Natural Products in the Discovery of Agrochemicals

810 CHIMIA 2017, 71, No. 12 Natural Products: source of INNovatIoN

doi:10.2533/chimia.2017.810 Chimia 71 (2017) 810–822 © Swiss Chemical Society Natural Products in the Discovery of Agrochemicals

Olivier Loiseleur*

Dedicated to John M. Clough

Abstract: Natural products have a long history of being used as, or serving as inspiration for, novel crop protection agents. Many of the discoveries in agrochemical research in the last decades have their origin in a wide range of natural products from a variety of sources. In light of the continuing need for new tools to address an ever-changing array of fungal, weed and insect pests, new agricultural practices and evolving regulatory requirements, the needs for new agrochemical tools remains as critical as ever. In that respect, nature continues to be an important source for novel chemical structures and biological mechanisms to be applied for the development of pest control agents. Here we review several of the natural products and their derivatives which contributed to shape crop protection research in past and present. Keywords: Agrochemicals discovery · Biopesticides · Crop protection agents · Leads for new agrochemicals · Natural products

Natural products have a long history resent a historical sources of insect biocon- ticidal properties, the carbamates for insect of use as pest management tools and have trol agents, peptides from plants and other pest control, and 2,4-dichlorophenoxyace- been intimately connected with agriculture proteinaceous natural products may offer tic acid (2,4 D) for weed management. The from its beginning to its most recent devel- next generation solutions. arrival of synthetic pesticides revolution- opments. This review article aims to pres- The chemical protection of crops ized the control of agricultural pests. Soon ent some of the important natural products probably began in the fertile crescent of after in the 1960s, chemistry research on and their derivatives, past and recent, used Mesopotamia with the application of ele- natural products with pesticidal properties as crop protection agents. A historical per- mental sulfur introduced by the Sumerians also started intensifying, in particular in in- spective is first given with the develop- in the earliest recorded instance of pest sect control with the unforeseen problems ment of the pyrethroids, a landmark in its management to control insects and mites.[1] of persistence and resistance encountered time for successful ligand-based design. It is not possible to describe the exact mo- with DDT and the organochlorines.[4,5] The Natural products are then discussed in the ment when humans started using plants and natural products brought bioinspiration context of contemporary agrochemical their products to control insects and micro- to laboratories for the discovery of new research, and some of the molecules that organisms, but it also has been historically weeds, plant pathogens, and insect pest contributed to shaping the field including associated with the onset of agriculture. control agents and played an important role those currently in late development are de- Natural products such as ground tobacco, in the advancement of crop protection re- scribed. Owing to their ecological role in essential oils and lime, used against aphids, search, uncovering new biology and mode adaptation processes and defense mecha- and ground pyrethrum flowers were some of actions. An illustrative example, which nisms, several secondary metabolites from of the earliest pest control agents used.[2] If inaugurated several approaches still in use microbials and plants have found use as initially the use of natural product extracts in modern discovery and optimization of effective starting points from which new was restricted to intuitive and naturalist agrochemicals[6] has been the work done lead areas of chemistry have been derived. procedures, the knowledge has spread and on the insecticidal pyrethroid esters.[7–10] Phytochemicals applied in their semi-pu- survived through different civilizations un- rified extract form are commercially used til the 19th and early 20th centuries, when for the biological control of pests, as is the the first scientific observations associated Pyrethroids for Control of Insect case for botanical insecticides. In that area, with empirical practices allowed the sig- Pests whereas plant secondary metabolites rep- nificant use of botanical extracts as pesticides.[3] In the same period, advances in The insecticidal properties of pyre- chemistry allowed the identification and thrum, the powder obtained from ground characterization of some plant secondary flowers of Chrysanthemum coccineum and compounds and better defined plant ex- Chrysanthemum cinerariaefolium, have tracts, such as derris (rotenone), pyrethrum been known for centuries and efficiently or nicotine came into use. The era of syn- applied for household use. In 1900, scien- thetic agrochemicals really took off in the tists in the viticulture research station of 1950s with the introduction of compounds the canton of Vaud in Switzerland tested such as dichlorodiphenyltrichloroethane in one of the first recorded large field tri- (DDT) which earned the industrial chemist als up to 80 different substances for their *Correspondence: Dr. O. Loiseleur Paul Müller the Nobel Prize in Physiology insecticidal activity against the European Syngenta Crop Protection AG Schaffhauserstrasse, CH-4332 Stein or Medicine in 1948 for his discovery at grape berry moth (Eupoecilia ambigu- E-Mail: [email protected] J.R. Geigy AG of its highly efficient insec- ella). Alongside sulfur, copper, arsenates Natural Products: source of INNovatIoN CHIMIA 2017, 71, No. 12 811

and soft soap, a large range of products reocenters took another 30 years enabled fast speed of kill, ease of application and a isolated from plants including spices as by advances in separation techniques and favorable human safety profile. well as pharmaceutically active ingredi- other new research methods (Fig. 1). The Hence pyrethrin I was used as starting ents were investigated. The best results absolute stereochemistry was confirmed point for the design of improved insecti- were obtained with an aqueous solution of in the 1970s by X-ray crystallography.[14] cides. The development of synthetic pyre- 1% of pyrethrum formulated with 3–5% of Pyrethrum is composed of six main com- throids targeted both modified alcohol and soft soap.[11] Due to the high cost of pro- ponents, the pyrethrins, which can be dicarboxylic acid moieties. A brief historical ducing pyrethrum, its subsequent appli- vided into two subgroups: ‘pyrethrins I’, perspective is given in Fig. 3.[7,8,14] cation on broad field crops proved to be which are chrysanthemic esters (R = Me, Early research by Laforge, a chem- unsustainable and eventually was limited Fig. 1); and ‘pyrethrins II’, which are py- ist at the bureau of entomology and plant only to certain highly valuable vineyards. rethric esters (R = MeO C, Fig. 1), with quarantine of the U.S. Department of 2 Nevertheless, this field trial allowed rec- pyrethrin I (R = Me, R' = CH=CH ) being Agriculture, focused on the alcohol varia- 2 ognition of the potential of this natural the most biologically active molecule. tion. The replacement of the complex cy- product for use as an effective insecticide With full structural information in clopentenolone Z-configured dienyl side for crop protection. These results and the hand in the 1950s, the synthesis of poten- chain by a simpler allyl brought the first increasing demand for pyrethrum attracted tial analogues with improved insecticidal improvements leading to allethrin (3), the the attention of far-sighted chemists, ini- activity could be envisaged. Despite many first synthetic pyrethroid to be equipotent tially in Japan where commercial produc- similarities between drug and agrochemi- to pyrethrin I.[16] Sumitomo Chemical Ltd., tion existed since the 1880s and later in cal discovery, the latter has unique char- who would become later a major player in Germany and Switzerland. In a landmark acteristics due to the biodiversity of pests the pyrethroid discovery process, devel- series of publications appearing in 1924, and the conditions of use. Because crop oped on the basis of the work of LaForge Staudinger and Ružicˇka described the protection agents are used outdoors, pho- a technical synthesis and brought to the isolation of the odor- and colorless active tostability is an important property to be market Allethrin as the first pyrethroid ingredient through an ingenious proce- considered and in some cases is the key in 1954, followed later by S-bioallethrin dure involving a self-developed bioassay- parameter to optimize. The limited stabil- (4), the isolated most active stereoisomer. guided fractionation using the German ity of pyrethrins toward oxidation and pho- Allethrin represented an important step as cockroach (Blattella germanica) and dis- todegradation (Fig. 2) as well as their high it proved it was possible to find potent syn- closed the main structural features of two cost of manufacturing impeded their use thetic analogues of the pyrethrins. The next pyrethrin esters 1 and 2 (Fig. 1).[12,13] The in crop protection but they otherwise pos- structural modifications were initially very full elucidation of the structures and the sessed attractive features as an insecticide incremental but the weakly active piperonyl determination of configuration of the ste- including high toxicity to the target pest, ester (5) from earlier work of Staudinger and Ružicˇka provided a new avenue, point- ing towards the potential of replacing the alcohol part by simpler and more economi- 1954 1958 1942 cal achiral moieties. The use of aromatic Staudinger and Ruzicka 1924 Crombie et al. Inouye et al. Gilam et al. alcohols as in furamethrin (6) provided the Pyrethrin I (1) first potent pyrethroids in this new class pyrethrolon and improved photostability. During the (R)(S) O O development of the technical synthesis of trans (Z) O O furamethrin, scientists at Sumitomo pre- O chrysanthemic acid CH2CH=CH=CH-CH3 O pared compound 7 as a reference to study 1947 1956 impurity profiles in the process and discov- Pyrethrin II (2) LaForge et al. Crombie et al. ered that α-substitution with ethynyl sig- nificantly increased potency over furame- O thrin. This modification proved to be appli- O MeO C MeO2C 2 (E) cable to other pyrethroids. Further optimi- O O O O zation led to the exchange of ethynyl with pyrethric acid CH2CH=CH=CH-CH3 1955 the more stable cyano substituent, which Inouye et al. eventually would become an element of design applied in many pyrethroids. The later introduction of the 3-phenoxybenzyl Pyrethrins I(R=Me) R' Pyrethrins II (R =MeO C) R' 2 (Z) Pyretrhin I CH=CH Pyretrhin II CH=CH alcohol or its cyanohydrin analog led to the 2 2 O (S) Cinerin I Me Cinerin II Me R (S)(R) R' discovery of even more stable and safer in- Jasmolin I Et Jasmolin II Et O secticides (compared to contemporary ag- O rochemicals) such as ciphenothrin (8).[15] Modification of the chrysanthemic acid Fig. 1. The arduous structure elucidation of pyrethrin I and II. moiety also was needed because of its poor stability towards oxygen, heat and light. The two methyl groups in the isobutenyl, Fig. 2. Hydrolytic and Metabolic soft spots of Cinerin Iinrat oxidative liabilities of in particular the one in trans-(E) position, the pyrethrins. are sensitive to radical attack leading to rapid photodecomposition upon sun lamp irradiation in laboratory stability tests.[16] O Oxidation -CYP450 Hundreds of analogues with different sub- Hydrolysis -esterase stituents at the double-bond were synthe- O sized and eventually the exchange of the O methyl groups with halogen atoms proved 812 CHIMIA 2017, 71, No. 12 Natural Products: source of INNovatIoN

regulatory landscape, in particular with the O O requirements for improved environmental O O O O trans O and toxicological profiles and the de-reg- O O O O istration of older active ingredients.[6,25,26] O O Allethrin (3) S-Bioallethrin (4) Piperonylester (5) Furamethrin (6) Typical for agrochemical research is LaForge (1949) Sumitomo from earlier work of Staundinger Katsuda (1966) Diastereoisomeric mixture and Ruzicka the testing of compounds directly on agro- 1st incremental modification Isolated most active 1st weakly active benzylester Potent benzylester nomically relevant whole organisms, that 1st pyrethroid on the market (1954) allethrin stereoisomer Simpler structure unstable to air and light is, the weed, fungus, or insect. Such meth- Br O Cl odology allows the generation of in vivo O O O O Br OPh OPh Cl OPh data on target species to control, very early O O O O N N in discovery. Scientists in crop protection α-Ethinyl furamethrin (7) Ciphenothrin (8) Permethrin (9) Deltamethrin (10) research use a variety of chemical inputs Serendipity at Sumitomo Matsuo (1971) Elliott (1972) Elliott (1972) from which new lead areas of chemistry Increased activity through Increased photostability Dichlorovinyl replacement Commercialized for better in vivo hydrolytic stability Stable enough for agricultural use agricultural use (Bayer) are derived:[27,28] Designed libraries based (Syngenta) through α-substitution on molecular target hypotheses,[29,30] com- Cl petitor-inspired chemistry, library acquisi- O O F3C OPh OPh tion from universities or chemical vendors, O O rac Cl N N project compounds and intermediates in

Lambda-Cyalothrin (11) Esfenvalarate (12) other indications, collections exchanged Huff(1977) Ohno (1973) with pharmaceutical companies and natu- Commercialized for Commercialized insecticide agricultural use (Syngenta) for foliar treatment (Sumitomo) ral products.[31,32] Utilizing natural products as a source Fig. 3. The evolution of the pyrethroids and selected compounds commercialized for agricultural use. of input for the discovery of new agrochemicals offers a number of advantages. Natural product chemotypes offer the abil- to be the solution of choice.[17] Elliott and degradation of the insecticide in mammals ity to target underexplored areas of the his team at the Rothamsted Experimental by means of esterases and cytochrome P , biologically relevant chemical space[33,34] 450 Station investigated this option with the also contributes to selective toxicity.[24] and their biological activity resulting best benzylic alcohols described in the Pyrethroid insecticides hence evolved from Darwinian selection increases the previous paragraph and discovered new in a now classical sequence of natural prod- likelihood of discovering a structure that insecticides with unprecedented potency at uct research: activity observed in a natural has utility as a pesticide or as a molecu- the time. This research culminated with the extract, isolation and identification of the lar scaffold for pesticide design. As such, discovery of permethrin (9) and deltame- active compounds serving as template to natural product hits in screenings can re- thrin (10) which were the first pyrethroids generate optimized agrochemicals. The li- veal new molecular target sites generating with sufficient stability toward oxidative or gand-based design for the optimization of new potential intervention points for pest photodegradation to be suited for agricul- the pyrethrins, the importance of efficient control ultimately leading to novel modes tural use.[18,19] A further refinement was the synthesis to maintain cost of manufactur- of action, which is particularly valuable replacement of a chlorine atom of cyper- ing of these chiral molecules in profitable for resistance management. Natural prod- methrin by a CF group to give lambda- range, smart exploitation of serendipities ucts and their semi-synthetic derivatives 3 cyhalothrin (11).[20] Eventually, to simplify and, for natural products, collaboration be- can have lower environmental half-lives. the pyrethroid scaffold further, scientists tween academic and industrial partners,[9] Owing to their structural complexity, they at Sumitomo, designed fenvalerate and its are still illustrative of many of the features also often show high target specificity and enantio-enriched form esfenvalerate (12), in today’s agrochemical discovery. Most low toxicity towards non-target organisms a synthetic pyrethroid that no longer con- importantly, the pyrethroids inaugurated and their shorter persistence offer oppor- tained a cyclopropyl carboxylic ester. the era of synthetically more complex, but tunities for differential in vivo metabolism Pyrethroids proved to be broad spec- toxicologically and environmentally more and exposure. Taken altogether, these fea- trum insecticides effective against a broad friendly molecules used in modern crop tures also provide an advantage for public range of foliar pests, including Coleoptera protection. and regulatory acceptance. However there (beetles and weevils), Diptera (flies are a number of problems and limitations and mosquitoes), Heteroptera (bugs), associated with natural product-based Homoptera (aphids, whiteflies, and leaf- Natural Products in Agrochemical discovery programs in agrochemical re- hoppers), Lepidoptera (moths and but- Research search, in particular with the screening of terflies), Thysanoptera (thrips), and compounds on whole organisms. Orthoptera (cockroaches and grasshop- For nearly three-quarters of a century, The data generated from screenings pers). Pyrethroids exert their insecticidal there has been a constant need for inno- driven by symptomology read-out on action through an interaction with the vations in crop protection technology that whole organisms is complex, multifacto- insect voltage-gated sodium channels.[21] helps to provide a sustainable food sup- rial and is tainted with uncertainty due They preferentially bind and stabilize the ply for an increasing global demand. The to the variability of the tests. This is es- open state of sodium channels, producing continued search for selective, safe and pecially challenging when a mode of ac- prolonged channel opening and conduc- cost-effective new agrochemical classes tion hypothesis is lacking and the in vitro tance of sodium, which ultimately results is stimulated by a number of important biochemical assay or structural biology in permanent axonal membrane depolar- factors: resistance development to exist- tools which help reduce complexity are not ization, eventually paralysis and death of ing crop protection products, new agri- available. In addition, for a compound to the insect.[22] Mammalian (rat) sodium cultural practices and technologies such give a reliable signal in a high-throughput channels are up to 1000-fold less sensi- as integrated pest management programs screening on a whole organism, its affinity tive than insect channels to pyrethroids.[23] and seed treatment applications, the shift- for the putative target protein generally has Differential metabolism, i.e. preferential ing of pest populations and the changing already to be at very high level, typically Natural Products: source of INNovatIoN CHIMIA 2017, 71, No. 12 813

nanomolar, meaning that confirmed hits The Mectins and the Spinosyns The spinosyns and the mectins pos- from screening are very rare. On the oth- sess many of the features required nowa- er hand, the attrition rate is lower than in Natural products isolated from micro- days for performance and product safety pharmaceutical research where the initial bial fermentation and their semi-synthetic in particular with regards to environmental in vitro optimization may be hard or im- derivatives have been most important to safety (Fig. 4). Starting in the 1970s, selec- possible to translate to the target. Because the development of new insect control tivity requirements initially were focused of the low initial hit rates, testing small col- agents. Abamectin (13, Scheme 1), a natu- on mammalian versus pest selectivity, the lections of purified natural products plays rally occurring mixture of avermectins B a desire being for products that were less 1 against the odds. The natural products’ and B b (16-member ring macrolides), is toxic to mammals. The development of 1 physicochemical properties[35] can often effective against a large number of mite the pyrethroids saw the introduction of in- be inadequate and their in vivo half-lives and lepidopteran pest insect species and secticides that possessed overall improved excessively short, which hides their true acts via the γ-aminobutyric acid (GABA)/ mammalian toxicological profiles on a per potential in a primary screening on whole glutamate-gated chloride channels.[45] gram/Kg basis coupled with an increase organisms where absorption, distribution, Spinosad (15), also a naturally occurring in overall insecticidal activity. Gradually, metabolism, and excretion play a role. mixture (12-member ring macrolides; with the implementation of integrated Prioritization of such hits is not easy, in spinosyns A and D), is active against a pest management programs[47] address- particular when the activity is only moder- wide range of lepidopteran, dipteran and ing the regulatory needs for an improved ate and could possibly just be due to gen- thyasnopteran pests and functions as an toxicological and environmental profile in eral toxicity. Moreover, the bioassays used allosteric nicotinic acetylcholine recep- insect control, emphasis also was put on are also sensitive to pan-assay interference tor (nAChR) agonist.[46] Abamectin and better environmental safety, starting with compounds (PAINS) or other promiscuous spinosad have been synthetically modi- less persistent molecules and subsequently groups including some frequently found fied, leading to insecticides that have an moving to low toxicity towards non-target in natural products such as the catechols, altered spectrum, improved efficacy and/ organisms and low mobility in soil. In quinones, Michael acceptors, 1,3-dioxo- or improved duration of activity. For this journey to modern agrochemicals, the lanyl and siderophores.[36,37] Screening of example, synthetic modification to the mectins, spinosad and its semi-synthetic crude or semi-purified extracts, as done in 4''-hydroxy group of abamectin to form derivative spinetoram provided early ex- bioassay-guided fractionation processes, 4''-N-methylamine yielded emamectin amples of favorable mammalian and en- brings additional complications due to fre- benzoate (14), which possesses greatly vironmental safety profile coupled with quent unwanted interferences between the improved efficacy for lepidopteran spe- excellent utility and efficacy. screened materials and the bioassays.[38] cies (spectrum of activity shift). Likewise, These products are degraded rapidly in Nevertheless, natural product-directed synthetic modifications (ethoxylation of the environment after application though a discovery programs have over the years the 3'-hydroxy on the rhamnose and reduc- combination of routes.[41,42] Photolysis on delivered a significant number of valuable tion of the 5,6-double bond of spinosyn J) plant surfaces is fast, and the compounds starting points and some of the most im- of a mixture of spinosyns J and L resulted bind tightly to soil, where they are rap- portant modes of action in agrochemicals. in spinetoram (16), a semi-synthetic spi- idly degraded by soil microorganisms. No The output of such effort is threefold: the nosyn insecticide with improved potency leaching or bioaccumulation occurs due identification of new ingredients that be- and extended residual activity. to rather high lipophilicity, low to moder- come commercial crop protection chemicals without further transformation, active molecules that require some synthetic modification (semi-synthesis) and active 4''-N-methylamine mectin OMe spectrum shift OMe molecules that inspire development of HO increased potency HN 4" OMe 4" OMe purely synthetic solutions (synthetic mim- 4steps: O O C -OH silylation O O [32,39] 5 ics). Other valuable outputs include C4''-OH swern oxidation O O O C4''-OH reductive amination O O O the possible discovery of a new target site C5-OH deprotection O CO2H O H H of action leading to target-based screening R R efforts or the identification of a new struc- O O O O OH OH ary 1ary component: R=C H tural motif that may inspire new classes 1 component: B1a,R=C2H5 2 5 minor component: B b,R=CH minor component: R=CH 1 3 5 3 O 5 of chemical structures. The identification O 5 of a molecule that has all of the required OH OH Abamectin (13) Emamectin benzoate (14) properties to be effective as an agrochemi- Merck -Syngenta Merck -Syngenta cal product directly from the screening of acaricide and insecticide broad spectrum of lepidoptera natural products, as was the case for insec- [40,41] [42] ticidal spinosyns and the mectins is Me N Me2N very infrequent. Rather, it is the identifi- 2 cation of active molecules with intriguing O O O O O O HH activity profiles that can serve as a starting HH O O O O 3' 3' point for further optimization. This can ei- H 5 6 MeO OMe H 5 6 MeO OMe H H OEt ther involve a few steps of modifications of O O R OMe O O R 5-6-dihydro spinosyn the naturally occurring molecule, as exem- more photostable 3'-O-ethyl spinosyn [43] intrinsically more active plified by spinetoram, emamectin ben- ary ary 1 component Spinosyn A:R=H 1 component: single bond at C5-C6,R=H zoate and more recently afidopyropen[44] minor component Spinosyn D:R=CH3 minor component: double bond at C5-C6,R=CH3 in insect control and fenpicoxamid[45] in Spinosad (15) Spinetoram (16) Dow Dow plant pathogen control, or an overall re- (semi-synhtetically derived from Spinosyn Jand L) configuration of the core structure of the insecticide improved potency and duration of activity natural product, as in the case of the strobilurins. Scheme 1. Mectins and spinosyns natural products and their modified analogues. 814 CHIMIA 2017, 71, No. 12 Natural Products: source of INNovatIoN

Fig. 4. Profile of a combination of favorable molecular and modern agrochemical. biological properties, strobilurins were •Highefficacyand selectivity immediately seized upon as starting points Biological •Bioavailability(plant,pest) for optimization as synthetic mimics. A performance •Right metabolic stability(plant,soil) clear attraction was the simplicity of their •Nocross resistancetoexisting products structures, which is a rarity in bioactive natural products. Independent programs of research aimed at optimizing strobilurin A started first within Syngenta (ICI at the •Low application rate time) and BASF which led after several Environmental •Low toxicity to non-target organisms safety •Fastdegradation in theenvironment years’ work to the discovery and almost •Low mobilityinsoil (noleaching) simultaneous launch of the first synthetic strobilurins: azoxystrobin (21, Scheme 2) and kresoxim-methyl (22). Many other compounds made it to the market despite •Favourable toxicological profile similar structure and identical mode of ac- •Safefor operatorsand bystanders Health safety tion, demonstrating the creative power of •Convenientapplication method chemical design and synthesis as tools to •Safepackaging deliver multiple compounds in a successful class. Milestones in the chemical optimization of azoxystrobin are shown in Scheme ate water solubility and short persistence. defense is widespread in plants but also in 2. The (E)-methyl-β-methoxyacrylate unit Their impact on populations of beneficial fungi.[48] Chemical defense compounds are in strobilurin is the main enthalpic con- predacious insects and mites is much lower usually effective against animals, insects, tributor for binding, important for activ- than on target pests. This wide margin of plants or fungi, thus exhibiting toxic, pun- ity and it was conserved in the design of safety is due to the rapid uptake into the gent, bitter, herbicidal or fungicidal prop- analogues. The phenylpentadienyl unit of foliage after application, combined with erties. To date at least three fundamentally strobilurin A is to a great extent respon- the fast degradation of surface residues different chemical defense mechanisms sible for photoinstability and volatility and under sunlight, which makes the com- are known. While constitutive chemical needed to be modified. Computer model- pound less bioavailable to beneficials than defense relies on permanently present ling shows that the β-methoxyacrylate and to pests under field condition. The combi- bioactive secondary metabolites, wound- the phenylpentadienyl units of strobilurin nation of insecticidal efficacy and safety activated chemical defense is based on the A are cross-conjugated, being almost or- profile of the spinosyns was recognized conversion of an inactive precursor into a thogonal with respect to each other in by the U.S. Presidential Green Chemistry bioactive defense compound that is only the lowest energy conformation.[53] This Award in 1999 for spinosad and in 2008 for generated upon injury. In induced chemi- unusual and fortunate structural feature spinetoram. The application rates for the cal defense, compounds are synthesized means that the interactions between the highly potent mectin insecticides and acar- de novo on demand. The identification of two parts of the molecule are minimized, icides are in the range 10–30 g-a.i./ha (10 a constitutive chemical defense metabolite so that structural changes to the phenyl- g/ha means that only one teaspoon of an is experimentally the simplest approach as pentadienyl unit have little effect on the active ingredient (a.i.) is required to protect it requires only a bioassay-guided screen- β-methoxyacrylate and vice versa. In the the area of a soccer pitch) which consti- ing against potential enemies or competi- efforts to stabilize the dienyl system while tutes a landmark in the dramatic reduction tors without additional method develop- retaining the same molecular shape, it was of use rates of crop protection considering ment as would be the case for wound- first discovered that the (Z)-olefinic bond that as recently as in the 1960s more than 1 activated and induced chemical defense. could be replaced by an ortho-disubstitut- kg of a crop protection chemical was typi- The very successful strobilurin class of ed benzene ring to give 18, which is less cally applied per ha. fungicides originates from such an effort volatile and much more stable in light. with the isolation in the 1970s of strobi- Compound 18 is active in the greenhouse lurin A (17, Scheme 2) from the pinecone but still degrades too quickly to express Higher Fungi and Plant Defense cap (Strobilurus tenacellus) in a research good activity in the field. The diphenyl Metabolites as Source of program targeted to grow mycelial cul- ether which removed the extended stilbene Inspiration for Crop Protection: The tures of basidiomycetes higher fungi.[49– conjugation 19 proved to be sufficiently Strobilurins and Stemofoline 51] Fungal mycelia have to compete with stable. Furthermore, 19 was shown to be other fungi for nutrition and space and are systemic in plants, an important property Whereas one natural product-based ap- potential victims of mycoparasitic fungi. of many modern fungicides which im- proach includes assaying large collections Therefore, several fungi, including S. te- proves field performance by redistribution of extracts obtained from taxonomically, nacellus contain fungicidal secondary me- of the compound within plant tissue after geographically and environmentally biodi- tabolites, which suppress other competing foliar application. However, the required verse inputs and leads to unanticipated dis- organisms in the same environment, and high application rate in the field (750g/ha) covery of pesticidal natural products such by doing so give themselves an advantage. was not economically viable and created as described above, study of chemical ecol- The strobilurins inhibit respiration in fungi some phytotoxicity issues (crop damage). ogy offers a more targeted alternative. The selectively by binding at the so-called Q Probing substitution around the aromatic o evolutionary forces driving the survival of site of cytochrome b which is part of the rings of 19 to increase binding affinity species include positive interactions such bc complex (complex III) located in the led to analogues such as 20. The tricyclic 1 as mutualistic and symbiotic relationships inner mitochondrial membrane of fungi compound 20 had improved fungicidal po- and negative interactions such as competi- and other eukaryotes, leading to intracel- tency but was too lipophilic to demonstrate tive and parasitic relationships. Chemical lular deficiency in ATP. [52] With their rare systemic movement. Further extensive ex- Natural Products: source of INNovatIoN CHIMIA 2017, 71, No. 12 815

perimentation (ca. 1400 compounds made overall in the project) aimed at tailoring lipophilicity and other important physical properties by careful combinations of suitable rings and substituents on them led finally to the discovery of azoxystrobin, a compound with remarkable biological activity against plant pathogenic fungi and still the world’s biggest-selling fungicide. In the search for new chemical scaf- folds leading to novel chemical classes of agrochemicals, constitutive defense systems in plants is another source of inter- esting leads. Amongst these, the alkaloids exert their effects on insects through anti- feeding, repelling or neurotoxic mechanisms. The natural product stemofoline (28, Scheme 3), isolated from the stems and leaves of the oriental medicinal plant Stemona japonica and known as a potent agonist of insect nicotinic acetylcholine receptors (nAChRs),[54] was considered as a good starting point. Stemofoline shows fast-acting insecticidal, antifeedant and re- pellent activities, but its activity is signifi- cantly lower than that of commercial products acting on insect nAChRs. The natural material is not readily accessible and its complex structure is troublesome for the synthesis of analogues. Therefore, 28 was used as a lead structure in order to identify novel active ingredients, particularly for sucking pest control. Scientists at Syngenta designed smaller molecules focusing on the stemofoline 2,6-methanofuro[2,3,4- gh]pyrrolizine cage structure. Based on this structure, a first class of tropane ethers (Scheme 3) was identified. Although they had weaker insecticidal activity, their Scheme 2. Discovery of Azoxystrobin through chemical optimization of Strobilurin A and selected lower complexity made them synthetically commercial strobilurins. more accessible. Optimization of this hit and implementation of a prodrug strategy eventually led to the class of pyridinyl-cyanotropanes and for example compound 31 LogP, molecular volume and pKa of agro- Natural Products Currently in Late (R = CH CF ), highly active in vivo against chemicals dictate their distribution in the Development: Afidopyropen and 2 2 aphids and whitefly, which is bioactivated plant’s compartment. When a compound Fenpicoxamid to compound 30 in insects by cleavage of is basic, an ion trap occurs that leads to the N-(2,2-difluoroethyl) residue.[55,56] its sequestration in vacuoles located inside Mectins, spinosyns, the herbicide the leaf owing to the low pH there (pH Bialaphos[61] and the fungicidal polyox- 5.5).[58] Trapping into vacuoles is not fa- ins are currently the only natural products ADME for Agrochemicals: Tuning vorable for insect control, when sap-feed- and semi-synthetic derivatives introduced the Physicochemical Properties of ing pests such as a hemipteran are target- to the agromarket but two more microbial Cyanotropanes ed. Vacuoles are not their preferred food fermentation products are in late devel- source, nor do they participate in systemic opment and have received an interna- The optimization of agrochemicals is a transport. In the case of aphicides, the tional standardization organization (ISO) multi-parameter endeavor (Fig. 4). Similar compounds should have low lipophilicity name.[62] Afidopyropen (34, Scheme 4) is to drugs, agrochemicals are optimized to (LogP < 2.5) for good kinetics of transfer a new natural product-derived insecticide interact with their target receptors at low through the leaf tissue and be non-basic in active against piercing and sucking insect concentrations via the same molecular order to allow optimal bioavailability. To pests such as aphids.[44,63] The molecule is recognition processes. However, although tune the physical properties, a pro-form of based on the natural product pyripyropene ADME is vital to both pharmaceuticals the highly basic active cyanotropane was A (33) that was isolated by the group of and agrochemicals,[57] the compounds en- designed using the effect of fluorine sub- Oˉ mura at the Kitasato Institute for Life counter different chemical environments stitution in β-position to reduce basicity Science in Japan, famous for its discovery from the site of application (foliar spray and adjust lipophilicity (Fig. 5).[59,60] The not only of avermectin but also of many or application in soil for root uptake) to optimum was reached by derivatization of other experimental natural products with the biochemical target. After uptake of a the cyanotropane nitrogen with the meta- pesticidal activity.[64] The group of Gloer sprayed agrochemical into the leaf, the bolically labile 2,2-difluoroethyl group. subsequently reported its insecticidal 816 CHIMIA 2017, 71, No. 12 Natural Products: source of INNovatIoN

Fenpicoxamid (38, Scheme 5) is a nAChR agonist (α-BgTx EC50 1.7nM) nAChR agonist (α-BgTx EC50 310nM) nAChR agonists novel picolinamide fungicide currently developed by Dow AgroSciences in col- OMe Substructure search of the Hit to Lead O Stemofoline “cage” in the R1 laboration with Meiji Seika Pharma for use company collection O N optimization O N [45,68] O R2 primarily in cereals. Fenpicoxamid is N O CN an acyloxymethyl ether of the natural an- O tifungal compound UK-2A (37) originally Stemofoline (28) Tropane ether (29) Cyanotropanes isolated from fermentation broths of a -not aviable commercial product in -weak insecticidal activity in vivo -new class of insecticides Streptomyces sp., extracts of which dem- its own right: weak activity in vivo and -easy synthetic access -two main variations points to be natural material not readily accessible explored onstrated strong antifungal activity against -complex structure troublesome for -structural similarity with natural synthesis of analogues products acting at nAChR a broad spectrum of fungi in in vitro as- (anatoxin, epibatidine) says. UK-2A and its derivatives inhibit the respiration of fungi at the mitochondrial Cl Cl Cl complex III site through binding to the Qi CHF CF ubiquinone site rather than to the Qo site NH N 2 N 3 N N N targeted by the strobilurin. CN CN CN UK-2A is structurally related to the [69] 30 (cide) 31(procide) 32 (procide) antimycin antibiotics (39, Scheme 5) and differs mainly by the presence of a in vivo activity against green peach activity on leaf disc picolinamide moiety. Whereas both natural 30:noactivity at 3mg/L 32:full effect at 3mg/L products exhibit their antifungal activity by specifically binding to the same Qi ubiqui- in vitro activity: [3H]- a-BgTx displacement on insect membranes 30:nAChR IC50 =1nM none site, antimycin in contrast to UK-2A, 32:nAChR IC50 =58900 nM is toxic to mammals.[70] Although the pico- Biokinetics :injection of 30 and 32 in Heliothis virescens linic moiety in UK-2A may be responsible 32 is bioactivated to 30 by oxidative cleavage of N-CH2-CF3 to NH for selectivity compared to the antimycins, Scheme 3. From Stemofoline to the cyanotropane procides. it bears electron-donating substituents including an ionizable OH group (pKa 8.5) and is prone to oxidative degradation, which is problematic for application in crop protection. These liabilities are apparent in the drop in activity observed when translat- Cl Search target ing from in vitro studies of fungitoxicity to -low lipophilicity (LogP <2.5) CF greenhouse tests in which the natural prod- N 3 -non basic N Cl uct is weaker than might be expected.[45] CN CHF The abiotic degradation of UK-2A when N 2 N pKa =2.7 Cl exposed as thin films on surfaces (e.g. on LogP =2.9 CN leaves) to air and light (e.g. formation of 40, NH Scheme 6)[71] is significant, and exposure to ΔpKa =-2.1 pKa =4.8 N

lipophilicity LogP =2.3 UV light for 24 h results in complete oxi- CN NH dative photodegradation. Scientists at Dow ΔpKa =-3.7 pKa =8.5 sought to stabilize the picolinamide and LogP =1.5 the protection of the OH group drove the synthetic effort that led to the identification ΔpKa =approx. -2.5 pKa =11 LogP =1 of fenpicoxamid 38 as a development can- (predicted) didate. Clearly the picolinamide hydroxyl group plays a role in predisposing UK- basicity 2A to degradation phenomena, since its derivatization to the isobutyryloxymethyl Fig. 5. Tuning of the physicochemical properties of cyanotropanes. ether addressed the issue very effectively. Fenpicoxamid was designed to act as a procide and short term metabolism stud- properties.[65] A collaboration between the treatment or prevention of atheroscle- ies show that fenpicoxamid 38 is almost the Oˉ mura group and Meiji Seika Pharma, rosis and hypercholesterolemia with an completely converted to UK-2A 37 within also confirmed pyripyropene A to be a po- IC value of 58 nM.[64] In 2017, scientists a few hours of incubation in fungal isolate 50 tent insecticide,[66] which was the starting at BASF reported that afidopyropen acti- cultures and wheat cell suspensions. On the point for the discovery of afidopyropen. vates the vanilloid-type transient recep- other hand the metabolically more stable The molecule went under development tor potential (TRPV) channels expressed OH-methylated ether of UK-2A is 1000 through collaboration between Japan’s exclusively in insect chordotonal stretch times less active than fenpicoxamid. Efforts Meiji Seika Pharma and BASF and the reg- receptor neurons (EC = 2nM in pea at Dow have not ended with the develop- 50 ulatory dossier was submitted in 2016.[67] aphid), hence controlling the insect plant ment of fenpicoxamid. Subsequent struc- The pyropyropenes are a rare example of pests by disturbing their motor coordina- ture–activity relationship investigations on a natural product family for which at least tion and ability to feed.[44] Interestingly the bislactone of the natural product led to two unrelated target receptors are known. the structurally unrelated synthetic pyme- the discovery of several macrocyclic com- Oˉ mura reported that pyripyropene A is ac- trozine and pyrifluquinazon commercial pounds possessing chemical and biological tive on acylcoenzyme A cholesterol acyl insecticides control insects via the same properties unique to this class of chemistry transferase, (ACAT), a research target for mechanism.[44] (Scheme 6).[72] Natural Products: source of INNovatIoN CHIMIA 2017, 71, No. 12 817

have emerged from soil (post-emergence control).[76] Bialaphos is a proherbicide O O N O O N O O N that is bioactivated into phosphinothricin HO HO HO by plants before exerting its herbicidal 1. aq. NaOH (50%) effect. It is obtained from fermentation O MeOH, 30 °C O O H H H cultures of a Streptomyces. Glufosinate, O O OH 2. 3.2 eq. Cl O O OH O O O the racemic form of phosphinothricin, is H H H O O O O O synthetically produced and is the commercially relevant version of the chemical.[73] O DMAC, 0°C O O This class of natural products are unique Pyripyropene A (33) Afidopyropen (34) 36 inhibitors possessing a methylphosphinic 41% acid unit. This unusual P-methylated amino acid is a structural analogue of glutamate and acts as an inhibitor of glutamine Scheme 4. Afidopyropen, an insecticidal semi-synthetic natural product currently in late develop- [77] ment. synthetase. Glutamine synthetase is required for the production of glutamine and for ammonia detoxification. Inhibition of this enzyme results in a reduction of the

O O cellular amount of glutamine and an in- O O crease in ammonia to toxic levels. This O O O O

O OH O O NH OH (CH2)5CH3 interrupts photosynthesis in the weed and O Cl O O HN HN HN O O O leads to death within a few days. O Na2CO3,NaI O O N O O acetone N O O O O For the herbicides in which natural O O O products played a crucial role in the dis- UK-2A (37) Fenpicoxamid (38) Antimycin A2a (39) procide covery and optimization process, two classes stand out; the auxin herbicides (e.g. Scheme 5. Fenpicoxamid, a fungicidal semi-synthetic natural product currently in late develop- 2,4-dichlorophenoxyacetic acid (2,4-D) ment. (46)) and the triketones (e.g. 48 and 49). 2,4-D, commercially available since 1945, provides effective post-emergence control of broadleaf weeds in a large variety of crops and was the first herbicide found O areobic O to be capable of selectively killing weeds O OH O O O conditions H HN N O but not crops. It acts by mimicking the ac- O O N O H O tion of the auxin plant growth hormone, N O O O O O O indoloacetic acid (47), which results in O UK-2A (37) metabolite isolated from uncontrolled growth and eventually death surface abiotic degratation (40) in susceptible plants. The auxin herbicides have their origin in the study of the plant growth-regulating activity of 47. As this O compound was too unstable to work with, O O OH O OH O OH O O O O O synthetic mimics were prepared and at O O O O O O N N N H O H O H O some point in time, their herbicidal poten- N N N tial was realized. 2,4-D was one of the two

DAS-598 (41) DAS-381(42) DAS-385 (43) first auxin herbicides to be developed.[78] removal of one C=O group to removal of two C=O groups to tested in field trials, [79,80] achieve better metabolic stability achieve even better metabolic stability enhanced acropetal mobility compared The triketones are a more recent class to fenpicoxamid of herbicides with both pre-emergence and post-emergence weed-control which exert Scheme 6. Sensitivity of the picolinamide towards aerobic conditions and new synthetic UK-2A their herbicidal activity by inhibition of the derivatives from Dow. enzyme 4-hydroxyphenylpyruvate dioxy- genase (HPPD). Inhibition of this enzyme by chelation of the iron in the active site Natural Products as Herbicides high rates to be effective. For instance, the by the 1,3-dicarbonyl motif disrupts the recommended rate for glufosinate (45, vide biosynthesis of carotenoids and causes There are numerous reports of second- infra) is in the range of 500 g/ha, compared bleaching (loss of chlorophyll) of the fo- ary compounds derived from plants and mi- to 100 g/ha or less for many modern her- liage followed by necrosis and death of croorganisms that are phytotoxic[73,74] and bicides. This relatively low activity of the the treated weeds.[81,82] Among the several the use of natural products as herbicides or herbicidal natural products combined with products which have been commercialized as lead structures for herbicide discovery their structural complexity is the reason for in this class, Syngenta’s contribution was programs is an approach that has also been their smaller market presence. The topic of mesotrione (48), launched in 2001 and exploited. However, relatively few natural natural-product herbicides cannot be cov- bicyclopyrone (49), belonging to the new products or semi-synthetic analogues have ered without featuring the success story class of nicotinoyl cyclohexane diones, made it to the market compared to insecti- of phosphinothricin (glufosinate) (44, launched in 2015.[83] Early in the course cides and fungicides.[75] Phytotoxic natural Scheme 7). Phosphinothricin and the tri- of the research which eventually would products are in general structurally more peptide analogue bialaphos (45) are broad lead to mesotrione, scientists at Stauffer, a complex than synthetic herbicides and ma- spectrum herbicides that can be used to fore-runner company of Syngenta, realized ny of them need to be applied at relatively control a wide range of weeds after they that the bottlebrush plant (Callistemon ci- 818 CHIMIA 2017, 71, No. 12 Natural Products: source of INNovatIoN

Scheme 7. Herbicidal the structure of the diacid. Access to this Commercial natural products natural products hav- natural product through synthesis is not ing achieved com- O OH OH O OH practicable and fermentation remains the H mercial status or in P N P method of choice. To support bioprocess HO O O N O H development. NH2 O NH2 engineering, several studies have been reported on the elucidation of the biosynthe- Phosphinothricin (44) Bialaphos (45) racemate: Glufosinate Meiji Seika (1984) sis of cornexistin and the characterization Hoechst (1970) of the corresponding gene cluster.[86,89]

Commercial herbicides inspired from natural products O OH Natural Products as Biopesticides O OH O OH O O OH NO2 Cl Certain natural compounds used N O SO Me O O for pest management are recog- H 2 Cl nized by state organizations such as 2,4-D (46) indoloacetic acid (47) Mesotrione (48) Leptospermone (50) the U.S. Environmental Protection (1945) Syngenta (2001) Agency (EPA) as biopesticides.[90] O OH O O They include insect repellants and attrac- N tants, biochemical insecticides, fungicides, O CF3 herbicides, nematicides. Although there Bicyclopyrone (49) are many definitions throughout the world Syngenta (2015) what a biopesticide is, their desirable prop- Example of experimental natural product erties are: • Naturally occurring chemicals or their OH OH derivatives

O O • Reduced toxicity to non-target organ- O -O C isms OH fast 2 OH - O O2C • Reduced persistence in the environment O • Low mammalian toxicity

Cornexistin (51) 52 • Safe for farmworkers and nearby resi- maleic anhydride form dianion form dents pH 2.5 pH 7 • Green technology possibly in development at BASF Amongst the various biopesticides in use nowadays, botanical pesticides trinus) was repressing the growth of other responding ring-opened diacid, as shown are used for insect control in agriculture, plants in its surroundings, suggesting that in Scheme 7. NMR experiments show that mainly on fruits, vegetables and green it might produce a strong phytotoxic agent. the anhydride form is preferred in organic house flowers. They consist of secondary Bioassay-guided isolation work led to the solvents and in water at low pHs, while metabolites from plants performing use- discovery of leptospermone (50), a previ- the ring-opened form is preferred in water ful functions against insect herbivores as ously characterized acyl syncarpic acid at neutral pHs. Hence, these forms could repellents, feeding inhibitors or toxins and plant metabolite with no known biological be in rapid equilibrium at physiological they often are applied as semi-purified ex- activity. The natural product caused bleach- pH allowing cornexistin to move easily in tracts rather than in their pure form.[91] Up ing symptoms indicative of what would plants, being able to penetrate both aque- to now there are only a handful of effective later become to be known as the HPPD ous and lipophilic phases.[87] Colton and commercial botanical products: the pyre- mode of action. The syncarpic acid unit of Kazlauskas have studied a similar phenom- thrins, which represent economically the leptospermone would then be incorporated enon in the use of dicarboxylic acids as pro- most important group, alkaloids such as in the triketones optimization program, ton transfer agents across membranes.[88] nicotine or the sabadilla alkaloids, the iso- combined with the benzoyl moiety and The ability of the acid to move through flavone rotenone (53), the limonoid azadi- further evolved to obtain compounds with the membrane was found to depend upon rachtin (54) and essential oils (Fig. 6). much higher overall herbicidal potencies a number of factors but at ambient tem- Azadirachtin provides a good example and weed-controlling spectra.[80] perature, the rate of anhydride formation of the advantages brought by botanicals Cornexistin (51) is a maleidride iso- was generally rate-limiting, varying with but also the complexities they carry. The lated by Sankyo in the late 1980s from the fungus Paecilomyces variotii.[84] It is a potent wide-spectrum herbicide against weeds but with low activity against maize OH and thus shows promise as a commercial O O O herbicide. The natural product is possibly H O OH O H OMe OH OH [85,86] O HO OH O OH in development at BASF. The mode of O OH O O O H action is the inhibition of transketolase and O O O O OH it is the only known inhibitor of this tar- O OH O N get with good herbicidal activity.[86] This MeO O feature may be explained by the molecular O O adaptability of the natural product. Indeed, Rotenone (53) Azadirachtin (54) Ryanodine (55) Linallol (56) Limonene (57) cornexistin equilibrates between the maleic anhydride form and the dianion of the cor- Fig. 6. Secondary metabolites responsible for activity in some botanical insecticides. Natural Products: source of INNovatIoN CHIMIA 2017, 71, No. 12 819

natural product, which is a complex tetra- 2017). For this perennial plant, harvesting cals that are their mainstays. State agencies nortriterpenoid, is the predominant insec- is once per year. The yield is about 2000 have helped to catalyze the introduction of ticidal active ingredient derived from the kg seed kernels per hectare with a content biopesticides by offering some regulatory neem tree and is considered to be environ- in active ingredients of 0.45%, represent- relief for their registration. mentally friendly because it is relatively ing 9.0 Kg of product per year. Moreover, Botanical insecticides have not only safe to mammals (LD rats > 3540 mg/ azadirachtin is hampered by the high cost benefited biocontrol strategies; they also 50 Kg), fish and pollinators and has a short of raw material due to the complex and ex- have provided mode of action knowledge persistence on crops. It is derived from the pensive extraction processing of the seeds in the discovery of commercially very seed kernels of the neem tree, which has compared with pyrethrum, meaning that successful synthetic compounds. The ry- been used for centuries in India as a po- the cost for treating one hectare (20–60 anodine alkaloid (55), which is the active tent antimicrobial and insecticide, either g of the active ingredient are required) is principle of the extract from Ryania stems, directly by cold-pressing or by hydroal- relatively high, which can raise some prof- is a prime example of this. Ryanodine and coholic extraction. Azadirachtin affects a itability issues.[101] The main bottleneck related alkaloids affect muscles by binding broad spectrum of insect species includ- in the conventional process of extraction to calcium channels in the sarcoplasmic re- ing aphids, mealybugs, caterpillars, beetle of azadirachtin is the variability imposed ticulum. This activates the uncontrolled re- and weevils, whiteflies, mites and thrips. by the heterozygosity in seeds resulting in lease of calcium stores necessary for mus- Azadirachtin’s potent biological action ap- fluctuating secondary metabolite content cle cell function, leading to Ca2+ depletion, pears to come from a combination of be- and uncontrollable seasonal and geogra- feeding cessation, muscle paralysis, and havioral and physiological effects leading phy constraints. Despite being nontoxic to ultimately insect death.[103] Ryania extracts to feeding deterrence.[92] Lepidopterians mammals, fish and pollinators, the influ- have found limited use as insecticides and are very sensitive to azadirachtin which ence of azadirachtin on beneficial insects ryanodine itself is not selective towards the shows effective antifeedancies from < is highly variable and requires special at- corresponding mammalian receptors which 1–50 ppm depending upon the species. tention. leads to moderate toxicity. Recently a new The antifeedant effects observed in these Lately, companies are relying on the class of insecticides, the diamides, has species are correlated with the sensory re- commercial development of essential oils- been discovered and provides exceptional sponse of chemoreceptors (taste receptors) based insecticides as a consequence of control through selective action on the ry- in the insects,[93–95] resulting in starvation some facilities in registration procedure, anodine receptors in insects.[104] Products and death.[92,93] Coleoptera, Hemiptera easy access to raw material and relatively on the market are flubendiamide (58)[105] and Homoptera are less sensitive to the low cost.[102] Essential oils are obtained jointly developed by Nihon Nohyaku and antifeedant azadirachtin. For these other by hydrodistillation of aromatic plants Bayer, chlorantraniliprole (59) and cyan- species, crop protection results from a (leaves, flowers, fruits) and are rather traniliprole (60)[106] developed by Dupont combination of physiological effects after complex mixtures containing terpenoids, (Fig. 7). In their works on the elucidation ingestion of azadirachtin.[96,97] An ED of alcohols, ketone, aldehydes and phenolic of the mode of action of flubendiamide, sci- 50 around 1 mg/g body weight is consistently compounds (given examples are 56 and entists at Nihon Nohyaku assayed several seen through these insect species.[97] The 57, Fig. 6). Essential oils are used as repel- active substances with a known mode of ac- mode of action of azadirachtin is a com- lents or contact spray. They are generally tion by injection into the larvae of lepidop- plex research topic and recent reports have lipophilic and hence can preferentially in- terians.[105] The larvae injected with ryano- indicated that the action of azadirachtin teract with membrane-bound receptors and dine showed sustained body contraction at the cellular level is to block microtu- enzymes in the insects and show effect similar to the larvae treated with fluben- bule formation[98] and a possible binding through fumigation of insecticidal contact diamide, which provided key information protein has been identified in Drosophila action. Because of variabilities in large eventually leading to the elucidation of the Kc167 cells.[99] scale production, agricultural practices and target receptor for this important class of The use of biopesticides has grown with environmental conditions, their effects are agrochemicals.[107] increasing restrictions on persistent pesti- relatively difficult to standardize but the cides and the growth of the organic food interest in these solutions and the experi- and farming movement. Biopesticides, ments on this efficacy has been increasing Peptides with Insecticidal introduced in integrated pest management since the 1990s. Properties Produced by Plants systems, are ideal for pre- or post-harvest In the area of biopesticides in gen- treatments of fruits and vegetables as they eral, smaller, specialty companies such Certain peptide compounds are envi- do not leave residues on food. However, as Trécé, Marrone Bio Innovations or ronmentally more stable and persistent from a commercial point of view, many of Vestaron are leading the technology, but than secondary metabolites and may be these chemicals are not really used on large larger companies such as Dow, DuPont, strong candidates for the next generation scale because of the lack of technology to Monsanto, Syngenta, Merck, BASF, and of botanical pesticides.[91] This comprises produce them in sufficient quantities and Bayer are developing or marketing biopes- for instance the cyclotides which constitute the time consuming and laborious proce- ticides along with the conventional chemi- the largest class of ribosomally synthe- dures to produce them. Currently, the demand for pyrethrins, for instance, is greater Fig. 7. The diamides than world production. David Morgan re- O O insecticides binding [92] S Br Br ported in 2009 an average yield per hect- I HN to insects ryanodine are of 55 kg of dried flowers containing N N receptors. O O O 1–2% of active ingredient. Thus, 0.55 Kg N N O Cl Cl to 1.1 Kg of pyrethrins can be obtained per NH NH N N HN hectare for each harvest (every two weeks O O Cl NC in the season). Sales forecast for Pyrethrins CF3 HN HN in 2017 is US$68 million.[100] Azadirachtin F CF3 products from neem, by comparison, rep- Flubendiamide (58) Chlorantraniliprole (59) Cyantraniliprole (60) resent lower figures (US$29 million in 820 CHIMIA 2017, 71, No. 12 Natural Products: source of INNovatIoN

sized cyclic peptides produced by plants (Fig. 8). Cyclotides are typically consti- tuted of 27–37 amino acid residues orga- nized in a backbone stabilized by disulfide bonds which confers exceptional stability against enzymatic degradation, tempera- ture variation and chemical stress.[108] Due to these properties, cyclotides have been evaluated for their biological functions, including antibacterial, nematicidal, and [109–111] insecticidal activities. The defen- Fig. 8. Cyclotides family members. Disulfide bonds are highlighted as yellow sticks. sins represent another class of insecticidal proteinaceous compounds from plants. Defensins comprise a family of cysteine- and they are the major contributors to the rich, basic peptides with 45–54 amino venom’s insecticidal activity.[117] They acids in length forming an α-helix and a have evolved to target a wide range of pre- triple-stranded antiparallel β-sheet stabi- synaptic ion channels or postsynaptic re- lized by four disulfide bonds.[112,113] Such ceptors either at peripheral neuromuscular biomolecules may find application in the junctions or at synapses in the insect cen- field of transgenic crops rather than being tral nervous system. These peptides can act applied as formulated biopesticides. All individually or in combination, to rapidly the peptides mentioned above exert their immobilize the envenomated prey either mechanism of action in the insect midgut by desensitizing its nervous system and mostly by interfering with the absorption causing flaccid paralysis or by over acti- of nutrients and affecting the integrity of vating it and causing convulsive paralysis. Fig. 9. Protein crystals (bipyramidal) mixed with insect’s peritrophic membrane or the epi- The overall effect induced by these neu- spores from Bt strain. thelial cells beneath. rotoxins is complex and involves groups of peptides that act at different times and Bacillus thuringiensis Toxins sites following envenomation. The natu- in greenhouses.[119,120] The active ingredi- Entomopathogenic bacteria and fungi ral prey of most spiders are invertebrates, ent claimed to have a dual mode of action have potential for insect control and have mainly insects. Because most spiders are has been registered by the United States provided a wide variety of insecticidal pro- polyphagous, their venoms have evolved Environmental Protection Agency in 2015 teins active against larvae of diverse insect to contain an array of compounds that and commercial availability is expected orders.[114] By far, the most widely used target a broad spectrum of insect prey. in 2018 under the trademark Spear T. The and best-known of these proteins are the Moreover, although some large spiders company has three more products in pipe- proteins from Bacillus thuringiensis (Bt), consume small vertebrates, very few are line: Spear P for potato beetle in combi- a Gram-positive, spore-forming bacterium toxic to humans.[118] Hence, the primary nation with Bt, Spear C for lep control in which is the richest source of insecticidal rationale for investigating spider venoms combination with Bt and Spear O for or- genes.[115] Bt strains synthesize Crystal as a potential source of bioinsecticides is namentals. (Cry) (Fig. 9) and cytolytic (Cyt) toxins as that their venoms are expected to contain parasporal crystalline inclusions during the a wide range of insecticidal peptides that pathogenic process at the time of sporula- mostly have little or no mammalian activ- Conclusion and Outlook tion. Once ingested by insects, these crys- ity. The research on spider venoms has talsaresolubilizedinthemidgut.Thetoxins largely validated this hypothesis. Most Growing consumer demand, pest resis- are then proteolytically activated by mid- of the insecticidal spider-venom peptides tance pressure and an ever-changing regu- gut proteases and bind to specific receptors contain a unique arrangement of disulfide latory environment necessitate the discov- located in the insect epithelial cell mem- bonds that provides them with a strong ery of new crop protection agents for grow- brane, leading to pore formation followed level of protease resistance. As a result, ers of today and tomorrow. Lead genera- by cell disruption and insect death.[116] these highly stable peptides are likely to tion and optimization is the critical engine Bt crystal and secreted soluble toxins are have long residence times in the insect gut for any agrochemical chemical company highly specific for their hosts and have and in the hemolymph, and therefore even wishing to maintain a robust pipeline of gained worldwide importance as bio- low rates of intestinal absorption will make new high-value products. A wide variety control agents. Several natural Bt strains them active through oral ingestion. Many of approaches exist for the generation of have been incorporated in the production of them have desirable properties, includ- new leads, with many having demonstrated of sprayable Bt-based bioinsecticides ing high potency, rapid speed of kill, lack success. In that respect, natural products wherein the active ingredient is a mixture of vertebrate toxicity, and activity against certainly had a broad influence over the of spores and protein crystals. Moreover, a wide range of crop pests. Moreover, they past decades and will continue to do so in some cry toxin genes have been introduced should be stable in the field owing to their the 21st century. As noted, natural product- into transgenic crops providing an effec- disulfide-rich molecular architecture, and based discovery can be challenging in that tive way to control certain lepidopteran their degradation is unlikely to produce finding new chemical classes with the ac- pests as well as some coleopteran. toxic residues. Thus, spider-venom pep- tivity and properties needed to be effective tides may have potential for use as stand- against agricultural pests is a rare event. alone bioinsecticides. Vestaron based in Frontiertechnologiesareincreasinglygain- Spider-venom Peptides the United States is currently developing ing momentum in modern crop protection the first sprayable formulation contain- discovery and natural products accompany Disulfide-rich peptides are the domi- ing a spider toxin (GS‐omega/kappa‐ the movement.[121,122] However, the devel- nant compounds in most spider venoms Hxtx‐Hv1a) for use in controlling thrips opment of a scalable fermentation process Natural Products: source of INNovatIoN CHIMIA 2017, 71, No. 12 821

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