Natural Products for Pesticides Discovery: Structural Diversity Derivation and Biological Activities of Naphthoquinones Plumbagin and Juglone

Plant diseases and insect pests seriously affect the yield and quality of crops and are difficult to control. Natural products are an important source for the discovery of new pesticides. In this work, naphthoquinones plumbagin and juglone were selected as parent structures, and a series of their derivatives were designed, synthesized and evaluated for their fungicidal activities, antiviral activities and insecticidal activities. We found that the naphthoquinones have broad-spectrum anti-fungal activities against 14 types of fungus for the first time. Some of the naphthoquinones showed higher fungicidal activities than pyrimethanil. Compounds I, I-1e and II-1a emerged as new anti-fungal lead compounds with excellent fungicidal activities (EC50 values: 11.35–17.70 µg/mL) against Cercospora, arachidicola Hori. Some compounds also displayed good to excellent antiviral activities against the tobacco mosaic virus (TMV). Compounds I-1f and II-1f showed similar level of anti-TMV activities with ribavirin, and could be used as new antiviral candidates. These compound also exhibited good to excellent insecticidal activities. Compounds II-1d and III-1c displayed a similar level of insecticidal activities with matrine, hexaflumuron and rotenone against Plutella xylostella. In current study, plumbagin and juglone were discovered as parent structures, which lays a foundation for their application in plant protection.


Introduction
With the continuous growth of the population, the improvement of crop yields and quality has gradually become a very challenging issue [1]. It is estimated that the annual economic losses caused by plant diseases exceed $220 billion worldwide, that almost all crops are attacked by diseases and pests, and about one third of agricultural products depend on the use of agricultural chemicals [2][3][4]. In addition, traditional pesticides are prone to produce drug resistance in the long-term use process. Therefore, the continuous innovation of modern agrochemicals has always been an important strategy to deal with this challenge [5,6].
Botanical natural products refer to derivatives of plants, crude plant extracts or active ingredients. Due to their advantages such as biodegradability, diverse structures, extensive sources and low susceptibility to drug resistance, developing botanical natural products into pesticides is recommended as an eco-chemical and sustainable strategy for agricultural pest management. At the same time, using natural products as active leads and optimizing their molecular structure through structural diversity synthesis has also gradually become a direction in the creation of green and efficient new pesticides [7][8][9][10][11][12][13].  Our research group has been committed to the discovery of new and efficient agrochemical lead compounds based on natural products for a long time. Through our research, a series of alkaloids, sesquiterpenes, amino acids and quaternary ammonium salts were found to have good to excellent fungicidal activities, antiviral activities or insecticidal activities, by which we accumulated rich experience in natural product selection and structural optimization [17][18][19][20][21].
In this work, plumbagin and juglone were selected as parent structures, and a series of their derivatives were designed (Figure 1), synthesized and evaluated for their fungicidal activities, antiviral activities and insecticidal activities.

Chemistry
Based on the scaffold hopping strategy [22], a series of structure modified derivatives of juglone and plumbagin were designed and synthesized. The selection of substituents was mainly based on aromatic rings, supplemented by long-chain alkyl, cycloalkyl and oxyalkyl. In the O-acylation reaction of juglone and plumbagin, different acyl chlorides and sulfonyl chlorides were used, with triethylamine as a base to give the corresponding esters I-1a-I-1g and II-1a-II-1g in good to high yields (Scheme 1). In order to study the effect of chain-like alkyl substituents at the 5-OH on the activities of juglone and plumbagin derivatives, these O-alkalation products (I-1h, II-1h-II-1j) were obtained by reacting juglone and plumbagin with corresponding haloalkanes or Me 2 SO 4 (Scheme 2). In order to study the effect of different substituents at the 3-position of juglone on the activity, compounds II-1k-II-1m, III and III-1b-III-1f with different substituents were designed and synthesized (Schemes 3 and 4).

Chemistry
Based on the scaffold hopping strategy [22], a series of structure modified derivatives of juglone and plumbagin were designed and synthesized. The selection of substituents was mainly based on aromatic rings, supplemented by long-chain alkyl, cycloalkyl and oxyalkyl. In the O-acylation reaction of juglone and plumbagin, different acyl chlorides and sulfonyl chlorides were used, with triethylamine as a base to give the corresponding esters I-1a-I-1g and II-1a-II-1g in good to high yields (Scheme 1). In order to study the effect of chain-like alkyl substituents at the 5-OH on the activities of juglone and plumbagin derivatives, these O-alkalation products (I-1h, II-1h-II-1j) were obtained by reacting juglone and plumbagin with corresponding haloalkanes or Me2SO4 (Scheme 2). In order to study the effect of different substituents at the 3-position of juglone on the activity, compounds II-1k-II-1m, III and III-1b-III-1f with different substituents were designed and synthesized (Schemes 3 and 4). Scheme 1. Synthesis of compounds I-1a-I-1g and II-1a-II-1g. Scheme 2. Synthesis of I-1h and II-1h-II-1j. Scheme 1. Synthesis of compounds I-1a-I-1g and II-1a-II-1g.

Chemistry
Based on the scaffold hopping strategy [22], a series of structure modified derivatives of juglone and plumbagin were designed and synthesized. The selection of substituents was mainly based on aromatic rings, supplemented by long-chain alkyl, cycloalkyl and oxyalkyl. In the O-acylation reaction of juglone and plumbagin, different acyl chlorides and sulfonyl chlorides were used, with triethylamine as a base to give the corresponding esters I-1a-I-1g and II-1a-II-1g in good to high yields (Scheme 1). In order to study the effect of chain-like alkyl substituents at the 5-OH on the activities of juglone and plumbagin derivatives, these O-alkalation products (I-1h, II-1h-II-1j) were obtained by reacting juglone and plumbagin with corresponding haloalkanes or Me2SO4 (Scheme 2). In order to study the effect of different substituents at the 3-position of juglone on the activity, compounds II-1k-II-1m, III and III-1b-III-1f with different substituents were designed and synthesized (Schemes 3 and 4).

Fungicidal Activity Result and Structure-Activity Relationship (SAR)
The aforementioned process is an effective approach to find new type fungicidal lead compounds based on natural products [23]. We first investigated the fungicidal activities of naphthoquinones I-III, I-1a-I-1h, II-1a-II-1m and III-1a-III-1f with commercial fungicides carbendazim, chlorothalonil and pyrimethanil as controls. These naphthoquinones exhibited broad-spectrum fungicidal activities against 14 types of phytopathogenic fungi at 50 µg/mL (Table 1). Some derivatives displayed more than 60% inhibitory effects. Compounds I and I-1e both had excellent inhibitory rates against Fusarium oxysporum f. sp. cucumeris. I, I-1e and II-1a showed a better inhibition rate than three commercial fungicides against Cercospora arachidicola Hori. I-1e also showed a comparable activity to commercial fungicides against Rhizoctonia cerealis and Fusarium moniliforme. Plumbagin (I) was effective against Cercospora arachidicola Hori, Phytophthora capsici and Rhizoctonia solani with a 100% inhibitory rate.

Fungicidal Activity Result and Structure-Activity Relationship (SAR)
The aforementioned process is an effective approach to find new type fungicidal lead compounds based on natural products [23]. We first investigated the fungicidal activities of naphthoquinones I-III, I-1a-I-1h, II-1a-II-1m and III-1a-III-1f with commercial fungicides carbendazim, chlorothalonil and pyrimethanil as controls. These naphthoquinones exhibited broad-spectrum fungicidal activities against 14 types of phytopathogenic fungi at 50 µg/mL (Table 1). Some derivatives displayed more than 60% inhibitory effects. Compounds I and I-1e both had excellent inhibitory rates against Fusarium oxysporum f. sp. cucumeris. I, I-1e and II-1a showed a better inhibition rate than three commercial fungicides against Cercospora arachidicola Hori. I-1e also showed a comparable activity to commercial fungicides against Rhizoctonia cerealis and Fusarium moniliforme. Plumbagin (I) was effective against Cercospora arachidicola Hori, Phytophthora capsici and Rhizoctonia solani with a 100% inhibitory rate.  Compounds I, I-1e and II-1a with excellent antifungal activities against Cercospora arachidicola Hori were further tested to determine their EC 50 values, with pyrimethanil as a control (Table 2). It can be seen that the EC 50 values of I, I-1e and II-1a (11.35-17.70 µg/mL) are lower than that of pyrimethanil (EC 50 value: 19.17 µg/mL), which indicates that I, I-1e and II-1a have better fungicidal activities than pyrimethanil and can be used as novel antifungal candidates for further investigation. SAR against Cercospora arachidicola Hori: Natural product plumbagin (I) displayed excellent fungicidal activity. Jonlone (II) or the introduction of bromine at the 3-position of II (i.e., III) leads to extremely reduced activity (inhibition effect: I > II, III). The introduction of an aliphatic functional group onto the 5-hydroxyl of plumbagin (I) is more advantageous than the introduction of an aromatic functional group, as compound I-1e containing a methoxyacetyl group displayed better antifungal activity than I (EC 50 values: I > I-1e). On the other hand, among ten 5-hydroxy modified derivatives of juglone (II), all showed higher fungicidal activities than II, except compound II-1i, having a long decyl chain (inhibition effect: II-1a-II-1h, II-1j > II > II-1i). The activities of the aryl formyl-substituted derivatives II-1a and II-1e were more prominent, and the 3-position of II substituted derivatives II-1k-II-1m showed higher antifungal activities than II, but the activity level was moderate, except for compound II-1d. The overall activities of the derivatives containing bromine at the 3-position of II (III and III-1a) are poor, indicating that the introduction of a bromine atom at the 3-position of II is very unfavorable to the activity.

Antiviral Activity Result and Structure-Activity Relationship (SAR)
There are many types of plant viruses, and their control is extremely difficult since one virus can infect one or several plants, and one plant can be infected by one or several viruses. There are few practical and efficient antiviral agents for plants. As a commonly used antiplant virus agent, ribavirin can only give an inhibition effect of less than 50% at 500 µg/mL. Therefore, it is particularly important to find new antiviral lead compounds [24,25].
Tobacco mosaic virus (TMV) is the earliest discovered and most deeply studied plant virus. It can infect a variety of Solanaceae plants, and is often used as a model virus for screening new plant antiviral agents [17][18][19].
As shown in Table 3, naphthoquinones I-III, I-1a-I-1h, II-1a-II-1m and III-1a-III-1f were also found to have good anti-TMV activities for the first time. Compounds I-1f, II-1f, II-1g, II-1l, III-1b and III-1d exhibited a similar or slightly higher level of antiviral activities. The introduction of benzenesulfonyl on the 8-hydroxyl can greatly improve the antiviral activities of these compounds. I-1f and II-1f emerged as new antiviral candidates with excellent inhibitory effects.
It can be seen from Table 4 that naphthoquinones I-III, I-1a-I-1h, II-1a-II-1m and III-1a-III-1f showed different degrees of insecticidal activities against various pests, especially against Plutella xylostella. Compounds I-1h, II-1b, II-1e, II-1h, II-1j and III-1c exhibited good mortality against Tetranychus cinnabarinus at a specific concentration (600 µg/mL or 200 µg/mL). The insecticidal activities of II-1b, II-1d, II-1h, III-1c and III-1d against Plutella xylostella were better than those of matrine and rotenone, furthermore, II-1d (phenylpropionionic acid ester of II) and III-1c, with a similar level of insecticidal activities as hexaflumuron, emerged as new insecticide candidates against Plutella xylostella. Table 4. Insecticidal activities of compounds I, II, III, I-1a-I-1h, II-1a-II-1m, III-1a-III-1f, matrine, hexaflumuron and rotenone against eight common crop pests a .    SAR against Plutella xylostella: The modification of the 5-hydroxyl of plumbagin (I) is disadvantageous to its activity. However, the introduction of p-methylbenzoyl, cyclopropyl, phenylpropionyl, n-hexyl or benzyl into the 5-hydroxyl of juglone (II) is beneficial to the improvement of its activity. The introduction of an aryl, alkyl, bromine or o/p-substituted aniline group at the 3-position of juglone (II) leads to the reduction of its activity, the introduction of m-substituted phenyl amino group (III-1c, III-1d) can improve the activity, and the introduction of phenoxy group (III-1f) basically maintains the activity.

Chemicals
The reagents (including ultra-dry dichloromethane) were purchased from commercial sources and were used as received. All anhydrous solvents were dried and purified by standard techniques prior to use. Naphthoquinones plumbagin (I) and juglone (II) were prepared using reported method [16].

Instruments
The melting points of the compounds were tested on an X-4 melting point apparatus (Beijing Tech Instruments Company, Beijing, China) without correction. NMR spectra were obtained through a 400 MHz (100 MHz for 13 C) instrument (Bruker, Billerica, MA, USA) at room temperature with either CDCl 3 or DMSO-d 6 as the solvent. Chemical shifts were referenced to tetramethylsilane as an internal standard, or to solvent peaks of DMSO-d 6 ( 1 H: δ = 2.50 ppm; 13 C: δ = 39.52 ppm) or CDCl 3 ( 1 H: δ = 7.26 ppm; 13 C: δ = 77.16 ppm). The following abbreviations are used to designate chemical shift multiplicities: s = singlet, d = doublet, dd = doublet of doublets, t = triplet, m = multiplet and brs = broad singlet. High-resolution mass spectra were obtained with an Ionspec, 7.0 T Fourier transform ion cyclotron resonance mass spectrometer (Bruker, Saarbrucken, Germany).

Preparation of 2-Bromo-8-hydroxynaphthalene-1,4-dione (III)
A mixture of juglone (4.00 g, 23 mmol), glacial AcOH (60 mL), Br 2 (1.2 mL, 23.4 mmol) in a 250 mL single-neck flask was stirred at room temperature for 15 min, then quenched with crushed ice and filtered. The solid was immediately transferred to a 100 mL singleneck bottle containing preheated ethanol. Then, the reaction mixture was refluxed for 10 min, cooled to room temperature, filtered and air-dried to obtain a reddish-brown solid, yield 80%, mp 172-174 • C. 1 I-1a-I-1g, II-1a-II-1g, III-1a A 0.10 mol/L solution of triethylamine (0.6 mL, 2 equiv.) in ultra-dry dichloromethane was added to a 50 mL round bottom flask containing substrate I, II or III. Then corresponding acyl chloride (2.6 mmol, 1.2 equiv.) was slowly added under ice bath condition. The mixture was stirred at room temperature for 1 h, quenched by H 2 O (100 mL) and then extracted with dichloromethane (50 mL × 3). The combined organic layer was washed with brine, dried over anhydrous Na 2 SO 4 and concentrated. The residue was subjected to column chromatography (petroleum ether: ethyl acetate, 10:1, v/v) to obtain the corresponding product.

Preparation of Compounds I-1h and II-1h-II-1j
To a solution of I or II (0.6 mmol) in acetone was added the corresponding iodide, Me 2 SO 4 or benzyl bromide (2 equiv.) and potassium carbonate (2 equiv.). The mixture was stirred at room temperature overnight and then concentrated. The residue was taken into water (100 mL) and extracted with ethyl acetate (30 mL × 3). The combined organic layer was washed with brine, dried over anhydrous Na 2 SO 4 and concentrated. The residue was subjected to column chromatography (petroleum ether: ethyl acetate, 10:1, v/v) to obtain the corresponding product.