposure at 96 hpf; (H) The mortality of zebrafish embryos exposed to 6c at 96 hpf; (I) The main phenotypic changes of zebrafish embryos and larvae under 6c exposure from 24 to 72 hpf. "*" represents significant differences at * p ≤ 0.05, ** p ≤ 0.01 and *** p ≤ 0.001 by one-way ANOVA followed by a Dunnet test.

Fish Husbandry and Embryo Collection
The zebrafish (D. rerio) used for the experiments were from the Institute of Hydrobiology. According to standard protocols, they were raised and adapted to the laboratory with a light/dark, 14 h/10 h cycle in a circulation system with dechlorinated tap water (pH 6.5-7.5) at a constant temperature (27 ± 0.5 • C). In order to raise the conductivity to 450-1000 µS/cm, the ocean salt was added to the water [30,31].
The eggs of the zebrafish that were acquired from spawning adults in tanks overnight, with a sex ratio of 2:2, were used for exposure experiments and collected within half an hour of light exposure [30]. By means of a stereomicroscope, the fertilized and normal embryos were inspected and staged for subsequent experiments. When at 6 hpf, they were distributed into 12-well plates [32] (10 embryos per well) for exposure, and three replicate experiments were performed.

Ethics Statement
The Institutional Animal Care and Use Committee (IACUC) at Wenzhou Medical University approved our study plan for the proper use of zebrafish. All studies were carried out in strict accordance with the guidelines of the IACUC. All dissections were performed on ice, and all efforts were made to minimize suffering.

Fungicidal Activity and Toxicity Determination
The fungicidal activity was investigated on the basis of reference [33], and the results were shown in Tables 1 and 2. Furthermore, compound 6b and 6c were selected to be assessed through the zebrafish embryo acute toxicity test. Based on the mortality rates of zebrafish, a series of gradient concentrations of the compound 6b and 6c were set according to the environmentally relevant concentrations and preliminary experiments.

Conclusions
In conclusion, the bioassay results indicated that at a concentration of 100 mg/L, the target compound had a high inhibitory effect on Botrytis cinereal, of which 6a-6e, 6g and 6h had a 100% inhibitory rate. Furthermore, most of the target compounds had a high inhibitory effect on Rhizoctonia solani. 6b (100%) had a high inhibition rate on Rhizoctonia solani. Additionally, the acute toxicity of compound 6b (4.878 mg/L) and 6c (6.257 mg/L) indicated that 6b and 6c exposure had an apparent influence on the normal development of the zebrafish process. By comparing their data, we also found that the toxicity of 6b to zebrafish embryos was higher than that of 6c. Therefore, we could draw the conclusion that, compared with mono-substitution, the structure of di-substitution was more beneficial to reduce the toxicity to zebrafish embryos, which merits further study.  Informed Consent Statement: Not applicable.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.