Abstract:
The present invention provides compositions and methods for controlling an helminth or arthropod pest. In a preferred embodiment of the invention provided herein, the compositions comprise compounds which alter the 5-HT 3  receptor of the pest. Also claimed are various esters of N-methyl 8-azabicyclo[3.2.1]octan-3-ol (tropan-3-yl esters) and an assay for identifying and/or assessing a helminth and/or arthropod control compound by determining the ability of a candidate compound to modulate the activity of a helminth or arthropod 5-HT 3  receptor.

Description:
FIELD OF THE INVENTION  
       [0001]     The present invention relates to methods and compositions for controlling a pest selected from helminths and arthropods.  
       BACKGROUND TO THE INVENTION  
       [0002]     Many species of nematodes are parasites of considerable medical, veterinary and agricultural significance. For example, nematodes of the Orders Strongylida,  Strongyloides , Ascaradida, Oxyurida and Trichocephalida include many species that cause disease in humans, sheep, cattle, pigs and other species. Further, nematodes of the Orders Tylenchida and Aphelenchida, and others, include species which are parasitic of important crop plants and fungi.  
         [0003]     It has been conservatively estimated that plant parasitic nematodes cause US $77 billion worth of damage to major food crops annually (Evans and Haydock, 1999). There are, unfortunately, very few control options for plant parasitic nematodes. Fumigants such as methyl bromide are generally being withdrawn from sale and use, because of their detrimental effects on the ozone layer, whilst the remaining available agents are among the most toxic and undesirable pesticides in current use.  
         [0004]     Animal parasitic nematodes infect humans, companion animals and livestock and cause serious morbidity and economic loss worldwide. This group of parasites includes hookworms and roundworms. They can cause anaemia, loss of weight, hyperimmune reactions and other complications, including death of livestock. There are currently a small number of human and veterinary drugs available for their treatment but, particularly in the veterinary field, the efficacy of a number of existing drugs is reducing because of resistance development.  
         [0005]     For the reasons described, there is an ongoing need for the identification of new nematicidal compounds.  
         [0006]     Pharyngeal pumping is the basis of nematode feeding and the ability of nematodes to maintain their “hydrostatic skeleton” (Brownlee et al., 1997). The pharyngeal pump of nematodes is already a well-established target organ for anthelmintics and nematicidal agents. In particular, inhibition of pharyngeal pumping is a major mode of action of ivermectin, an extremely successful modern nematicide and insecticide. Ivermectin acts on inhibitory glutamate receptors present in the pharynx and other tissues of nematodes and insect (Brownlee et al. 1997). Novel compounds that inhibit pumping of the nematode pharynx would have significant potential benefit for the control of plant and animal parasitic nematodes, and other helminth, arthropod and other invertebrate pests. They would also serve as lead molecules to facilitate the discovery process for other nematicidal compounds and insecticidal compounds.  
         [0007]     The macrocyclic lactone nematicides (avermectins) exemplify the potential utility of nematicides in controlling other invertebrate pests and parasites. Avermectins were originally registered as anthelminthics but these and related compounds are now being used increasingly as insecticides.  
         [0008]     The damage caused by arthropod pests is better known and characterised than that caused by nematodes. For example, the worldwide market for chemical insecticides is about US $12 billion, mostly in crop protection, but also in animal and public health. The market is growing at about 5% p.a. These costs of control are only a fraction of the economic costs of losses to crops and livestock worldwide. In particular, sucking plant pests such as aphids and plant-hoppers are second only to caterpillars in their economic importance and their value as a market for insecticides. They are particularly important in Europe and Asia. Whilst there are some existing insecticides active against these pests, a number of them are highly toxic and development of resistance is also causing problems. Thus, there is a great need for new classes of insecticides active on these pests.  
         [0009]     Also, insects with piercing and sucking mouthparts are the main vectors of diseases to humans and livestock. These vectors include mosquitoes (e.g. malaria, Japanese encephalitis, dengue fever etc.), higher flies (e.g. onchocerciasis) and true bugs (e.g. trypanosomiosis). Existing control measures are increasingly reliant on pesticides (e.g. permethrin-treated mosquito nets), because of the absence or failure of drug treatments. Therefore, there is also a need for new classes of insecticides active against these pests.  
         [0010]     Serotonin (5-Hydroxytryptamine, 5-HT) has a number of profound effects on the behaviour of  Caenorhabditis elegans  and other nematodes. In  C. elegans , exogenously applied 5-HT results in reduced locomotion, increased pharyngeal pumping, increased egg-laying and decreased defaecation. It is also involved in male mating behaviour. These effects of exogenous 5-HT are believed to occur because 5-HT is a natural nematode neurotransmitter that serves these behaviours. For example, two serotonergic neurones (NSM) are located over the pharynx whilst the HSNL and HSNR serotonergic neurones connect with the vulva. It is quite likely, based on the known biology of 5-HT in vertebrates, that each of these behaviours is controlled by serotonin action on different receptors present in different cells.  
         [0011]     Vertebrate serotonin receptors are known to fall into two distinct multigene superfamilies. One of these, the rhodopsin/β-adrenergic receptor superfamily, includes 7-transmembrane G-protein-linked receptors of the 5-HT 1 , 5-HT 2 , 5-HT 4 , 5-HT 5 , 5-HT 6 , and 5-HT 7  classes. Receptors of the other, 5-HT 3 , class belong to the nicotinic-acetylcholine receptor (nAChR), GABA-, glycine- and glutamate-gated ion channel superfamily and are pentameric, 4-membrane-spanning ligand-gated ion channels. It is generally observed that physiologically expressed pentameric receptors of this family comprise two or more types of subunits, with each type being the product of a distinct gene. While functioning ion channels may be obtained experimentally with a pentamer composed of identical subunits, these do not behave identically with respect to their channel conductance properties, to the heteropentameric ion channel that is present in vivo. In the case of the mammalian 5-HT 3  gated ion-channel, faithful electrophysiology has only been obtained with a heteromeric ion channel containing both 5-HT 3A  and 5-HT 3B  subunits (Davies et al., 1999). The mammalian 5-HT 3  receptors are known to form channels that gate the passage of cations across the cell membrane and when activated they tend to excite the cell. In this respect, as in many others, their closest relatives are the nicotinic acetylcholine receptors. GABA A -gated, glycine-gated, and the invertebrate-specific glutamate-gated ion channels all gate the passage of anions and their activation generally hyperpolarises the cell membrane.  
         [0012]     WO 01/6100 (the entire disclosure of which is to be regarded as incorporated herein by reference) is the first disclosure of the cloning of a cationic 5-HT 3  receptor subunit from an invertebrate species. In addition, WO 01/6100 shows that two 5-HT 3  antagonists (tropanyl dichlorobenzoate and ondansetron) profoundly inhibited pharyngeal pumping while the 5-HT 3  specific agonist 2-methyl-5-hydroxytryptamine hydrochloride strongly and specifically stimulated pharyngeal pumping. Moreover, tropanyl dichlorobenzoate caused dose-dependent mortality and other detrimental effects in  C. elegans , and tropanyl dichlorobenzoate and ondansetron caused significant detrimental effects, including some mortality, in insects.  
         [0013]     There is a need for further compounds, compositions and methods for controlling helminth and arthropod populations.  
       DISCLOSURE OF THE INVENTION  
       [0014]     The present inventors have identified compounds which can be used in methods for controlling helminth and/or arthropod populations. Thus, in a first aspect, the present invention provides a method for controlling a pest selected from helminths and arthropods, said method comprising exposing said pest to an effective amount of a compound comprising one of the following formulae:  
                         
        in which,     X is selected from substituted and unsubstituted cyclic rings,     Y is absent or otherwise selected from substituted of unsubstituted alkyl, substituted or unsubstituted alkyloxy, optionally interrupted by one or more heteroatoms,     Z is selected from substituted or unsubstituted alkyl, O, N, NH, S and SH, and 
 
 A is selected from nitrogen-containing substituents;  
                         
 
 in which, 
    X, Y and A are as defined above, and     D is selected from C, CH, CH 2 , O and N; and  
                         
 
 in which, 
    A and D are as defined above,     R is H or alkyl;     with the proviso that said compound is not ondansetron or tropanyl dichlorobenzoate.        
 
         [0027]     As used herein, the term “controlling a pest selected from helminths and arthropods” refers to the ability of the compound to have a detrimental effect on the pest. Preferably, the compound has a detrimental effect on pest development, feeding, neural function, reproduction or digestion. More preferably, the compound kills the pest.  
         [0028]     In one embodiment, the compound inhibits the activity of a 5-HT 3  receptor of said pest. In another embodiment, the compound stimulates the activity of a 5-HT 3  receptor of said pest.  
         [0029]     In one embodiment, X of formula (I) or (II) is a mono- or bicyclic ring. Preferably, X comprises at least one heteroatom.  
         [0030]     In another embodiment, X of formula (I) or (II) comprises at least one substituted or unsubstituted aromatic and/or heterocyclic rings which may be fused or non-fused. Preferably X is selected from mono-, di- and tri-substituted phenyl. Preferably, the substituents of such phenyls are selected, independently, from halogens (especially Cl and F), NH 2 , N 2 O, N(CH 3 ) 2 , lower alkyl (especially methyl and ethyl), lower haloalkyl (especially —CH 2 C 1  and CH 2 F), lower alkylamino (especially methylamino and ethylamino), lower alkylester (especially methanoate and ethanoate), lower alkyloxy (especially —OCH 3  and —OCH 2 CH 3 ).  
         [0031]     In a further embodiment, Y is absent and X is bonded directly to the carbon of the C═O group.  
         [0032]     In another embodiment, Y is a substituted or unsubstituted lower alkyl.  
         [0033]     In another embodiment, Y is a substituted or unsubstituted lower alkyloxy. More preferably, the lower alkyloxy is selected from —O—CH 2 — or —O—CH(CH 3 )—.  
         [0034]     In a further embodiment, Y is a heteroatom selected from the group consisting of: O, N, NH, S and SH.  
         [0035]     Preferably, Z of formula (I) is O or NH.  
         [0036]     In a further embodiment, Z of formula (I) is a substituted or unsubstituted lower alkyl.  
         [0037]     Preferably, A comprises nitrogen-containing substituents with a basic characteristic. More preferably, A comprises a substituted or unsubstituted 6-8 membered ring. Even more preferably, A is selected from a bridged ring or bicyclic ring, for example an imino bridged ring. More preferably, the imino bridge is —N(CH 3 )—.  
         [0038]     Alternatively, A may be an alkylamine, for example, —CH 2 CH 2 N(CH 3 ) 2 , —CH 2 CH 2 N(Et) 2  etc.  
         [0039]     Preferably, A in the compound of formula (III) is a heterocyclic or heterocyclicalkyl. The hetercyclic ring may be a fused or joined directly or indirectly to another heterocyclic or saturated or unsaturated carbocyclic ring. Preferably the heterocylic ring comprises at least one nitrogen atom.  
         [0040]     Preferably, D of formula (II) is CH or N.  
         [0041]     Preferably, R is lower alkyl.  
         [0042]     Particularly preferred compounds are those according to the formula below:  
                         
        where X is as defined above.        
 
         [0044]     Preferably, X of formula (IV) is selected from substituted and unsubstituted phenyl, phenoxyalkyl (eg phenoxypropyl), phenyl alkyl (eg phenylmethyl), cubanyl carboxylate, cycloalkyl (eg cyclopropane), cycloalkyl carboxylate (eg cydohexane carboxylate), benzylcarboxylate (eg 2-benzyl carboxylate, 3-benzyl carboxylate, 4 benzyl carboxylate), pyridine carboxylate, indolyl (eg 1H-indol-3-yl). The substituents may be any suitable substituent, such as those described above. The substituent(s) may be one or more of a halogen, for example, F and/or Cl.  
         [0045]     Particularly preferred is when X of formula (IV) is selected from substituted and unsubstituted phenyl, benzylcarboxylate (eg 2-benzyl carboxylate, 3-benzyl carboxylate, 4 benzyl carboxylate), and indolyl (eg 1H-indol-3-yl). The substituents may be any suitable substituent, such as those described above. The substituent(s) may be one or more of a halogen, for example, F and/or Cl.  
         [0046]     More preferably, the compound is selected from the group consisting of 3-chloro-benzoic acid tropan-3-yl ester, 3,4-dichloro-benzoic acid tropan-3-yl, 2-fluoro-benzoic acid tropan-3-yl ester, phthalic acid methyl ester tropan-3-yl ester, and 1H-indole-3-carboxylic acid tropan-3-yl.  
         [0047]     Suitable “lower alkyl” and lower alkyl moieties in the terms “lower haloalkyl”, “lower alkylamino”, “lower alkylester”, and “lower alkyloxy may be straight or branched such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, hexyl or the like.  
         [0048]     The formulae given herein are intended to extend to all possible geometrical and optical isomers as well as racemic mixtures thereof.  
         [0049]     Preferably the helminth is selected from the group consisting of: nematodes, cestodes and flatworms. More preferably, the nematode is an animal parasitic nematode or a plant parasitic nematode. Preferably, the arthropod is an insect. More preferably, the pest has a feeding mechanism involving sucking plant or animal fluids by means of a muscular pump, such as those generally referred to as sucking insects. Examples of sucking insects which can be controlled by the methods of the present invention include, but are not limited to,  Thrips  (Thysanoptera) such as  Frankliniella occidentalis, Thrips palmi ; Plant/Leaf bugs, Lace Bugs, Kissing bugs (Hemiptera:Reduviidae) such as  Rhodnius prolixus, Triatoma  spp; Leaf/Plant/Tree hoppers (Hemiptera) such as  Nilaparvata lugens  (rice planthopper),  Empoasca fabae ; Psyllids (Hemiptera) such as  Diaphorina citri ; Aphids (Hemiptera) such as  Myzus persicae, Aphis gossypii, Aphis craccivora, Acyrthosiphon pisum, Acyrthosiphon kondoi, Diuraphis noxia, Schizaphis graminum, Sitobion avenae, Rhopalosiphum padi, Rhopalosiphum maidis, Diuraphis noxia, Schizaphis graminum, Sitobion avenae, Macrosiphum euphorbiae, Brevicoryne brassicae, Lipaphis erysimi, Theriaphis trifolii ; Whiteflies (Hemiptera) such as  Bemisia tabaci, Bemisia argentifolii, Trialeurodes vaporariorum ; Mealybugs; Scales; Mosquitoes (Diptera) such as  Anopheles gambiae, Anopheles stephensi, A. maculipennis  and other  Anopheles  spp.  Culex pipiens  and other  Culex  spp.  Aedes aegyptae, Aedes albopictus  and other  Aedes  spp,  Haemagogus equinus ; Blackflies (Diptera) such as  Simulium damnosum . Examples of non-insect arthropods which can be controlled by the methods of the present invention include, but are not limited to, Mites (Acarina) such as Spider mites (eg  Tetranychus urticae ) and Eriophyid mites; and Ticks such as  Boophilus  spp. Examples of nematodes (Nematoda) which can be controlled by the methods of the present invention include, but are not limited to,  Onchocerca volvulus, Dracunculus  spp,  Trichuris  spp.,  Wuchereria bancrofti, Strongyloides stercoralis , other  Strongyloides  spp,  Brugia malayi, Angiostrongylus vasorum, Toxocara canis, T. cati, Baylisascaris procyoni, Ancylostoma duodenal  and other  Ancylostoma  spp.,  Necator americanus  and other  Necator  spp,  Haemonchus contortus  and other  Haemonchus  spp,  Ostertagia  spp,  Trichostrongylus colubriformis, Ascaris suum, A. caninum, A. braziliense , and  A. tubaeforme  and other  Ascaris  sp.  Trichinella  spp.,  Meloidogyne incognita  and other  Meloidogyne  species,  Globodera pallida  and other  Globodera  spp,  Pratylenchus thomei, P. neglectus  and other  Pratylenchus  sp.,  Ditelenchus  sp.  
         [0050]     An effective amount of said compound may be in the range of 100 μM or less, preferably 10 μM or less, more preferably 1 μM or less at the whole organism level.  
         [0051]     In a second aspect, the present invention provides a composition for controlling a pest selected from helminths and arthropods, said composition comprising a compound as defined in the first aspect in combination with a pharmaceutically/veterinary/agriculturally-acceptable carrier and/or excipient.  
         [0052]     The present inventors have also synthesised numerous new compounds. Thus, in a third aspect the present invention provides a compound of the formulae:  
                         
 
 in which, 
    X is selected from substituted and unsubstituted cyclic rings,     Y is absent or selected from lower alkyl, lower alkyloxy, O, N, NH, S and SH,     Z is O and     A is tropanyl, 
 
 with the proviso that compound is not tropanyl dichlorobenzoate. 
   
 
         [0059]     Preferably, Y is absent.  
         [0060]     Preferably the compound is of formula:  
                         
        where X is as defined above.        
 
         [0062]     Preferably, X of formula (IV) is selected from substituted and unsubstituted phenyl, phenoxyalkyl (eg phenoxypropyl), phenyl alkyl (eg phenylmethyl), cubanyl carboxylate, cycloalkyl (eg cyclopropane), cycloalkyl carboxylate (eg cyclohexane carboxylate), benzylcarboxylate (eg 2-benzyl carboxylate, 3-benzyl carboxylate, 4 benzyl carboxylate), pyridine carboxylate, indolyl (eg 1H-indol-3-yl). The substituents may be any suitable substituent, such as those described above. The substituent(s) may be one or more of a halogen, for example, F and/or Cl.  
         [0063]     Particularly preferred is when X of formula (IV) is selected from substituted and unsubstituted phenyl, benzylcarboxylate (eg 2-benzyl carboxylate, 3-benzyl carboxylate, 4 benzyl carboxylate), and indolyl (eg 1H-indol-3-yl). The substituents may be any suitable substituent, such as those described above. The substituent(s) may be one or more of a halogen, for example, F and/or Cl.  
         [0064]     Preferably, the compound is selected from the group consisting of 3-chloro-benzoic acid tropan-3-yl ester, 3,4-dichloro-benzoic acid tropan-3-yl ester, 2-fluoro-benzoic acid tropan-3-yl ester, phthalic acid methyl ester tropan-3-yl ester, and 1H-indole-3-carboxylic acid tropan-3-yl.  
         [0065]     The present invention also allows for the compounds defined herein to be screened for their ability to be used as a helminth and/or arthropod control compound. Thus, in a fourth aspect, the present invention provides an assay for identifying and/or assessing an helminth and/or arthropod control compound, the method comprising determining the ability of a compound defined herein to modulate the activity of a 5-HT 3  receptor of the helminth or arthropod.  
         [0066]     The assay can be performed in an in vitro or in vivo system. In an example of an in vitro system, the 5-HT 3  receptor, or functionally equivalent fragment thereof, is expressed in a recombinant host cell and the ability of the candidate modulate compound is measured on the host cell. An example of an in vivo avenue of performing the assay is the “automated feeding assay” described herein.  
         [0067]     Preferably, the modulation of 5-HT 3  activity is determined by measuring changes in cell membrane potential or Ca 2+  levels.  
         [0068]     Preferably, the 5-HT 3  receptor(s) is contacted simultaneously with a serotonergic ligand and the candidate compound, and the modulation of 5-HT 3  activity is determined by measuring the amount of bound and/or unbound labelled serotonergic ligand. More preferably, said serotonergic ligand is 5-hydroxytryptamine.  
         [0069]     In one embodiment, the compound inhibits the activity of a 5-HT 3  receptor of said helminth and/or arthropod. In another embodiment, the compound stimulates the activity of a 5-HT 3  receptor of said helminth and/or arthropod.  
         [0070]     In a fifth aspect, the present invention provides an helminth or arthropod control compound identified according to the fourth aspect.  
         [0071]     A lead compound is an helminth or arthropod control compound which is subject to trials with the goal of ultimately being formulated in, for example, a composition and sold as an agent for controlling helminth or arthropod pest populations. The lead compound, when exposed to a helminth or arthropod, more preferably an insect, disrupts 5-HT 3  receptor activity leading to a reduction in reproduction rates, a reduction in feeding rates or death etc.  
         [0072]     As will be apparent, preferred features and characteristics of one aspect of the invention are applicable to many other aspects of the invention.  
         [0073]     Throughout this specification the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.  
         [0074]     Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.  
         [0075]     The invention will hereinafter be described with reference to the following non-limiting examples and accompanying figures. 
     
    
     BRIEF DESCRIPTION OF THE ACCOMPANYING FIGURES  
       [0076]      FIG. 1 : Structural formulae of compounds used or mentioned in Example 1.  
         [0077]      FIG. 2 : Log dose responses for bead ingestion by nematodes. Compounds identified as strongly reversing the stimulatory effects of 0.325 mM serotonin on bead ingestion (see Table 3) were tested over a range of doses. The results were plotted as percentage of the fluorescence due to bead ingestion in the presence of 1 mM serotonin alone. Data were fitted to the equation y=a/(1+Exp(b−c*x)) by iterative least squares regresssion in DeltaGraph version 4.05 and 4.5 for the Macintosh (SPSS Australasia Pty Ltd). 
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [heading-0078]     5-HT 3  Receptor  
         [0079]     The term “5-HT 3  receptor” as used herein refers to a receptor having one or more of the following features (1)-(3): 
    (1) Is a serotonin-gated molecular ion-channel which gates the conductance of cations and is composed of five receptor subunits each of which has a nicotinicoid transmembrane topology (N-terminus, large extracellular domain, 3 transmembrane helices, large intracellular domain, 1 transmembrane helix, C-terminus).     (2) Is a helminth or arthropod receptor composed of subunits with a higher level of amino acid homology to mammalian 5-HT 3  receptor subunits (such as those described by Maricq et al. (1991); Miyake et al. (1995) and U.S. Pat. No. 6,365,370) than to known mammalian nicotinic acetylcholine receptor subunits.     (3) Has a characteristic pharmacological profile in that it binds to a subset of known 5-HT 3 -specific agonists and antagonists with greater selectivity than it binds to agonists and antagonists of other 5-HT receptor classes.    
 
         [0083]     Examples of 5-HT 3  receptors, the activity of which is altered by the compounds described herein, are provided in co-pending application WO 01/61000. These receptors include those naturally occurring in pest species which posses a sequence which is at least 50% identical, more preferably at least 60% identical, more preferably at least 70% identical, more preferably at least 80% identical, more preferably at least 90% identical, more preferably at least 95% identical, more preferably at least 97% identical, and even more preferably at least 99% identical to the those 5-HT 3  receptors disclosed in WO 01/61000 and provided herein as SEQ ID NO&#39;s 1 to 3.  
         [0084]     As used herein a “functionally equivalent fragment” of a 5-HT 3  receptor is a portion of the receptor which has at least one of features (1) to (3) as outlined above.  
         [0085]     5-HT 3  receptors useful for assays of the present invention can either be naturally occurring or mutants and/or fragments (especially functionally equivalent fragments) thereof.  
         [0086]     The % identity of a polypeptide is determined by GAP (Needleman and Wunsch, 1970) analysis (GCG program) with a gap creation penalty=5, and a gap extension penalty=0.3. The query sequence is at least 15 amino acids in length, and the GAP analysis aligns the two sequences over a region of at least 15 amino acids. More preferably, the query sequence is at least 50 amino acids in length, and the GAP analysis aligns the two sequences over a region of at least 50 amino acids. Even more preferably, the query sequence is at least 100 amino acids in length and the GAP analysis aligns the two sequences over a region of at least 100 amino acids. More preferably, the query sequence is at least 250 amino acids in length and the GAP analysis aligns the two sequences over a region of at least 250 amino acids. Even more preferably, the query sequence is at least 500 amino acids in length and the GAP analysis aligns the two sequences over a region of at least 500 amino acids.  
         [heading-0087]     Compositions  
         [0088]     The composition may be in any form known in the art including, but not limited to, a solid form (e.g. oral dosage forms for pharmaceutical or veterinary use, or pellets for agricultural or horticultural use which may be cast or spread onto an area or surface affected by the target pest), in liquid forms for application by, for example, spraying techniques, or as suspensions or syrups.  
         [0089]     Typical pharmaceutically/veterinary/agriculturally-acceptable carriers and/or excipients which may be employed in the compositions according to the invention include, but are not limited to, preservatives, buffering systems, viscosity enhancing agents, flavouring aids, colouring aids, sweeteners, and mixtures thereof.  
         [0090]     Suitable carriers for the compounds provided as formulae (I) and (IV) include dimethyl sulfoxide (DMSO).  
         [0091]     Suitable preservatives include one or more alkyl hydroxybenzoates such as methyl, ethyl, propyl and/or butyl hydroxybenzoates; sorbic acid or a salt thereof; benzoic acid or a salt thereof; and mixtures thereof.  
         [0092]     Suitable buffering systems include combinations of citric acid and salts and solvates thereof, for example citric acid (anhydrous or monohydrate) combined with sodium citrate dihydrate.  
         [0093]     Suitable viscosity enhancing agents include gums (e.g. Xanthan gum); glycerol; polyvinyl alcohol; polyvinylpyrrolidine; cellulose derivatives, such as carboxymethylcellulose or a salt thereof, C 1-4  alkyl and/or hydroxy C 2-4  alkyl ether of cellulose, such as methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxyethylmethylcellulose and hydroxypropyl-methylcellulose; and mixtures thereof.  
         [0094]     Liquid compositions according to the invention conveniently have a viscosity which lies in the range 1 to 100 cps, such as 10 to 75 cps, for example about 15 to 50 cps.  
         [0095]     Suitable additional sweeteners include, for example, sugars such as glucose; and cyclamate and salts thereof.  
         [0096]     Preferably, the compositions contain compounds which attract target pests, and/or repel non-target organisms (such as beneficial insects, mammals etc).  
       EXAMPLE  
       [0097]     It had been previously observed that mammalian 5-HT 3  specific drugs affect the rate of pharyngeal pumping in the nematode  C. elegans  indicating the presence of an excitatory serotonin-gated ion channel in nematodes which controls the rate and strength of pharyngeal pumping (WO 01/61000). Specifically, it was observed that the drugs 3,5-dichlorobenzoic acid tropan-3-yl ester (tropanyl dichlorobenzoate, MDL 72222) and ondansetron (also known as zofran), both at concentrations of 0.325 mM, antagonised the stimulatory effect of 0.325 mM serotonin on the rate of pharyngeal pumping. The drug 2-methyl-5-hydroxytryptamine at 3 mM strongly stimulated pharyngeal pumping but, unlike a similar dose of serotonin, did not inhibit locomotion. It had also been observed that tropanyl dichlorobenzoate had a range of dose-dependent detrimental effects, including lethality, on  C. elegans  and the aphid  Myzus persicae  and also caused reduced growth rates in the larvae of the lepidopteran  Helicoverpa armigera.    
         [0098]     Pharyngeal pumping is a vital function for nematodes since it is necessary for feeding and maintenance of the hydrostatic skeleton. Furthermore, the pharynx, or a functionally equivalent pump at the anterior end of the gut, is vital for many other invertebrates since the pump is required for ingestion of food (e.g. aphids) attachment to the host (e.g. trematodes).  
         [0099]     An automated screening assay has been developed and described in the present Applicants&#39; co-pending International patent application No. PCT/AU00/01476 (WO 01/40500). Using the screening assay, twenty novel tropanyl ester compounds as well as tropanyl dichlorobenzoate were screened for nematicidal activity.  
         [heading-0100]     Materials and Methods  
         [heading-0101]     Drugs  
         [0102]     Serotonin. (5-Hydroxytryptamine creatinine sulphate, C 14 H 19 N 5 O 2 .H 2 SO 4 1H 2 O) was obtained from Sigma-Aldrich Corporation, Castle Hill, NSW.  
         [0103]     Tropanyl dichlorobenzoate. (3-Tropanyl-3,5-dichlorobenzoate, C 15 H 17 Cl 2 NO 2 ) was obtained from RBI Research Biochemicals International, Natick, Mass., USA.  
         [0104]     Ondansetron HCl (C 18 H 19 N 3 O.HCl 2H 2 O) was donated by Dr. Paul Cooper of the Division of Botany and Zoology, Australian National University.  
         [0105]     2-methyl-5-hydroxytryptamine hydrochloride (C 11 H 14 N 2 O.HCl H 2 O) was obtained from Tocris Cookson Ltd., Avonmouth, UK.  
         [heading-0106]     Synthetic Tropanyl Ester Compounds  
         [0107]     The tropanyl esters shown in Table 1 were synthesised as described below.  
                         TABLE 1                       Tropanyl esters.                                DCP-45221 (1)   benzoic acid tropan-3-yl ester       DCP-45222 (2)   2-chloro-benzoic acid tropan-3-yl ester       DCP-45223 (3)   3-chloromethyl-benzoic acid tropan-3-yl ester       DCP-45224 (4)   3-chloro-benzoic acid tropan-3-yl ester       DCP-45225 (5)   4-chloro-benzoic acid tropan-3-yl ester       DCP-45226 (6)   2,5-difluoro-benzoic acid tropan-3-yl ester       DCP-45227 (7)   2,6-difluoro-benzoic acid tropan-3-yl ester       DCP-45228 (8)   3,4-dichloro-benzoic acid tropan-3-yl ester       DCP-45229 (9)   2-fluoro-benzoic acid tropan-3-yl ester       DCP-45230 (10)   2-phenoxy-propionic acid tropan-3-yl ester       DCP-45231 (11)   phenyl-acetic acid tropan-3-yl ester       DCP-45232 (12)   cubane-1,4-dicarboxylic acid methyl ester tropan-3-yl           ester       DCP-45233 (13)   cyclopropanecarboxylic acid tropan-3-yl ester       DCP-45234 (14)   cyclobutanecarboxylic acid tropan-3-yl ester       DCP-45235 (15)   trans-cyclohexane-1,4-dicarboxylic acid methyl ester           tropan-3-yl ester       DCP-45236 (16)   terephthalic acid methyl ester tropan-3-yl ester       DCP-45237 (17)   isophthalic acid methyl ester tropan-3-yl ester       DCP-45238 (18)   phthalic acid methyl ester tropan-3-yl ester       DCP-45239 (19)   pyridine-3,5-dicarboxylic acid methyl ester tropan-           3-yl ester       DCP-45240 (20)   1H-indole-3-carboxylic acid tropan-3-yl ester                  
 
 Preparation of Tropanyl Esters 
 
         [0109]     Acid chlorides were used where commercially available (Table 2). Where only a carboxylic acid was commercially available, it was first converted to the acid chloride before use by well known techniques (March, 1985).  
         [0110]     The tropanyl esters were prepared using parallel synthesis techniques in 4×6 microtitre plate arrays (Bunnin, 1998). Into each well of the microtitre plate was placed tropan-3-ol (100 mg) followed by dichloromethane (4 ml) then triethylamine (0.5 ml). Each well was treated with a solution or suspension of an acid chloride (1.05 molar equivalents) in dichloromethane (2 ml). The reactions were allowed to react overnight. Water (50 μl) was added into each well, and the reaction plate agitated for 2 h. The reactions were concentrated under a vacuum at 45° C. Each sample was purified by LC/MS (ESI mode) on a Reverse Phase C-18 ODS-AL 20 mm×50 mm column from YMC, then concentrated to afford a solid or oil.  
         [heading-0111]     Automated Feeding Assay  
         [0112]     The assay used was essentially that described in WO 01/40500 with minor modifications. The protocol was as follows:  
         [0113]     (1.) Plate Culture.  C. elegans  of the Bristol N2 strain (Brenner, 1974) were cultured at room temperature on HMS174  E. coli  bacteria (Campbell et al., 1978) spread on a 150 mm diameter petri dish containing enhanced NGM 1.7% (w/v) (Avery and Horvitz, 1990) agar until a good population of adult nematodes was present, but food reserves had not been exhausted.  
         [0114]     (2.) The nematodes were collected by washing each plate with 10 mL and then 5 mL of M9 buffer plate and filtering over a 20 μm nylon mesh (Nytal—Catalogue no. BCNY-HDO02-20) to retain adult nematodes. The nematodes from 7-8 of these 150 mm plates were collected together and placed on a 63 μm mesh filter (Nytal—product code PA-25-43) and washed with 200-250 mL of M9 buffer or MilliQ water. This procedure allows the passage of nematodes whilst retaining larger debris. Finally the nematodes were collected over a 20 μm mesh.  
         [0115]     (3.) Adult nematodes were collected from the mesh using a pasteur pipette and allowed to settle in an eppendorf tube at 1×g for 10 minutes. This packed volume was used to calculate quantities of nematodes for experiments and assays, although nematodes were resuspended in 2-10 volumes of M9 buffer for transfer. Quantitative transfers of nematodes were performed using a Gilson Pipetman P200 or equivalent and a yellow tip with 5 mm cut off the sharp end.  
                                                   TABLE 2                           Synthesis of the tropan-3-yl esters in this study.                Product Information                Name of the carboxylic acid           mol           parent of the acid chloride reacted   formula   mol weight   weight   tropanyl ester       with tropan-3-ol   (calc)   (calc)   (obs) a     designation               benzoic acid   C 15 H 19 NO 2     245.14   246.4   DCP-45221 (1)       2-chloro-benzoic acid   C 15 H 18 CINO 2     279.10   280.0   DCP-45222 (2)       3-chloromethyl-benzoic acid   C 16 H 20 CINO 2     293.12   294.2   DCP-45223 (3)       3-chloro-benzoic acid   C 15 H 18 CINO 2     279.10   280.0   DCP-45224 (4)       4-chloro-benzoic acid   C 15 H 18 CINO 2     279.10   280.0   DCP-45225 (5)       2,5-difluoro-benzoic acid   C 15 H 17 F 2 NO 2     281.12   282.0   DCP-45226 (6)       2,6-difluoro-benzoic acid   C 15 H 17 F 2 NO 2     281.12   282.0   DCP-45227 (7)       3,4-dichloro-benzoic acid   C 15 H 17 Cl 2 NO 2     313.06   314.0   DCP-45228 (8)       2-fluoro-benzoic acid   C 15 H 18 FNO 2     263.13   264.2   DCP-45229 (9)       2-phenoxy-propionic acid   C 17 H 23 NO 3     289.17   290.0   DCP-45230 (10)       phenyl-acetic acid   C 16 H 21 NO 2     259.16   260.0   DCP-45231 (11)       cubane-1,4-dicarboxylic acid   C 19 H 23 NO 4     329.16   330.0   DCP-45232 (12)       monomethyl ester       cyclopropanecarboxylic acid   C 12 H 19 NO 2     209.14   210.2   DCP-45233 (13)       cyclobutanecarboxylic acid   C 13 H 21 NO 2     223.16   224.0   DCP-45234 (14)       trans-Cyclohexane-1,4-dicarboxylic   C 17 H 27 NO 4     309.19   310.2   DCP-45235 (15)       acid monomethyl ester       terephthalic acid monomethyl ester   C 17 H 21 NO 4     303.15   304.2   DCP-45236 (16)       isophthalic acid monomethyl ester   C 17 H 21 NO 4     303.15   304.2   DCP-45237 (17)       phthalic acid monomethyl ester   C 17 H 21 NO 4     303.15   304.2   DCP-45238 (18)       pyridine-3,5-dicarboxylic acid   C 16 H 20 N 2 O 4     304.14   305.2   DCP-45239 (19)       monomethyl ester       1H-indole-3-carboxylic acid   C 17 H 20 N 2 O 2     284.15   285.0   DCP-45240 (20)                   a Molecular ions obtained in +ESI mode, giving M + H ions, on a PE-Sciex API-150EX.             
 
         [0116]     (4.) Assays were performed in 24 well tissue-lture clusters (Nunc multidish). The final assay volume was 230 μL comprising the following sequential additions: 
        (i) M9 buffer—sufficient to bring the final volume to 230 μL     (ii) Serotonin—11.5 μL of a 20 mM stock solution in water     (iii) Any other pharmacological agents—≦1.5 μL of drug in DMSO (or water)     (iv) Nematodes—150 μL of a 10% (v/v) stock in M9     (v) Fluorescent beads—23 μL of a 1% w/v solution in M9.        
 
         [0122]     Where serotonin or other pharmacological agents were omitted, the volume was adjusted with M9 buffer. Control experiments included 1.5 μL of DMSO except in the case of ondansetron, which was added from an aqueous stock.  
         [0123]     The fluorescent beads were 1.30 μm uniformly dyed microsphere beads with a hydrophilic surface-coating. The fluorescence excitation maximum was 420 nm and the emission maximum was 485 nm (catalogue code FC04F, carboxylate-modified polystyrene/polyvinyl copolymer, manufactured and supplied by Bangs Laboratories, Inc. 9025 Technology Drive, Fishers, INDIANA 46038-2886, USA).  
         [0124]     (5.) The nematodes were incubated for 60 minutes after the addition of the fluorescent beads at room temperature (≈21° C.) without shaking. The assay was terminated by the addition of 20 μL of 100 mM sodium azide.  
         [0125]     (6.) Two aliquots of 100 μl each were transferred from each well of the 24 well cluster to a pair of wells in a pre-blocked mesh-bottomed plate (see below for details of the mesh-bottomed plate) and the buffer/bead suspension was immediately aspirated away through the filter mesh using a Millipore Multiscreen vacuum manifold.  
         [0126]     (7.) A total of eight washing steps were performed. Each of the first five washes used 200 μL of 1%(w/v) sodium lauryl sulphate (SDS—Sigma) in M9 buffer. The third and fifth washes involved pipetting the solution up and down in the wells of the plate ten times each wash. The SDS washes were followed by three washes with 200 μL of M9 buffer only. At the end of each washing step, solutions were aspirated away through the mesh using a Millipore Multiscreen vacuum manifold.  
         [0127]     (8.) 50 μL M9 was added to the wells of the mesh plate and it was placed on a piece of black cardboard before being read in the PolarStar fluorimeter with excitation at 420 nm and emission at 485 nm and with the gain set at 30.  
         [0128]     (9.) Preparation of 96-well mesh-bottomed plates. Millipore Multiscreen N20 plates, (custom order no. SE3R090M6) were blocked prior to performing the assay in order to minimise non-specific adherence of fluorescent microsphere beads. To block, 200 μL of a 0.1% (w/v) suspension of dark blue-dyed, non-fluorescent microsphere beads with a mean diameter of 0.95 μm and a hydrophilic surface coating (Catalogue code DC03B, carboxylate-modified polystyrene/polyvinyl copolymer) (manufactured and supplied by Bangs Laboratories, Inc. 9025 Technology Drive, Fishers, INDIANA 46038-2886, USA) were added to the plate and incubated overnight at room temperature with orbital shaking at 70 r.p.m. Prior to addition of the nematode sample, the bead suspension was removed under vacuum using a Millipore Multiscreen manifold. The plate was washed three times by pipetting 200 μL of M9 buffer into each well, allowing the plate to sit for 5 minutes and then sucking off the suspension.  
         [0129]     (10.) The fluorescence reading in the presence of 0.325 mM MDL 72222 was defined as background. When measuring the efficacy of antagonists the fluorescence reading in the presence of 1 mM added 5-HT was defined as 100% (note that higher concentrations of serotonin are capable of eliciting higher responses).  
         [heading-0130]     Results  
         [0131]     The results of screening of twenty novel tropanyl esters and tropanyl dichlorobenzoate for nematicidal activity using the automated screening assay are shown at Table 3. Compounds, each at 0.325 mM, were tested for their effects in isolation and in combination with 0.325 mM serotonin. DCP-45224 (4), DCP-45228 (8), DCP-45229 (9), DCP-45238 (18), and tropanyl dichlorobenzoate strongly reversed the stimulatory effect of 0.325 mM serotonin. Of these DCP-45228 (8) and tropanyl dichlorobenzoate also reduced the unstimulated level of bead ingestions. The compounds that exhibited strong effects at 0.325 mM concentration compounds were investigated further.  
         [0132]     The selected compounds were tested over a range of doses for their effects on bead ingestion in the presence of 1 mM serotonin. Tropanyl dichlorobenzoate was also tested in the absence of added serotonin, i.e. for its  
                                 TABLE 3                           Effects of tropanyl esters on nematode pharyngeal pumping measured by the bead       ingestion assay in the presence and absence of serotonin.                        Effect of 0.325 mM               Effect of    compound on serotonin-               0.325 mM compound   stimulated (0.325 mM)       Compound no.   Compound name   on bead ingestion   bead ingestion               MDL 72222   3,5-dichlorobenzoic acid tropan-3-yl ester   inhibits   reverses strongly       DCP-45221 (1)   benzoic acid tropan-3-yl ester   stimulates marginally   potentiates marginally       DCP-45222 (2)   2-chlorobenzoic acid tropan-3-yl ester   no effect   no effect       DCP-45223 (3)   3-chloromethyl-benzoic acid tropan-3-yl   no effect   no effect           ester       DCP-45224 (4)*   3-chloro-benzoic acid tropan-3-yl ester   no effect   reverses strongly       DCP-45225 (5)   4-chloro-benzoic acid tropan-3-yl ester   no effect   no effect       DCP-45226 (6)   2,5-difluoro-benzoic acid tropan-3-yl ester   no effect   reverses marginally       DCP-45227 (7)   2,6-difluoro-benzoic acid tropan-3-yl ester   no effect   reverses marginally       DCP-45228 (8)*   3,4-dichloro-benzoic acid tropan-3-yl ester   inhibits   reverses strongly       DCP-45229 (9)*   2-fluoro-benzoic acid tropan-3-yl ester   no effect   reverses marginally       DCP-45230 (10)   2-phenoxy-propionic acid tropan-3-yl ester   inhibits   reverses marginally       DCP-45231 (11)   phenyl-acetic acid tropan-3-yl ester   inhibits   reverses marginally       DCP-45232 (12)   cubane-1,4-dicarboxylic acid methyl ester   no effect   reverses marginally 1             tropan-3-yl ester       DCP-45233 (13)   cyclopropanecarboxylic acid tropan-3-yl   no effect   potentiates marginally           ester       DCP-45234 (14)   cyclobutanecarboxylic acid tropan-3-yl ester   no effect   no effect       DCP-45235 (15)   trans-cyclohexane-1,4-dicarboxylic acid methyl   no effect   no effect/reverses           ester tropan-3-yl ester       marginally       DCP-45236 (16)   terephthalic acid methyl ester tropan-3-yl   no effect   no effect           ester       DCP-45237 (17)   isophthalic acid methyl ester tropan-3-yl   no effect   no effect           ester       DCP-45238 (18)*   phthalic acid methyl ester tropan-3-yl ester   no effect   reverses markedly       DCP-45239 (19)   pyridine-3,5-dicarboxylic acid methyl ester   no effect   potentiates marginally           tropan-3-yl ester       DCP-45240 (20)*   1H-indole-3-carboxylic acid tropan-3-yl ester   no effect   reverses markedly                   1 Observed with an unpurified mixture            *Samples selected for further study             
 
 ability to depress the basal level of pumping due to endogenous serotonin release by the serotonergic cells of the nematode. The data were converted into percentages of the response in the presence of 1 mM serotonin and a log dose response curve was plotted ( FIG. 2 .). Where possible, the data were fitted with a logistic equation. 
 
         [0134]     IC 50  (inhibitory concentration—50%) values were estimated from the curves. In the presence of 1 mM serotonin these were: 
        DCP-45228 (8)=25 μM     MDL 72222=40 μM     DCP-45238 (18)=56 μM     DCP-45240 (20)=316 μM     ondansetron=562 μM        
 
         [0140]     It was impossible to estimate the IC 50  values for DCP-45224 (4) and DCP-45229 (9) under these conditions because of the lower potency of these compounds. In addition DCP-45229 (9) exhibited anomalous activity with very strong stimulatory effects at lower doses.  
         [0141]     In the absence of added serotonin, the IC 50  value for tropanyl dichlorobenzoate (i.e. the dose that depressed the basal bead ingestion rate by 50%) was estimated at 11 μM ( FIG. 2 —dotted black line). This indicates an effective endogenous level of serotonin equivalent to approximately 0.5 mM exogenous serotonin. Interestingly, 11 μM is very close to the lowest effective concentration in the chronic toxicity assay reported previously (see WO 01/61000). The correspondence of the effective doses in the bead ingestion and chronic toxicity assays supports the conclusion that the toxicity of tropanyl dichlorobenzoate is mediated primarily by its effect on pharyngeal pumping.  
         [0142]     The differential factor between the IC 50  values for tropanyl dichlorobenzoate in the presence and absence of exogenous serotonin also implies that DCP-45228 (8) may have an IC 50  and therefore a chronic EC 50  approximately equal to 6 μM in the absence of added serotonin.  
         [0143]     It is clear from these results that with regard to inhibition of nematode pharyngeal pumping, a number of tropanyl esters, with both minor and major variations or substitutions in the carboxylic acid moiety, have activity superior to ondansetron and comparable with, and in one case superior to, tropanyl dichlorobenzoate. It is apparent that a range of additional substitutions, both in the carboxylate and tropanyl moieties, have efficacy in inhibiting nematode pharyngeal pumping.  
         [0144]     The activity of the phthalic acid derivative DCP-45238 (18) and also of the indole derivative DCP-45240 (20) shows that a range of other substituted cyclic rings are effective. Similarly, such substituted ring structures can be combined with any of the other basic side chains which, like tropane, have been shown to produce 5-HT 3  antagonists with mammalian efficacy. These combinations would include the side chains and rings present in other known 5-HT 3  receptor-antagonists including metoclopramide (21), BMY 25801 (22), dimethpramide (23), zacopride (24), renzapride (25), and dazopride (26), and pancopride (27), MDL 72422 (28), BRL 24682 (29), Y-25130 (30), ADR 851 (31), ADR 847 (32), LY 277359 (33), ICS 205,930 (34), MDL 73,147 (35), granisetron (36), BRL 46470 (37), DAU 6215 (38), BIMU 0001 (39), L683,877 (40), AS-5370 (41), ondansetron (42), GR68755 (43) and GR65630 (44) (King, 1994). Similarly, while the ester linkage is clearly compatible with high levels of potency, other linkages between the basic and aromatic groups, such as imino, epithio and epoxy linkages are also useful.  
         [heading-0145]     Discussion  
         [0146]     The data presented herein, as well as the data provided in co-pending WO 01/61000, indicate that the stimulation or inhibition of nematode pharyngeal pumping by 5-HT 3  selective agents occurs across three chemical classes, namely simple substitutions of 5-hydroxytryptamine (2-methyl-5-hydroxytryptamine hydrochloride), an indolyl with a basic imidazole side chain (namely ondansetron) and a range of tropanyl esters of carboxylic acid-substituted rings. It is concluded that inhibition of pharyngeal pumping and therefore toxicity to helminths and arthropods (particularly arthropods that use a muscular pump for feeding such as aphids), is a general feature of any compound which specifically inhibits 5-HT 3  receptors. All such known compounds are therefore potential nematicides and insecticides. Relative potency and specificity towards the invertebrate specific receptor is related to, but not identical to, the potency and specificity for mammalian 5-HT 3  receptors. However, using the bead ingestion assay, it is a straightforward matter to screen through known mammalian 5-HT 3  antagonists and related compounds such as those listed in King (1994) and derivatives thereof for those with suitable potency and specificity.  
         [0147]     It is also of note that the mammalian toxicity of many 5-HT 3  antagonists is well-characterised and that a number are relatively well-tolerated (e.g. granisetron and ondansetron) and are registered for human therapeutic use (i.e. ondansetron). It is therefore feasible, by judicious choice of compounds, to achieve the selective toxicity which would be required for animal and human anthelmintics. It is believed that this is because the serotonergic signalling in the mammalian gut is involved in the emetic response rather than being critical to the normal healthy functioning of the gut as it is in nematodes and some other invertebrates.  
         [0148]     All publications discussed above are incorporated herein in their entirety.  
         [0149]     It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.  
       REFERENCES  
       [0150]    
       
          Avery, L., and Horvitz, H. R. (1990). Effects of starvation and neuroactive drugs on feeding in  Caenorhabditis elegans . J. Exp. Zool., 253, 263-70.  
          Brenner, S. (1974). The genetics of  Caenorhabditis elegans . Genetics, 77, 71-94.  
          Brownlee, D. J. A. et al. (1997). Actions of the anthelmintic ivermectin on the pharngeal muscle of the parasitic nematode,  Ascans suum . Parasitol., 115, 553-61.  
          Bunnin, B. A. (1998). “The Combinatorial Index”, Academic Press, San Diego.  
          Campbell, J. L. et al. (1978). Genetic recombination and complementation between bacteriophage T7 and cloned fragments of T7 DNA. Proc. Natl. Acad. Sci. USA, 75, 2276-80.  
          Davies, P. A. et al. (1999). The 5-HT 3B  subunit is a major determinant of serotonin receptor function. Nature, 397, 359-63.  
          Evans, K and Haydock, P. (1999). Control of plant parasitic nematodes. Pesticide Outlook, 10, 107-11.  
          King, F. D. (1994). “Structure activity relationships of 5-HT 3  receptor antagonists, in 5-hydroxytryptamine-3 receptor antagonists”. Ed. F. D. King, B. J. Jones and G. J. Sanger pp. 1-43. CRC Press, Boca Raton, Fla.  
          March, J. (1985). “Advanced Organic Chemistry”, John Wiley, NY, 3 rd  edition.  
          Maricq, A. V. et al. (1991). Primary structure and functional expression of the 5-HT 3  receptor. A serotinin-gated ion channel. Science, 254, 432-37.  
          Miyake, A. et al. (1995). Molecular cloning of human 5-hydroxytryptamine3 receptor: heterogeneity in distribution and function among species. Mol. Pharmacol., 48, 407-16.  
          Needleman, S. B. and Wunsch, C. D. (1970). A general method applicable to the search for similarities in the amino acid sequence of two proteins. J. Mol. Biol., 48, 443-53.