An appraisal of natural products active against parasitic nematodes of animals

Phenols

Phenolic compounds are widespread in nature and are often components of natural product extracts. They include monophenols or terpenoids (thymols, cresols and carvacrol) (Fig. 2), benzene diols (catechols and resorcinols), flavonoids (e.g. quercetin) (Fig. 3), substituted benzoic acid (gallic acids and vanillic acids) and cinnamic acids. Many of these classes frequently appear in the natural product literature, particularly in studies using extracts of plants and in organisms hosting symbionts able to photosynthesize. Photosynthesis underpins key synthetic routes, such as the acetate-malonate and shikimate pathways that generate the malonyl-CoA and shikimic acid synthons utilised in the biosynthesis of a large number of phenolic compounds [38].

The reported biological activities of natural product extracts commonly result from the intrinsic antioxidant character of phenolic compounds. This property originates from the ability of the phenol group to form a phenoxy radical upon donating a hydrogen atom. This phenoxy anion can scavenge free radicals and participate in metal chelation reactions. The types of chemical functionalities and their position on the phenol ring modulate its antioxidant potential and the capacity to act as a radical scavenger [39, 40]. Several assays have been developed to measure antioxidant activity and the most commonly used with natural product extracts is the 1,1-diphenyl-2-picrylhydrazyl radical scavenging assay (DPPH) [41]. The DPPH assay is commercially available, inexpensive and technically simple to execute, being one of the main reasons the antioxidant activity of natural product extracts is frequently reported in the literature, and in the general media, as a source of benefits such as healthy ageing and cancer prevention. It is also probable that the antioxidant activity of phenols is largely responsible for the broad, promiscuous activity of this class of compounds reported from screening assays. In addition to antioxidant potential, ortho-dihydroxy aryl derivatives possess metal chelating properties, and high order polyphenols have the capacity to bind macromolecules through multiple, non-specific hydrogen bond interactions, thus causing aggregation, particularly when tested at near millimolar concentrations. These mechanisms are nearly impossible to optimise chemically to reach pharmacologically useful potency and selectivity, and they are largely responsible for the view of phenolic compounds as promiscuous, “nuisance assay positives” by the drug discovery community [42]. Nevertheless, natural phenols keep being reported as anthelmintics in the literature, and herein we discuss their nematocidal activity. Cinnamoyl derivatives, polyphenols and flavanols will be reviewed in their own subsection below.

Carvacrol and thymol (Fig. 2) are commonly found in plant extracts and have displayed activity against the model nematode C. elegans [43]. When treating worms in vitro at a concentration of 670 µM for 24 h, the two compounds caused 100% mortality, unlike p-cymene, which displayed little efficacy in this assay. Microscopy showed that thymol-treated C. elegans displayed burst or bent morphologies. Carvacrol and thymol were also shown to be effective against the pig roundworm, Ascaris suum, in vitro. Both compounds killed 80% of A. suum worms after treatment for 24 h at a concentration of 330 µM, while p-cymene at 370 µM killed only 20% after 24 h [43]. Thymol has also been described as an inhibitor of H. contortus egg hatching (IC₅₀ = 2.4 mM), L1 to L3 larval development (IC₅₀ = 0.83 mM), and L3 motility (IC₅₀ = 3.3 mM) [44].

Carvacrol and thymol are more toxic to human cells than their acetate derivatives. It was hypothesised that the difference was due to acetylation suppressing hydroxy radical formation, and both carvacrol acetate and thymol acetate (Fig. 2) were compared with their non-acetylated parent compounds in a nematocidal study using H. contortus. In the carvacrol study [45], it was found that the acetate had a somewhat weaker anthelmintic activity than free carvacrol against H. contortus in vitro (egg hatching EC₅₀ = 11.3 mM and 1.1 mM; larval development EC₅₀ = 2.0 mM and 1.3 mM, respectively). Both carvacrol- and carvacrol acetate-treated adult H. contortus displayed morphological alterations in the cuticle and the vulvar flap, suggesting that both have the same mechanism of action. Dosed at 250 mg/kg (once), presumably p.o., carvacrol acetate reduced gastrointestinal nematode faecal egg counts in infected sheep by 66%, while its LD₁₀ and LD₅₀ in mice by the same route were 567 mg/kg and 1545 mg/kg, respectively. A metabolite study was not undertaken, so it was not known whether carvacrol acetate was possibly metabolised to carvacrol under the study conditions and, therefore, the real active constituent is unknown. The thymol acetate study [46] produced very similar results. Acetylation of thymol reduced its acute toxicity to mice, but it also caused a 10-fold reduction of potency in an egg-hatch assay and a slightly decreased potency in a larval development assay when compared with the parent thymol. These results suggest that the modest nematocidal activity of these phenolic molecules is likely derived from their intrinsic antioxidant character.

Cinnamoyl derivatives and polyphenols (tannins, flavonoids and isoflavonoids)

Cinnamic acid is an intermediate in the biosynthesis of other natural phenolic compounds, such as coumarates, flavonoids and tannins (Fig. 3). Several cinnamic acid derivatives were isolated from fractions of natural extracts with anthelmintic activity. One such study found that leaves of Acacia cochliacantha collected in Mexico contained a high percentage of caffeic acid, ferulic acid and p-coumaric acid, and their respective esters (Fig. 3) [47]. Fractions containing these cinnamic acid-like derivatives were tested in a H. contortus egg-hatching inhibition assay. At a concentration of 1 mg/ml (~ 5 mM), all compounds tested displayed an egg hatch inhibition of 71–98%, in agreement with a previous field study that found that goats fed A. cochliacantha foliage excreted less H. contortus eggs than those on a different feed (cf. [47]), suggesting that the reduction in egg counts was due to the presence of cinnamic acid derivatives in this plant.

As introduced in the subsection on phenolic compounds, polyphenols (also named hydrolysable or condensed tannins) are able to bind non-specifically to macromolecules and aggregate them when the compounds are present at near millimolar concentrations, a process central to the leather tanning process (cf. [48, 49]). Condensed tannins, such as procyanidins (Fig. 3), were the primary constituents isolated from fractions of Tilia flowers and goat willow leaves, while prodelphinidins (Fig. 3) were the main components isolated from fractions of black and red currant leaves. These fractions were tested in an exsheathment inhibition assay using the L3 stage of the Juan strain of H. contortus [50] and compared with the activities of the flavonoids naringenin, quercetin and luteolin (Fig. 3). In this assay, the prodelphinidin-rich fractions showed similar activity to the flavonoids (IC₅₀ = 60–90 µg/ml; ~ 100–150 µM) and were 2-fold more potent than the procyanidin-rich fractions (IC₅₀ = 140–176 µg/ml; ~ 250–300 µM). Luteolin was also found to possess anthelmintic activity against Trichuris muris (IC₅₀ = 9.7 µg/ml; ~ 34 µM) (and against S. mansoni, not reviewed here) [51]. Electron microscopy revealed that luteolin-induced cuticular or tegumental damage in all worms tested.

A series of 33 hydrolysable tannins were isolated from cold-acetone plant extracts from Finland and evaluated against H. contortus, measuring inhibition of egg hatching and motility of L1 and L2 larvae [52]. Of the tannins evaluated, pentagalloylglucose, tellimagrandin II and hippophaenin B were the most potent at reducing egg hatching (> 50% inhibition at 0.5 mM), while pentagalloylglucose, heptagalloylglucose, tellimagrandin II and casuarinin (Fig. 3) all had an IC₅₀ of < 0.5 mM when inhibiting the motility of L1 and L2 larvae. Perhaps worryingly, cryo-scanning electron microscopy showed that pentagalloylglucose formed aggregates on the surface of the L1 and L2 larvae and eggs, profoundly affecting the buccal capsule and the anterior amphidial channels. This suggests that its anthelmintic effects may be due to tannin deposition on biological surfaces, rather than to interactions with specific targets. The degradation of pentagalloylglucose and casuarinin was also measured following incubation in phosphate-buffered saline (PBS) or in ‘egg matrix’ solution, and it showed considerable hydrolysis of the gallic acid residues plus oxidation. The authors determined that there was an optimal molecular weight for activity (940 Da) and used this parameter, together with the number and linkage type of gallic acid residues, to propose an empirical equation predicting the anthelmintic potency of hydrolysable tannins [52].

Gallic acid analogues, the ellagic and gentisic acids, were isolated from the axlewood tree, Anogeissus leiocarpus [53]. Ellagic acid exhibited the greatest activity against the free-living nematode C. elegans and the bovine filarial nematode Onchocerca ochengi, with an IC₅₀ = 90 µM against the adult and an IC₅₀ = 30 µM against the microfilariae of O. ochengi. The potency of ellagic acid against wildtype C. elegans (IC₅₀ = 85 µM) was unchanged in strains resistant to levamisole and ivermectin. However, this compound displayed 10-fold less activity against the CB3474 strain, resistant to albendazole, suggesting that ellagic acid activity is affected by some albendazole resistance mechanisms. On the positive side, ellagic acid showed a good safety profile in rats and, therefore, may represent a reasonable starting point for development as an anthelmintic agent. The multiple hydroxyl groups are, however, frequent sites of metabolism and would probably need to be chemically protected.

Other gallic acid analogues were isolated from the fruits of Acacia nilotica from the northern areas of Cameroon [54]. (+)-catechin-3-O-gallate, and four related proanthocyanidins: (+)-epicatechin-3-O-gallate, (+)-gallocatechin, (−)-epigallocatechin and (−)-epigallocatechin-3-O-gallate were all isolated and evaluated against C. elegans and O. ochengi. All derivatives displayed IC₅₀ = 1–11 µM against adult and microfilariae of O. ochengi, a reasonable potency, even though they were all less active against wildtype C. elegans (IC₅₀ = 34–350 µM). Importantly, C. elegans strains resistant to albendazole, levamisole or ivermectin were equally susceptible to these compounds, implying that the gallic acid analogues tested were not susceptible to resistance mechanisms affecting these commercial anthelmintics. However, all derivatives also displayed toxicity against a human cell line (Caco-2) at concentrations comparable with their potency against C. elegans (see [53]). This raises questions about the pharmacological developability of this set of proanthocyanins.

Polyphenols are also found modified with other natural synthons. Socolsky et al. [55] isolated several unusual terpenylated acylphloroglucinols, wallichin A-D, filixic acids ABA and ABB, and albaspidin AA and AB (Fig. 4), from the rhizomes and scales of the fern Dryopteris wallichiana, traditionally used to treat helminthiases. The authors then evaluated these derivatives in a viability assay against the L4 larval stage of the rat nematode Nippostrongylus brasiliensis. It was found that wallichins A and B as well as flilixic acids displayed higher potency than unmodified polyphenols, with LD₅₀ = 22–37 µM, while wallichin C and the albaspidins displayed 5-fold weaker activity. The authors hypothesised that a free hydroxy group at the C-4′ position of wallichins A and B was important for modulating activity.

The isoflavonoids deguelin and rotenone (Fig. 4) were discovered from a screen of a small natural product library against exsheathed third-stage (xL3) larvae of H. contortus [56]. The two compounds are commonly isolated from plants of the family Leguminosae [57] and have been reported previously to display a range of activities, such as anti-inflammatory [58], apoptotic [59] and insecticidal [60]. However, rotenone exhibits potent mammalian cytotoxicity [61, 62] and, therefore, its nematocidal activity was not further investigated. Deguelin was relatively benign to mammalian cells in the screening study (neonatal foreskin fibroblast cells; IC₅₀ > 50 µM), while showing an IC₅₀ = 14 µM against xL3 and an IC₅₀ = 0.004 µM against L4 in a 72-h motility assay. Deguelin also blocked development of xL3s to the L4 stage after 7 days, with an IC₅₀ = 3.2 µM [56]. Transcriptomic responses of xL3 H. contortus to deguelin treatment identified differential gene transcription associated with energy metabolism, particularly oxidative phosphorylation and mito-ribosomal protein production, supporting an inhibition of the respiratory complex I as the mode of anthelmintic action, as also proposed for the compound’s effects on mammalian cells [63]. In mammals, complex I inhibition by deguelin seems to act in parallel to the disruption of the PI3K-Akt kinase signalling pathway [64], which has been linked to neurotoxicity [63, 65]. Further analyses are needed to determine whether deguelin targets the same pathways in mammals and nematodes, which would enable a better understanding as to whether it is possible to refine the selectivity of this potent natural nematocide.

Terpenes

From a formal point of view, terpenes are the products of condensation of at least two isoprene monomers (as in monoterpenes), and, more frequently, three (sesquiterpenes), four (diterpenes) or six (triterpenes) five-carbon units. Terpenoids (=isoprenoids) are terpenes modified by the addition of heteroatoms, frequently oxygen. Chemical formalisms aside, there are two main routes of terpene and terpenoid biosynthesis. Many organisms produce them utilising mevalonic acid as a key intermediate (the mevalonate pathway). An alternative, non-mevalonate pathway results in the production of isopentenyl pyrophosphate and dimethylallyl pyrophosphate, which are key synthons for the production of higher order terpenes, such as the prenyl derivatives and triterpenoids discussed in their own subsections below. Terpenes are susceptible to oxidation by cytochrome P450 enzymes, a main route for the production of oxygenated terpenoids [66]. Oxidation events include the formation of lactones, sesquiterpene quinones and hydroxyl quinones and epoxides, all of them reactive species that can endow this chemical class with biological activity in the correct molecular environment.

In recent literature, several terpenoid natural products were found to possess nematocidal activity. Terpinen-4-ol (Fig. 5), commonly isolated from conifers and Melaleuca alternifolia, the source of tea tree oil, is well known for its modest antimicrobial activity [67]. A recent study [68] also demonstrated some activity of terpinen-4-ol against H. contortus. In this study, in which a “nanostructured” formulation of the oil was also tested, terpinen-4-ol was the most active test article, with an IC₅₀ = 4.1 mM in the egg hatch inhibition assay, and an 82% inhibition of L3 migration at 22 mM. These in vitro potencies were too low to consider in vivo testing in animals.

A prickly shrub found in China and Taiwan and used in traditional medicine, Zanthoxylum simulans, produces an essential oil containing a variety of alkaloids, coumarins and flavonoids, including a significant amount of the terpenes borneol and β-elemene (Fig. 5). The two terpenes were evaluated against larval stages of H. contortus [69]. Borneol inhibited H. contortus egg hatching (IC₅₀ = 9.7 mM) and larval development (IC₅₀ = 12.9 mM), and at 130 mM it suppressed larval migration by 98%, while β-elemene was poorly active or completely inactive in all assays, despite the high millimolar concentrations used; these results essentially led to the elimination of these terpenoids as anthelmintic starting points.

Warburganal and polygodial are structurally related sesquiterpenes containing two aldehyde moieties (Fig. 5). The two compounds were isolated from the medicinal plant Warburgia ugandensis, native to eastern and southern Africa [70]. An ethanolic plant extract was fractionated, guided by the detection of anthelmintics activity against C. elegans, and warburganal and polygodial were recovered among the most active natural constituents, with respective IC₅₀ values of 28.2 µM and 13.1 µM. The two sesquiterpenes were equally active against several drug resistant strains of C. elegans, suggesting that the compounds were not susceptible to drug resistance mechanisms affecting commonly used anthelmintics. Several analogues of the two sesquiterpenes were obtained and evaluated for preliminary structure-activity relationship studies. It was found that at least one of the two aldehyde groups present in these structures was required for activity, suggesting that these chemically-reactive motifs contribute to the observed anthelmintic effect. Mechanism of action studies were also undertaken, based on similar studies of polygodial using the yeast Saccharomyces cerevisiae and mammalian cells, where this compound was found to act as an uncoupler of mitochondrial oxidative phosphorylation [71]. In C. elegans, polygodial was found to inhibit mitochondrial ATP synthesis with an IC₅₀ = 1.8 µM and a phenotype consistent with inhibition of mitochondrial ATP synthesis. Collectively, these data suggest that polygodial is a worthwhile compound to be considered for further investigation as a nematocide. However, selectivity will need to be addressed, since polygodial inhibits mammalian ATP synthesis to a similar degree as in C. elegans (see [71]).

Prenyl derivatives

The final biosynthetic products from the non-mevalonate pathway of isoprenoid biosynthesis, geranyl pyrophosphate and farnesyl pyrophosphate, are precursors to linear mono-, di- and sesquiterpenes, called prenyl groups, which are frequently isolated as natural products with some additional modifications. The monoterpene alcohol linalool (Fig. 6), present in hundreds of plant species, exists as a mixture of two enantiomers to which numerous biological activities have been ascribed, including significant toxicity against transformed cells (cf. [72]). Linalool, presumably the racemate, has recently been put forward as a chemical starting point for anthelmintic drug development, along with other natural products [73]. The compounds in this study were selected from phytochemical databases and filtered chemoinformatically for favourable ADME and drug-like properties. Compounds were further screened computationally for binding to a modelled structure of glutathione S-transferase (GST) from the filarial worm Brugia malayi, and the best hits were biochemically tested at 1 µg/ml against a crude GST preparation from the heartworm Dirofilaria immitis (due to a lack of access to B. malayi). Linalool inhibited DiGST by 98% at 1 µg/ml (~ 6.5 µM) in this assay, and it was thereby assumed to possess anthelmintic efficacy, although this was not directly tested, a problem when interpreting the results. Linalool is sensitive to an extensive metabolism that generates multiple derivatives with biological activity [72] and its purported biological target in nematodes and insects, GST, is usually present as a family of multiple isoenzymes in metazoans; the H. contortus genome, for example, has at least 8 distinct genes encoding GST isozymes (ParaSite@WormBase; release 11) [74]. Presently, it is unknown whether any of these enzymes is singularly essential and thus represents a good anthelmintic target, or how many functionally redundant isozymes can be simultaneously inhibited to a sufficient extent by a single compound. These are some of the biological and chemical questions that need to be addressed ahead of a drug development effort with these compounds.

Three prenyl alcohols present in the essential oil from Matricaria chamomilla and multiple other plants were described as active in vitro and in vivo against L3 larvae of Anisakis simplex [75, 76]. Compounds were tested in vitro at three different concentrations and scored as active down to 15.6 µg/ml (70 µM) for farnesol and nerolidol (presumably a racemate in the case of nerolidol) and down to 32.5 µg/ml (146 µM) for α-bisabolol (Fig. 6). At those concentrations, these compounds arrested motility, abolished infectivity of treated Anisakis larvae in rats and caused extensive tissue damage to the worms. In vivo, they protected rats from Anisakis-induced gastric lesions when dosed at ~ 300 mg/kg simultaneously with infection, although larval mortality in vivo was lower with the pure compounds than that achieved when using complete plant essential oil [75, 76], suggesting the existence of beneficial synergistic interactions within the unfractionated extract. To a similar or even greater extent than linalool, these prenyl alcohols are all very hydrophobic compounds, with chemical structures prone to being extensively metabolised and with numerous known biological activities, although always with relatively low potency [77, 78].

The activity of these and other chemically related prenyl alcohols has been confirmed in C. elegans (see [79]), albeit with the same low potencies (IC₅₀ = 50–100 μM). Activity against this free-living nematode facilitates, in principle, the use of genetic and biochemical techniques to identify the mode of anthelmintic action. However, low potencies may make these expectations difficult to achieve in practice, because high compound concentrations will be needed to observe unambiguous results, and this creates problems of compound solubility and non-specific effects.

Despite the low chemical developability of prenyl compounds, their biology makes them enticing scaffolds for the generation of derivatives able to disrupt essential pathways in target organisms. Aliphatic prenyl derivatives are signalling molecules in multiple cellular pathways and old models of them acting by simply “sticking” to lipid bilayers are giving way to observations of specific interactions with cognate protein domains [80]. Prenyl compounds can affect cells by interfering with the synthesis or transfer of essential farnesyl and geranylgeranyl groups to proteins [81]. Their signalling roles are also worth considering when thinking of anthelmintic analogues. It is noteworthy in this context that insect juvenile hormone is a linear sesquiterpene, and, although not yet isolated from other Ecdysozoa, this hormone and related farnesol esters interfere with ecdysis in nematodes at relatively high micromolar concentrations [82]. This suggests that it may be possible to usefully disrupt endocrine signalling in nematodes if a more potent and water-soluble prenyl mimic can be found.

Triterpenoids and triterpenoid glycosides (saponins)

Six studies discussed below reported triterpenoid compounds with some measure of inhibitory activity against animal parasitic nematodes. Triterpenoids constitute a large class of natural lipids synthesised from a 30-carbon precursor, modified by addition of heteroatoms (generally oxygen) and cyclizations that commonly generate tetracyclic (e.g. steroids) or pentacyclic (e.g. oleanoates) structures. Their conjugation to sugar residues produces amphiphilic glycosides, termed saponins, present in most plant species and displaying the surfactant (detergent) properties expected from their chemical structure [83]. The physicochemical properties of saponins allow them to disrupt molecular associations that depend on hydrophobic interactions (although it is apparently their bitter taste that deters herbivores). Triterpenoids have large hydrophobic surfaces able to bind, with biologically relevant affinity, to hydrophobic patches in proteins, sterol-rich membrane domains and binding sites for steroid or isoprenoid ligands. Therefore, they are promiscuous binders, particularly at the relatively high concentrations required for anti-infective use and, consequently, triterpenoids have been linked to numerous biological activities [84–88]. While saponins are water-soluble, and generally toxic, the unglycosylated free triterpenoids tend to be less toxic but are challenging to develop into oral drugs, mainly because of their low aqueous solubility. This difficulty has been overcome with sophisticated formulations for price-insensitive applications [89].

Recent reports on triterpenoids active against parasitic nematodes described compounds extracted from four different tropical plants, a common crop (oats) and an edible mushroom. An ethyl acetate extract from the latex of the shrub Calotropis procera (Ait.) was shown to have activity against H. contortus, with a reasonably potent EC₅₀ against egg-hatching (1.6 mg/ml), larval development (0.22 mg/ml) and adult motility (~ 0.05 mg/ml) [90]. Three major components were identified upon fractionation of the crude extract: urs-19(29)-en-3-yl acetate (Fig. 7), (3β)-Urs-19(29)-en-3-ol and 1-(2′,5′-dimethoxyphenyl)-glycerol. Surprisingly, even though they were obtained in pure form, no individual activities were reported, and it is thus unknown at this stage whether the anthelmintic properties of the extract can be attributed to either of the three compounds, to their combination or, less probably, to a minor component of Calotropis spp. latex that was not considered in the study.

The saponin fraction extracted from the bark of Zizyphus joazeiro, a shrub or small tree native to Brazil, was shown to have activity against caprine gastrointestinal nematodes (> 90% Haemonchus, with Oesophagostomum and Trichostrongylus species making up the rest) [91]. Intriguingly, the preparation displayed ovicidal activity with promising potency (EC₅₀ = 1.3 mg/ml), but no activity against L3 larvae. This could be mainly due to permeability differences between egg-shell and larval cuticle, as the extract seemed to possess clear cytotoxic activity, with IC₅₀ = 0.2–0.3 mg/ml against Vero cells in culture. Joazeiroside B and lotoside A (Fig. 7) were identified among the saponins in the preparation, an identification supported by an earlier report of their presence in this plant species [92]. The authors proposed that joazeiroside B and lotoside A are responsible for most of the observed activity.

Saponin cytotoxicity was also encountered when analysing active methanolic extracts from seeds, bark and leaves from the Sri Lankan tree Dipterocarpus zeylanicus. All extracts displayed activity against the adult and microfilarial stages of the bovine filarial nematode Setaria digitata [93]. Considering toxicity to human cells, the extract from seeds seemed to have better selectivity (although no more than 2-fold) than extracts from other plant parts, and it was fractionated to identify its active components. Four pure compounds, representing ~ 0.4% by weight of the total methanolic extract, were identified and studied. The two most abundant and potent, although also cytotoxic, were the 3-glucosyl and 3-arabinosyl saponins of oleanolic acid (Fig. 7). Two minor components also identified were free triterpenoids: a regio-isomer of oleanolate and betulinic acid, both found to be inactive in this study. Given the multiple biological activities attributed to oleanolic acid (cf. [94]), it was considered interesting to hydrolyse the saponins and test the released oleanolate. Senathilake et al. [93] measured an EC₅₀ of ~ 40 µM for inhibition of motility by oleanolic acid, both in microfilariae and adult worms, with selectivity indices > 10-fold relative to cytotoxicity. At concentrations of more than twice the EC₅₀, signs of tissue damage in worms were observed, with apoptotic bodies, DNA fragmentation and increased levels of oxidative stress markers, concordant with previously described effects of oleanolate in rapidly growing cells. Interestingly, a search for the free terpenoid in Dipterocarpus seeds failed, and, thus, the basis for the higher selectivity of the seed extract relative to those from other plant parts remained unexplained.

Different terpenoids were detected in organic solvent extracts from roots of the West African plant Dichapetalum filicaule, which had ovicidal activity against the human hookworm Necator americanus [95]. Silica gel column-fractionation and analysis by NMR and MS revealed the presence of numerous triterpenoids, and the nematocidal activity of two of them, dichapetalins A and X (Fig. 7), was investigated. Hatching of hookworm eggs was inhibited by both compounds, with an EC₅₀ of 162 µg/ml and 523 µg/ml for the A and X forms, equivalent to 277 µM and 745 µM, respectively. Other triterpenoids detected in the extract, such as pomolic acid, have been described previously to have multiple biological activities, but they were not tested in the assay.

A more sustainable plant source than those referred to above is oats (Avena sativa); the saponins avenacosides A and B (Fig. 7) were extracted from seedlings using methanol [96]. The authors also sought to emulate the plant response to fungal infection by treating the saponins with avenacosidase, a β-glucosidase that produces the 26-desglucosyl form of the compounds. The products of the enzymatic treatment still retain their glycolipid character due to a tetrasaccharide at position 3 of the triterpenoid core, but the removal of glucose at position 26 reportedly makes them effective disruptors of fungal membranes [97]. Given the use of oats as a traditional remedy for humans and cattle, avenacoside B and its 26-desglucosyl form were each tested against fungi, as well as against eggs and larvae of the parasitic nematode Heligmosomoides bakeri. Although growth of the fungus Trichoderma harzianum was inhibited in vitro, anthelmintic activity was apparently confounded by the presence of 2% ethanol in the preparations, which claimed much of the effect observed relative to medium-only controls in most tests, perhaps with the exception of Rho-123 dye accumulation, which would indicate an interference with a P-glycoprotein-type efflux pump [96].

The only non-plant source of an anthelmintic triterpenoid reported since 2010 was the edible fungus Pleurotus djamor. A hydro-ethanolic extract of fruiting bodies (mushrooms) was fractionated by direct phase silica gel chromatography, and one fraction with ovicidal activity against H. contortus was analysed by gas chromatography-mass spectrometry (GC–MS). It was found to contain mostly free fatty acids plus ≤ 1% of the triterpenoid β-sitosterol (Fig. 7), as determined by ion counts [98]. It is, however, to be expected that the abundant free fatty acids present in the most active fraction were responsible for the largest share of the extract’s ovicidal activity, given that their concentrations greatly exceeded the 241 µM of β-sitosterol present.

In summary, no new nematocidal triterpenoids were reported in the reviewed literature, and the reported ones were previously known and displayed relatively low potencies and narrow selectivity windows in anthelmintic assays, making their development as nematocidal compounds challenging. Nevertheless, it is possible to develop triterpenoids into anti-infective drugs (e.g. fusidic acid), and nematodes are known to use steroid-hormone signalling (cf. [99]), which could potentially be disrupted to obtain an anthelmintic effect if developable analogues can be identified.

Macrocycles

Studies have described anti-nematode activities of macrocyclic lactones or related compounds. Macrocycles are a large class of bioactive natural products that includes the macrolide and glycolipopeptide antibacterials and the cyclosporin and rapamycin immunosuppressants. Nematocidal macrocycles (avermectins and their aglycons, the milbemycins) are produced by several Streptomyces species and were discovered in the 1970s [100]. The discovery and development of the avermectins were awarded the 2015 Nobel Prize in Physiology or Medicine, rewarding the large contribution of a semi-synthetic derivative, ivermectin, to the control of lymphatic filariasis and river blindness. Although not compliant with several drug-likeness rules, avermectins are amongst the most potent pesticides in use, with efficacious doses against nematode and arthropod infections well below 1 mg/kg, even as a single administration (reviewed in [101]). Their recognised mode of action is the activation of a neuronal glutamate-gated chloride ion channel [102].

More recently, Xiang et al. [103] extracted mycelium from Streptomyces microflavus with methanol and fractionated the resultant product by chromatography on silica gel, thus purifying a compound active against C. elegans and adult two-spotted spider mites (Tetranychus urticae). In vitro potency was surprisingly high (EC₅₀ = 15.4 µg/ml or 26 µM against C. elegans), taking into account that, although the compound shares structural motifs with the macrocyclic lactone nemadectin (a milbemycin), it lacks the lactone ring itself (Fig. 8). Activity was confirmed with four similarly open structures obtained from Streptomyces bingchenggensis, showing that these much more flexible compounds possess biological activity found previously only on the cyclic milbemycins [103]. It is not known at this early stage whether the new compounds share the mode of action of the closed macrocycles and whether they possess pharmacological advantages or disadvantages. It would be especially interesting to learn whether they also utilise the unexpected route of entry into nematodes recently described for ivermectin [104].

One study on macrocyclic anthelmintics confirmed the clinical efficacy of the semi-synthetic oxime of a milbemycin (Fig. 8) in treating dogs infected with Toxocara canis, Toxascaris leonina, Trichuris vulpis or Ancylostoma caninum [105]. Milbemycin oxime and another semisynthetic milbemycin, moxidectin, are part of numerous pesticide combinations used in animals. The latter has also recently received FDA approval for use in humans in the treatment of river blindness [106].

Although new nematocidal macrocycles have not been reported between 2010 and 2018, related open analogues that expand the possibilities for semi-synthesis, and that can be readily obtained from culturable bacteria, may contribute to the development of future commercial drugs. These novel analogues will be particularly interesting if their targets and entry routes are distinct from those utilised by the macrocyclic lactone anthelmintics, thus avoiding cross-resistance.

Miscellaneous compounds

The vesicant agent cantharidin (Fig. 9) from beetles of the family Meloidea has been chemically synthesised and used as an anti-cancer chemotherapy for many years [107]. Its highest affinity molecular target in humans was identified as protein phosphatase 2A (PP2A) [108]. In nematodes, some protein phosphatases are among the gene products with sex-specific expression [109–111]. This may be in apparent agreement with the purported aphrodisiac properties of cantharidin in the folk remedy “Spanish fly”, but more importantly, it has brought attention to protein phosphatases as tractable nematocidal drug targets [112]. Fifty-four analogues of the less chemically reactive norcantharidin (Fig. 9), belonging to three different chemical families, were made and tested for inhibition of H. contortus larval development, expecting to see a correlation between anthelmintic potency and biochemical inhibition of PP2A [112]. Unfortunately, no such correlation was observed, suggesting that chemical modification had optimised whole-animal activity by acquiring a new and unknown target. This is a relatively common occurrence in anti-infective research that confounds structure–activity relationships (SARs).

In the same study that proposed linalool as a nematocidal drug starting point (see subsection on prenyl derivatives) [73], the alkaloids piperine (from Piper nigrum and other Piper species) (Fig. 9) and strychnine (from Strychnos nux-vomica) (Fig. 9) were also put forward. As described for linalool, these alkaloids were also chemoinformatically selected as possible GST inhibitors and inferred to have anthelmintic activity. Piperine and strychnine were found to inhibit a crude preparation of DiGST by 80% and 87% at 1 µg/ml (3.5 µM and 3 µM, respectively). Piperine, responsible for the pungency of pepper seeds, is a known low-potency inhibitor of multiple targets [113, 114], possibly because of its chemically reactive α,β-unsaturated group. Strychnine is a known potent non-selective poison and one of the time-honoured challenges in synthetic chemistry. The known properties of the two compounds indicate that it will be difficult to develop potent and selective analogues devoid of activity against non-target organisms. Additionally, there is the problem of the multiple GST isoforms in metazoans, assuming that the enzyme is indeed their lethal anthelmintic target.

Another promiscuous molecule, a non-protein l-α-amino acid from Mimosa pudica and Leucaena glauca (mimosine) (Fig. 9), has been described previously to possess anti-cancer, anti-viral and even herbicidal properties, perhaps related to its ability to chelate metal ions (cf. [115]), a mechanism that is hard to optimise for selectivity. A complex inference was made that since phosphoramidothionates of some l-amino alcohols have insecticidal activity, and acetylcholinesterase and tyrosinase can be taken to be good insecticidal targets, phosphoramidothionates of mimosinol should be prepared and tested as inhibitors of both enzymes and as insecticides or nematocides [116]. The results were disappointing, in that activity against C. elegans was higher in the parent compound, mimosine (reported IC₅₀ = 17 µM), than in any of its derivatives, including bis-deuterated analogues that displayed potency differences in several assays, an unexpected result which was not discussed in the report [116].

Closing this subsection, the SAR of the hydrophobic azoxy compounds jietacins (Fig. 9) has been explored and reported recently [117]. This compound class, produced by Streptomyces bacteria, and its nematocidal activity were first described decades ago by workers at the Kitasato Institute [118], but no reports of chemical development had appeared until 2018. Interestingly, a Japanese patent application was filed a year before the first communication by the original authors (JP S6366155 A), but it seems to have been abandoned. However, a Chinese institution did obtain local intellectual property (IP) protection for methods to prepare the compounds in 1995 (CN 1027905 C). The SAR study now published by Sugawara et al. [117] indicates that a vinyl azoxy group, located at the end of a linear aliphatic chain of 9–14 carbon atoms, is most efficacious in vitro and in vivo. Compounds were tested in vitro for inhibition of motility of xL3 larvae of both Cooperia curticei and H. contortus, and for inhibition of enzyme secretion by adult Nippostrongylus brasilensis. The most active compounds exhibited complete inhibition in the three assays at concentrations of 1–5 µg/ml (3–16 µM) and showed in vivo efficacy in mice infected with Heligmosomoides polygyrus at 25–100 mg/kg, p.o., q.d., for 4 days [117]. The parent compound, jietacin A, was also efficacious in vivo under the same conditions and, unlike some of the simplified analogues, did not present tolerability issues in mice, although it lacked activity against H. contortus. These compounds could be regarded as simple amphiphiles, which are able to disrupt biological membranes and protein–protein interactions by virtue of their detergent-like physicochemical properties. However, the reported jietacins displayed some striking regio-isomerism and chiral effects, which would be difficult to explain if they acted as simple detergents. The vinyl azoxy group, in particular, appears to be worth investigating in other structural contexts.