Patent Application: US-48670204-A

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 - azabicyclooctan - 3 - ol 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:
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 . 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 . 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 . 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 . 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 . 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 . 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 . suitable carriers for the compounds provided as formulae ( i ) and ( iv ) include dimethyl sulfoxide ( dmso ). 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 . 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 . 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 . 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 . suitable additional sweeteners include , for example , sugars such as glucose ; and cyclamate and salts thereof . preferably , the compositions contain compounds which attract target pests , and / or repel non - target organisms ( such as beneficial insects , mammals etc ). 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 . 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 ). 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 . 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 . 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 . 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 . 2 - methyl - 5 - hydroxytryptamine hydrochloride ( c 11 h 14 n 2 o . hcl h 2 o ) was obtained from tocris cookson ltd ., avonmouth , uk . the tropanyl esters shown in table 1 were synthesised as described below . 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 ). 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 . the assay used was essentially that described in wo 01 / 40500 with minor modifications . the protocol was as follows : ( 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 . ( 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 . ( 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 . ( 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 . 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 . 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 ). ( 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 . ( 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 . ( 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 . ( 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 . ( 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 . ( 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 ). 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 . 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 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 ( fig2 .). where possible , the data were fitted with a logistic equation . 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 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 . 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 ( fig2 — 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 . 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 . 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 . 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 . 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 . 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 . 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 . avery , l ., and horvitz , h . r . 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