Design, Synthesis, and Anti-Fungal Evaluation of Heterocyclic Benzoxazole Derivatives

In order to discover more promising anti-fungal agents, a series of benzoxazole family was synthesized by PPA-catalyzed condensation and a Raney nickel/hydrazine reduction. Altogether 45 compounds were obtained in good to excellent yields and characterized by FT-IR, NMR, MS, and X-ray crystal diffraction. Moreover, the biological activity against eight phytopathogenic fungi was investigated. All in all, most of these compounds bear moderate antifungal activities. Among them, three candidates show the strongest activities, compound 4ac, 4bc provided over 50% inhibition rate against five fungi. Especially, the inhibitory rate of compound 4ah on Mycosphaerella melonis reached 76.4%.


Introduction
In recent years, pathogenic bacteria and plant viruses targeting agricultural crops have become increasingly difficult to control, leading to enormous losses in worldwide crop production each year. As such, the development of environmentally friendly pesticides with high selectivity, high efficiency, low toxicity, and suitable degradation are important challenges to both chemists and biologists [1]. Development of drug-like heterocyclic compounds from simple and facile substrates is one of the emerging areas in modern synthetic chemistry. Heterocyclic structures are important key features in natural products or synthetic medicines and pesticides because of their high-efficiency, low toxicity, and diversity of possible substituents [2]. As shown in Figure 1, the benzoxazole is an important heteroaromatic moiety found in many pharmaceuticals and agrichemicals [3][4][5]. Benzoxazole is a structural scaffold which can be found in nonsteroidal anti-inflammatory drugs such as flunoxaprofen [6], uniprofen [7], and Prinaberel [8]. Many benzo heterocycles compounds can be found in agrichemicals, such as carbendarim [9], fubridazole [10], and thiabendazole [11]. There are also other pharmaceutical applications such as antibiotic [12,13], anticancer, [14][15][16] anti-inflammatory applications [17]. Due to their structural characteristics, they can be used in material industry such as fluorescent chemosensor for special ions [18] and mesogenic applications [19].
Benzoxazoles are important members of the family of fused heterocycles that have attracted much attention because of their diverse biological activity. In order to find new potential pesticide molecules with antifungal activities, we designed and synthesized a series of benzoxazole derivatives and evaluated their potential antifungal activities against eight kinds of plant pathogenic fungus commonly found in agriculture systematically, including Fusarium solani, Colletotrichum gloeosporioides, Mycosphaerella melonis, Alternaria brassicae, Pyricularia grisea, Curvularia lunata, Alternaria solani, and Fusarium graminearum. As Scheme 1 shows, namely the synthesis of benzoxazoles can be classified into two kinds: [20,21] one is using PPA as solvent and catalyst at the presence of acid and aniline in one step [15,17,22], Benzoxazoles are important members of the family of fused heterocycles that have attracted much attention because of their diverse biological activity. In order to find new potential pesticide molecules with antifungal activities, we designed and synthesized a series of benzoxazole derivatives and evaluated their potential antifungal activities against eight kinds of plant pathogenic fungus commonly found in agriculture systematically, including Fusarium solani, Colletotrichum gloeosporioides, Mycosphaerella melonis, Alternaria brassicae, Pyricularia grisea, Curvularia lunata, Alternaria solani, and Fusarium graminearum. As Scheme 1 shows, namely the synthesis of benzoxazoles can be classified into two kinds: [20,21] one is using PPA as solvent and catalyst at the presence of acid and aniline in one step [15,17,22], the other is using condensation of aldehyde and aniline then an oxidation step using some oxidizer such as DDQ, Manganese oxide, air, etc. [23]. Our research group has been intensively studying the synthesis of heterocycles and their agricultural applications [24][25][26]. Pyridine, also known as azabenzene, has a certain aromatic structure and is widely used in sulfonamides and pesticides. Inspired by three molecules used as agrichemicals in Figure 1, we envisioned a combination of pyridine, benzoxazole, and amide might have good antifungal activities against plant pathogen ( Figure 2). Prompted by the above thoughts, we wish to report the synthesis, spectroscopic, singlecrystal diffraction, antifungal activity, of a series of benzoxazoles bearing a pyridine ring and amide moiety. In our devise, the two heterocyclic moiety contributes the biological activities, and the amide would tune the activities.   Benzoxazoles are important members of the family of fused heterocycles that have attracted much attention because of their diverse biological activity. In order to find new potential pesticide molecules with antifungal activities, we designed and synthesized a series of benzoxazole derivatives and evaluated their potential antifungal activities against eight kinds of plant pathogenic fungus commonly found in agriculture systematically, including Fusarium solani, Colletotrichum gloeosporioides, Mycosphaerella melonis, Alternaria brassicae, Pyricularia grisea, Curvularia lunata, Alternaria solani, and Fusarium graminearum. As Scheme 1 shows, namely the synthesis of benzoxazoles can be classified into two kinds: [20,21] one is using PPA as solvent and catalyst at the presence of acid and aniline in one step [15,17,22], the other is using condensation of aldehyde and aniline then an oxidation step using some oxidizer such as DDQ, Manganese oxide, air, etc. [23]. Our research group has been intensively studying the synthesis of heterocycles and their agricultural applications [24][25][26]. Pyridine, also known as azabenzene, has a certain aromatic structure and is widely used in sulfonamides and pesticides. Inspired by three molecules used as agrichemicals in Figure 1, we envisioned a combination of pyridine, benzoxazole, and amide might have good antifungal activities against plant pathogen ( Figure 2). Prompted by the above thoughts, we wish to report the synthesis, spectroscopic, singlecrystal diffraction, antifungal activity, of a series of benzoxazoles bearing a pyridine ring and amide moiety. In our devise, the two heterocyclic moiety contributes the biological activities, and the amide would tune the activities.  Benzoxazoles are important members of the family of fused heterocycles that have attracted much attention because of their diverse biological activity. In order to find new potential pesticide molecules with antifungal activities, we designed and synthesized a series of benzoxazole derivatives and evaluated their potential antifungal activities against eight kinds of plant pathogenic fungus commonly found in agriculture systematically, including Fusarium solani, Colletotrichum gloeosporioides, Mycosphaerella melonis, Alternaria brassicae, Pyricularia grisea, Curvularia lunata, Alternaria solani, and Fusarium graminearum. As Scheme 1 shows, namely the synthesis of benzoxazoles can be classified into two kinds: [20,21] one is using PPA as solvent and catalyst at the presence of acid and aniline in one step [15,17,22], the other is using condensation of aldehyde and aniline then an oxidation step using some oxidizer such as DDQ, Manganese oxide, air, etc. [23]. Our research group has been intensively studying the synthesis of heterocycles and their agricultural applications [24][25][26]. Pyridine, also known as azabenzene, has a certain aromatic structure and is widely used in sulfonamides and pesticides. Inspired by three molecules used as agrichemicals in Figure 1, we envisioned a combination of pyridine, benzoxazole, and amide might have good antifungal activities against plant pathogen ( Figure 2). Prompted by the above thoughts, we wish to report the synthesis, spectroscopic, singlecrystal diffraction, antifungal activity, of a series of benzoxazoles bearing a pyridine ring and amide moiety. In our devise, the two heterocyclic moiety contributes the biological activities, and the amide would tune the activities.

Chemistry
As shown in Scheme 2, the isomers (2-, 3-, 4-, respectively) of picolinic acid condensed with 4-nitro-2-aminophenol at the presence of PPA to provide the three types of pyridylbenzoxazole in 75-85% yield. Compared with the reported method, the PPA was preheated to 80 • C before use in order to be measured easily. Next, a mild reduction in the nitro group to amino group was adopted using hydrazine and Raney nickel in 75-80% yield. The other reduction methods proved unsuccessful, such as palladium/carbon catalytic hydrogenation, stannous chloride in common solvent (ethyl acetate and tetrahydro furan), and iron powder in acetic acid. A lot of black tar which can be solved in acidic condition was produced under these conditions. We suspected that the oxales ring was opened upon these conditions. As to the preparation of the nitro-benzoxazoles, there is another nitration method of benzoxazole, but the nitro group position is limited to the parato the OH group. Next, we condensed three pyridyl-benzoxazole compounds with 15 different types of acyl chlorides to obtain 45 amide derivatives (4aa-4co) in fairly good to excellent yield (Table 1) and were characterized by 1 H NMR and 13 C NMR. The stereochemistry of two compounds 4ac and 4bc with the best antifungal effect was further confirmed by the X-ray crystallographic analysis ( Figure 3).
As shown in Scheme 2, the isomers (2-, 3-, 4-, respectively) of picolinic acid condensed with 4-nitro-2-aminophenol at the presence of PPA to provide the three types of pyridylbenzoxazole in 75-85% yield. Compared with the reported method, the PPA was preheated to 80 °C before use in order to be measured easily. Next, a mild reduction in the nitro group to amino group was adopted using hydrazine and Raney nickel in 75-80% yield. The other reduction methods proved unsuccessful, such as palladium/carbon catalytic hydrogenation, stannous chloride in common solvent (ethyl acetate and tetrahydro furan), and iron powder in acetic acid. A lot of black tar which can be solved in acidic condition was produced under these conditions. We suspected that the oxales ring was opened upon these conditions. As to the preparation of the nitro-benzoxazoles, there is another nitration method of benzoxazole, but the nitro group position is limited to the para-to the OH group. Next, we condensed three pyridyl-benzoxazole compounds with 15 different types of acyl chlorides to obtain 45 amide derivatives (4aa-4co) in fairly good to excellent yield (Table 1) and were characterized by 1 H NMR and 13 C NMR. The stereochemistry of two compounds 4ac and 4bc with the best antifungal effect was further confirmed by the X-ray crystallographic analysis ( Figure 3).   As shown in Scheme 2, the isomers (2-, 3-, 4-, respectively) of picolinic acid condensed with 4-nitro-2-aminophenol at the presence of PPA to provide the three types of pyridylbenzoxazole in 75-85% yield. Compared with the reported method, the PPA was preheated to 80 °C before use in order to be measured easily. Next, a mild reduction in the nitro group to amino group was adopted using hydrazine and Raney nickel in 75-80% yield. The other reduction methods proved unsuccessful, such as palladium/carbon catalytic hydrogenation, stannous chloride in common solvent (ethyl acetate and tetrahydro furan), and iron powder in acetic acid. A lot of black tar which can be solved in acidic condition was produced under these conditions. We suspected that the oxales ring was opened upon these conditions. As to the preparation of the nitro-benzoxazoles, there is another nitration method of benzoxazole, but the nitro group position is limited to the para-to the OH group. Next, we condensed three pyridyl-benzoxazole compounds with 15 different types of acyl chlorides to obtain 45 amide derivatives (4aa-4co) in fairly good to excellent yield (Table 1) and were characterized by 1 H NMR and 13 C NMR. The stereochemistry of two compounds 4ac and 4bc with the best antifungal effect was further confirmed by the X-ray crystallographic analysis ( Figure 3).

X-ray Crystallographic Studies of 4ac, 4bc
Compound 4ac crystallizes in the monoclinic space group P2 1 . There are two crystallographic independent molecules in the asymmetric unit (only one of them is shown in Figure 3). In the molecule of 4ac, bond lengths and angles are very similar to those provided in the literature for benzoxazole derivative. The benzoxazole ring was approximately planar. Compared with the 4bc, the dihedral angle between the methylphenyl plane and the oxazole ring was 55.65 • . Compound 4bc crystallizes in the monoclinic space group C2/c. There are eight crystallographic independent molecules in the asymmetric unit (only one of them is shown in Figure 3). In the molecule of 4bc, bond lengths and angles are very similar to those provided in the literature for benzoxazole derivative. The whole molecule was approximately planar. The dihedral angle of the methylphenyl plane, the oxazole ring, was 8.61 • . X-ray diffraction reflection data of 4ac and 4bc are shown in the Supplementary Materials.

X-ray Crystallographic Studies of 4ac, 4bc
Compound 4ac crystallizes in the monoclinic space group P21. There are two crystallographic independent molecules in the asymmetric unit (only one of them is shown in Figure 3). In the molecule of 4ac, bond lengths and angles are very similar to those provided in the literature for benzoxazole derivative. The benzoxazole ring was approxi-a Reaction condition: each entry was performed at 3 mmol scale, in the presence of DMAP, in THF, r.t., 6 h.

In Vitro Anti-Fungal Activity
Meanwhile, the anti-fungal activities of these compounds were evaluated against eight common phytopathogenic fungi, (Fusarium solani, Colletotrichum gloeosporioides, Mycosphaerella melonis, Alternaria brassicae, Pyricularia grisea, Curvularia lunata, Alternaria solani, and Fusarium graminearum) using the inhibition zone method at the concentration of 100 µg/mL by the poisoned food technique. While thiophanate-methyl, which is a fungicide bearing similar heterocyclic structures and a commercially available agricultural fungicide, was used as a positive control at 100 µg/mL. As shown in Figure 4, the experiments revealed that most of these compounds bears moderate result to Colletotrichum gloeosporioides and Mycosphaerella melonis. Through the 3D -Bar of inhibition rate against eight fungi, we could see, compound 4ac, 4bc showed best results, providing over 50% inhibition rate against five fungi. Herein, we believe the methyl makes a rotation barrier, and to some extent enhances the combination of compound to the enzyme. Interestingly, some compounds could selectively inhibit some fungi. For example, the inhibitory rate of compound 4ah on Mycosphaerella melonis could reach 76.4%, but it had little inhibitory activity on other fungi. Another example was that compound 4bh could highly selectively inhibit the growth of Alternaria brassicae. However, 4be against Fusarium solani, 4cj and 4af against Colletotrichum gloeosporioides provided unsatisfactory results, even positive results. From the whole table, the effect of acyl ligand on antifungal activity was greater than that of pyridyl-benzoxazole skeleton. Typical examples are compounds 4ac and 4bc, and even compound 4cc, which have very good antifungal activity and all of them have the same acyl ligand.

Chemistry
All chemicals were purchased from Energy chemicals and Merck (used without further purification). The melting point was determined using a digital melting point meter WRS-1B. The 1 H NMR and 13 C NMR spectra were carried out by AV 500 MHz spectrometer. X-ray analysis of the samples were recorded on a Bruker APEX II area detector diffractometer at 296(2) K with a graphite-monochromatic Mo K radiation (λ = 0.71073A°).

Chemistry
All chemicals were purchased from Energy chemicals and Merck (used without further purification). The melting point was determined using a digital melting point meter WRS-1B. The 1 H NMR and 13 C NMR spectra were carried out by AV 500 MHz spectrometer. X-ray analysis of the samples were recorded on a Bruker APEX II area detector diffractometer at 296(2) K with a graphite-monochromatic MoK radiation (λ = 0.71073A • ).

5-Nitro-2-(pyridin-2-yl)benzo[d]oxazole (3a)
. To a solution of 2-picolinic acid (2.46 g 0.01 mol) condensed with 4-nitro-2-aminophenol (3.08 g, 0.01 mol) in polyphosphoric acid (30 mL) in a 100mL three-necked flask, the middle neck was installed a mechanical stirring, and the side neck was set for thermometer. The reaction mixture was heated 140 • C and the external temperature was kept at 150 • C. After 6 h, the reaction was completed through TLC. Then the reaction mixture was poured into water slowly and was adjusted to pH 6 using potassium carbonate. A quantity of solid precipitated and was collected by suction. This compound was used without further purification and drying. Because the next reduction was carried out in ethyl alcohol, the crude product 2a was used directly. The crude 2a was dissolved in 95% ethyl alcohol (40 mL), and then was added W-2 Raney nickel (ca. 0.20 g), and then hydrazine (1.96 g, 3 equiv.) was introduced drop wise. Moreover, the mixture was heated to 60 • C and monitored by TLC. After completion, the reaction mixture was filtered through a Celite pad. The filtration was condensed and purified by flash column chromatography (petroleum ether: ethyl acetate) to obtain 3a (1.94 g, 72% for two steps) as a brown solid

Conclusions
In conclusion, a series of benzoxazoles were synthesized efficiently using a combination of PPA and Raney nickel reduction. On the basis of syntheses, their anti-phytopathogenic activities were investigated as well. All the synthesized coumarin derivatives were characterized by NMR and MS. The single crystal of 4ac, 4bc were obtained and characterized by X-ray. These compounds showed moderate to good excellent inhibition rate using linear growth rate method. These results will not only be helpful in the synthesis of similar heterocycles, but also will enlighten the discovery of a new kind of agrochemicals in the future.