ralstonia solanacearum ( r. solanacearum ) , one of the most important bacterial diseases on plants , is a devastating , soil - borne plant pathogen with a global distribution and an unusually wide host range , which belongs to the -proteobacteria and is considered as a " species complex " . it causes a wilt disease with deadly effects in many important crops , such as in potato , tomato and eggplant firstly described by smith in 1896 and subsequently in tobacco in 1908 [ 1 - 3 ] . once the plants infected with bacterial wilt , the main symptoms of plants were rapid death , but stems and leaves remained green . tobacco bacterial wilt , caused by r. solanacearum , is one of the main plant diseases in tobacco production and occurred in the most common and serious form . the high incidence of plant mortality and the lack of effective control methods make r. solanacearum become one of the world 's most destructive plant pathogens . pyridine is an important class of heterocyclic ring , which have been attracted more and more attention in the pesticides areas due to their broad - activities , such as fungicidal activity [ 6 - 8 ] , insecticidal activity   and herbicidal activity . in fungicidal activity regard , some pyridine derivatives can prevent erysiphe graminis , botrytis cinerea , pyricularia oryzae   and phytophthora infestans . currently , some pyridine compounds have been developed and commercialized , for example , fluopicolide , boscalid   and picoxystrobin   ( figure 1 ) . in the recent literature , hydrazones demonstrated significant antimicrobial activity , antitubercular activity   and antitumoral activity   in medicinal areas . on the other hand , hydrazones also exhibit good fungicidal activity against phytophthora infestans , cladosporium cucumerinum and colletotrichum orbiculare   in the pesticides areas , some of them containing hydrazone structure have been commercialized , such as benquinox and ferimzone   ( figure 1 ) . controlling of plant bacterial diseases has long been a challenging mission in the agricultural sector . the application of traditional pesticides has not proved much effective and at the same time high residue level or negative impact on the environment were caused . copper formulation , a commercial bactericide can enhance resistance in host tobacco plant . despite being useful in the treatment of plants affected by tobacco bacterial wilt , the use of copper formulation for field trial is largely limited due to its phytotoxicity , strong alkali and low mobility . therefore , the search for new antibacterial agent still remains a daunting task in pesticide science . in order to discover new molecules and pesticide acting on tobacco bacterial wilt , we sought to combine the active structure of hydrazone and pyridine together to design and synthesize a class of novel hydrazone derivatives containing a pyridine moiety . thus , 2-chloronicotinic acid and 2-amino-5-chloro-3-methylbenzoic acid were used as start materials , 11 novel analogues of hydrazones containing pyridine were synthesized . the biological activities on r. solanacearum were tested , the results showed that most of the synthesized compounds exhibited antibacterial activity against r. solanacearum to a certain extent , compounds 3e and 3 g showed good antibacterial activity at 200 mg / l . especially compounds 3 g displayed excellent antibacterial activity ( 57.0% ) at 200 mg / l . to the best of our knowledge , this is the first report on the antibacterial activity of hydrazone derivatives containing a pyridine moiety . 6- chloro-2-(2-chloropyridin-3-yl)-8-methyl-4h - benzo[d ] oxazin-4-one 1 were prepared by treatment of 2-chloronicotinic acid with 2-amino-5-chloro-3-methylbenzoic acid using readily available starting materials and a simple procedure as describing in the literature . the further reaction of intermediates 1 with 80% hydrazine hydrate could proceed readily at room temperature to give intermediates 2 . subsequent treatment of intermediates 2 with different aldehydes in ethanol at room temperature afforded the desired compounds ( 3a to 3k ) . the synthetic route in scheme 1 had several advantages , which included short steps , short reaction times and excellent yields ( e.g. the yields for 3b , 3d , 3e and 3j were 93.3% , 96.6% , 96.6% and 93.6% , respectively ) , especially the mild conditions ( room temperature ) . additional file shows the structures , yields and elemental analysis data for title compounds in more detail [ see additional file 2 ] . all the synthesized compounds ( 3a-3k ) were characterized on the basis of their spectroscopic data . the ir absorption bands near 3260 - 3180 cm , 3100 - 2910 cm , 1666 - 1610 cm , 1600 - 1575 cmand 1367 - 1352 cmconfirmed the presence of two n - h , ar - h , amide , - c = n-   and -ch3 functional groups , respectively . in the h - nmr spectra of the title compounds , the -conhar proton appeared as a broad singlet at 12.17 - 10.53 ; the -conhn proton appeared as a broad singlet at 10.34 - 10.17 ; the -c = nh proton mainly appeared as a broad singlet at 8.73 - 8.20 ; the pyridine ring of 4 and 6 positions protons were occurred double - double peaks near 8.52 - 8.51 and 8.01 - 7.94 ; the methyl ( ar - ch3 ) proton signals were observed as a singlet near 2.34 - 2.30 . meanwhile , we observed the titles compounds possess e and z configuration in the h - nmr spectra , but the e configuration was mainly forms by the spectra analysis , such as e configuration of -conhar proton appeared at 12.11 , but z proton was occurred at 12.01 in 3e , and the e / z is approximately equal to 3.60 . the antibacterial activity of compounds 3a-3k against r. solanacearum was assayed by the reported method . kocide , one of the proven commercial agents for controlling r. solanacearum , was used as the reference of bactericides . the results provided in table 1 indicate that most of the prepared compounds have weak to good antibacterial activity against r. solanacearum at 500 mg / l . compounds 3e and 3 g displayed higher activities than compounds 3a-3d , 3f to 3h-3k at 500 for example , inhibition of 3e ( r2 = 3-trifluoromethylphenyl ) and 3 g ( r2 = n , n - dimethylamino ) on tobacco bacterial wilt was 80.9% and 100.0% at 500 mg / l , whereas 3a ( r2 = et ) , 3b ( r2 = 2-chlorophenyl ) and 3f(r = 4-tert - butylphenyl ) was only 5.0% , 6.0% and 39.0% at the same condition . primary structure- activity relationships revealed that when the conhn = chr2 was simultaneously substituted by n , n - dimethylamino ( at the r2 group ) and 3-trifluoromethylphenyl ( at the r2 group ) , the activity on r. solanacearum increased . however , the introduction of ch2ch3 , 2-chlorophenyl , 4-fluorophenyl , 2,6-dichlorophenyl , 4-tert - butylphenyl , 3 , 4-dichlorophenyl , 2 , 3-dimethoxyphenyl , 2 , 4-dimethoxyphenyl and 2 , 5-dimethoxy- phenyl groups into r2 groups decreased the activity of the compound . furthermore , the mono- substituent at the different positions on phenyl of the r2 group also affected the bioactivity of the compound . for instance , the compound with trifluromethyl group at 3-position on phenyl ring ( 3e ) displayed good activity , while the compounds with tert - butyl at 4-position on phenyl ring ( 3f ) exhibited moderate activity and the compound with chlorine at 2-position on phenyl ring ( 3b ) and the compound with fluorine at 4-position on phenyl ring ( 3c ) showed no antibacterial activities on r. solanacearum . compared with the same substituents on phenyl , the substituents at the 2 , 3-positions , the corresponding molecules always had higher inhibition rates for tobacco bacterial wilt . for example , the inhibition rates of 3i ( r2 = 2 , 3-dimethoxyphenyl ) and 3k ( r2 = 2,4-dimethoxyphenyl ) was 40.3% , 36.1% and the inhibition rates of 3j ( r2 = 2,5-dimethoxyphenyl ) on tobacco bacterial wilt was 25.2% . antibacterial activity of compounds 3a to 3 k , kocide against r. solanacearum unless otherwise stated , all the reagents and reactants were purchased from commercial suppliers ; melting points were uncorrected and determined on a xt-4 binocular microscope ( beijing tech instrument co. , china ) . the h - nmr and c - nmr spectra were recorded on a jeol ecx 500 nmr spectrometer ( jeol ltd . , japan ) at room temperature operating at 500 mhz for h - nmr and 125 mhz for c - nmr by using cdcl3 or dmso as solvents and tms as an internal standard ; infrared spectra were recorded in kbr on a ir pristige-21 spectrometer ( shimadzu corporation , japan ) ; elemental analysis was performed on an elemental vario - iii chn analyzer ( elementar , german ) . the course of the reactions was monitored by tlc ; analytical tlc was performed on silica gel gf 254 . intermediate 1 and 2 were prepared according to the reported methods   and used without further purifications [ additional file 3 ] . antibacterial activities of some title compounds against r. solanacearum were evaluated by the turbidimeter test , whereas kocide3000 was the positive control . the compounds were dissolved in 30 l dmso and diluted with water containing tween-20 ( 0.1 mg / l ) to generate a final concentration of 500 mg / l and 200 mg / l , which were added to the toxic nutrient broth ( nb ) liquid medium in 5 ml tubes , respectively . to the above tubes , 40 l nb liquid medium containing r. solanacearum pathogen was individually added , then shaken at 30c and 180 r.p.m . for 48 h , the relative inhibition rate of the circle mycelium compared to blank assay was calculated via the following equation . relative inhibition rate % = a0-a1a0100% a0 : corrected od values of the control medium of bacilli ; a1 : corrected od values of the medium of toxic . 6- chloro-2-(2-chloropyridin-3-yl)-8-methyl-4h - benzo[d ] oxazin-4-one 1 were prepared by treatment of 2-chloronicotinic acid with 2-amino-5-chloro-3-methylbenzoic acid using readily available starting materials and a simple procedure as describing in the literature . the further reaction of intermediates 1 with 80% hydrazine hydrate could proceed readily at room temperature to give intermediates 2 . subsequent treatment of intermediates 2 with different aldehydes in ethanol at room temperature afforded the desired compounds ( 3a to 3k ) . the synthetic route in scheme 1 had several advantages , which included short steps , short reaction times and excellent yields ( e.g. the yields for 3b , 3d , 3e and 3j were 93.3% , 96.6% , 96.6% and 93.6% , respectively ) , especially the mild conditions ( room temperature ) . additional file shows the structures , yields and elemental analysis data for title compounds in more detail [ see additional file 2 ] . all the synthesized compounds ( 3a-3k ) were characterized on the basis of their spectroscopic data . the ir absorption bands near 3260 - 3180 cm , 3100 - 2910 cm , 1666 - 1610 cm , 1600 - 1575 cmand 1367 - 1352 cmconfirmed the presence of two n - h , ar - h , amide , - c = n-   and -ch3 functional groups , respectively . in the h - nmr spectra of the title compounds , the -conhar proton appeared as a broad singlet at 12.17 - 10.53 ; the -conhn proton appeared as a broad singlet at 10.34 - 10.17 ; the -c = nh proton mainly appeared as a broad singlet at 8.73 - 8.20 ; the pyridine ring of 4 and 6 positions protons were occurred double - double peaks near 8.52 - 8.51 and 8.01 - 7.94 ; the methyl ( ar - ch3 ) proton signals were observed as a singlet near 2.34 - 2.30 . meanwhile , we observed the titles compounds possess e and z configuration in the h - nmr spectra , but the e configuration was mainly forms by the spectra analysis , such as e configuration of -conhar proton appeared at 12.11 , but z proton was occurred at 12.01 in 3e , and the e / z is approximately equal to 3.60 . the antibacterial activity of compounds 3a-3k against r. solanacearum was assayed by the reported method . kocide , one of the proven commercial agents for controlling r. solanacearum , was used as the reference of bactericides . the results provided in table 1 indicate that most of the prepared compounds have weak to good antibacterial activity against r. solanacearum at 500 mg / l . compounds 3e and 3 g displayed higher activities than compounds 3a-3d , 3f to 3h-3k at 500 for example , inhibition of 3e ( r2 = 3-trifluoromethylphenyl ) and 3 g ( r2 = n , n - dimethylamino ) on tobacco bacterial wilt was 80.9% and 100.0% at 500 mg / l , whereas 3a ( r2 = et ) , 3b ( r2 = 2-chlorophenyl ) and 3f(r = 4-tert - butylphenyl ) was only 5.0% , 6.0% and 39.0% at the same condition . primary structure- activity relationships revealed that when the conhn = chr2 was simultaneously substituted by n , n - dimethylamino ( at the r2 group ) and 3-trifluoromethylphenyl ( at the r2 group ) , the activity on r. solanacearum increased . however , the introduction of ch2ch3 , 2-chlorophenyl , 4-fluorophenyl , 2,6-dichlorophenyl , 4-tert - butylphenyl , 3 , 4-dichlorophenyl , 2 , 3-dimethoxyphenyl , 2 , 4-dimethoxyphenyl and 2 , 5-dimethoxy- phenyl groups into r2 groups decreased the activity of the compound . furthermore , the mono- substituent at the different positions on phenyl of the r2 group also affected the bioactivity of the compound . for instance , the compound with trifluromethyl group at 3-position on phenyl ring ( 3e ) displayed good activity , while the compounds with tert - butyl at 4-position on phenyl ring ( 3f ) exhibited moderate activity and the compound with chlorine at 2-position on phenyl ring ( 3b ) and the compound with fluorine at 4-position on phenyl ring ( 3c ) showed no antibacterial activities on r. solanacearum . compared with the same substituents on phenyl , the substituents at the 2 , 3-positions , the corresponding molecules always had higher inhibition rates for tobacco bacterial wilt . for example , the inhibition rates of 3i ( r2 = 2 , 3-dimethoxyphenyl ) and 3k ( r2 = 2,4-dimethoxyphenyl ) was 40.3% , 36.1% and the inhibition rates of 3j ( r2 = 2,5-dimethoxyphenyl ) on tobacco bacterial wilt was 25.2% . unless otherwise stated , all the reagents and reactants were purchased from commercial suppliers ; melting points were uncorrected and determined on a xt-4 binocular microscope ( beijing tech instrument co. , china ) . the h - nmr and c - nmr spectra were recorded on a jeol ecx 500 nmr spectrometer ( jeol ltd . , japan ) at room temperature operating at 500 mhz for h - nmr and 125 mhz for c - nmr by using cdcl3 or dmso as solvents and tms as an internal standard ; infrared spectra were recorded in kbr on a ir pristige-21 spectrometer ( shimadzu corporation , japan ) ; elemental analysis was performed on an elemental vario - iii chn analyzer ( elementar , german ) . the course of the reactions was monitored by tlc ; analytical tlc was performed on silica gel gf 254 . intermediate 1 and 2 were prepared according to the reported methods   and used without further purifications [ additional file 3 ] . antibacterial activities of some title compounds against r. solanacearum were evaluated by the turbidimeter test , whereas kocide3000 was the positive control . the compounds were dissolved in 30 l dmso and diluted with water containing tween-20 ( 0.1 mg / l ) to generate a final concentration of 500 mg / l and 200 mg / l , which were added to the toxic nutrient broth ( nb ) liquid medium in 5 ml tubes , respectively . to the above tubes , 40 l nb liquid medium containing r. solanacearum pathogen was individually added , then shaken at 30c and 180 r.p.m . for 48 h , the relative inhibition rate of the circle mycelium compared to blank assay was calculated via the following equation . relative inhibition rate % = a0-a1a0100% a0 : corrected od values of the control medium of bacilli ; a1 : corrected od values of the medium of toxic . unless otherwise stated , all the reagents and reactants were purchased from commercial suppliers ; melting points were uncorrected and determined on a xt-4 binocular microscope ( beijing tech instrument co. , china ) . the h - nmr and c - nmr spectra were recorded on a jeol ecx 500 nmr spectrometer ( jeol ltd . , japan ) at room temperature operating at 500 mhz for h - nmr and 125 mhz for c - nmr by using cdcl3 or dmso as solvents and tms as an internal standard ; infrared spectra were recorded in kbr on a ir pristige-21 spectrometer ( shimadzu corporation , japan ) ; elemental analysis was performed on an elemental vario - iii chn analyzer ( elementar , german ) . the course of the reactions was monitored by tlc ; analytical tlc was performed on silica gel gf 254 . intermediate 1 and 2 were prepared according to the reported methods   and used without further purifications [ additional file 3 ] . antibacterial activities of some title compounds against r. solanacearum were evaluated by the turbidimeter test , whereas kocide3000 was the positive control . the compounds were dissolved in 30 l dmso and diluted with water containing tween-20 ( 0.1 mg / l ) to generate a final concentration of 500 mg / l and 200 mg / l , which were added to the toxic nutrient broth ( nb ) liquid medium in 5 ml tubes , respectively . to the above tubes , 40 l nb liquid medium containing r. solanacearum pathogen was individually added , then shaken at 30c and 180 r.p.m . for 48 h , the relative inhibition rate of the circle mycelium compared to blank assay was calculated via the following equation . relative inhibition rate % = a0-a1a0100% a0 : corrected od values of the control medium of bacilli ; a1 : corrected od values of the medium of toxic . in conclusion , a series of the novel hydrazone derivatives containing a substituted pyridine ring were designed and synthesized . the reaction of intermediates 2 with different aldehydes in ethanol at room temperature used 2-chloronicotinic acid and 2-amino-5-chloro-3-methylbenzoic acid as start materials provides a ready access to a series of the novel hydrazone derivatives containing pyridine moiety . the antibacterial tests indicated that some of the synthesized compounds possessed of moderately high activity against r. solanacearum . the title compounds 3e and 3 g exhibited favourable activity against tobacco bacterial wilts in vitro compared to the commercial bactericides kocide 3000 . the antibacterial tests showed that when the r2 group of conhn = chr2 was 3-trifluoromethylphenyl or n , n - dimethylamino , the corresponding compounds presented good antibacterial activities . the structure of the target products needs to be optimized to enhance their antibacterial activity . future structural modification and biological evaluation should be carried out to explore the full potential of this novel class of antibacterial molecules . the current study is an outcome of constructive discussion with bas , sy and dyh who offered necessary guidance to jw and shk to carry out their synthesis and characterization experiments . mh performed the antibacterial tests ; lhj carried out the h nmr , c nmr spectral analyses and elemental analysis . synthetic sequence to the novel hydrazone analogues 3 containing pyridine ring from 1 and 2 . yield and elemental analyses data for title compounds 3a - k . which contains the a table about structure , yield and elemental analyses data for title compounds 3a - k . experimental details and data of title compounds 3a - k . which includes the experimental procedure , spectroscopic data of intermediate 1 , intermediate 2 , title compounds 3a - k , copies of h nmr and c nmr . 2010cb 126105 ) , key technologies r&d program ( no.2011bae06b05 - 6 ) and national natural science foundation of china ( no.21162004 ) , the special foundation of governor for outstanding talents in guizhou ( no.2011 - 38 ) .
backgroundralstonia solanacearum , one of the most important bacterial diseases on plants , is a devastating , soil - borne plant pathogen with a global distribution and an unusually wide host range . in order to discover new bioactive molecules and pesticides acting on tobacco bacterial 
 wilt , we sought to combine the active structure of hydrazone and pyridine together to design and synthesize a series of novel hydrazone derivatives containing a pyridine moiety.resultsa series of hydrazone derivatives containing a pyridine moiety were synthesized . 
 their structures were characterized by 1 h - nmr , 13 c - nmr , ir , and elemental analysis . 
 the preliminary biological activity tests showed that compound 3e and 3 g exhibited more than 80% activity against ralstonia solanacearum at 500 mg / l , especially compound 3 g displayed relatively good activity to reach 57.0% at 200 mg / l.conclusiona practical synthetic route to hydrazone derivatives containing a pyridine moiety by the reaction of intermediates 2 with different aldehydes in ethanol at room temperature using 2-chloronicotinic acid and 2-amino-5-chloro-3-methylbenzoic acid as start materials is presented . 
 this study suggests that the hydrazone derivatives containing a substituted pyridine ring could inhibit the growth of ralstonia solanacearum .