Prospects of Computer-Aided Molecular Design of Coumarins as Ecotoxicologically Safe Plant Protection Agents

Featured Application

Coumarin derivatives are promising candidates for developing novel plant-protection products of the new generation, which meet all requirements of modern integrated pest management.

Abstract

Coumarins are secondary plant metabolites widely distributed in higher plants, bacteria, fungi, and sponges. This great structural diversity of these natural compounds and their synthesized derivatives enables their wide range of pharmacological activities, such as antioxidant; antibacterial; antifungal; anti-human immunodeficiency infection; anti-tubercular; and anti-cancer activities, which were very well reviewed previously. There are also many reports about their effectiveness against plant pathogenic pests (phytopathogenic fungi, bacteria, nematodes, and insects). These secondary metabolites protect environmental enemies and competing plants. However, there is still limited literature on coumarins’ practical applications in agriculture, as well as their effects on beneficial populations of soil organisms. This review summarizes recent knowledge about the effects of natural and synthesized coumarins on phytopathogens and beneficial populations of soil organisms. A separate section is dedicated to poorly reviewed computer-aided molecular design (CAMD) methods of agrochemicals. It also reviewed CAMD techniques to develop low-toxicity and environmentally safe pesticides. Despite the many positive effects of coumarins related to plant protection, they do possess properties harmful to the environment and health. These properties are described in the last section. Prediction of coumarin hazardous properties using a quantitative structure-activity relationship (QSAR) approach was also reviewed.

1. Introduction

The control of fungal pathogens and pests has vital importance for the protection of crops and food provision worldwide. Organic compounds are still the major active components of plant protection products. Although pesticides in agronomy exert powerful effects on disease control management, the long-term their unreasonable use led to serious environmental problems inducing successive pesticide-resistant pathogen development, disrupting soil ecological balance, and causing environmental and human health. There are among the most common pollutants in one-fifth of the Earth’s land. Plant protection products pollute terrestrial and aquatic ecosystems, and every year millions of people are exposed to pesticides [1]. Daily exposure to pesticides has numerous consequences for human health. Pesticide exposure has numerous health consequences. Pesticides could induce tumors in the liver, lungs, stomach, kidneys, skin, and stomach [2,3]. Also, exposure to pesticides has been associated with dermatological, gastrointestinal, neurological, respiratory, reproductive, and endocrine symptoms of diseases [4]. Drug resistance and environmental and health hazards indicate the urgent need for novel active compounds. These compounds must be highly specific with a broad-spectrum mode of action, as well as environmentally and toxicologically acceptable [5]. In order to limit pesticide harmful effects, the European Parliament and the Council issued Directive 2009/128/EC that promotes integrated pest management with priority for plant protection products. This Directive has the fewest side effects on human health, non-target organisms, and the environment [6]. Coumarins are secondary plant metabolites widely distributed in higher plants, bacteria, fungi, and sponges [7]. Depending on the substitution of the 1-benzopyran-2-one skeleton, coumarins can be divided into several types: simple coumarins, furanocoumarins, pyranocoumarins, and other coumarins (Figure 1). This great structural diversity of these natural compounds and their synthesized derivatives enables their wide range of pharmacological activities, such as antioxidant [8,9,10]; antibacterial [11,12,13,14,15,16,17]; antifungal [18,19,20]; anti-human immunodeficiency virus (HIV) infection [21,22]; anti-tubercular [23]; cytotoxicity [24]; and anti-cancer activities [25,26,27,28,29].

There are also many reports about their effectiveness against plant pathogenic pests. These secondary metabolites are natural protection agents against environmental enemies and competing plants, therefore they are called allelochemicals. Allelochemicals are biocommunicators that act in a natural mixture of active components, while single compounds are not active [7,30,31]. Coumarin derivatives have been reported as strong agents against phytopathogen fungi, such as: Botrytis cinerea [32]; Moniliophthora perniciosa [33]; Colletotrichum gloeosporioides, Fusarium oxysporum, Valsa mali [34]; Macrophomina phaseolina and Sclerotinia sclerotiorum [35,36]. Coumarins have also antimicrobial potential against phytopathogens: Ralstonia solanacearum [37]; Agrobacterium tumefaciens [38]; Pseudomonas aeruginosa [39]. Nematicidal activity has been demonstrated for several simple coumarins, furanocoumarines, and dicoumarols, and their skeletons have been used for the development of new efficient nematicides against plant parasitic nematodes: Meloidogyne incognita, Ditylenchus destructor, Bursaphelenchus xylophilus, Bursaphelenchus mucronatus, and Aphelenchoides besseyi [40,41].

Despite numerous pieces of evidence in the literature about coumarins’ biological effects on plant pathogenic pests, there is still scarce information about their practical applications in agriculture. In addition, there is scarce information about their effects on beneficial soil organism populations [35,36].

The traditional approach to plant protection product discovery is an expensive and time-consuming process. This process includes many steps that include: the synthesis of tens of thousands of compounds, followed by biological evaluation, greenhouse evaluation, field experiments, toxicology, and environmental evaluation, approval of substances, and manufacture and commercialization of new plant protection products [42]. Computer-aided molecular design (CAMD) is a rational approach used for screening, optimization, and design of highly potent agents for plant protection. CAMD is a promising technique used in drug design and pesticide discovery. Due to advances in biochemistry and structural biology [43,44,45], it has become a promising rational approach in agrochemistry. During the past decade, many reports described the roles of in-silico approaches in novel molecule development. In silico techniques like quantitative structure-activity relationships (QSAR), pharmacophore, docking, and virtual screening play crucial roles in the design of “better” molecules that may later be synthesized and biologically assayed. QSAR techniques provide insight into the relationships between chemical structure and biological activity. They present an alternative pathway for the design and development of new molecules with improved activity.

Using this relationship, the QSAR model predicts novel compounds’ activity [46]. The QSAR approach aims to form a quantitative relationship between biological activity (or toxicity) and the structure of each chemical. The general purpose of the QSAR study is to predict the biological activity and physicochemical properties and rationalize the mechanisms of action within a series of chemicals [45]. Also, QSAR has the potential to estimate the risks of chemicals for the environment and human health, reducing time, monetary cost, and necessary animal testing [47]. In a mathematical sense, activity (toxicity) is a function of physiochemical and/or structural properties:

Activity (toxicity) = f (physiochemical properties and/or structural properties).

Information on chemical structure is encoded by molecular descriptors, numerical values obtained by various mathematical algorithms. In QSAR, activity data are correlated with molecular descriptors using a statistical approach. Various regression- and classification-based methods are used to derive a mathematical correlation between structural and activity/toxicity information. Regression-based methods are multiple linear regression (MLR) and partial least squares (PLS), Neural Networks (NN), and Support Vector Machine (SVM). Classification-based approaches are linear discriminant analysis (LDA) and cluster analysis [48,49]. Recently, regression methods were replaced by various mathematical methods that improved QSAR studies’ performance, such as Gene Expression Programming (GEP), Project Pursuit Regression (PPR), and Local Lazy Regression (LLR) [50].

The 3D-QSAR analysis permits correlations between a series of diverse molecular structures and their biological functions at a particular target. Introducing three-dimensional parameters allows the identification of the pharmacophoric arrangement of molecular features in space. This method takes into account the numerous field properties of each molecule, such as steric, lipophilic, and electrostatic interactions [51]. CoMFA is a widely used 3D QSAR method. [52]. The CoMFA model focuses on ligand properties favorable and unfavorable for receptor–ligand interactions. Molecular descriptors in CoMFA models are energies calculated by placing aligned ligands on the energy grid. Resultant energies correspond to ligand electrostatic and steric properties. CoMFA models are generated by correlating descriptors in 3D space with a biological response by PLS and validated by cross-validation. The CoMSIA method incorporates 3D information for the ligands by searching for conformation or spatial orientation of molecules capable of being modified into better specific ligands [53].

The agencies and official organizations in Europe that are involved in pesticide control, such as ECHA, European Food Safety Authority (EFSA), Joint Research Centre (JCR), European Centre for Ecotoxicology and Toxicology of Chemicals (ECTOC), Organisation for Economic Co-operation and Development (OECD) have promoted QSAR as an important tool in the pesticide risk assessment process. These organizations are responsible for the systematization and harmonization of computation tools, as well as the standardization of QSAR model validation [54]. The OECD announced guidelines that a valid QSAR model should have: a defined endpoint; an unambiguous algorithm; a defined domain of applicability; appropriate measures of goodness of fit, robustness, and predictivity; and a mechanistic interpretation [55]. CAMD has been generally accepted and extensively applied in ecotoxicological modeling and design of agrochemicals. This is due to its high efficiency in the design of novel compounds, saving both time and economic costs in large-scale experimental synthesis and biological tests. According to the REACH (Registration, Evaluation, and Authorization of Chemicals) guidelines of the European Chemicals Agency (ECHA) from 2011, animal tests can be avoided if the hazardous properties of a substance can be predicted using computer models of the QSAR approach [56].

Molecular docking is a valuable tool for drug discovery, but it has also been successfully used in the discovery of novel plant protection agents. The molecular docking technique reveals the mechanism of action of a potential drug or pesticide at the atomic level. This method allows insight into the interactions between a small molecule (ligand) and the binding site of target proteins (receptors) related to biological activity. Molecular docking involves the prediction of the ligand orientation within the binding site of the protein, as well as the evaluation of binding affinity between the receptor and the ligand by the scoring function (binding energy) [57,58]. The molecular dynamic simulation study provides more insights into ligand-receptor complex dynamics and structural properties. It evaluates the docked complex’s thermal and structural stability [59].

This paper reviews a recent advance in research into the biological activities of coumarins related to plant protection. It also discusses the use of CAMD techniques for the development of new pesticides.

2. Naturally Occurring Coumarins and Their Role in Plants

2.1. Biosynthesis and Distribution of Coumarins in Nature

As a family of benzopyrones (1,2-benzopyrones or 2H-1-benzopyran-2-ones), coumarins are widely distributed throughout nature. The benzopyrone framework is an electron-rich system with favorable charge-transport properties. Therefore, they are characterized by UV light absorption, resulting in a characteristic blue fluorescence. Besides their role in iron mobilization and uptake by plant roots, natural coumarins have a role in environmental stress responses. Also, they participate in the defense against plant pathogens, acting as phytoanticipins, or phytoalexins, which are produced upon infection and are typically not present in healthy tissues. Their increased accumulation on plant tissue is a response to the application of a molecule that triggers the hypersensitivity response in the plant (elicitor) or plant hormones [60].

Since coumarins act as signaling molecules that regulate the interaction between commensals, pathogens, and plants, they could be used as biopesticides. Endophytes such as bacteria or fungi have the ability to produce some of the secondary metabolites. Thus, coumarin isofraxidin was synthesized by the fungal endophyte Biscognia uxiacylindrospora, and identified in host plants Siberian ginseng (Acanthopanax senticosus) and Apium graveolens [61]. Infection by pathogenic bacteria can induce the synthesis of coumarin compounds around plant roots and stems to immunize the plant against pathogen invasion and propagation. Inoculation of Arabidopsis thaliana by the plant pathogen Dickeya spp. strains induced coumarin accumulation and plant resistance to pathogens [62].

Treatment of ryegrass (Lolium multiflorum Lam.) with coumarins stimulates the colonization of beneficial flora in the root rhizospheric microbial community. Rhizosphere microorganisms enhance plant nutrient absorption, coordinate growth, and improve environmental adaptability [63]. The secretion of coumarins from Arabidopsis thaliana roots under soil iron deprivation stimulates the bacterial root microbiota to improve plant adaptation to iron-limiting soils [64]. A strain of Aspergillus synthesizes 4-hydroxycoumarin and dicoumarol [65,66].

Naturally occurring coumarins are mostly distributed in plants seeds, flowers, leaves, roots, and stems in more than 40 different families including Apiaceae, Rutaceae, Asteraceae, Fabaceae, Oleaceae, Moraceae, and Thymelaeaceae [67]. The natural coumarins are derivatives of 2H-1-benzopyran-2-one, and they are classified into six groups: simple coumarins; furanocoumarins (linear and angular type); dihidrofuranocoumarins; pyranocoumarins (linear and angular type); phenylcoumarins; and bicoumarins (Figure 1) [68]. Angiospermaes are rich in simple coumarins, followed by furanocoumarins and pyranocoumarins. The most diverse sources of coumarins are plants families Apiaceae and Rutaceae containing five different types of coumarin derivatives (simple coumarins, lineal furocoumarins, angular furocoumarins, lineal pyranocoumarins, and angular pyranocoumarins) [30].

Simple coumarins are derived by biosynthesis from shikimic acid, via cinnamic acid. They are the most common in all angiosperms, especially in Oleaceae and Asteraceae. A key step in the biosynthesis of simple coumarins is ortho-hydroxylation of cinnamates that branch off from lignin biosynthesis. The gene required for the production of feruloyl coenzyme A (CoA) is CCoAOMT1. It also participates in the biosynthesis of lignin and simple coumarin scopoletin in Arabidopsis roots. A key enzyme involved in the biosynthesis of simple coumarins is 2-oxoglutarate-dependent dioxygenase (2OGD), which is encoded by 2OGD genes. Thus, the gene AtF6_H1 encodes otho-hydroxylase activity to feruloyl coenzyme A, and its deficient mutation causes a significant reduction in scopolin accumulation. 2OGD gene RgC2′H formation of furanocoumarins in Ruta graveolenes [69]. Beta-glucosidase (BGLU) genes are regulatory genes responsible for coumarin biosynthesis in Melilotus species and differences in their expression result in coumarin content diversity among Melilotus species [70]. Coumarin biosynthesis genes are also activated after foliar pathogen infection to create a microbial soil-borne legacy that primes plants for defenses. Coumarin biosynthesis genes, such as root-specific transcription factors myb72 and f6′h1 are also activated in the plant Arabidopsis thaliana after foliar pathogen infection with downy mildew pathogen Hyaloperonospora arabidopsis (Hpa), for the creation of a microbial soil-borne legacy (SBL) that primes plants for defenses [71]. Since scopoletin selectively inhibits the soil-borne fungal pathogens Fusarium oxysporum and Verticillium dahliae, the study of Stringlis et al. [72] has shown that during infection, probiotic root-associated microbes stimulate MYB72-dependent excretion of scopoletin. Armillarisin A (3-acetyl-5-hydroxymethyl-7-hydroxycoumarin) is a coumarin derivative extracted from the fungus Armillariella tabescens (Scop. ex Fr.) Sing [73]. Chlorinated coumarins, 6-chloro-4-phenyl-2H-chromen-2-one and ethyl 6-chloro-2-oxo-4-phenyl-2H-chromen-3-carboxylate, were identified in the polypore mushroom Fomitopsis officinalis [74].

2.2. Classification of Naturally Occurring Coumarins and Their Role in Plant Protection

2.2.1. Simple Coumarins

Simple coumarins act in plants’ interaction with biotic and abiotic environmental stress factors. Secretion of iron-mobilizing coumarins by plant roots is a crucial factor for improving iron bioavailability in crops, enabling them to grow in iron-depleted soils. In Arabidopsis, three coumarins with iron-mobilizing properties are found: fraxetin (7,8-dihydroxy-6-methoxycoumarin), sideretin (5,7,8-trihydroxy-6-methoxycoumarin), and esculetin (6,7-dihydroxycoumarin) (Figure 2 Their catechol moiety (two neighboring hydroxyl groups) is thought to be a crucial structural feature for iron mobilization in soil [75]. Scopoletin (7-hydroxy-6-methoxy coumarin) (Figure 2) is a simple coumarin that occurs in Arabidopsis thaliana [76], and many other plants [77]. Physiologically, scopoletin protects against stress. Thus, it was proved that scopoletin accumulates in Arabidopsis leaves after the attack of the fungus Phakopsora pachyrhizi, which causes Asian soybean rust disease [78].

In tobacco plants, scopoletin and its β-glucoside, scopolin, have physiological roles against stress, for example during tobacco mosaic virus infiltration [79]. Surangib B from Mammea longifolia inhibits mycelial growth of Rhizoctonia solani and Botrytis cinerea [80]. Umbelliferone (7-hydroxycoumarin) is a phytoalexin widely distributed within the Rutaceae and Apiaceae (Umbelliferae) families. It is an important phytoalexin that protects plants from pathogenic fungi, such as Fusarium culmorum [81], and the aerobic, Gram-negative, plant-pathogenic bacterium Ralstonia solanacearum [82].

Yang et al. [83] studied the antimicrobial activity of 18 natural compounds against Ralstonia solanacearum, the bacterium responsible for tobacco, tomatoes, and potatoes wilting in subtropical regions. The research showed that four coumarins, daphnetin, esculetin, umbelliferone, and xanthotol demonstrated stronger antibacterial effects than the standard treatment with thiadiazole and copper. A more detailed analysis showed that the enhanced antibacterial activity is due to the substitution at positions C-6, C-7, and C-8 of the coumarin nucleus. For this reason, they tested the activity of the hydroxycoumarins umbelliferone, esculetin, and daphnetin in the concentration range from 10 to 100 mg/L. Daphnetin (OH groups at positions C-7 and C-8) proved to be the most effective, esculetin (OH groups at positions C-6 and C-7) was somewhat weaker, while umbelliferone (OH group at positions C-7) showed the weakest activity. Thus, treatment of tobacco roots with umbelliferone prior to infection with R. solanacearum significantly reduced R. solanacearum biofilm formation, increasing resistance to disease [84]. Application of an elicitor of coumarin biosynthesis, salicylic acid to the roots of chamomile (Matricaria chamomilla) resulted in the accumulation of ambelliferone and herniarin (7-methoxy coumarin) in the leaves [85]. Herniarin suppressed R. solanacearum bacterial growth by destroying the bacterial cell membrane [86]. Also, plant-derived 6-methylcoumarin showed inhibitory effects against R. solanacearum, and suppressed tobacco bacterial wilt [87]. Derivatives of 3,4-dihydroisocoumarin isolated from endophytic fungus Lophiostoma sp., displayed antibacterial activities against Bacillus subtilis, Agrobacterium tumefaciens, R. solanacearum, and Xanthomonas vesicatoria [88].

Increased biosynthesis of coumarins, ayapin and scopoletin, has been observed in sunflowers (Helianthus annuus L.) during the attack of the sunflower beetle, Zygogramma exclamationis, which resulted in distracting further feeding of the beetle [89]. The coumarin (2H-1-benzopyran-2-one) proved effective against the green peach aphid Myzus persicae and friendly to the natural enemy of aphids, Harmonia axyridis and soil invertebrates, Eisenia fetida. The study implied that coumarin can be recommended as a selective and effective botanical aphicide friendly to non-target organisms. However, the environmental safety of a given insecticide must be estimated with further tests to clarify the mechanism of its action and efficacy [90]. Recently, five simple coumarin-based scaffolds (limetin-derivatives) were identified in Citrullus lanatus seeds, which possess significant bactericidal and fungicidal potential [91].

2.2.2. Furanocoumarins

Furanocoumarins are composed of a furan ring fused to a coumarin core (Figure 3).

They are mostly present in plant families, including Apiaceae, Fabaceae, Moraceae, and Rutaceae. Their increased level in plants is a response to stress conditions, such as exposure to pathogenic fungi or to physical damage caused by occasional lesions or insect bites. They are involved in plant defense, acting against microorganisms, nematodes, phytophagous insects, herbivores, and plant competitors [92]. Linear furanocoumarins, such as psoralen, bergapten, isopimpinellin, and xanthotoxin, together with the angular dihydrofuranocoumarin athamantin, are antifeedants. Peucedanin (Figure 3) inhibits the growth of neonate larvae of Spodoptera litlis (Boisduval) (Lepidoptera: Noctuidae) [93]. Plants are a prominent source of novel nematicidal chemicals. Thus, furanocoumarins 8-geranyloxy psolaren, imperatorin, and heraclenin from the root extract of Heracleum candicans Wall., exhibited nematicidal effects against Bursaphelenchus xylophilus (Steiner et Buhrer) Nickle and Pangrellus redivivus (Linn.) [94]. Also, bergapten and psoralen extract from Ficus carica L. leaves showed strong nematicidal activity against pine wood nematode (PWN), Bursaphelenchus xylophilus. Their nematicidal mechanism is probably based on the inhibition of amylase, cellulase, and acetylcholinesterase [95]. Ethanol extract from Chinese medicinal herb Notopterygium incisum rhizomes possessed strong nematicidal activity against two species of nematodes, Bursaphelenchus xylophilus and Meloidogyne incognita. The extract contained two furanocoumarins, colombienetin and isoimperatorin [96]. Essential oil and methanol extracts of parsley (Petroselinum crispum) [97], as well as ethanol extracts from Angelica pubescens Maxim. f. biserrata Shan et Yuan roots [98], exhibited promising nematicidal activity as a source of nematotoxic furanocoumarins.

A number of furanocoumarin compounds isolated from Semenovia transiliensis shoots have herbicidal activities [31]. Citrus plants produce simple coumarins and furanocoumarins to cope with herbivorous insects and pathogens [99]. Ramirez-Pelayo et al. [100] studied coumarin derivatives found in citrus peels. They isolated six coumarins (5-geranyloxy-7-methoxycoumarin, bergamottin, bergapten, isopimpineline, citropten, and oxypeucedanin hydrate) and tested their antifungal activity against Colletotrichum sp., a fungus causing fruit anthracnose. Their activity was compared with the simpler coumarins, umbelliferone, scoparone, and scopoletin. The test results showed that all six coumarins inhibited the growth of Colletotrichum sp. mycelia, and among them, bergapten and citropten proved to be the most effective. The research concluded that there is a synergistic effect between the individual coumarin components in the citrus peel extract.

2.2.3. Dihydrofuranocoumarins

The presence of dihydrofuranocoumarins in all plant parts, and especially the roots, is responsible for plants’ poisonous properties, such as Opopanax chironium (Apiaceae) [101] and Opopanax hispidus (Friv.) Griseb. [102]. It was found that dihydrofuranocoumarin xanthoarnol from the plant Xanthoxylum arnottianum (Rutaceae) showed an inhibitory effect on the germination of conidia of the parasitic fungus [103].

2.2.4. Phenylcoumarins

4-Phenylcoumarins protect plants against pests and fungi. Thus, 5,7-dimethoxy-4-p-methoxyphenylcoumarin and 5,7-dimethoxy-4-phenylcoumarin (Figure 4) were found in the microorganism Streptomyces aureofaciens, which was isolated from ginger root tissues (Zingiber officinale Rosc. (Zingiberaceae) were active against phytopathogenic fungi [104].

The naturally-occurring 3-phenylcoumarins were identified in plant species, mostly in the family Fabaceae, such as: mucodianin A from Mucuna birdwoodiana [105]; pterosonin A-F from heartwood of Pterocarpus soyauxii [106]; Sphenostylis marginata [68]; Pterocarpus soyauxii [107]; and selaginolide A from Vietnamese medicinal plant Selaginella rolandi-principis (Selaginellaceae) [108]. There is no evidence about their role in plants.

2.2.5. Pyranocoumarins

Pyranocoumarins are rare secondary metabolites of plants that contain a pyran core condensed with coumarin. These substances are distributed widely among the plant families Umbelliferae and Rutaceae. Although pyranocoumarins in the plant are very poorly studied, some studies indicate their protective role against phytopathogenic organisms [109]. However, pyranocoumarin isolated from the Rutaceae tree (Staurantus perforatus), xanthyletin, has shown significant phytotoxic effect on seed germination and root growth of Amarathus hypochondriacus (Amaranthaceae) [110]. Among the pyranocoumarins isolated from the roots of Ferulago campestris (Apiaceae), aegelinol and grandivittin have been shown to have cytotoxic properties [111]. Pyranocoumarin seselin isolated from Clausena anisata (Rutaceae) leaves acts as an antifeedant against Lucilia cuprina larvae [112].

2.2.6. Bicoumarines

Bicoumarines have been isolated from plants Triphasia trifolia (Rutaceae) [113], Dysoxylum parasiticum (Osbeck) Kosterm (Meliaceae) [114], and Pleurospermum rivulorum (Umbelliferae) [115]. The best-researched bicoumarin is dicoumarol (Figure 5). Dicumarol is generated by the hydroxylation of the 4-position of the coumarin. This is followed by capturing of a molecule of formaldehyde, and subsequently by condensation with another molecule of 4-hydroxycoumarin. Finally, the enolization of the keto group forms dicumarol [68]. Dicumarol is discovered as constituent of sweet clover hay that caused the death of cattle due to bleeding disorders. Dicoumarol is an anticoagulant that acts as a vitamin K antagonist [116]. The dicoumarol, also formed by bacterial fermentation of yellow sweet clover, was isolated for the first time from the decomposed leaves of Melilotus albus (Fabaceae/Leguminosae) [68].

The presence of natural coumarins in different species of plants and microbes, and their biological effects are summarized in

Table 1. The presence of natural coumarins in different species of microbes, spongi and plants with their biological effects.

Phylum/ Famillies	Species	Group of Coumarins	Specific Compounds	Known Biological Activities	Ref.
Bacteria
Streptomyces	Streptomyces roseochromogenes var. oscitans	3-amino-4,7-dihydroxycoumarins	clorobiocin, novobiocin, coumermycin	antibacterial	[15]
Porifera
Axinellidae	Axinella cf. corrugate	simple	esculetin-4-carboxylic acid esters	anti-SARS-CoV	[22]
Fungi
Pleosporaceae	Alternaria alternata	simple	isofraxidin	antibacterial	[61]
Trichocomaceae	Aspergillus fumigatus Fresenius	simple, bicoumarins	4-hydroxycoumarin, dicoumarol	biosynthesis of coumarin	[65,66]
Physalacriaceae	Armillariella tabescens	simple	armillarisin A	choleretic	[73]
Fomitopsidaceae	Fomitopsis officinalis	simple	6-chloro-2-oxo-4-phenyl-coumarins	anti-TBC	[74]
Lophiostomataceae	Lophiostoma sp. Sigrf10	3,4-dihydroisocoumarin	lophiostomin derivatives	antifungal, antibacterial	[88]
Plants
Citrus	C. maxima, C. medica, C. reticulata, C. micrantha	simple, furanocoumarin		unknown	[99]
	Citrus sinensis, C. reticulata, C. aurantifolia	simple, furanocoumarin	limettin, isopimpinellin, psoralen, bergamottin	antifungal	[100]
Cucurbitaceae	Citrullus lanatus	simple	derivates of 5,7-dimethoxycoumarin	antimicrobial	[91]
Apiaceae	Ferulago campestris	pyranocoumarin	aegelinol, grandivittin,	cytotoxicity	[111]
(or Umbelliferae)		furanocoumarin	bergapten, felamidin, isoimperatorin	antimicrobial, antioxidant	[16]
	Notopterygium incisum	dihidrofuranocoumarin	columbianetin	nematicidal	[96]
		linear furanocoumarin	isoimperatorin	nematicidal
	Petroselinum crispum	furanocoumarins	xanthotoxin, psoralen, bergapten	nematicidal	[97]
	Angelica pubescens Maxim. f. biserrata Shan et Yuan	simple, dihidrofuranocoumarin,	osthole, columbianadin	nematicidal	[98]
		furanocoumarin	bergapten, xanthotoxin	nematicidal	[115]
	Pleurospermum rivulorum	bicoumarin	rivulobirins	unknown
	Opopanax hispidus(Friv.) Griseb.	dihydrofuranocoumarin	3′-isobutyryl-3′-hydroxymarmesin	unknown	[102]
		simple, furanocoumarin	officinalin, oreoselon, peucedanin,	unknown
	Peucedanum sp.	simple, furanocoumarin	ostruthin, osthol; isoimperatorin	insecticidal	[93]
		dihydropyranocoumarin	xanthalin, peuarenarin	insecticidal
		dihydrofuranocoumarin	athamantin, columbianadin	insecticidal
	Semenovia transiliensis	furanocoumarin	Imperatorin, xanthotoxin	herbicidal	[31]
	Heracleum candicans Wall.	uranocoumarin	8-geranyloxy psolaren, imperatorin, heraclenin	nematicidal	[94]
Fabaceae	Melilotus officinalis	simple	dihydrocoumarin	cytotoxicity	[24]
		bicoumarin	dicoumarol	anticoagulant	[116]
	Mucuna birdwoodiana	phenylcoumarin	mucodianin A	unknown	[107]
	Sphenostylis marginata	phenylcoumarin	sphenostylisin A	anticancer	[68]
	Pterocarpus soyauxii	phenylcoumarin	pterosonins	anticancer	[106]
	Millettia thonningii	pyranocoumarin, furanocoumarin	robustic acid, thonningine-C	antifungal	[20]
Solanaceae	Nicotiana tabacum	simple	scopolin, scopoletin	antiviral	[79]
Lamiaceae	Baikal skullcap	simple	7.8-dihydroxy-4-methylcumarin	antibacterial	[17]
Brassicales	Arabidopsis thaliana	simple	scopoletin	antifungal	[78]
Moraceae	Ficus carica	furocoumarin	bergapten, psoralen	nematicidal	[95]
Meliaceae	Dysoxylum parasiticum (Osbeck) Kosterm	bicoumarin	bidysoxyletine	unknown	[114]
Rutaceae	Triphasia trifolia	simple, furocoumarin	umbelliferone, isopimpinellin,	unknown	[113]
	Xanthoxylum arnottianum	dihydrofuranocoumarin	xanthoarnol	antifungal	[103]
	Staurantus perforatus	pyranocoumarin	xanthyletin	phytotoxic	[110]
	Ruta angustifolia	furocoumarin, dihydrofuranocoumarin	chalepensin, chalepin	anticancer, antiviral	[28]
	Clausena anisata	pyranocoumarin	seselin	antifeedant	[112]
Thymelaeaceae	Wikstroemia indica (L.)	bicoumarin	daphnoretin	antiviral, antitumor	[29]
Calophyllaceae	Mammea longifolia	simple	surangib B	antifungal	[80]

. It is evident that the diversity and structural complexity of the coumarins constitute is a consequence of higher plant evolution. Simple coumarins are the most common in fungi and all angiosperms. They exhibit a wide range of biological effects related to plant protection from pathogen microbes, fungi, and nematodes. Furanocoumarin is the second most prevalent type of coumarin. Furanocoumarins are found in the family Citrus, Apiaceae, and Rutaceae, with most of them showing nematicidal properties. Pyranocoumarins are present in Apiaceace and Fabaceae, where they exhibit antifungal, phytotoxic, and antifeedant effects. Phenylcoumarins are the most abundant in the plant family Fabaceae, but there is no literature on their functions in plants.

3. Synthetic Coumarin Derivatives in Plant Protection

Since naturally occurring coumarins have shown biological and allelopathic potential in various organisms, their structural core is widely used as a scaffold in agricultural chemicals. Thus, osthol (7-methoxy-8-prenylcoumarin) (Figure 6), is a natural coumarin and lead compound that has been developed into commercial fungicides Osthol EW in China which exhibits effective activity against Magnaporthe oryzae, Sphaerotheca fuliginea, Fusarium graminearum, and Sphaerotheca fuliginea [32].

Synthetic coumarins are strong antifungal agents. Several coumarin-3-carboxamides/hydrazides have been shown to have antifungal activities against Botrytis cinerea, Colletotrichum capsica, Rhizoctorzia solani, Cucumber anthrax, and Alternaria cucumerina leaf spot [117]. These compounds exhibited equivalent antifungal activity to broad-spectrum carboximide fungicides Boscalid, against Botrytis cinerea. For example, compound 2-oxo-2H-chromene-3-carboxylic acid N-(2-chloro-phenyl)-hydrazide (Figure 6), exhibited much more effective activity (1.80 μg/mL) than the Boscalid (2.98 μg/mL) against Rhizoctorzia solani. A structure-activity relationship analysis revealed the following conclusions: the replacement of the amide bond with a hydrazide bond led to an increase in antifungal activity; derivatives with an amide bond showed better activity against B. cinerea and R. solani, compounds with electron-withdrawing groups had no effect on A. cucumerina; chlorinated and fluorinated phenylhydrazine derivatives were effective against C. anthrax.

Sodium 3-hydroxycoumarin (Figure 6), inhibited a causal agent of witches’ broom disease in Theobroma cacao L., Moniliophthora perniciosa fungus [33]. Copper (II) complexe with coumarins, [L₂Cu(OAc)], have showed strong inhibition against both, pathogen fungi Alternaria alternata, and Gram-positive bacteria (Bacillus subtilis). The activity of this complex was based on the leakage of sugars and electrolytes from microbial cells accompanied by collapsed hyphae of A. flavus and membrane blebbing of B. subtilis [118]. In study of Montagner et al. [119], 40 coumarins were tested against the most economically important phytopathogenic fungi Fusarium solani.

The most potent inhibitors of fungal growth were 6-nitrocoumarin (Figure 6) among the monosubstituted coumarins and natural prenylated coumarin, 7-hydroxy-8-prenylcoumarin or osthenol (Figure 6) (with a minimal inhibitory concentration (MIC) of 125 μg/mL, both). A series of synthesized 8-substituted coumarin derivatives exhibited moderate to high antifungal activity against four phytopathogenic fungi: Botrytis cinerea, Colletotrichum gloeosporioides, Fusarium oxysporum, and Valsa mali. The strongest fungal inhibition has been demonstrated by 8-chloro coumarin and ethyl 8-chloro-coumarin-3-carboxylate (Figure 6) [34].

Study of Kovač et al. has shown that 7-substituted-coumarinyl thiosemicarbazides possess a better antifungal activity than 4-substituted ones against mycotoxin producer, Aspergillus flavus Link [120].. The 38 simple coumarin derivatives were synthesized in environmentally safe organic solvents [121], and their antifungal activities were assayed on four cultures of phytopathogenic fungi (Fusarium oxysporum f. sp. lycopersici, Fusarium culmorum, Macrophomina phaseolina, and Sclerotinia sclerotiourum). In order to validate their environmental impact, the compounds were assessed against soil-beneficial nematodes and bacteria. Coumarin derivative, which possesses 3-CN and 6-OH groups at the coumarin scaffold, has shown antifungal activities against all fungi tested, is nontoxic, and is not harmful to beneficial bacteria and nematodes [35]. Coumarinyl Schiff bases proved to be promising candidates for inhibition of M. phaseolina, especially derivatives with an aromatic nucleus substituted with a bromine atom (71.51% inhibition), or a methoxy group (70.36% inhibition) (Figure 7).

Also, neither compound exhibited inhibitory effects against two beneficial bacteria (Bacillus mycoides and Bradyrhizobium japonicum) and two entomopathogenic nematodes (Heterorhabditis bacteriophora and Steinernema feltiae) [36]. Among the novel series of 4-methylumbelliferone derivatives, coumarin esters exhibited significant inhibitory activity against Botrytis cinerea, especially those without hydroxyl groups. A phenolic aldehyde with hydroxyl group in ortho position had showed the most effective activity against Fusarium oxysporum f. sp. lycopersici [122]. Coumarin derivatives synthesized using Brønsted acidic pyridinium-based ionic liquid displayed antifungal activities against M. phaseolina comparable to reference fungicide mancozeb [123].

Plant pathogenic bacteria invade host plants through root wounds, and colonize and multiply profusely in living plant tissues. Disease symptoms are pretty characteristic of the pathogen/host combination, but infection is usually followed by wilting and death of the host plant. Infection reduces plant growth and yield and lowers product quality. Ralstonia solanacearum is one of the most devastating plant bacterial pathogens that affect tobacco production [37]. 3-Acetyl coumarin and benzo-4-methyl coumarin have shown bactericidal activity against bacteria Erwinia amylovora and R. solanacearum [124]. Novel chalcone derivatives containing a coumarin moiety exhibited excellent antibacterial activity against R. solanacearum [125]. Feng et al. [126] designed and synthesized an isopropanolamine-decorated coumarin derivative that exhibited better activity against Xanthomonas oryzae pv. oryzae (Xoo) than standard bactericide, bismerthiazol.

Plant parasitic nematodes (PPN) cause yield losses by disrupting water and nutrient transport and acting as vectors for viruses [127]. Due to environmental side effects and health concerns, many synthetic nematicides have been banned (Directive 91/414/EEC) [128], against the phytopathogenic nematode, Bursaphelenchus xylophilus, and industry hardly supports the development of novel nematicides. Nematicides that have been used before the establishment of the Environmental Protection Agency (EPA) were soil sterilants, organophosphates or carbamates (neural toxins), and animal health drugs (abamectin), therefore many of them have been banned [129]. Currently, there are only a few nematicides left in use, and repeated applications of the same formulation are inevitable, leading to resistance in nematodes. Several new coumarin derivatives reported activities against plant-parasitic nematodes. Alkoxycoumarins, and especially 5-ethoxycoumarin, exhibit high nematicidal activity phytopathogenic nematode B. xylophilus [130]. It has been shown that tin(IV) complexes are more effective than their parent ligands, 3-formyl-4-chlorocoumarin semicarbazones (L1H) and 3-formyl-4-chlorocoumarin thiosemicarbazones (L2H) [131]. Also, newly synthesized complexes of lanthanide(III) with 3-formyl-4-chlorocoumarin hydrazinecarbothioamide (L1H) and 3-formyl-4-chlorocoumarin hydrazinecarboxamide (L2H) have been found to be more active against M. incognita than the parent ligands themselves [132]. A series of coumarin derivatives were synthesized with targeted derivatization of the C-4 and C-7 hydroxyl groups. They were assayed for nematicidal activity against plant-parasitic nematodes. The modification of the hydroxyl at C4 and C7 positions led to the identification of promising lead compounds against M. incognita, Ditylenchus destructor, Bursaphelenchus mucronatus, and B. xylophilus. The most effective analog was one whose structure combines a coumarin moiety, bromine atoms, and a butyl chain [41]. In

Table 2. The biological activities of synthetic coumarins related to the plant protection.

Coumarin Derivatives	Biological Activity	Phytopathogenic Organism	Reference
coumarin-3-carboxamides/hydrazides	antifungal	Botrytis cinerea, Colletotrichum capsica, Rhizoctorzia solani, Cucumber anthrax, and Alternaria leaf spot	[117]
sodium 3-hydroxycoumarin	antifungal	Moniliophthora perniciosa	[33]
copper (II) complexe with coumarins, [L2Cu(OAc)]	antifungal antibacterial	Alternaria alternata Bacillus subtilis	[118]
8-substituted coumarins	antifungal	Botrytis cinerea, Colletotrichum gloeosporioides, Fusarium oxysporum, Valsa mali	[34]
7-substituted-coumarinyl thiosemicarbazides	antifungal	Aspergillus flavus Link	[120]
-CN and 6-OH simple coumarins	antifungal	Fusarium oxysporum f. sp. lycopersici, Fusarium culmorum, Macrophomina phaseolina, and Sclerotinia sclerotiourum	[35]
coumarinyl Schiff bases	antifungal	Macrophomina phaseolina	[36]
4-methylumbelliferone, coumarin esters	antifungal	Fusarium oxysporum f. sp. lycopersici	[122]
4-(chloromethyl)-7-hydroxycoumarin; 4-(chloromethyl)-7,8-dihydroxycoumarin	antifungal	Macrophomina phaseolina	[123]
benzo-4-methyl coumarin	antibacterial	Erwinia amylovora, Ralstonia solanacearum	[124]
isopropanolamine coumarin derivative	antibacterial	Xanthomonas oryzae pv. oryzae (Xoo)	[126]
5-ethoxycoumarin	nematicidal	Bursaphelenchus xylophilus	[130]
tin(IV) complexes with 3-formyl-4-chlorocoumarin hydrazinecarbothioamide and 3-formyl-4-chlorocoumarin thiosemicarbazone	nematicidal	Meloidogyne incognita, Ditylenchus destructor, Bursaphelenchus mucronatus, B. xylophilus	[41]

, we summarized the biological activities of synthetic coumarins related to plant protection.

4. Computer-Aided Molecular Design (CAMD) of Coumarins for Potential Plant Protection Application

This approach enables deeper insight into the structure-activity relationship (SAR) of newly discovered natural and synthetic compounds with biological activity, as well as the prediction of future compounds’ activity using quantitative structure-activity relationship (QSAR) models. Computational methods have also been used to discover pesticide mechanisms at the molecular level. CAMD approach includes several computational methods: QSAR, three-dimensional quantitative structure-activity relationships (3D-QSAR), including comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA); high throughput screening (HTS); molecular docking, and molecular dynamics (MD) simulations [57].

4.1. QSAR

There is a limited number of published QSAR studies about coumarins as agents against phytopathogenic fungi. Song et al. [32] performed simple structure–activity relationship (SAR) analyses of the antifungal activity of 35 coumarin derivatives against S. sclerotiorum and B. cinerea, without quantitative evaluation. SAR study revealed that the coumarins with esters at their C-5 position showed higher antifungal activities than the corresponding compound with a hydroxyl group at the same position. Luo et al. [133] evaluated the acaricidal potency of thirty phenolic ether derivatives of scopoletin against female adults of Tetranychus cinnabarinus, the most economically important arthropod pest. They derived a QSAR model with five descriptors calculated by the Dragon program (three GETAWAY descriptors; one drug-like indice; one 2D autocorrelation indice; and one topological indice [134]. The published model has satisfactory fitting parameters (high squared correlation coefficient, R²_train = 0.875), with a high internal predictivity for chemicals in the data set. However, its external predictivity was unreliable considering the low R²_ext (0.583).

A predictive MLR QSAR model [35] was obtained for the antifungal activity against M. phaseolina (R²_train = 0.78; R²_ext = 0.67; Q²_loo = 0.67) for 31 of coumarin derivatives:

log % inhibition = 1.82 + 5.55 JGI6 − 0.72 Mor28v − 0.05 L2e

(1)

where molecular descriptor JGI6 belongs to the topological charge indices, Mor28v is 3D-MoRSE (Molecular Representation of Structures based on Electronic diffraction), and L2e is WHIM (Weighted Holistic Invariant Molecular). According to the obtained QSAR model, multiple electron-withdrawal groups, especially at position C-3, promote, while benzoyl groups and Br atoms at C-8 reduce inhibition. Although the predictivity of the QSAR model for antifungal activity against S. sclerotiourum failed, internal validation confirmed the model’s stability. This makes it relevant for explaining the relation of the structure to observed activity. The model indicates that the hydrophobic benzoyl group at the pyrone ring, and –Br, –OH, –OCH₃, at the benzene ring, may increase inhibition of S. sclerotiourum.

Du et al. [44] developed linear and nonlinear QSAR models by three machine learning methods, GA-MLR, LS-SVM, and PPR, for predicting the fungicidal activities of 100 thiazoline derivatives against rice blast caused by Magnaporthe grisea. The obtained models demonstrated strong correlations between the 3D and conformational structures of the molecules and the fungicidal activities of these compounds.

Bingchuan et al. [135] have generated a QSAR model for 25 coumarin derivatives, which exhibited acaricidal activities against Tetranychus cinnabarinus Bois. using stepwise regression analysis method (R² = 0.967 and F = 155.176). Although the model performed well in fitting, internal and external validation was not conducted, so it cannot be characterized as predictive.

Wei et al. [34] developed 3D-QSAR models for antifungal activities against Valsa mali of coumarin derivatives using the CoMFA/CoMSIA method. Derived CoMFA and CoMSIA models (R² = 0.918 and 0.949, respectively) revealed small, electron-withdrawing and hydrophilic groups on C-3 and C-8 enhanced antifungal activity.

CoMFA and CoMSIA analysis was performed on the scopoletin and coumarine derivates to study the relationship their structure and inhibition of Tetranychus cinnabarinus plasma membrane Ca²⁺-ATPase 1 gene (TcPMCA1), which is responsible for the development of various life stages of carmine spider mite T. cinnabarinus. The results of 3D-QSAR models indicate that substitutions at C-3, C-6, and C-7 positions of coumarins are important for their acaricidal activity [136].

4.2. Molecular Docking, and Molecular Dynamics (MD) Simulations

Molecular docking and MS simulation study of 3-hydroxycoumarin as inhibitors of the fungus Moniliophthora perniciosa, the causal agent of witches’ broom disease in Theobroma cacao L., shows that their antifungal activity is based on the inhibition of chitin synthase (CS). CS’ active site predominates residues for hydrogen bond acceptors. Also, the low hydrophobicity of the active site of CS, favors 3-hydroxycoumarin, due to the hydroxyl groups [33].

In order to determine the possible mechanism of action of coumarins [35] and coumarinyl Schiff bases [36] against pathogenic fungi Rastija et al. have performed molecular docking studies on three enzymes responsible for the fungal growth: demethylase (sterol 14α-demethylase (CYP51), pdb ID: 5eah) [137]; chitinase (pdb ID: 4txe) [138]; transferase (N-myristoyltransferase, pdb ID: 2p6g) [139]; and the three plant cell wall-degrading enzymes: endoglucanase I (pdb ID:2ovw) [140]; proteinase K (pdb ID: 2pwb) [141]; pectinase (endopolygalacturonase, pdb ID:1czf) [142]. The results of molecular docking suggest that tested coumarins may act against S. sclerotiorum as inhibitors of proteinase K and pectinase. The results of the molecular docking study are in agreement with the results of the study conducted by Zhu et al. [143], which proved that S. sclerotiorum, destroys plant tissues during infection by various enzymes, such as proteinases.

The most active coumarin forms four strong hydrogen bonds in the binding site of proteinase K (Figure 8): oxygen atoms from the 6-OH group with Ala172; oxygen atoms from the 3-carbonyl group with Ser224 and Thr223, and oxygen atoms from the 2-keto group with Asn161, which indicates the importance of groups with electronegative atoms from hydroxyl and acetyl groups for enhanced antifungal effects of coumarin derivatives. Molecular docking has shown that coumarins [35] and coumarinyl Schiff bases [36] possibly act against M. phaseolina as inhibitors of endoglucanase and pectinase.

The binding modes between acetylcholinesterase (AChE, pdb:1odc), one of the targets of nematode Meloidogyne incognita, and the novel chromone derivatives were defined using molecular docking. The docking results indicated that the two most active compounds interact with amino acid residues Tyr121, Trp279, Tyr70, Trp84, and Phe330 of AChE via hydrogen bond and π-π stacking [144]. Also, molecular docking and molecular dynamics show that the strong and stable binding of coumarin derivatives to AChE can be attributed to their strong inhibitory potential [35,36,145]. DNA gyrase (EC 5.6.2.2) is a type II topoisomerase that catalyses changes in topology of DNA i.e., an enzyme that catalyzes the ATP-dependent negative super-coiling of double-stranded closed-circular DNA. It is an essential bacterial enzyme but absent from higher eukaryotes, making it an attractive target for antibacterial activities. Targeting DNA gyrase with an inhibitor disrupts DNA synthesis, leading to cell death [146]. Using in silico analysis, heterocyclic compounds containing coumarin scaffolds have been proposed as bacterial DNA gyrase inhibitors [147].

The molecular docking has demonstrated a high affinity of scopoletin to the nucleotide-binding pocket of TcPMCA1, forming five hydrogen bonds: between the 7-hydroxy with Sre297, 6-methoxy with Ala298, oxygen at position 1 with Lys301, and oxygen at position 2 with Lys301 and Ser300 [136]. Molecular docking of phenolic ether derivatives of scopoletin with TcPMCA1 demonstrates that derivatives with shorter side chains at the 7-position interact with more key amino acid residues than scopoletin [133]. Results of computational methods helped to elucidate TcPMCA1-mediated detoxification mechanisms of scopoletin and other coumarin derivatives, providing valuable information for the design of novel PMCA-inhibiting acaricides [136].

4.3. Quantitative Estimation of Pesticide-Likeness Properties

With progress in the parallel synthesis of large numbers of compounds, large chemical spaces have been created. This has conditioned the need for fast screening of leading compounds in both the pharmaceutical and agrochemical industries. However, many studies have shown that most leading compounds are unsuitable for further development into drug candidates due to their inappropriate absorption, distribution, metabolism, excretion, and toxicity (ADMET). Therefore, Lipinski and co-workers published structural properties necessary for drug-like qualities of hit molecules [148]. The famous “rule of 5” proposes the prediction of poor absorption or permeation of a compound as a future active drug component if it has: more than 5 hydrogen-bond donors (HBD), 10 hydrogen-bond acceptors (HBA), the molecular weight (MW) is greater than 500, and the calculated Log P (CLogP) is greater than 5 (or MlogP 4.15). Compared to drugs, pesticides have significantly different ADME properties, therefore the „drug-likeness“ properties were adapted to agrochemicals and changed to „pesticide-likeness“ properties. A pesticide-like compound should have the following characteristics: MW 435 Da; CLOGP 6; HBA 6; HBD 2; rotatable bonds (RB) 9; and aromatic bonds (AB) 17 [149]. The pesticide-likeness studies revealed that a decrease in MW is associated with the toxic reduction of the pesticide, while aromatic double bonds in the structure are associated with pesticide photostability [150,151]. The pesticide-likeness molecular descriptors of coumarinyl Schiff bases have shown that only two compounds have a satisfactory molecular weight. These two compounds are phenyl derivatives of 2-((4-methyl-2-oxo-2H-chromen-7-yl)oxy)acetohydrazide: (E)-N′-(4-(dimethylamino)benzylidene)-2-((4-methyl-2-oxo-2H-chromen-7-yl)oxy)acetohydrazide (MW = 366) and (E)-N′-(4-methoxybenzylidene)-2-((4-methyl-2-oxo-2H-chromen-7-yl)oxy)acetohydrazide (MW = 379). Interestingly, the molecular docking study has shown that only these two compounds bind directly to the active site of acetylcholinesterase, and consequently, the derivative with the lowest molecular weight only exhibited nematicidal activity. However, their number of hydrogen-bond acceptors is higher than the recommended 6 due to additional oxygen and nitrogen atoms in their structure [36].

There are several software programs available for estimating pesticide-like properties. Commercial software is ADMEWORKS (Version 7.9.1.02011) ModelBuilder (Fujitsu Kyushu Systems Limited, Krakow, Poland), and freely available on the web are ADMETlab 2.0 (https://admetmesh.scbdd.com/ (accessed on 15 November 2022)), [152] and SwissADME (http://www.swissadme.ch/index.php (accessed on 15 November 2022)) [153].

4.4. Environmental and Health Hazards Properties of Coumarins

Pesticides and their residues have detrimental effects on the environment, non-target soil organisms and microorganisms, and human health. Plant protection products must first be approved at the EU level before being authorized at the national level. By adopting the 2009 “pesticide package,” the EU proposed a common approach to limiting pesticide harmful effects, promoting integrated pest management, and the progressive replacement of the most dangerous pesticides with low-risk alternatives [154]. The EU proposed a common approach to limiting pesticide harmful effects. The new pesticides should be: effective at an extremely low dosage; readily degradable and less residual in the environment; selective against toxic agrochemicals. Also, active components of plant protection products, including their residues in food, must be safe for people’s and animal health, as well as for the environment [155].

Entomopathogenic nematodes (EPNs) are beneficial organisms used in insect pest management programs and are often combined with plant stimulants, inorganic and organic plant fertilizers, or evaluated on chemical pesticide compatibility [156]. EPN family Heterorhabditidae has evolved mutual associations with insect pathogenic Photorhabdus symbiotic bacteria, while Steinernematididae with Xenorhabdus bacteria, to deliver that symbiotic bacterium into the insect hemocoel causing rapid insect mortality [157].

Testing of entomopathogenic nematodes’ compatibility with pesticides under laboratory conditions may contribute to cost-effective and sustainable pest management. Thus, the mortality, infectivity, and reproductive capacity of EPNs H. bacteriophora and Steinernema feltiae were tested on six registered pesticides in Turkey. Pesticides affect nematodes differently due to diverse insect physiology and specific feeding patterns [158]. Although coumarins and their derivates as botanical nematicides have attracted considerable interest due to their favorable biorational profile, the methods are still not standardized [159]. However, the study of Rastija et al. [35] has shown that coumarin derivatives with the highest antifungal effects were not harmful against infective juveniles (IJs) of two beneficial nematode species, H. bacteriophora and S. feltiae, as well as, against beneficial soil bacteria Bacillus mycoides and Bradyrhizobium japonicum. Most of the coumarinyl Schiff bases did not exhibit nematicidal activity against the same infective juveniles, except Schiff base with 4-methoxybenzylidenen which was also most effective against fungi S. sclerotiorum [36].

Although coumarins have an important role in plant growth regulation and defense against phytopathogens, they also have adverse effects on plant germination. Inhibitory effect of coumarin on the germination of durum wheat (Triticum turgidum ssp. durum, cv. Simeto) [160], and rice seeds [161] was observed. Coumarins is also a potentially toxic for livestock. Dicoumarol toxicosis was confirmed in blood from Friesian cattle fed with wrapped, bailed silage containing approximately 90% sweet vernal grass (Anthoxanthum odoratum) [162]. The fodder plant, sweet clover (Melilotus spp.), or weeds such as bracken fern (Pteridium aquilinum), and giant fennel (Ferula communis), contain coumarins that cause hemorrhagic disease in animals) [163].

Active components of plant protection products, including food residues, must be proven safe for people’s health, and their effects on animal health and the environment. Plant protection products are registered following extensive laboratory testing on animals to assess their short-term and long-term effects on health. Chronic coumarin administration resulted in liver lesions, liver tumors, and cholangiocarcinoma in rats and mice, as well as lung tumors in mice [164].

A recently demonstrated hepatotoxic effect of psoralen in oral doses of 80 mg/kg bw in rats and 320 mg/kg in mice [165]. Results of an acute toxicity study suggested that osthole is a moderately toxic substance when administered intraperitoneally to mice (lethal dose that causes the death of 50% of a group of test animals (LD₅₀) = 710 mg/kg), but results of the subchronic study revealed histopathological changes in organs, especially in the kidney [166].

Studies of toxicity and carcinogenicity from the European Food Safety Authority in 2004 rejected the possibility of natural coumarins being genotoxic. In addition, exposure to coumarin from food and/or cosmetic products poses no health risk to humans [167]. However, clinical studies performed on patients treated with coumarin as a medicinal drug demonstrated its hepatotoxic effect. The hepatotoxic effect appeared among breast cancer patients taking coumarin for chronic lymphedema [168]. Several natural coumarins like psoralen, bergapten, and xanthotoxin, present in large concentrations in celery or lime, may cause a limited number of photoallergic reactions in humans [169]. No data has been published on natural coumarin’s reproductive and developmental toxicity [170].

The REACH guidelines [56] suggest “in-silico” prediction methods. In order to reduce these expensive and time-consuming experiments, the quantitative structure-toxicity relationship (QSTR) method is valuable. Only robust QSTR models with an adequate external predictive ability could be used for the prediction of experimentally undermined toxicity of known pesticides, as well as new pesticides. Devillers et al. [47] derived a QSAR model for estimating the acute toxicity of pesticides on honey bee by means of a three-layer feedforward neural network trained by the back-propagation algorithm based on the experimentally determined toxicity of 100 pesticides. Thus Moreira-Filho et al. [171] developed a predictive (R²_external = 0.75) QSAR model using Feedforward Neural Networks (FNNs) and implemented it in the publicly available BeeToxAI web application (http://beetoxai.labmol.com.br/ (accessed on 27 April 2023)) for prediction of acute toxicity of chemicals in honey bees. Also, Como et al. [172] generated a k-Nearest Neighbor (k-NN) QSTR model for pesticide toxicity prediction in bees. Coumarin derivatives [35] were estimated by the Toxicity Estimation Software Tool (T.E.S.T.) program (v.4.1) [173]. Among 38 derivatives only five compounds are characterized as “toxic” for rats, and three of them have a benzoyl radical at position C-3. Also, the same compounds exhibit the highest aquatic toxicity against Tetrahymena pyriformis. Nine coumarinyl Schiff bases were also estimated by the program T.E.S.T. Eight of the nine compounds are characterized as “harmful if swallowed” by rats. Only the most lipophilic compounds have been estimated as not mutagenic but lethal for water organisms. Tested compounds have no significant high bioaccumulation factor, which means organisms have difficulty absorbing them from the environment [36].

5. Conclusions

The basic scaffold of coumarins, 1,2-benzopyrone, is present in many biologically active agents, and their activities are closely related to their structure. Naturally occurring coumarins are effective in controlling plant pathogens (invertebrate pests, pathogenic fungi, and other microorganisms and weeds), but have limited applications in agriculture as biopesticides. Despite isolated cases of harmful effects of both natural and synthesized coumarins on plants and animals, there are numerous positive biological effects related to plant protection. These biological properties, in particular, make coumarin compounds more attractive for further investigation as novel agrochemicals. Since both natural and synthesized coumarins have shown their negative effects, further research on this scaffold should be focused on the study of the relationship between structure and active biological activity, and molecular docking studies, which aims to understand their mode of action.

Since the biological properties of coumarins depend upon the pattern of substitution of their core, it gives the possibility of designing new coumarin-based compounds and investigating their potential as an active component in plant protection. Synthesis of novel coumarin derivatives should be aim at developing active agrochemicals with ultra-high efficacy, low toxicity, and environmentally safe properties. To satisfy the requirements of integrated pest and disease management, novel active components of future plant protection products based on coumarin scaffolds must be synthesized using green chemistry principles. Application of computer-aided molecular design presents a faster and lower-cost way to discover new pesticides enabling the prediction of activities and properties of untested coumarins, and giving a direction for the synthesis of novel derivatives with higher potency in plant protection, as well as safe for beneficial, non-target organisms and humans.

Reviewed studies have shown that coumarin derivatives are promising candidates for developing novel plant-protection products of the new generation, which meet all requirements of modern integrated pest management. These novel coumarin compounds must be highly specific to be environmentally and toxicologically acceptable. Computational design of future compounds and their synthesis, evaluation of their effectiveness on harmful and beneficial organisms in the soil, as well as detailed research into mechanisms of action at the molecular level, represents an initial stage in the long-lasting and expensive process of designing plant protection products. Newly developed active compounds will be potential candidates for further phases of developing plant protection products until their final registration.