Patent Application: US-67815308-A

Abstract:
methods for overcoming multiple herbicide resistance in plants using inhibitors of gst suppression of formula , novel chemical inhibitors of formula , compositions comprising compounds of formula , and uses and methods relating thereto .

Description:
fig1 gsts in mhr black - grass . ( a ) 2d - gel electrophoresis of hydrophobic protein fraction from wt and peldon plants , with polypeptides corresponding to amgstf1 arrowed . ( b ) western blot of extracts from wt and peldon plants using an anti - gstl - serum ( c ) the effect of 0 . 1 mm inhibitors on the activity of recombinant black - grass amgstf1 - 1 ( 100 %= 640 nkat . mg − 1 protein ), amgstl ( 100 %= 83 nkat . mg − 1 ), amgstu1 ( 100 %= 685 nkat . mg − 1 ) with activities determined using the assays described in table 1 . fig2 hplc analysis of flavonoid metabolites in the foliage of arabidopsis ( a ) wt plants and ( b ) amgstfl over - expressors ( line 12 ). flavonoids showing altered accumulation were identified by hplc - ms with reference to published data ( s17 ). compound 1 = kaempferol - 7 - o -[ rhamnosyl - glucosyl - rhamnoside ] ( m − h ) − = 739 ; compound 2 = cyanidin - 3 - o -[ 2 - 0 ( 2 - o -( sinapoyl )- xylosyl )- 6 - o -( 4 - o -( β - d - glucosyl )- p - coumaryl - β - d - glucosyl ] 5 - o -[ 6 - o -( malonyl ) β - d - glucoside ] ( m − h ) − = 1341 ; compound 3 kaempferol - 7 - o -[ rhamnosyl - rhamnoside ] ( m − h ) − = 577 4 — unidentified flavonol glycoside ( m − h ) − = 679 . fig3 . effect of transgenic over - expression of amgstf1 in arabidopsis . ( a ) amgstfl over - expressors ( lines 8 , 12 ) and vector - only controls germinated and grown on agar containing 10 μm herbicides for 30 days . ( b ) activities of detoxifying enzymes ( substrates in parentheses ) and antioxidant contents . ( c ) western blot of leaf proteins from vector - only control and lines 8 and 12 probed with maize anti - gstf - serum . seed of the black - grass mhr populations ‘ peldon ’ and spain and from an herbicide - susceptible wild - type ( wt ) line were obtained from herbiseed , twyford , uk . plants were grown as described previously ( 1 ) under two lighting intensity regimes ( low = 100 μe . m − 2 . s − 1 or high = 220 μe . m − 2 . s − 1 ). for biochemical studies , plants were harvested after 30 days ( 2 to 3 leaf stage ), weighed and frozen in liquid nitrogen . for spray trials , herbicides were dissolved in acetone and then diluted ( 1 : 100 ) in 0 . 1 % v / v tween 20 and applied to 14 - day - old plants with a hand - held sprayer at doses equivalent to field rates as expressed as grams active ingredient applied per hectare ( chlortoluron = 500 g a . i . ha − 1 , fenoxaprop ethyl = 85 g a . i . ha − 1 ). for studies with inhibitors , plants were pre - treated with chemicals formulated as described for the herbicides , at rates equivalent to 80 g a . i . ha − 1 . arabidopsis thaliana ( columbia ) plants were grown and maintained as described previously ( 2 ) for transgenic studies , amgstf1 was cloned into the xhoi and kpni sites of the vector prt107 after introducing the respective restriction sites by pcr , with a 35s promoter driving construct expression and a polyadenylation signal added ( 2 ). after cloning into the binary vector pcambia 3300 ( cambia , canberra , australia ), the plasmid was electroporated into agrobacterium tumefaciens and used to transform arabidopsis by the floral dip method ( 3 ). homozygote lines were selected after spraying with gluphosinate ammonium ( basta ) as described ( s2 ) and analysed for amgstf1 expression by western blotting using an anti - gstf - serum ( 1 ). seed from two lines of amgstfl - transformants showing intermediate ( line 8 ) and high levels ( line 12 ) of transgene expression were selected for further study . plants were either grown in growth rooms ( irradiance 85 μe . m − 2 . s − 1 ), or in the glass - house ( up to 1500 μe . m − 2 . s − 1 ), and harvested for analysis when 30 days old . controls were transformed with pcambia 3300 alone . seeds from lines 8 , 12 and controls were germinated on agar containing 2 μm or 10 μm herbicides . plants were maintained in growth rooms for 30 days and assessed for phytotoxic damage . all extractions were carried out on ice at 5 v / w of buffer using a pestle and mortar , with samples clarified by centrifugation ( 10 , 000 g , 10 min , 4 ° c .) and protein contents determined using dye binding reagent ( biorad ) with γ - globulin as the reference . enzymes were isolated and assayed as crude extracts at 30 ° c . using the published methods given in table 1 , with non - enzymic reactions corrected for by using boiled protein controls . glutathione and hydroxymethylglutathione ( 4 ), were quantified in both reduced and oxidised forms by hplc ( table 1 ), while ascorbic acid content was determined enzymically ( table 1 ). phenolic metabolites were solvent extracted from plant foliage and resolved by hplc with the eluant analysed by photodiode array detection ( pda ) and time - of - flight mass spectrometry following ionization by electrospray ionization time - of - flight mass spectrometry ( esi tof ms ) as described ( 5 ). the major flavonoid present in black - grass was purified from wt shoots by preparative hplc and incubated with 6 % v / v conc hcl for 1 h at 95 ° c ., then re - analysed by hplc esi tof ms as above . sugars released in the hydrolysate were identified by co - chromatography with reference standards on a carbo - pak pa - 100 hplc column ( 4 mm × 250 mm , dionex ) coupled with electrochemical detection using a mobile phase of 100 mm naoh . crude protein extracts were precipitated with ammonium sulfate ( 40 %- 80 % saturation ) and the proteins applied to a phenyl sepharose column ( 6 ). the hydrophobic proteins eluted with 50 mm potassium phosphate were desalted and precipitated with acetone ( 5 v / v ). proteins were analysed by two - dimensional gel electrophoresis pellets using ph 4 - 7 isoelectric focusing strips in the first dimension followed by sds - page on large format gels ( 7 ). polypeptides of interest were identified by staining in sypro ruby prior to excision , digestion with trypsin and hplc ms - ms sequencing ( 6 ). two dimensional , differential , gel electrophoresis of peldon and wt foliage using fluorescent dyes was carried out on total and hydrophobic protein extracts by the cambridge proteomics centre ( http :// www . bio . cam . ac . uk / proteomics /). the expression of specific classes of gsts was monitored by sds - page and western blotting using antisera raised against maize gstfs and gstus ( 1 ) and wheat gstls ( 8 ). quantitative pcr was used to determine the abundance of gst transcripts ( 9 ). based on a normalisation with actin ( relative abundance = 1 ) values in the control plants were atgstl1 ( 0 . 0021 ± 0 . 0002 ), atdhar3 ( 1 . 3 ± 0 . 1 ) and atgstu19 ( 1 . 8 ± 0 . 2 ). in the amgstfl - over - expressing lines atgstl1 transcript abundance was doubled ( 0 . 0047 ± 0 . 0003 ), while atdhar3 and atgstul19 were unaffected . alignment of dna sequences encoding gstls from wheat ( accession y17386 ), maize ( accession x58573 ) and rice ( accession af237487 ) directed the design of oligonucleotides for the initial amplification of a partial sequence of amgstl1 prepared from black - grass cdna ( 1 ). amplifications were performed using the primers amgstlf1 ( 5 ′ atggccgcagctgcagca 3 ′), which contained the start codon , together with primers to conserved internal regions , namely amgstlf2 , ( 5 ′ ggtgccttccctggagcacgac 3 ′ ( seq id no . 1 )), amgstlr1 ( 5 ′ gtcgtgctccagggaagg 3 ′( seq id no . 2 )) and amgstlr2 , ( 5 ′ ccaagctaaattggccaaggaagaa 3 ′ ( seq id no . 3 )). amplification products derived from pcr using amgstlf1 with the reverse primer generated products , were cloned , sequenced and confirmed to be gstl - like . the 3 ′ end of the gstl sequence was then obtained by 3 ′ race , using amgstlf1 and amgstlf2 in conjunction with an oligo ( dt )- containing primer . the full - length black - grass lambda sequence was then amplified , with the addition of 5 ′ nde1 and 3 ′ xho 1 sites to allow cloning into pet 24 . after confirming its identity , the his - tagged recombinant protein was expressed and affinity purified from e . coli and determined to be active as a thiol transferase ( 83 nkat . mg − 1 pure protein ) when assayed with hydroxyethyldisulfide ( 9 ). to investigate the biochemical basis of mhr and possible roles for amgstf1 - 1 , the resistant peldon ( 27 ) and spanish ( 24 ) populations and a wt line were subjected to metabolic profiling and enzyme assay screens ( 34 ). as compared with wts , when aqueous extracts were analysed for antioxidants , both mhr lines contained elevated concentrations of the tripeptide glutathione and its derivative hydroxymethylglutathione ( 29 ), but identical levels of ascorbic acid ( table 1 and table 2 ). these changes did not affect the ratio of oxidised : reduced thiols ( redox ratio ), but were associated with an enhancement in the activities of catalase and five glutathione - dependent enzymes ( table 1 ). in contrast , ascorbate peroxidase , guiacol peroxidase and superoxide dismutase were unaffected ( table 2 ). of the glutathione - dependent enzymes , thioltransferase activity was particularly enhanced in peldon plants . while this increase was partly explained by the induction of dehydroascorbate reductases , which can also catalyse thiol transfer ( 33 ), it was also clear that additional glutathione - dependent enzymes with this activity were present . plant lambda ( l ) class gsts have been shown to be highly active thiol transferases ( 33 ). western blotting of protein extracts with an antibody raised to a gstl from wheat ( 35 ) identified an immunoreactive 27 kda polypeptide in peldon plants which was absent in the wts . subsequent cloning of the black - grass amgstl1 confirmed this identification and the activity of the protein as a thioltransferase ( 34 ). plants were then solvent - extracted and analysed by lc - ms ( fig1 ). as compared with wts , the foliage of mhr plants contained more anthocyanin pigments and twice the amount of the major flavonoid , tentatively identified as apigenin - 6 - c -( 2 ″- o - arabinosyl )- glucoside ( table 3 ). the changes in the proteome in black - grass foliage underlying associated with mhr were then investigated by two - dimensional differential gel electrophoresis following clean - up by hydrophobic interaction chromatography ( 34 ). the proteome of peldon and wt plants were virtually identical , except for 9 polypeptides which were strongly up - regulated in the mhr plants . seven of these polypeptides had molecular masses ( 28 kda ) characteristic of gst subunits . when analysed by maldi tof ms after digestion with trypsin , all seven gave identical peptide fingerprints , with tandem ms of a 1038 da fragment identifying the sequence vfgpamstnv . this identified all 7 up - regulated polypeptides as amgstf1 subunits . this appears to be due to multiple genes encoding variants of amgstf1 in peldon ( 32 ). our results demonstrated that the up - regulation in expression of amgstf1 polypeptides was the dominant change associated with the proteome of mhr in black - grass . to investigate the role of this protein , amgstf1 - 1 was constitutively over - expressed in the model plant arabidopsis thaliana ( 34 ) two independent lines ( 8 and 12 ) of homozygous transformants were screened for over - expression of amgstf1 by western blotting using an anti - gstf - serum . these studies confirmed transgene expression , together with the accumulation of immunoreactive arabidopsis atgstfs which were present at low concentration in the vector - only controls . when gst and gpox activities were determined in the amgstfl - over - expressors ( fig3 ), the observed increases in atrazine - conjugating activity were found to be due to increased expression of atgsts and not amgstf1 , with this enzyme having no detectable activity toward this substrate . amgstf1 expression also caused an enhancement in other detoxifying enzymes in arabidopsis , notably ogt activity ( fig3 ). while the over - expressors were indistinguishable from controls when grown in growth - rooms , when transferred to glasshouses exposed to full sunlight they became visibly pigmented due to the accumulation of anthocyanins . lc - ms analysis of phenolic metabolites showed 3 - fold to 4 - fold increases in 2 major anthocyanins and a doubling in the content of the major flavonoids ( fig3 ). upon analysis for antioxidants and associated enzymes ( fig3 table 2 ), glutathione was found to be modestly enhanced in the over - expressing lines without any disturbance of the redox ratio . quantitative pcr showed that amgstf1 expression doubled the expression of arabidopsis gstls , but had no effect on dehydroascorbate reductases or the tau class enzyme atgstu19 ( 33 ). similarly , enzyme assays also showed that other enzymes of antioxidant metabolism which were up - regulated by mhr in black - grass were unaffected by amgstfl - expression in arabidopsis ( table 2 ). these results demonstrated that the ectopic expression of amgstf1 had selectively replicated part of the mhr phenotype , by enhancing detoxification enzymes , glutathione and flavonoid metabolism . to determine how these changes had affected resistance , amgstf - expressors were germinated on agar plates containing herbicides with differing modes of action ( fig3 ). the transgenics showed enhanced resistance toward the cell division inhibitor alachlor ( a chloroacetanilide ) and the psii inhibitors atrazine ( chloro - s - triazine ) and chlortoluron ( phenylurea ). these results showed that expression of amgstf1 - 1 in arabidopsis conferred tolerance to three distinct classes of herbicide , acting on two target sites . tolerance to atrazine and alachlor could be partially explained by the direct enhancement in gst - mediated conjugation , and hence detoxification ( table 3 ). in contrast , resistance to chlortoluron could not be due to enhanced glutathionylation , as this herbicide is inactivated by cyps and ogts ( 26 ). cumulatively , these experiments showed that when expressed in arabidopsis , amgstf 1 orchestrated a series of changes in primary and secondary metabolism , including the enhancement of two distinct pathways of xenobiotic detoxification , which resulted in resistance to multiple herbicides . the identification of such a regulator of mhr , suggested that it would be possible to use chemical intervention to suppress the associated phenotype and restore sensitivity to herbicides in black - grass . because of their importance in detoxifying chemotherapeutic agents used in cancer therapy , gsts are a well - recognised target for medicinal chemistry , with a range of inhibitors selective toward different enzymes having been developed ( 37 ) eight different inhibitor chemistries were tested for their ability to disrupt the activity of the black - grass enzymes amgstf1 , amgstl and the herbicide - detoxifying tau ( u ) class amgstu1 ( 32 ) amgstf1 and amgstl1 were totally inhibited by 4 - chloro - 7 - nitro - 2 , 1 , 3 - benzoxadiazole ( cnbd ) ( fig2 ). bromoenol lactone ( bel ) totally inhibited amgstl1 , but not amgstf1 . based on this differential inhibition of two gsts which were selectively up - regulated in peldon , the effect of cnbd and bel on mhr in black - grass was determined . when applied 48 h prior to an application of chlortoluron , or fenoxaprop ethyl , cnbd but not bel , reduced resistance to both types of herbicides . treatment with cnbd also reduced the flavonoid content in mhr peldon plants to wt levels ( fig2 ). the results of the transgenesis studies in arabidopsis and selective inhibitor trials have identified a central role for amgstf1 in mhr and a potential mechanism for restoring chemical control . gsts have long been implicated in tolerance mechanisms to drugs and pesticides due to their well - studied role in conjugating xenobiotics with glutathione , thereby detoxifying them ( 26 , 38 ). gsts are also known to exert broad - ranging antioxidant protection to a range of biotic and abiotic stresses in animals , plants and microbes ( 39 , 40 ). in the case of amgstf1 we had previously proposed that this enzyme could counter herbicide action by acting as a gpox , thereby detoxifying cytotoxic lipid oxidation products formed as a secondary consequence of chemical action ( 32 ). the current results demonstrate that amgstf1 exerts a much more profound protective effect than previously thought , acting as a causative agent of mhr rather than being part of the protective response . while the mechanisms by which amgstf1 co - ordinates signalling events leading to mhr are currently unknown , there are two potential clues arising from the signalling roles of gsts in animals , which may be relevant . several mammalian gsts are known to bind and hence inactivate a c - jun n - terminal kinase ( jnk ), which regulates apoptosis and responses to oxidative stress ( 41 ) these gsts attenuate the responsiveness of jnk to oxidative stress and interestingly , thioether derivatives of 2 , 1 , 3 - benzoxadiazoles , structurally related to cnbd , have been shown to selectively interfere with this interaction and promote apoptosis ( 42 ). another clue may lie in the unusual antioxidant activity of amgstf1 - 1 . the lipid oxidation product , 2 - hydroxynonenal ( hne ) is known to modulate apoptosis as well as cell differentiation and growth in mammalian cells and it has been proposed that gsts control this activity ( 43 ). thus , amgstf1 would regulate the supply of the hydroperoxide precursors of hne ( 32 ), thereby modulating oxidative stress signalling leading to mhr . the regulatory role of amgstf1 in herbicide resistance is currently under investigation . importantly , the identification of chemical agents which disrupt its signalling function provide both a valuable new research tool to study mhr in wild grasses as well as leads for new agrochemicals to counteract this threat to sustainable arable crop protection . compound i ) available from aldrich chemicals compound ii ) available from new horizons laboratories compound iii ) available from maybridge chemicals compound iv ) synthesized as described herein compound v ) synthesized as described herein compound vi ) available from fisher scientific , uk compound vii ) available from scientific exchange inc . compound viii ) available from alfa aesar compound ix ) synthesized as described herein compound x ) synthesized as described herein compound xi ) synthesized as described herein compound xii ) synthesized as described herein compound xiii ) synthesized as described herein compound xiv ) synthesized as described herein compound xv ) synthesized as described herein compound xvi )* synthesized as described herein compound xvii )* synthesized as described herein compound xviii )* synthesized as described herein compound xix )* synthesized as described herein compound xx )* synthesized as described herein compound xxi ) synthesized as described herein compound xxii ) synthesized as described herein compound xxiii ) synthesized as described herein compound xxiv )* synthesized as described herein compound xxv ) synthesized as described herein compound xxvi ) synthesized as described herein compound xxvii )* synthesized as described herein compound xxviii ) synthesized as described herein compound xxix ) synthesized as described herein compound xxx ) available from aurora screening library compound xxxi )* synthesized as described herein compound xxxii ) synthesized according to the methodology described in analytica chimica acta 344 ( 1997 ) 227 - 232 compound xxxiii ) synthesized as described herein compound xxxiv ) synthesized according to the teaching of wo2000 / 076972 compound xxxv ) available from the maybridge chemicals compound xxxvi ) available from fluorochem compound xxxvii ) available from princeton compound xxxviii ) available from maybridge chemicals compound xxxix ) available from scientific exchange product list compound xl ) available from timtec stock library compound xli ) available from chembridge screening library compound xlii ) available from ryan scientific inc compound xliii ) available from ryan scientific inc compound xliv ) available from ryan scientific inc compound xlv ) available from sci . exchange product list compound xlvi ) available from ryan scientific inc compound xlvii ) available from aurora compound xlviii ) synthesized as described herein compound xlix ) synthesized as described in zhurnal obshchei khimii ( 1966 ) 26 ( 7 ) 1268 - 74 compound l ) available from ryan scientific inc compound li ) synthesized as described in zhurnal obshchei khimii ( 1964 ) 34 ( 1 ) 261 - 72 compound lii ) available from ambinter stock screening collection compound liii ) synthesized as described herein compound liv ) available from tci laboratory chemicals compound liv ) available from ryan scientific inc compound lvi ) synthesized as described herein compound lvii ) synthesized as described herein compound lviii ) available from ryan scientific inc compound lvix ) available from ryan scientific inc compound lx ) synthesized as described herein compound lxi ) available from ryan scientific inc compound lxii ) available from ryan scientific inc compound lxiii ) available from ryan scientific inc compound lxiv ) available from timtec building blocks and reagents compound lxv ) synthesized as described herein compound lxvi ) available from ryan scientific inc compound lxvii ) available from alfa aesar selected compounds ( indicated as compounds i ) to lxvii ) herein ) were applied to 14 day old mhr black - grass plants at a rate equivalent to 80 g a . i . ha − 1 . the plants were then treated 48 h later with chlortoluron at 500 g a . i . ha − 1 as described previously . after 10 days the plants were scored for phytotoxic injury , with +++= full injury ( 100 %) determined with chlortoluron in the presence of cnbd ( compound i ) of formula ( i )); ++= approximately 50 % damage as compared with cnbd ; += minor but measurable injury . where tested , the score is shown next to the compound . the halogenated derivatives are all known compounds ( cl and f commercially available )— the other two specific compounds shown are already described in the literature — preparation of all the other derivatives follows from the halo ( chloro ) analogues following well established literature methods ( see r . m . paton 1 , 2 , 5 - oxadiazoles in science of synthesis , volume 13 chapter 7 p185 , thieme , stuttgart ). the 7 - halo derivatives are prepared by standard condensation reactions between amines or alcohols and the appropriate sulfonyl chloride recognisable by those skilled in the art of organic chemical synthesis — eg see following procedures and data for the two new compounds iv ) and v ). further variation of substituents is achieved following analogous procedures as used for the nitro analogues as outlined above . to a solution of 4 - chloro - 7 - chlorosulfonyl - 2 , 1 , 3 - benzoxadiazole ( obtainable from aldrich chemicals ) ( 0 . 127 g , 0 . 50 mmol ) in anhydrous acetonitrile ( 6 ml ) was added a solution of 40 % wt methylamine in water ( 0 . 052 ml , 0 . 60 mmol ) followed immediately by triethylamine ( 0 . 084 ml , 0 . 60 mmol ) under argon , with stirring . the reaction was stirred at room temperature for 10 min . before removing the solvent in vacuo . purification by flash column chromatography on silica gel ( pe 40 - 60 : etoac , 8 : 2 ) gave the title product as a white solid ( 0 . 046 g , 0 . 38 mmol , 37 %). 1 h nmr ( 500 mhz , cdcl 3 ): δ 8 . 01 ( 1h , d , j = 7 . 3 , h - 5 ), 7 . 57 ( 1h , d , j = 7 . 3 , h - 6 ), 5 . 06 ( 1h , br q , j = 5 . 2 , nh ), 2 . 75 ( 3h , d , j = 5 . 2 , ch 3 ); 13 c nmr ( 125 mhz , cdcl 3 ): δ 148 . 8 ( c7a ), 144 . 9 ( c3a ), 134 . 1 ( c5 ), 129 . 1 ( c6 ), 127 . 9 ( c7 ), 126 . 9 ( c4 ), 29 . 4 ( ch 3 ). to a solution of 4 - chloro - 7 - chlorosulfonyl - 2 , 1 , 3 - benzoxadiazole ( obtainable from aldrich chemicals ) ( 0 . 127 g , 0 . 50 mmol ) in anhydrous acetonitrile ( 4 ml ) was added a solution of 1 - azido - 4 - aminobutane ( 0 . 068 g , 0 . 60 mmol ) in anhydrous acetonitrile ( 2 ml ) followed immediately by triethylamine ( 0 . 084 ml , 0 . 60 mmol ) under argon , with stirring . the reaction was stirred at room temperature for 10 min . before removing the solvent in vacuo . purification by flash column chromatography on silica gel ( pe 40 - 60 : etoac , 7 : 3 ) gave the title product as a white solid ( 0 . 099 g , 0 . 30 mmol , 60 %). 1 h nmr ( 500 mhz , cdcl 3 ): δ 8 . 00 ( 1h , d , j = 7 . 3 , h - 5 ), 7 . 57 ( 1h , d , j = 7 . 3 , h - 6 ), 5 . 19 ( 1h , t , j = 6 . 0 , nh ), 3 . 30 - 3 . 27 ( 2h , m , ch 2 - 4 ′), 3 . 11 - 3 . 06 ( 2h , m , ch 2 - 1 ′), 1 . 62 - 1 . 59 ( 4h , m , ch 2 - 2 ′, 3 ′); 13 c nmr ( 125 mhz , cdcl 3 ): δ 148 . 8 ( c7a ), 144 . 9 ( c3a ), 133 . 6 ( c5 ), 129 . 1 ( c6 ), 127 . 9 ( c7 ), 127 . 7 ( c4 ) 50 . 7 ( c4 ′), 42 . 9 ( c1 ′), 27 . 1 ( c3 ′), 25 . 8 ( c2 ′); anal . calcd for c 10 h 11 cln 6 o 3 s : c , 36 . 31 ; h , 3 . 35 ; n , 25 . 41 . found : c , 36 . 53 ; h , 3 . 39 ; n , 25 . 33 . for compounds where r 1 = co 2 r 7 ; conhr 4 ; conr 4 r 5 ; — cn ; r 2 ═ f , cl , or br , nhr 4 , nr 4 r 5 , or 4 , sr 4 ; and r 3 ═ h the carboxylic acid precursors are either commercially available ( see below ) or may be prepared by oxidation of the known aldehydes ( see below ) using one of the many known oxidants for such a conversion including kmno 4 , k 2 cr 2 o 7 , pdc / dmf , ag 2 o , naclo 2 , naio 4 / rucl 3 , ruo 4 . the carboxy ester and amide derivatives may be prepared by standard condensation reactions between amines or alcohols as employed in the art . incorporation of r 2 substituents to replace the halogen may be achieved following analogous procedures as used for the nitro analogues — see above nitriles may be introduced from the respective amino - 2 , 1 , 3 - benzothiadiazole or benzoxadiazole either via sandmeyer chemistry or by reaction of the bromoheterocycle with cuprous cyanide in dmf . the nitrile may subsequently be hydrolysed to the carboxylic acid or converted to the thioamide with h 2 s ( see j . agric . food chem , 1975 , 23 , 392 and references cited therein ) the core structures are all well characterised — routes are summarised in science of synthesis volume 13 chapters 7 , 11 , 27 , thieme , stuttgart 2004 . this can provide access to the 4 halo derivatives . nitration or chlorosulfonylation then provides the key intermediates — procedures for these are also documented in this volume 4 - bromo - 7 - nitro - 2 , 1 , 3 - benzoxadiazole / 4 - bromo - 7 - chlorsulfonyl - 2 , 1 , 3 - benzoxadiazole may be prepared by nitration / chlorosulfonylation of the known parent 4 - halobenzoxadiazoles ( j . chem soc b 1971 , 2209 and references cited therein ). the benzoselenadiazole and benzothiadiazole series may be generated in a similar fashion from the corresponding 4 - halo precursors ( for preparation of these see j . mater . chem ., 2005 , 15 , 2865 ; synth commun , 1992 , 22 , 73 , j . heterocyclic chem . 1970 , 7 , 629 and references cited therein ). 4 - chloro - 7 - formylbenzoxadiazoles may be prepared by a multi - step sequence involving formation of 6 - chloranthanil ( tetrahedron supp 1966 , 49 ), nitration and thermal rearrangement to afford formylbenzofurazan oxide ( j . org . chem ., 1980 , 45 , 1653 ; 1977 , 42 , 897 ; 1970 , 35 , 1662 ) and subsequent reduction with triphenyl phosphine or tributylphosphine . all reactions were carried out under an argon atmosphere in glassware dried under high vacuum by a heat - gun unless otherwise stated . 40 - 60 pet . ether refers to the fraction of petroleum ether boiling between 40 and 60 ° c . and was redistilled before use . ether refers to diethyl ether . solvents were distilled from the following reagents under nitrogen atmosphere : ether and thf ( sodium benzophenone ketyl ); dcm , xylene and benzene ( calcium hydride ); chloroform ( phosphorus pentoxide ) and methanol ( sodium methoxide ) or obtained from innovative technology solvent purification system . in cases where mixtures of solvents were utilised , the ratios refer to the volumes used . reagents were used as supplied unless otherwise stated . lithium bromide was made anhydrous by heating at 100 ° c . at 0 . 06 mmhg for 3 h . magnesium bromide was synthesised by addition of 1 , 2 - dibromoethane to an equivalent amount of magnesium in ether . aldehydes and dienes were distilled , immediately prior to use , from anhydrous calcium sulphate and sodium borohydride , respectively . flash chromatography was carried out using silica gel 40 - 63μ . analytical thin layer chromatography ( tlc ) was performed using precoated glass - backed plates ( silica gel 60 f 254 ) and visualised by uv radiation at 254 nm , or by staining with phosphomolybdic acid in ethanol or potassium permanganate in water . all melting points were determined using a gallenkamp melting point apparatus and are uncorrected . gas chromatography was carried out on a hewlett - packard 5890 series ii fitted with a 25 m column . detection was by flame ionisation . infrared spectra were recorded using a diamond atr ( attenuated total reflection ) accessory ( golden gate ) or as a solution in chloroform via transmission ir cells on a perkin - elmer ft - ir 1600 spectrometer . 1 h nmr spectra were recorded in cdcl 3 on varian mercury 200 , varian unity - 300 , varian vxr - 400 or varian inova - 500 instruments and are reported as follows ; chemical shift δ ( ppm ) ( number of protons , multiplicity , coupling constant j ( hz ), assignment ). residual protic solvent chcl 3 ( δ h = 7 . 26 ) was used as the internal reference . 13 c nmr spectra were recorded at 63 mhz or 126 mhz , using the central resonance of cdcl 3 ( δ c = 77 . 0 ppm ) as the internal reference . all chemical shifts are quoted in parts per million relative to tetramethylsilane ( δ h = 0 . 00 ppm ) and coupling constants are given in hertz to the nearest 0 . 5 hz . assignment of spectra was carried out using cosy , hsqc , hmbc and noesy experiments . gas chromatography - mass spectra ( ei ) were obtained using a thermo trace mass spectrometer . electrospray mass spectra ( es ) were obtained on a micromass lct mass spectrometer . high resolution mass spectra were obtained using a thermo ltq mass spectrometer ( es ) at the university of durham , or performed by the epsrc national mass spectrometry service centre , university of wales , swansea . a solution of 2 , 6 - dibromoaniline ( 1 . 0 g , 4 . 0 mmol ) in chcl 3 ( 8 ml ) was treated with a suspension of m - cpba ( 2 . 1 g , 12 . 0 mmol ) in chcl 3 ( 8 ml ) and the resulting mixture stirred overnight . after 24 h the solution was diluted with chcl 3 and washed successively with sat . aq . na 2 s 2 o 3 , sat . aq . nahco 3 and brine . the organic layers were dried over na 2 so 4 , filtered , concentrated and dried in vacuo to afford 1 , 3 - dibromo - 2 - nitrosobenzene ( 1 . 0 g , 100 %); the resultant solid was used directly in the next stage . 1 , 3 - dibromo - 2 - nitrosobenzene ( 1 . 0 g , 3 . 8 mmol ) was suspended in dmso ( 15 ml ) and treated with a solution of sodium azide ( 0 . 3 g , 4 . 2 mmol ) in dmso ( 15 ml ) at room temperature . the resultant solution was stirred for 2 h , until nitrogen evolution had ceased , then was warmed to 120 ° c . for 5 mins . after cooling to room temperature the solution was poured onto crushed ice and the resulting precipitate was filtered and dried in vacuo to afford 4 - bromobenzo [ c ][ 1 , 2 , 5 ] oxadiazole ( 0 . 7 g , 93 %); v h ( 400 mhz , cdcl 3 ) 7 . 83 ( 1h , d , j9 , ar — h ), 7 . 64 ( 1h , d , j7 , ar — h ), 7 . 30 ( 1h , dd , j9 , 7 , ar — h ); the resultant solid ( compound 34 ) was used directly in the next stage . 4 - bromobenzo [ c ][ 1 , 2 , 5 ] oxadiazole ( 0 . 7 g , 3 . 5 mmol ) was redissolved in h 2 so 4 ( 5 ml ) and treated dropwise with a solution of kno 3 ( 0 . 5 g , 4 . 7 mmol ) in 50 % h 2 so 4 ( 3 ml ). the resulting solution was heated to 85 ° c . for 30 mins and then poured onto crushed ice . the aqueous material was then extracted with etoac ( 3 × 15 ml ), dried over na 2 so 4 , filtered , concentrated and dried in vacuo . flash chromatography ( n - hexane , n - hexane / etoac [ 9 : 1 ], [ 4 : 1 ]) afforded the title compound as a tan solid ( 0 . 47 g , 49 %); r f 0 . 5 ( n - hexane / etoac 4 : 1 ); m . p . 92 - 94 ° c . ; v max ( thin film ) 1509 , 1443 , 1322 , 1042 , 997 , 933 , 858 , 801 , 728 , 604 cm − 1 ; δ h ( 400 mhz , cdcl 3 ) 8 . 38 ( 1h , d , j 8 , ar — h ), 8 . 67 ( 1h , d , j 8 , ar — h ); δ c ( 126 mhz , cdcl 3 ) 150 . 5 ( c ═ n ), 142 . 3 ( c ═ n ), 136 . 2 ( ipso - ar — c ), 132 . 1 ( ar — c ), 130 . 4 ( ar — c ), 119 . 1 ( ipso - ar — c ); m / z ( ei ) 245 ([ 81 br ] mh + , 2 %), 243 ([ 79 br ] mh + ), 215 ( 2 %), 213 ( 2 %), 185 ( 6 %); hrms ( ei ) found m + , 242 . 9271 ( c 6 h 2 79 brn 3 o 3 requires 242 . 9274 ). nbd - cl ( 0 . 4 g , 2 . 0 mmol ) was dissolved in ethanol : 0 . 1m sodium phosphate buffer ( 1 : 1 v / v , 40 ml ). then sodium methanethiolate ( 0 . 17 g , 2 . 4 mmol ) was added and the reaction was stirred for 3 h after which time the resultant precipitate was filtered . the precipitate was then subjected to flash chromatography ( chcl 3 ) to afford the title compound as a orange solid ( 0 . 33 g , 77 %); r f 0 . 4 ( chcl 3 ); m . p . 118 - 120 ° c . ; v max ( thin film ) 1497 , 1418 , 1308 , 1280 , 1122 , 1044 , 957 , 847 , 733 cm − 1 ; δ h ( 700 mhz , cdcl 3 ) 8 . 43 ( 1h , d , j 8 , ar — h ), 7 . 10 ( 1h , d , j 8 , ar — h ), 2 . 77 ( 3h , s , ar — sch 3 ); δ c ( 176 mhz , cdcl 3 ) 149 . 0 ( ipso - ar — c ), 142 . 5 ( c ═ n ), 142 . 4 ( c ═ n ), 132 . 7 ( ipso - ar — c ), 130 . 6 ( ar — c ), 119 . 5 ( ar — c ), 14 . 7 ( ar — sch 3 ); m / z ( ei ) 211 ( m + ); hrms ( es + ) found mnh 4 + , 229 . 0387 ( c 7 h 9 n 4 o 3 s requires 229 . 0390 ). nbd - cl ( 0 . 2 g , 1 . 0 mmol ) was dissolved in ethanol : 0 . 1m sodium phosphate buffer ( 1 : 1 v / v , 20 ml ). then 4 -( trifluoromethyl ) thiophenol ( 0 . 17 ml , 1 . 2 mmol ) was added and the reaction was stirred for 2 h , after which time the resultant precipitate was filtered . the precipitate was then washed with water and ethanol to afford the title compound as a yellow solid ( 0 . 20 g , 57 %); m . p . 134 - 138 ° c . ; v . ( thin film ) 1509 , 1315 , 1169 , 1130 , 1099 , 1050 , 952 , 835 cm − 1 ; δ h ( 500 mhz , cdcl 3 ) 8 . 30 ( 1h , d , j 8 , ar — h ), 7 . 85 ( 4h , s , ar — h ), 6 . 76 ( 1h , d , j 8 , ar — h ); δ c ( 126 mhz , cdcl 3 ) 148 . 5 ( c ═ n ), 142 . 5 ( c ═ n ), 140 / 4 ( ipso - ar — c ), 135 . 8 ( ipso - ar — c ), 133 . 3 ( ar — cf 3 , m ), 131 . 2 ( ipso - ar — c ), 130 . 4 ( ar — c ), 127 . 5 ( 4ar — c ), 124 . 5 ( ipso - ar — c ), 122 . 7 ( ar — c ); δ f ( 188 mhz , cdcl 3 )- 63 . 5 ( 3f , s , ar — cf 3 ); m / z ( ei ) 341 ( m + , 100 %), 322 ( 20 %), 272 ( 50 %), 265 ( 40 %), 253 ( 45 %), 242 ( 43 %), 195 ( 70 %), 176 ( 25 %), 157 ( 35 %), 145 ( 40 %), 119 ( 35 %); hrms ( ei ) found m + , 341 . 0080 ( c 13 h 6 f 3 n 3 o 3 s requires 341 . 0076 ) nbd - cl ( 0 . 2 g , 1 . 0 mmol ) was dissolved in ethanol : 0 . 1m sodium phosphate buffer ( 1 : 1 v / v , 20 ml ). then morpholine ( 0 . 1 ml , 1 . 2 mmol ) was added and the reaction was stirred for 2 h , after which time the resultant precipitate was filtered . the precipitate was then washed with water and ethanol to afford the title compound as a red solid ( 0 . 22 g , 90 %); m . p . sublimes ° c . ; v . ( thin film ) 2368 , 2342 , 2240 , 1601 , 1548 , 1482 , 1438 , 1292 , 1259 , 1211 , 1163 , 1115 , 1035 , 993 cm − 1 ; δ h ( 500 mhz , cdcl 3 ) 8 . 45 ( 1h , d , j 9 , ar — h ), 6 . 33 ( 1h , d , j 9 , ar — h ), 4 . 08 ( 4h , m , ( ch 2 ) 2 ), 3 . 96 ( 4h , m , ( ch 2 ) 2 ); δ c ( 126 mhz , cdcl 3 ) 145 . 2 ( ipso - c ), 144 . 9 ( c ═ n ), 144 . 7 ( c ═ n ), 134 . 9 ( ar — c ), 102 . 6 ( ar — c ), 66 . 3 (( ch 2 ) 2 ), 49 . 4 (( ch 2 ) 2 ); m / z ( es + ) 251 ( mh + ), 523 ( 2mna + ); hrms ( es + ) found mh + , 251 . 0775 ( c 10 h 11 n 4 o 4 requires 251 . 0775 ). nbd - cl ( 0 . 4 g , 2 . 0 mmol ) was dissolved in ethanol : 0 . 1m sodium phosphate buffer ( 1 : 1 v / v , 40 ml ). then ethylene glycol ( 0 . 1 ml , 2 . 2 mmol ) was added and the ph adjusted to 7 with 1m aq . naoh . the reaction was stirred for 3 h then mixed with sat . aq . nh 4 cl ( 10 ml ) and extracted with etoac ( 3 × 15 ml ). the organic layers were dried over mgso 4 , filtered , concentrated and dried in vacuo . flash chromatography ( n - hexane / etoac [ 9 : 1 ], [ 4 : 1 ], [ 7 : 3 ]) afforded the title compound as a light brown solid ( 0 . 01 g , 2 %); r f 0 . 6 ( n - hexane / etoac 1 : 1 ); δ h ( 400 mhz , cdcl 3 ) 8 . 53 ( 1h , d , j 8 , ar — h ), 6 . 67 ( 1h , d , j 8 , ar — h ), 4 . 47 ( 2h , q , j 7 , och 2 ch 3 ), 1 . 63 ( 3h , t , j 7 , och 2 ch 3 ); δ c ( 126 mhz , cdcl 3 ) 154 . 8 ( ipso - ar — c ), 145 . 2 ( c ═ n ), 143 . 9 ( c ═ n ), 134 . 2 ( ar — c ), 129 . 5 ( ipso - ar — c ), 104 . 3 ( ar — c ), 67 . 1 ( och 2 ch 3 ), 14 . 2 ( och 2 ch 3 ); m / z ( es + ) 232 ( mna + ), 264 ( mna + meoh + ). 4 - chloro - 7 - nitrobenzo [ c ][ 1 , 2 , 5 ] oxadiazole ( nbd - c1 , 1 . 0 g , 5 . 0 mmol ) was dissolved in meoh ( 25 ml ) and added dropwise at room temperature via cannula to a freshly made 1n solution of naome ( 0 . 23 g na in 10 ml meoh ). the solution was stirred for 2 h then treated carefully with sat aq . nh 4 cl . the mixture was then extracted with et 2 o ( 3 × 20 ml ). the organic layers were dried over mgso 4 , filtered , concentrated and dried in vacuo to afford 4 - methoxy - 7 - nitrobenzo [ c ][ 1 , 2 , 5 ] oxadiazole as an off white solid ( 0 . 76 g , 66 %); δ h ( 400 mhz , cdcl 3 ) 8 . 56 ( 1h , d , j 8 , ar — h ), 6 . 68 ( 1h , d , j 8 , ar — h ), 4 . 24 ( 3h , s , och 3 ); the resultant solid was used directly in the next stage . 45 4 - methoxy - 7 - nitrobenzo [ c ][ 1 , 2 , 5 ] oxadiazole ( 0 . 76 g , 3 . 9 mmol ) ( compound 34 ) was added to hot 1 % naoh ( 10 ml ). the resultant solution was refluxed for 30 min then cooled to room temperature . the aqueous material was then acidified with c . hcl and extracted with et 2 o ( 3 × 20 ml ). the organic layers were dried over mgso 4 , filtered , concentrated and dried in vacuo to afford the title compound as a brown solid ( 0 . 5 g , 71 %); r f 0 . 4 ( dcm / meoh 4 : 1 ); v . ( thin film ) 3260 - 2788 ( broad - oh ), 1638 , 1560 , 1518 , 1310 , 1084 , 1002 , 913 , 875 , 840 cm − 1 ; δ h ( 200 mhz , cd 3 od ) 8 . 60 ( 1h , d , j 8 , 6 - h ), 6 . 73 ( 1h , d , j 8 , 5 - h ). δ c ( 176 mhz , cdcl 3 ) 156 . 4 ( c ═ no ), 146 . 9 ( ipso - ar — c ), 145 . 7 ( ipso - ar — c ), 136 . 8 ( ar — c ), 108 . 7 ( ar — c ); m / z ( ei ) 181 ( m + , 45 %), 121 ( 25 %), 99 ( 40 %), 80 ( 80 %), 76 ( 100 %), 75 ( 65 %), 71 ( 50 %), 64 ( 75 %), 52 ( 85 %); hrms ( ei ) found m + , 181 . 0119 ( c 6 h 3 n 3 o 4 requires 181 . 0118 ); elemental analysis [ found c , 40 . 23 %; h , 1 . 75 %; n , 23 . 01 % required for c 6 h 3 n 3 o 4 : c , 39 . 79 %; h , 1 . 67 %; n , 23 . 20 %]; all data agree with those reported in the literature . 45 nbd - c1 ( 0 . 2 g , 1 . 0 mmol ) was dissolved in ethanol : 0 . 1m sodium phosphate buffer ( 1 : 1 v / v , 20 ml ). then 3 - mercapto - 1 - propionic acid ( 0 . 1 ml , 1 . 1 mmol ) was added and the ph adjusted to 7 with 1m aq . naoh . the reaction was stirred for 6 h after which time the resultant precipitate was filtered and washed with water ( 2 × 15 ml ) and dried to afford the title compound as a light brown solid ( 0 . 16 g , 61 %); δ h ( 700 mhz , ( cd 3 ) 2 c0 ) 8 . 58 ( 1h , d , j 8 , ar — h ), 7 . 63 ( 1h , d j 8 , ar — h ), 3 . 64 ( 2h , t , j 7 sch 2 ch 2 cooh ), 2 . 93 ( 2h , t , j 7 , sch 2 ch 2 cooh ); δ c ( 175 mhz , ( cd 3 ) 2 c0 ) 171 . 7 ( c ═ o ), 149 . 8 ( c ═ n ), 143 . 2 ( c ═ n ), 139 . 9 ( ipso - ar — c ), 133 . 3 ( ipso - ar — c ), 131 . 9 ( ar — c ), 122 . 2 ( ar — c ), 32 . 3 ( sch 2 ch 2 cooh ), 26 . 4 ( sch 2 ch 2 cooh ); m / z ( es ) 196 ( m − - co 2 h , — ch 2 ch 2 ), 268 ( m ), 536 ( 2m − ). nbd - cl ( 0 . 4 g , 2 . 0 mmol ) was dissolved in ethanol : 0 . 1m sodium phosphate buffer ( 1 : 1 v / v , 40 ml ). then 6 - mercaptohexan - 1 - ol ( 0 . 3 ml , 2 . 2 mmol ) was added and the ph adjusted to 7 with 1m aq . naoh . the reaction was stirred for 3 h after which time the resultant precipitate was filtered and washed with water ( 2 × 15 ml ) and dried to afford the title compound as a light brown solid ( 0 . 44 g , 74 %); δ h ( 400 mhz , cdcl 3 ) 8 . 40 ( 1h , d , j 8 , ar — h ), 7 . 14 ( 1h , d , j 8 , ar — h ), 3 . 67 ( 2h , t , j 6 , ch 2 oh ), 3 . 28 ( 2h , t , j7 , ch 2 s ), 1 . 88 ( 2h , q , j7 , ch 2 ch 2 ch 2 ), 1 . 63 - 1 . 45 ( 6h , m , ( ch 2 ) 3 ); δ c ( 126 mhz , cdcl 3 ) 149 . 2 ( ipso - ar — c ), 142 . 5 ( c ═ n ), 141 . 9 ( c ═ n ), 130 . 6 ( ar — c ), 120 . 2 ( ar — c ), 62 . 7 (( ch 2 ) 6 ), 32 . 4 (( ch 2 ) 6 ), 31 . 7 (( ch 2 ) 6 ), 28 . 6 (( ch 2 ) 6 ), 27 . 8 (( ch 2 ) 6 ), 25 . 3 (( ch 2 ) 6 ); m / z ( es + ) 320 ( mna + ); following the standard procedure outlined hereinabove , 7bromobenzo [ c ][ 1 , 2 , 5 ] thiadiazole - 4 - carboxylic acid ( 0 . 06 g , 0 . 23 mmol ) was transformed into the title compound which was isolated as a white solid ( 0 . 008 g ); r f 0 . 5 ( n - hexane / etoac 7 : 3 ); m . p . 110 - 115 ° c . ; vmax ( thin film ) 3350 ( nh ), 2955 , 2920 , 2869 , 1636 ( c ═ o ), 1520 , 1477 , 1305 , 1261 , 1190 , 886 cm − 1 ; δh ( 700 mhz , cdcl 3 ) 8 . 97 ( 1h , bs , nh ), 8 . 49 ( 1h , d , j 8 , ar — h ), 8 . 00 ( 1h , d , j 8 , ar — h ), 3 . 56 ( 2h , q , j 7 , nhch 2 ch 2 ch 3 ), 1 . 75 ( 2h , sextet , j 7 , nhch 2 ch 2 ch 3 ), 1 . 05 ( 3h , t , j 7 , nhch 2 ch 2 ch 3 ); δc ( 176 mhz , cdcl 3 ) 162 . 6 ( c ═ o ), 153 . 6 ( c ═ n ), 151 . 5 ( c ═ n ), 133 . 5 ( ar — c ), 132 . 3 ( ar — c ), 124 . 6 ( ipso - ar — c ), 118 . 4 ( ipso - ar — c ), 41 . 9 ( nhch 2 ch 2 ch 3 ), 22 . 8 ( nhch 2 ch 2 ch 3 ), 11 . 6 ( nhch 2 ch 2 ch 3 ); m / z ( es + ) 302 ([ 81 br ] mh + ), 300 ([ 79 br ] mh + ), 324 ([ 81 br ] mna + ), 322 ([ 79 br ] mna + ), 625 ([ 81 br ] 2mna + ), 623 ([ 79 br , 81 br ] mna + ), 621 ([ 79 br ] 2mna + ); hrms ( es + ) found mh + , 299 . 98013 ( c 10 h 11 on 3 79 brs requires 299 . 98007 ) following the standard procedure outlined herein , 7 - bromobenzo [ c ][ 1 , 2 , 5 ] thiadiazole - 4 - carboxylic acid ( 0 . 06 g , 0 . 23 mmol ) was transformed into the title compound which was isolated as a white solid ( 0 . 012 g , xx %); r f 0 . 2 ( n - hexane / etoac 7 : 3 ); m . p . 156 - 160 ° c . ; vmax ( thin film ) 3330 ( nh ), 2944 , 2896 , 2854 , 1637 ( c ═ o ), 1555 , 1520 , 1475 , 1303 , 1264 , 1197 , 856 cm − 1 ; δh ( 700 mhz , cdcl 3 ) 8 . 91 ( 1h , bs , nh ), 8 . 49 ( 1h , d , j 8 , ar — h ), 8 . 00 ( 1h , d , j 8 , ar — h ), 3 . 15 ( 3h , d , j 5 , nhch 3 ); δ c ( 176 mhz , cdcl 3 ) 163 . 3 ( c = 0 ), 153 . 5 ( c ═ n ), 151 . 4 ( c ═ n ), 133 . 5 ( ar — c ), 132 . 2 ( ar — c ), 124 . 4 ( ipso - ar — c ), 118 . 5 ( ipso - ar — c ), 26 . 8 ( nhch 3 ); m / z ( es + ) 274 ([ 81 br ] mh + ), 272 ([ 79 br ] mh + ), 296 ([ 81 br ] mna + ), 294 ([ 79 br ] mna + ); hrms ( es + ) found mh + , 271 . 94879 ( c 8 h 7 79 brn 3 os requires 271 . 94877 ) following the standard procedure outlined herein , 7 - bromobenzo [ c ][ 1 , 2 , 5 ] thiadiazole - 4 - carboxylic acid ( 0 . 06 g , 0 . 23 mmol ) was transformed into the title compound which was isolated as a colourless oil ( 0 . 006 g ); r f 0 . 2 ( n - hexane / etoac 7 : 3 ); vmax ( thin film ) 2922 , 2856 , 2809 , 1629 ( c ═ o ), 1532 , 1396 , 1141 , 876 , 841 cm − 1 ; δh ( 700 mhz , cdcl 3 ) 7 . 90 ( 1h , d , j 7 , ar — h ), 7 . 54 ( 1h , d , j 7 , ar — h ), 3 . 24 ( 3h , s , n ( ch 3 ) 2 ), 2 . 89 ( 3h , s , n ( ch 3 ) 2 ); δc ( 176 mhz , cdcl 3 ) 166 . 9 ( c ═ o ), 153 . 2 ( c ═ n ), 151 . 1 ( c ═ n ), 131 . 8 ( ar — c ), 129 . 6 ( ipso - ar — c ), 128 . 6 ( ar — c ), 115 . 6 ( ipso - ar — c ), 38 . 9 ( n ( ch 3 ) 2 ), 35 . 2 ( n ( ch 3 ) 2 ); m / z ( es + ) 287 . 9 ([ 81 br ] mh + ), 285 . 9 ([ 79 br ] mh + ), 309 . 9 ([ 81 br ] mna + ), 307 . 9 ([ 79 br ] mna + ), 596 . 8 ([ 81 br ] 2mna + ), 594 . 8 ([ 81 . 79 br ] 2mna + ), 592 . 8 ([ 79 br ] 2mna + ); hrms ( es + ) found mna + , 307 . 94640 ( c 9 h 8 brn 3 naos requires 307 . 94637 ) following the standard procedure outlined herein , 7 - bromobenzo [ c ][ 1 , 2 , 5 ] thiadiazole - 4 - carboxylic acid ( 0 . 06 g , 0 . 23 mmol ) was transformed into the title compound which was isolated as a white solid ( 0 . 011 g ); r f 0 . 7 ( n - hexane / etoac 7 : 3 ); m . p . 122 - 126 ° c . ; vmax ( thin film ) 2956 , 2928 , 2848 , 1708 ( c = 0 ), 1523 , 1329 , 1303 , 1260 , 1194 , 1165 , 939 , 891 , 853 cm 1 ; δh ( 700 mhz , cdcl 3 ) 8 . 25 ( 1h , d , j 8 , ar — h ), 7 . 95 ( 1h , d , j 8 , ar — h ), 4 . 07 ( 3h , s , — cooch 3 ); δc ( 176 mhz , cdcl 3 ) 164 . 6 ( c ═ o ), 154 . 1 ( c ═ n ), 151 . 5 ( c ═ n ), 133 . 8 ( ar — c ), 131 . 2 ( ar — c ), 122 . 5 ( ipso - ar — c ), 120 . 5 ( ipso - ar — c ), 52 . 8 ( cooch 3 ); m / z ( es + ) 275 ([ 81 br ] m + ), 273 ([ 79 br ] m + ), 297 ([ 81 br ] mna + ), 295 ([ 79 br ] mna + ), 571 ([ 81 br ] 2mna + ), 569 ([ 81 br , 79 br ] 2mna + ), 567 ([ 79 br ] 2mna + ); hrms ( es + ) found mh + , 272 . 93264 ( c 8 h 6 79 brn 2 o 2 s requires 272 . 93279 ) 4 , 7 - dibromobenzo [ c ][ 1 , 2 , 5 ] thiadiazole ( 0 . 25 g , 0 . 85 mmol ), 4 -( methoxycarbonyl ) phenylboronic acid ( 0 . 15 g , 0 . 85 mmol ), pd ( pph 3 ) 4 ( 0 . 014 g , 0 . 01 mmol ) and na 2 co 3 ( 0 . 09 g , 0 . 85 mmol ) were weighed into a round bottom flask and dissolved with toluene ( 1 ml ), thf ( 1 ml ) and h 2 o ( 0 . 2 ml ). the solution was then refluxed for 24 h , cooled and poured in to h 2 o . the aqueous layer was extracted with et 2 o ( 3 × 15 ml ). the organic layers were dried over mgso 4 , filtered , concentrated and dried in vacuo . flash chromatography ( n - hexane / dcm [ 4 : 1 ], [ 6 : 4 ], [ 4 : 6 ]) afforded the title compound as a pale yellow solid ( 0 . 08 g , 26 %); r f 0 . 3 ( n - hexane / dcm 6 : 4 ); m . p . 188 - 192 ° c . ; vmax ( thin film ) 1732 ( c ═ o ), 1608 , 1480 , 1430 , 1317 , 1274 , 1185 , 1151 , 1110 , 944 , 881 , 830 , 765 , 700 cm − 1 ; δh ( 500 mhz , cdcl 3 ) 8 . 20 ( 1h , d , j 8 , ar — h ), 7 . 98 ( 1h , d , j 8 , ar — h ), 7 . 96 ( 1h , d , j 8 , ar — h ), 7 . 64 ( 1h , d , j 8 , ar — h ), 3 . 97 ( 3h , s , och 3 ); δc ( 126 mhz , cdcl 3 ) 166 . 7 ( c ═ o ), 153 . 8 ( ipso - ar — c ), 152 . 8 ( ipso - ar — c ), 140 . 9 ( ipso - ar — c ), 132 . 8 ( ipso - ar — c ), 132 . 2 ( ar — c ), 130 . 1 ( ipso - ar — c ), 129 . 9 ( ar — c ), 129 . 1 ( ar — c ), 128 . 8 ( ar — c ), 114 . 2 ( ipso - ar — c ), 52 . 3 ( och 3 ); m / z ( ei ) 350 ([ 81 br ] m + , 100 %), 348 ([ 79 br ] m + , 90 %), 319 ([ 81 br ] m + - och 3 , 90 %), 317 ([ 79 br ] m + - och 3 , 80 %), 291 ([ 81 br ] m + - och 3 , — c ═ o , 40 %), 289 ([ 79 br ] m + - och 3 , — c ═ o , 35 %), 209 ( 70 %); hrms ( ei ) found [ 79 br ] m + , 347 . 9560 ( c 14 h 9 79 brn 2 o 2 s requires 347 . 9563 ) further elution of the column gave 4 , 7 - dibromobenzo [ c ][ 1 , 2 , 5 ] thiadiazole ( 0 . 1 g , 50 %) and the title compound as a mixture with the di - coupled product ( 0 . 05 g , 25 %). 4 , 7 - dibromobenzo [ c ][ 1 , 2 , 5 ] thiadiazole ( 1 . 0 g , 3 . 4 mmol ) was suspended in 70 % hno 3 ( 6 ml ). the reaction was then heated to reflux for 2 h until a solution had formed . the reaction mixture was then poured onto ice , warmed back to room temperature and the precipitate that has formed was filtered . flash chromatography of the solid material ( chcl 3 ) afforded the title compound as a pale yellow solid ( 0 . 06 g , 7 %); r f 0 . 3 ( chcl 3 ); m . p . 216 - 218 ° c . ; vmax ( thin film ) 3051 , 1502 , 1342 , 1314 , 1195 , 1001 , 939 , 653 , 815 , 730 , 571 cm − 1 ; δh ( 700 mhz , cdcl 3 ) 8 . 48 ( 1h , d , j 7 , ar — h ), 8 . 04 ( 1h , d , j 8 , ar — h ); δc ( 176 mhz , cdcl 3 ) 154 . 6 ( c ═ n ), 145 . 8 ( c ═ n ), 139 . 0 ( ipso - ar — c ), 130 . 4 ( ar — c ), 127 . 7 ( ar — c ), 123 . 1 ( ipso - ar — c ); m / z ( ei ) 261 ([ 81 br ] mh + , 70 %), 259 ([ 79 br ] mh + , 65 %), 231 ( 100 %), 229 ( 85 %), 203 ( 45 %), 201 ( 45 %); hrms ( ei ) found m + , 258 . 9048 ( c 6 h 2 79 brn 3 o 2 s requires 258 . 9046 ). 4 , 7 - dibromobenzo [ c ][ 1 , 2 , 5 ] selenadiazole ( 0 . 5 g , 1 . 5 mmol ) was suspended in 70 % hno 3 ( 6 ml ) and h 2 o ( 2 ml ). the reaction was then heated to reflux for 5 h , cooled to room temperature and filtered . flash chromatography of the solid material ( chcl 3 ) afforded the title compound as a pale yellow solid ( 0 . 01 g , 3 %); r f 0 . 4 ( chcl 3 ); m . p . & gt ; 300 ° c . ; vmax ( thin film ) 1508 , 1471 , 1321 , 1257 , 1090 , 992 , 920 , 855 , 814 , 766 , 727 cm − 1 ; δh ( 400 mhz , cdcl 3 ) 8 . 36 ( 1h , d , j 8 , ar — h ), 7 . 96 ( 1h , d , j 8 , ar — h ); 4 - bromo - 7 - methylbenzo [ c ][ 1 , 2 , 5 ] thiadiazole ( 0 . 25 g , 1 . 1 mmol ) was dissolved in acoh ( 12 ml ) and h 2 so 4 ( 1 . 7 ml ). [ caution : exothermic ]. the solution was then treated with chromium ( vi ) oxide ( 1 . 5 g ) portionwise and stirred for 30 mins . the reaction was then poured onto ice and allowed to warm to room temperature . the aqueous layer was then extracted with dcm ( 3 × 30 ml ). the aqueous layer was left overnight and the precipitate filtered and washed with water ( 3 × 2 ml ) to afford the title compound as a white solid ( 0 . 085 g , 30 %); vmax ( thin film ) 3100 - 3300 ( broad — oh ), 1682 ( c ═ o ), 1680 , 1525 , 1312 , 1289 , 1193 cm − 1 ; δh ( 500 mhz , ( cd 3 ) 2 c0 ) 8 . 31 ( 1h , d , j 8 , ar — h ), 8 . 15 ( 1h , d , j 8 , ar — h ), 2 . 09 ( 1h , s , co 2 h ); δc ( 126 mhz , ( cd 3 ) 2 c0 ) 165 . 1 ( c ═ o ), 154 . 8 ( c ═ n ), 152 . 6 ( c ═ n ), 134 . 7 ( ar — c ), 132 . 6 ( ar — c ), 124 . 2 ( ipso - ar — c ), 120 . 1 ( ipso - ar — c ); m / z ( es ) 259 ([ 81 br ] m ), 257 ([ 79 br ] m ); hrms ( es − ) found m − , 256 . 90248 ( c 7 h 2 79 brn 2 o 2 s requires 256 . 90258 ). diazole carboxylic acid ( 0 . 23 mmol ) and a large excess of thionyl chloride were mixed and heated to 55 ° c . for 1 h . after cooling the solution was mixed with toluene and evaporated to dryness . the residue was then suspended in chloroform ( 4 ml ) and treated with a large excess of amine , alcohol or thiol . the solution was stirred for 1 h and then evaporated to dryness . the residue was then subjected to flash chromatography ( n - hexane / etoac 4 : 1 , 7 : 3 , 1 : 1 ) to afford the desired carboxamides . 4 , 7 - dibromobenzo [ c ][ 1 , 2 , 5 ] thiadiazole ( 0 . 25 g , 0 . 85 mmol ) and pd ( oac ) 2 ( 0 . 004 g , 0 . 017 mmol , 2 mol %) were dissolved in toluene ( 10 ml ) and treated successively with butyl acrylate ( 0 . 12 ml , 0 . 85 mmol ), dipea ( 0 . 33 ml , 1 . 87 mmol ) and pph 3 ( 0 . 004 g , 0 . 017 mmol , 2 mol %). the resulting solution was then refluxed for 24 h , cooled and poured in to h 2 o . the aqueous layer was extracted with et 2 o ( 3 × 15 ml ). the organic layers were dried over mgso 4 , filtered , concentrated and dried in vacuo . flash chromatography ( n - hexane / dcm [ 1 : 1 ], [ 3 : 7 ], [ 1 : 9 ], dcm ) afforded the title compound as a dark green solid ( 0 . 05 g , 15 %); r f 0 . 2 ( n - hexane / dcm 3 : 7 ); m . p . 90 - 94 ° c . ; vmax ( thin film ) 2959 , 2932 , 2872 , 1706 ( c ═ o ), 1631 ( c ═ c ), 1564 , 1537 , 1392 , 1308 , 1227 , 1162 , 983 , 841 cm − 1 ; δh ( 200 mhz , cdcl 3 ) 8 . 02 ( 2h , d , j 16 , ch ═ ch ), 7 . 73 ( 2h , s , ar — h ), 7 . 52 ( 2h , d , j 16 , ch ═ ch ), 4 . 27 ( 2h , d , och 2 ch 2 ch 2 ch 3 ), 1 . 80 - 1 . 73 ( 2h , m , och 2 ch 2 ch 2 ch 3 ), 1 . 54 - 1 . 40 ( 2h , m , och 2 ch 2 ch 2 ch 3 ), 0 . 98 ( 3h , m , och 2 ch 2 ch 2 ch 3 ); δc ( 175 mhz , cdcl 3 ) 167 . 1 ( c ═ o ), 153 . 6 ( q - c ), 139 . 3 ( c ═ c ), 131 . 1 ( ar — c ), 129 . 1 ( q - c ), 124 . 6 ( c ═ c ), 64 . 7 ( och 2 ch 2 ch 2 ch 3 ), 30 . 8 ( och 2 ch 2 ch 2 ch 3 ), 19 . 2 ( och 2 ch 2 ch 2 ch 3 ), 13 . 7 ( och 2 ch 2 ch 2 ch 3 ); m / z ( ei ) 332 ( m + - bu , 25 %), 259 ( m + - bu , - buo , 100 %), 249 ( 80 %). 3 - methyl - 1 , 2 - phenylenediamine ( 2 . 0 g , 16 . 4 mmol ) was dissolved in toluene ( 40 ml ), treated with selenium oxychloride ( 1 . 1 ml , 16 . 4 mmol ) dropwise and refluxed for 12 h . once cooled to room temperature the solvent was removed and the residue subjected to flash chromatography ( n - hexane , n - hexane / etoac 9 : 1 , 4 : 1 , 7 : 3 ) to afford the title compound as a light brown solid ( 1 . 42 g , 53 %); r f 0 . 7 ( n - hexane / etoac 7 : 3 ); m . p . 90 - 94 ° c . ; vmax ( thin film ) 1533 , 1378 , 1071 , 1012 , 859 , 839 , 794 , 741 , 705 cm − 1 ; δh ( 700 mhz , cdcl 3 ) 7 . 65 ( 1h , d , j 9 , ar — h ), 7 . 35 ( 1h , dd , j 9 , 6 , ar — h ), 7 . 18 ( 2h , d , j 6 , ar — h ), 2 . 68 ( 3h , s , ar — ch 3 ); δc ( 175 mhz , cdcl 3 ) 161 . 4 ( c ═ n ), 161 . 0 ( c ═ n ), 133 . 3 ( ipso - ar — c ), 130 . 1 ( ar — c ), 128 . 0 ( ar — c ), 121 . 4 ( ar — c ), 18 . 4 ( ar — ch 3 ); m / z ( ei ) 198 ([ 79 se ] m + , 90 %), 196 ([ 77 se ] m + , 50 %), 170 ( 30 %), 117 ( m - 79 se , 45 %), 91 ( 60 %), 80 ( 50 %), 64 ( 55 %), 39 ( 100 %); hrms ( ei ) found m + , 197 . 9691 ( c 7 h 6 n 2 79 se requires 197 . 9691 ) 4 - bromo - 7 - methylbenzo [ c ][ 1 , 2 , 5 ] thiadiazole ( 0 . 5 g , 2 . 2 mmol ) was suspended in methanol ( 7 ml ) and warmed to 45 ° c . ( internal temperature ). the suspension was then treated with magnesium turnings ( 0 . 42 g , 17 . 6 mmol ) and stirred for 30 mins . the reaction was then cooled and the methanol removed in vacuo . the residue was then mixed with aq . nh 4 cl and extracted with etoac ( 3 × 30 ml ). the organic layers were dried over mgso 4 , filtered , concentrated and dried in vacuo to afford 3 - bromo - 6 - methylbenzene - 1 , 2 - diamine ( 0 . 39 g , 94 %); δh ( 400 mhz , cdcl 3 ) 6 . 88 ( 1h , d , j 8 , ar — h ), 6 . 47 ( 1h , d , j 8 , ar — h ), 3 . 60 ( 4h , bs , ar — nh 2 ), 2 . 16 ( 3h , s , arch 3 ); m / z ( ei ) 202 ([ 81 br ] m + , 95 %), 200 ([ 79 br ] m + , 100 %), 120 ( m + − br , 75 %); the resultant solid was used directly in the next stage . 46 3 - bromo - 6 - methylbenzene - 1 , 2 - diamine ( 0 . 39 g , 2 . 1 mmol ) was dissolved in toluene ( 10 ml ) and treated with selenium oxychloride ( 0 . 14 ml , 2 . 1 mmol ). the resultant suspension was refluxed overnight , cooled and the solvent removed in vacuo . the residue was then subjected to flash chromatography ( chcl 3 ) to afford the title compound as a yellow solid ( 0 . 27 g , 50 %); r f 0 . 4 ( chcl 3 ); m . p . 223 - 227 ° c . ; vmax ( thin film ) 3025 , 2911 , 1595 , 1476 , 1372 , 1071 , 913 , 831 , 755 , 710 , 578 cm − 1 ; δh ( 700 mhz , cdcl 3 ) 7 . 65 ( 1h , d , j7 , ar — h ), 7 . 12 ( 1h , dq , j7 , 1 , ar — h ), 2 . 65 ( 3h , d , j 1 , ar — ch 3 ); δc ( 176 mhz , cdcl 3 ) 160 . 4 ( c ═ n ), 158 . 1 ( c ═ n ), 132 . 9 ( ipso - ar — c ), 132 . 2 ( ar — c ), 128 . 2 ( ar — c ), 114 ( ispo - ar — c ), 18 . 1 ( ar — ch 3 ); m / z ( ei ) 278 ([ 81 br 79 se ] m + , 30 %), 276 ([ 79 br 77 se , 79 br 79 se ] m + , 40 %), 274 ([ 79 br 77 se ] m + , 20 %), 197 ( 40 %), 170 ( 40 %), 117 ( 70 %), 90 ( 100 %); hrms ( ei ) found m + , 275 . 8796 ( c 7 h 5 n 2 81 br 77 se requires 275 . 8796 ) 4 , 7 - dibromobenzo [ c ][ 1 , 2 , 5 ] selenadiazole ( 0 . 25 g , 0 . 74 mmol ) and cucn ( 0 . 07 g , 0 . 74 mmol ) were dissolved in dmf ( 4 ml ) and stirred at reflux for 2 h . the solution was then cooled and poured into nh 4 oh and extracted with toluene ( 3 × 10 ml ). the organic layers were dried over mgso 4 , filtered , concentrated and dried in vacuo to afforded the title compound as a beige solid ( 0 . 04 g , 25 %); r f 0 . 3 ( dcm / n - hexane 6 : 4 ); vmax ( thin film ) 2235 ( c ═ n ), 877 , 765 , 634 cm − 1 ; δh ( 400 mhz , cdcl 3 ) 8 . 04 ( 2h , s , ar — h ); δc ( 126 mhz , cdcl 3 ) 156 . 6 ( c ═ n — se ), 134 . 5 ( ar — c ), 114 . 2 ( cn ), 112 . 4 ( ipso - ar — c ); m / z ( ei ) 234 ([ 79 se ] m + , 100 %), 232 ( 40 %), 230 ([ 76 se ] m + , 25 %); hrms ( ei ) found [ 76 se ] m + , 229 . 9463 ( c 8 h 2 n 4 76 se requires 229 . 9466 ). benzo [ c ][ 1 , 2 , 5 ] oxadiazole ( 2 . 5 g , 21 mmol ) was heated to 90 ° c . with iron powder ( 0 . 23 g , 4 . 2 mmol ). the molten liquid was then treated with bromine ( 3 . 2 ml , 62 mmol ) and refluxed for 2 h . the resultant solution was poured onto ice and the precipitate filtered . the precipitate was then mixed with aq . nahco 3 , stirred for 15 min and filtered . flash chromatography ( n - hexane / etoac 9 : 1 ) afforded the title compound as an orange solid ( 1 . 5 g , 25 %); m . p . 88 - 92 ° c . ; vmax ( thin film ) 1514 , 1344 , 1201 , 1026 , 954 , 871 , 841 cm − 1 ; δh ( 500 mhz , cdcl 3 ) 7 . 51 ( 2h , s , ar — h ); 6c ( 126 mhz , cdcl 3 ) 149 . 6 ( c ═ n ), 134 . 4 ( ar — c ), 108 . 9 ( ipso - ar — c ); m / z ( ei ) 280 ([ 81 , 81 br ] m + , 6 %), 278 ([ 81 , 79 br ] m + , 8 %), 276 ([ 79 , 79 br ] m + , 6 %), 250 ( 5 %), 248 ( 10 %), 246 ( 7 %), 197 ( 5 %); all data agree with those reported in the literature . nbd - cl ( 1 . 0 g , 5 . 0 mmol ) was suspended in ethylene glycol ( 10 ml ) and treated with a solution of naoh ( 0 . 4 g , 10 mmol ) in ethylene glycol ( 20 ml ) at room temperature . the reaction mixture was stirred for 1 h and then acidified with 5 m hcl ( 20 ml ). the resultant aqueous layer was extracted with etoac ( 3 × 20 ml ). the organic layers were dried over mgso 4 , filtered , concentrated and dried in vacuo . flash chromatography ( chcl 3 / acetone [ 9 : 1 ]) afforded the title compound as an orange oil ( 0 . 9 g , 80 %); δh ( 400 mhz , cd 3 od ) 8 . 64 ( 1h , d , j 8 , ar — h ), 6 . 97 ( 1h , d , j 8 , ar — h ), 4 . 50 ( 2h , t , j 4 , och 2 ch 2 oh ), 4 . 04 ( 2h , t , j 4 , och 2 ch 2 oh ); δc ( 126 mhz , cd 3 od ) 153 . 9 ( ipso - ar — c ), 144 . 6 ( c ═ n ), 143 . 3 ( c ═ n ), 133 . 9 ( ar — c ), 128 . 6 ( ipso - ar — c ), 104 . 3 ( ar — c ), 71 . 6 ( och 2 ch 2 oh ), 58 . 7 ( och 2 ch 2 oh ); m / z ( es + ) 248 ( mna + ). 4 , 7 - dibromobenzo [ c ][ 1 , 2 , 5 ] selenadiazole ( 0 . 25 g , 0 . 74 mmol ), 4 -( methoxycarbonyl ) phenylboronic acid ( 0 . 15 g , 0 . 74 mmol ), pd ( pph 3 ) 4 ( 0 . 014 g , 0 . 01 mmol ) and na 2 co 3 ( 0 . 09 g , 0 . 74 mmol ) were weighed into a round bottom flask and dissolved with toluene ( 1 ml ), thf ( 1 ml ) and h 2 o ( 0 . 2 ml ). the solution was then refluxed for 24 h , cooled and poured in to h 2 o . the aqueous layer was extracted with et 2 o ( 3 × 15 ml ). the organic layers were dried over mgso 4 , filtered , concentrated and dried in vacuo . flash chromatography ( dcm / etoac [ 99 : 1 ], [ 98 : 2 ], [ 95 : 5 ]) afforded the title compound as a green solid ( 0 . 02 g , 7 %); r f 0 . 1 ( n - hexane / dcm 1 : 1 ); δ h ( 400 mhz , cdcl 3 ) 8 . 17 ( 1h , d , j 8 , ar — h ), 7 . 90 ( 1h , d , j 8 , ar — h ), 7 . 89 ( 1h , d , j 7 , ar — h ), 7 . 47 ( 1h , d , j 7 , ar — h ), 3 . 95 ( 3h , s , och 3 ). to a solution of 4 - chloro - 7 - chlorosulfonyl - 2 , 1 , 3 - benzoxadiazole ( 0 . 127 g , 0 . 50 mmol ) in anhydrous acetonitrile ( 6 ml ) was added a solution of dimethylamine in thf ( 2 . 0 m , 0 . 30 ml , 0 . 60 mmol ) followed immediately by triethylamine ( 0 . 15 ml , 1 . 10 mmol ) under argon , with stirring . the reaction was stirred at room temperature for 10 min . before removing the solvent in vacuo . purification by flash column chromatography on silica gel ( pe 40 - 60 : etoac , 8 : 2 ) gave the title product as a white solid ( 0 . 098 g , 0 . 38 mmol , 75 %). 1 h nmr ( 500 mhz , cdcl 3 ): δ 7 . 97 ( 1h , d , j = 7 . 3 , h - 5 ), 7 . 56 ( 1h , d , j = 7 . 3 , h - 6 ), 2 . 96 ( 6h , s , 2 × ch 3 ); 13 c nmr ( 125 mhz , cdcl 3 ): δ 148 . 9 ( c7a ), 145 . 7 ( c3a ), 134 . 4 ( c5 ), 129 . 1 ( c6 ), 127 . 7 ( c7 ), 126 . 1 ( c4 ); ms - es + ( m / z ) 262 ([ m + h ] + , 35 c1 , 100 %); hrms - es + ( m / z ) calcd for c 8 h 9 n 3 o 3 35 cl 32 s [ m + h ] + : 262 . 0048 , found 262 . 0048 ; anal . calcd for c 8 h 8 cln 3 o 3 s : c , 36 . 72 ; h , 3 . 08 ; n , 16 . 06 . found : c , 37 . 11 ; h , 3 . 16 ; n , 15 . 85 . compound xlvii ) may be prepared by an analogous route to that used for compound x ) replacing 4 -( trifluoromethyl ) thiophenol with 2 - benzothiazolethiol ( available from sinochemexper product list ) compounds lvi ) and lvii ), may be prepared by reacting 2 , 1 , 3 benzothiadiazole - 4 - sulfonylchloride ( commercially available from apollo ) with hydrazinecarboxylic acid , 2 -( aminoacetyl )-, 9h - fluoren - 9 - ylmethyl ester ( maybridge ) deprotection of the fmoc group and subsequent imine formation with an aldehyde ( in this case 2 - chlorobenzaldehyde ) compound lvii ), may be prepared by reacting 2 , 1 , 3 benzothiadiazole - 4 - sulfonylchloride ( commercially available from apollo ) with hydrazinecarboxylic acid , 2 -( aminoacetyl )-, 9h - fluoren - 9 - ylmethyl ester ( maybridge ) deprotection of the fmoc group and subsequent acylation with an acid chloride ( in this case with 2 - chlorobenzoyl chloride ) compound lx ) may be prepared by combining the required piperazine ( in this specific case 1 -[ 2 -( 4 - chlorophenyl ) ethyl ]- piperazine which is available from aurora screening library ) with 2 , 1 , 3 benzothiadiazole - 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