Patent Application: US-25649405-A

Abstract:
the present invention relates to the determination that a group of trinervitadiene compounds possess antimicobial activity . the invention provides pharmaceutical and / or veterinary formulations comprising these compounds , as well as methods for treating a microbial infection or disease , and methods for disinfecting a surface .

Description:
at least some of the compounds of the present invention are naturally occurring trinervitanes which have been obtained from native australian termites , and are likely to be present in the same and possibly in related species in other countries . access to larger quantities of these and related natural trinervitanes , which would be needed for therapeutic use , could be obtained from cultured colonies of the appropriate termites , or alternatively by laboratory synthesis of the desired compounds . analogues of the natural materials , of the types described herein , could be obtained by chemical conversion from the natural materials , or alternatively by total synthesis in cases where such a route would be more efficaceous . the synthesis of the basic tricyclic nucleus of the trinervitane diterpenes has been accomplished by means of robinson annelation and mcmurry coupling to yield oxygenated trinervitadiene products carrying olefinic functionality at 1 ( 15 ), 8 ( 9 )- or 1 ( 15 ), 8 ( 19 )- positions ( dauben et al ., 1998 ). furthermore , a trinervitatriene - 2 , 3 - diol carrying olefinic functionality at the 7 ( 8 ), 11 ( 12 ), 15 ( 17 )- positions has been synthesised by chemically simulating the proposed biogenetic route to such natural products ( hirukawa et al ., 1994 ; kato et al ., 1998 and 2001 ). extension or adaptation of these routes using chemical reactions well known and described in the art ( cf ., for example , trost , 1989 ; apsimson , 1973 - 1992 ), or the development of purpose designed synthetic routes again using chemical reactions well known and described in the art ( cf ., for example , trost , 1989 ; apsimson , 1973 - 1992 ), would provide convenient access to the natural trinervitanes and their analogues . throughout this specification , unless the context requires otherwise , 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 . 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 in australia before the priority date of each claim of this application . the invention will hereinafter be further described by reference to the following non - limiting examples . a sample comprising 11 . 59 g wet weight of nasutitermes triodiae ( isoptera : termitidae ) ( froggatt ) adults ( mixed castes ; mainly soldiers ) was collected manually in the field and the sample was snap frozen in a dry - shipper containing liquid nitrogen . the specimen was stored at − 80 ° c . before being freeze - dried to constant weight ( 1 . 39 g ). the sample was ground to a powder and dispersed in 49 ml of 70 % ( v / v ) methanol in water and shaken at room temperature overnight . the sample was filtered and centrifuged and the supernatant was recovered . the combined residues were then re - extracted with a further 20 ml of 70 % methanol . the supernatants were combined to a total of 47 ml . antimicrobial activity was detected by saturating a ¼ inch diameter filter paper disk ( bacto ) with the methanolic extract , evaporating the solvent in a cool air stream and placing the disk onto a bacteriological plate containing bacillus subtilis which is known in the art to be a model organism for gram positive bacteria ( van welt et al ., 2001 ; harwood et al ., 1992 ; ferreira et al ., 2004 ). the bacillus subtilis atcc strain 6633 was used at 9 . 2 ml of a log phase culture with abs 60nm = 1 per 200 ml of luria - bertani medium containing 1 . 5 % ( w / v ) agar . the plate was incubated at 28 ° c . for 24 hours and the diameter of the clearing zone was measured . dilutions of the extract and fractions from hplc chromatography were tested in the same way . in some cases , fractions from the column were tested by evaporating them to dryness , redissolving in methanol and simply spotting 10 μl of the methanolic sample directly onto the bacteriological plate and proceeding as described . for the purification of the compound of fraction 23 , ( 6 ) 6 ml of the methanolic extract was purified in 12 × 0 . 5 ml batches by semi - preparative reverse - phase hplc over a ymc ods - aq capped c18 column ( 250 mm × 10 mm ) ( sapphire biosystems ) under the following conditions : 0 . 5 ml of extract for each batch solvent a = 99 . 95 % water + 0 . 05 % ( v / v ) trifluoroacetic acid solvent b = 100 % acetonitrile 0 - 2 minutes 100 % a 2 - 22 minutes linear gradient 0 - 100 % b 22 - 35 minutes 100 % b flow rate 4 ml / minute fractions were collected by time ( 1 minute per fraction ). the absorbance of the effluent was monitored at 230 nm . corresponding fractions were pooled across all 12 batches and the eluate in each of the pooled fractions was evaporated to dryness under nitrogen . the residues were weighed and taken up again in small volumes of appropriate solvents — methanol for preparative or analytical hplc and electrospray mass spectroscopy , deuterated chloroform for nuclear magnetic resonance ( nmr ) spectroscopy , etc . for compounds in fractions other than fraction 23 , a similar protocol was used but with the following additional steps . an extra 4 ml of extract was used and the eluates from all 20 batches were pooled and processed as described above . the material in fractions 24 and 26 was a mixture after this first preparative hplc step , therefore the pooled active fractions were further purified using one of the two following isocratic chromatographic procedures . in both isocratic purifications the same ymc ods - aq capped c18 column ( 250 mm × 10 mm ) ( sapphire biosystems ) was used as in the first step . for each batch , 0 . 5 ml of fraction 24 ( 7 ) in methanol ( used to purify fraction 26 , ( 8 and 9 ) in 2 batches ) for each batch 0 . 5 ml of fraction 26 ( 8 and 9 ) in methanol the purified fractions were examined by analytical hplc using similar gradient elution conditions to the preparative procedure , i . e . a water - acetonitrile gradient , followed by 100 % acetonitrile . the only differences were that the analytical column was a ymc ods - aq capped c18 column ( 250 mm × 3 mm ), the flow rate was 0 . 55 ml / minute and 20 μl of sample was loaded onto the column for each run . the purity and composition of the active pooled fractions were determined using a range of standard spectroscopic techniques including electrospray mass spectroscopy ( esms ), high resolution electron impact mass spectroscopy ( hreims ), electron impact mass spectroscopy ( eims ) and 300 and 500 mhz proton and carbon nuclear magnetic resonance in one and two dimensional modes . effects on mammalian cell growth were determined by exposing cultures of two mammalian neoplastic cell lines ( sp2 / 0 — ag8 , a non - secreting mouse myeloma cell line derived from balb / c mice , and ncl - h460 , a human - derived small cell lung carcinoma line ) to fixed dilutions of the methanolic extract of n . triodiae or to fixed concentrations of fraction 23 , ( 6 ) for 19 hours at 37 ° c . cells were grown in wells of sterile 96 - well tissue culture cluster plates by standard methods . cell growth was estimated using the cell proliferation reagent wst - 1 ( roche diagnostics ) according to the manufacturer &# 39 ; s instructions and proliferation data were compared with those from untreated control wells . minimum inhibitory concentrations with bacillus subtilis atcc strain 6633 were determined using the national committee for clinical laboratory standards broth microdilution test ( nccls , 2000 . nccls document m7 - a5 — methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically , approved standard — fifth edition ). the test was standardised using penicillin g and gentamicin with staphyloccous aureus atcc strains 29213 and 25923 and enterococcus faecalis atcc strain 29212 . results of standardisation were assessed according to the nccls standards ( nccls , 2000 . nccls document m100 - s10 ( m7 )— performance standards for antimicrobial susceptibility testing ; tenth informational supplement ( aerobic dilution ). nccls , wayne , pa .). the crude 70 % methanol extract of n . triodiae displayed antimicrobial activity against b . subtilis ( clear zone diameter 9 mm in the standard filter disk test ) and moderate inhibitory activity against mammalian cells ( 37 % of control at a concentration of approximately 30 μg / ml ). there was no activity against a test strain ( acm 3221 ) of escherichia coli ( a gram negative bacterium ) in a similar test protocol . after chromatographic fractionation of the methanolic activity , antimicrobial activity was detected in the fraction eluting between 22 and 23 minutes ( fraction 23 ) and also the fractions eluting between 23 and 24 minutes ( fraction 24 ) and between 25 and 26 minutes ( fraction 26 ). a total of 9 mg of the pure compound was purified from 6 ml of crude extract , indicating a starting concentration of 1 . 5 mg / ml . the molecular formula of the compound in fraction 23 ( 6 ) was determined as c 20 h 32 o 3 by esms which show sodiated ions at m / z 343 ( mna + ) and m / z 663 ( m 2 na + ) and by hreims which showed m + - h 2 o at m / z 302 . 2243 where m / z calculated for c 20 h 3 o 2 is 302 . 2246 . the 1 h nmr and 13 c nmr chemical shift data for the compound ( 6 ) in fraction 23 are shown in table 1 . the compound in fraction 23 was determined to be 1 ( 15 ), 8 ( 19 )- trinervitadiene - 3α , 5α , 18 - triol ( 6 ). this compound has not been reported previously . the minimum inhibitory concentration of compound ( 6 ) against b . subtilis was estimated as ≦ 50 μg / ml . purified compound ( 6 ) had no detectable inhibitory effect on the proliferation of ncl - h460 cells at concentrations up to 100 μg / ml . compound ( 6 ) had no detectable inhibitory effect on the proliferation of sp2 / 0 cells at concentrations up to 30 μg / ml . the material in fraction 24 , which also showed antimicrobial activity , was a mixture of at least two compounds after the first chromatographic step . it was therefore submitted to “ isocratic procedure 1 ” as described above and the single biologically active u . v .- absorbing peak which eluted between 14 and 17 minutes was collected . a total of 4 mg of the pure biologically active compound present in fraction 24 was purified from 10 ml of starting material indicating an approximate starting concentration of 0 . 4 mg / ml in the crude extract . the molecular formula of the compound in fraction 24 ( 7 ) was determined as c 20 h 32 o 2 by esms which show sodiated ions at m / z 327 ( mna + ) and by hreims which showed m + at m / z 304 . 2403 where m / z calculated for c 20 h 32 o 2 is 304 . 2402 . the 1 h nmr and 13 c nmr chemical shift data for the compound ( 7 ) in fraction 24 are shown in table 2 . the compound in fraction 24 was determined to be 1 ( 15 ), 8 ( 19 )- trinervitadiene - 3α , 5α - diol ( 7 ). this compound has not been reported previously . 5 μg of compound ( 7 ) gave a clear zone of diameter 7 mm in the disc diffusion assay . the minimum inhibitory concentration of compound ( 7 ) against b . subtilis was estimated as ≦ 50 μg / ml . the material in fraction 26 was a mixture of at least two biologically active compounds after the first chromatographic step . it was therefore submitted to “ isocratic procedure 2 ” as described above and two active fractions were collected . the first , which eluted from the column at approximately 12 minutes is designated fraction 26a , the second , which eluted from the column at approximately 14 minutes is designated fraction 26b . a total of 0 . 5 mg of the pure biologically active compound present in fraction 26a was purified from 10 ml of starting material indicating an approximate starting concentration of 0 . 05 mg / ml in the crude extract . the molecular formula of the compound in fraction 26a ( 8 ) was determined as c 22 h 34 o 4 by esms which showed ions at m / z 345 ( mh + - h 2 o ) 363 ( mh + ), 385 ( mna + ), 747 ( m 2 na + ) and by hreims which showed m + at m / z 362 . 2460 , where c 22 h 34 o 4 requires 362 . 2457 ; and m + - h 2 o at m / z 344 . 2350 where c 22 h 32 o 3 requires 344 . 2351 . the 1 h nmr chemical shift data for the triol monoacetate ( 8 ) in fraction 26a are shown in table 3 . the identity of the triol monoacetate was further confirmed by partially acetylating the triol ( 6 ) and confirming the presence in the acetylation mixture of a major component with identical retention time and 1 h nmr spectrum to the natural triol monoacetate . the protocol used was as follows : the triol ( 6 ) ( 1 mg ) and acetic anhydride ( 10 μl ) in dry pyridine ( 100 μl ) were kept for 3 hours at room temperature . the reaction was diluted with water , extracted with dichloromethane , and the extract subjected to preparative hplc on the standard column using gradient elution in acetonitrile / water from 50 : 50 to 100 : 0 . the fraction with retention time 18 min when analysed under normal gradient elution conditions contained a major component with identical retention time to the natural triol monoacetate ( 8 ). the 1 h nmr spectrum of this major component ( table 3 ) also matched that of the natural triol monoacetate ( 8 ). the compound in fraction 26a was therefore determined to be 1 ( 15 ), 8 ( 19 )- trinervitadiene - 3α , 5α , 18 - triol 5 - acetate ( 8 ). this compound has not been reported previously . an unknown concentration and mass of the compound ( 8 ) gave a clear zone of diameter 15 mm in the disc diffusion assay . the minimum inhibitory concentration of compound ( 8 ) in the standard broth microdilution assay against b . subtilis was estimated as & gt ; 50 μg / ml . a total of 1 . 5 mg of the pure biologically active compound present in fraction 26b was purified from 10 ml of starting material indicating an approximate starting concentration of 0 . 15 mg / ml in the crude extract . the molecular formula of the compound in fraction 26b ( 9 ) was determined as c 20 h 32 o 2 by esms which showed sodiated ions at m / z 327 ( mna + ), 631 ( m 2 na + ) and by hreims which showed m + at m / z 304 . 2404 where c 20 h 32 o 2 requires 304 . 2402 . the 1 h nmr chemical shift data for the trinervitadiene diol ( 9 ) in fraction 26b are shown in table 4 . the compound in fraction 26b was therefore determined to be 1 ( 15 ), 8 ( 9 )- trinervitadiene - 2β , 3α - diol ( 9 ) by comparison of the measured chemical shifts with previously published 1 h nmr data ( goh , chuah et al ., 1984 ; braekman , daloze et al ., 1983 ; prestwich & amp ; collins , 1981 ; prestwich et al ., 1976b ) for this compound ( table 4 ). an unknown concentration and mass of the compound ( 9 ) gave a clear zone of diameter 15 mm in the disc diffusion assay . the minimum inhibitory concentration of compound ( 9 ) in the standard broth microdilution assay against b . subtilis was estimated as ≦ 25 μg / ml . although the structure of this compound has been published previously , it has not previously ( e . g . goh , chuah et al ., 1984 ; braekman , daloze et al ., 1983 ; prestwich & amp ; collins , 1981 ; prestwich et al ., 1976b ) been reported that it has potent antimicrobial activity . 1 ( 15 ), 8 ( 19 )- trinervitadiene - 3α , 5α , 18 - triol 3 , 5 , 18 - triacetate ( 10 ) was synthesised by acetylation of the triol ( 6 ). the triol ( 6 ) ( 1 mg ) and acetic anhydride ( 30 μl ) in dry pyridine ( 100 μl ) were kept for 3 days at room temperature . the reaction was diluted with water , extracted with dichloromethane , and the extract subjected to preparative hplc under the standard conditions . the major fraction was collected and filtered through a short silica column in dichloromethane to afford the triacetate ( 10 ) ( 1 mg ), which eluted as a single major peak at 28 minutes when chromatographed under the standard analytical hplc conditions described in the above materials and methods section . the molecular formula of the reaction product was confirmed as c 26 h 38 o 6 by esms which showed sodiated ions at m / z 469 ( mna + ) and by hreims which showed ( m + - acoh ) at m / z 386 . 2449 where c 24 h 34 o 4 requires 386 . 2457 . the 1 h nmr chemical shift data for the trinervitadiene triol triacetate ( 10 ) reaction product are shown in table 5 . the acetylation product was therefore determined to be 1 ( 15 ), 8 ( 19 )- trinervitadiene - 3α , 5α , 18 - triol 3 , 5 , 18 - triacetate ( 10 ). this compound has not been reported previously . 5 μg of compound ( 10 ) gave a clear zone of diameter 11 mm in the disc diffusion assay . the minimum inhibitory concentration of compound ( 10 ) against b . subtilis was estimated as & gt ; 50 μg / ml . in studies on the antimicrobial activity of other australian termites of the genus nasutitermes ( family : termitidae ; sub - family : nasutitermitinae ) it was noted that extracts of three other species , nasutitermes exitiosus ( hill ) and two unidentified species of the same genus , exhibited antimicrobial activity which eluted at the same retention time as 1 ( 15 ), 8 ( 9 )- trinervitadiene - 2β , 3α - diol ( 9 ) and shared identical 1 h nmr chemical shift data ( table 4 ) with the diol ( 9 ) purified from fraction 26b . it was also noted that extracts made exclusively from workers of the species n . exitiosus exhibited no antimicrobial activity and did not exhibit any of the characteristic u . v . absorbing peaks attributed to trinervitadiene derivatives , which elute in the 22 - 28 minute region of the standard gradient hplc chromatogram . on the other hand , an extract of soldiers from the same nest as the aforementioned workers exhibited antimicrobial activity . when the extract of soldier termites was subjected to the standard gradient hplc chromatography , the biologically active u . v .- absorbing peaks characteristic of trinervitadiene derivatives were observed . this indicates that separation of soldier termites from workers prior to their extraction may improve the efficiency both of detecting and purifying biologically active trinervitadiene derivatives . a range of compounds with the trinervitadiene carbon skeleton have previously been reported from termites ( for example : prestwich , tanis et al . 1976a , b ; vrkoc , budesinsky et al . 1978a , b ; dupont , braekman et al . 1981 ; prestwich , spanton et al , 1981 ; baker & amp ; walmsley , 1982 ; braekman , daloze et al . 1986 ). trinervitadiene derivatives have been found in extracts of a number of species of termites belonging to nine genera of termites within the subfamily nasutitermitinae of the family termitidae . however , this is the first time that the isolation of compounds ( 6 , 7 , and 8 ) have been reported or that the triacetate ( 10 ) of compound ( 6 ) has been prepared . the novelty of these compounds is underlined by the fact that no trinervitadiene derivatives have previously been reported with hydroxylation or other substitutions at positions 5 and 18 on the carbon skeleton ( e . g . compound ( 6 )). furthermore , no data have been reported previously regarding antimicrobial activity of any trinervitadiene or derivative thereof including the compounds whose isolation or preparation is described herein ( 6 , 7 , 8 , 9 and 10 ). it has been determined that compound ( 6 ) has a minimum inhibitory concentration in the range ≦ 50 parts per million and that it is at least twice as toxic to microbial cells as it is to human cells and potentially significantly more selective than this . compound ( 9 ) has a minimum inhibitory concentration in the range of ≦ 25 parts per million and has significant inhibitory activity as low as 12 parts per million . compounds ( 7 ), ( 8 ) and ( 10 ) have reduced but detectable antimicrobial activity . for example , acetylation of the hydroxyl groups seems to reduce but not abolish activity , whilst it is also clear that the number and arrangement of groups on the trinervitadiene carbon skeleton modulates the level of antimicrobial activity . compounds ( 6 , 7 , 8 , 9 and 10 ) and a range of other trinervitadiene derivatives where the number , position and nature of groups is varied ( such as the specified derivatives of compounds 11 - 16 ) can therefore be expected to have utility as antibiotics or for some of the other purposes mentioned above . alternatively , they may be useful lead compounds for the development of derivatives with enhanced antibiotic activities . it is interesting that prolonged investigations of the role of soldier defensive secretions has led to the classification of the trinervitadiene derivatives as “ defensive compounds ” ( i . e . the assumption has been that their function is solely in defence of the termite colony against attack by invertebrate or vertebrate predators ). however , the present discovery that these compounds have antimicrobial activity raises the possibility that they also function naturally to suppress microbial parasites within the termite colony . 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 . apsimon , j . 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