imidazole / benzimidazole and their derivatives are important class of organic compounds in coordination chemistry , photophysics , photochemistry , bioinorganic chemistry , and bioorganic chemistry [ 17 ] .
2-(2-hydroxyphenyl ) benzimidazole ( hpbm ) is an important benzimidazole - derived n , o - donor ligand .
hydroxyl benzimidazole shows excited - state intramolecular transfer ( esipt ) properties due to acidic protons of phenol and imidazole nitrogen ( tautomerism phenomenon ) .
it has been reported that hydroxyl benzimidazole and benzoxazole behave as a structural mimic of dna base pair for which tautomerism may be initiated at a definite time and position within duplex dna .
structurally similar natural product bis(benzoxazole ) uk-1 has been reported to posses anticancer activity , and the metal - binding studies of uk-1 indicates that benzoxazole - like compound are capable of binding a variety of biologically important metal ions .
benzimidazole derivatives exhibit significant activity against several viruses such as hiv , human cytomegalovirus ( hcmv ) [ 11 , 12 ] , herpes ( hsv-1 ) , and influenza .
benzimidazole derivatives are unique and broad - spectrum class of antirhino / enteroviral agents such as antihistaminic , antipyretic , antiulcerative , antihypertensive , antiviral [ 18 , 19 ] , antitumor [ 2024 ] , antihistaminic , and antiallergic   and are also efficient selective neuropeptide y y1 receptor antagonists .  
several derivatives are reported in literature for the synthesis of benzimidazole - metal complexes for fluorescent probes and bioorganic application , but antimicrobial activities of these classes of compounds have received little attention . in the literature , there are no reports available describing synthesis and antimicrobial activities of metal complexes of 5-(diethylamino)-2-(5-nitro-1h - benzimidazol-2-yl)phenol . in this paper , we have synthesized novel ligand 5-(diethylamino)-2-(5-nitro-1h - benzimidazol-2-yl)phenol and its metal complexes and studied their antimicrobial activities .
the synthetic route for the preparation of 5-(diethylamino)-2-(5-nitro-1h - benzimidazol-2-yl)phenol metal complexes is shown in scheme 1 .
m-(n , n - diethylamino)phenol ( 1 ) on formylation by using vilsmeier - haack reaction with dmf : pocl3 at 60c yielded p - n , n - diethyl amino salicylaldehyde ( 2 ) .
the p - n , n - diethyl amino salicylaldehyde on further reaction with 4-nitrobenzene-1,2-diamine in ethanol and pcl3 at 60c yielded the 5-(diethylamino)-2-(5-nitro-1h - benzimidazol-2-yl)phenol ( 3 ) .
the 5-(diethylamino)-2-(5-nitro-1h - benzimidazol-2-yl)phenol was reacted with different metal salt in methanol in the presence of catalytic amount of triethyl amine at room temperature to form 5-(diethylamino)-2-(5-nitro-1h - benzimidazol-2-yl)phenol - metal complexes .
the purity of the compounds was confirmed by tlc using precoated silica gel as stationary phase , using appropriate solvent system as mobile phase and visualized under uv - light .
structures of the title compounds were confirmed by ft - ir , h - nmr analysis , and metal ligand complex formation is confirmed by atomic absorption spectroscopy .
intermediate p - n , n - diethyl amino salicylaldehyde formation was confirmed by its melting point .
ft - ir and h nmr spectrum of 5-(diethylamino)-2-(5-nitro-1h - benzimidazol-2-yl)phenol showed absence of absorption band at 1670  cm confirming the absence of aldehydic functional group by way of conversion into the corresponding benzimidazole , and absence of peak at 9.90    ppm confirmed the conversion of formyl functional group into 5-(diethylamino)-2-(5-nitro-1h - benzimidazol-2-yl)phenol .
the ft - ir spectra of compounds 4a4d are also in complete agreement with their structure .
there was sharp modification between the ft - ir spectra of the metal complexes 4a4d and the ligand 5-(diethylamino)-2-(5-nitro-1h - benzimidazol-2-yl)phenol ( 3 ) , most of the bands change their pattern in the region due to coordination of the phenolic oxygen atom of oh group and nitrogen atom of imidazole ring to the metal ions .
the complexation of biologically important metals with 5-(diethylamino)-2-(5-nitro-1h - benzimidazol-2-yl)phenol was further explored with the evaluation of their antimicrobial activity .
all compounds were evaluated for in vitro antibacterial activities against e. coil and s. aureus strains and in vitro antifungal activity tested against c. albicans and a. niger strains by using serial dilution method .
incubator at 35 and 37c ; pipettes of various sizes ( gilson ) ; sterile tips , 100 , 200 , 500 , and 1000  l ; sterile normal saline ; sterile isosensitest agar ( southern group laboratory , sgl ) ; antibiotic solutions ( sigma - aldrich ) ; sterile solution of 10% ( v / v ) dmso in water ( sigma - aldrich ) .
although nccls recommends the use of mueller hinton medium for susceptibility testing , the isosensitest medium had comparable results for most of the tested bacterial strains .
a volume of 100  l of test material in 10% ( v / v ) dmso ( usually a stock concentration of 4  mg / ml ) was pipetted into the first row of the plate . to all other wells , 50  l of nutrient broth was added .
tips were discarded after use such that each well had 50  l of the test material in serially descending concentrations .
to each well , 10  l of resazurin indicator solution was added . using a pipette ,
30  l of 3.3x strength isosensitised broth added to each well to ensure that the final volume was single strength of the nutrient broth .
finally , 10  l of bacterial suspension ( 5  10  cfu / ml ) was added to each well to achieve a concentration of 5  10  cfu / ml .
each plate was wrapped loosely with cling film to ensure that bacteria did not become dehydrated .
each plate had a set of controls : a column with a broad - spectrum antibiotic as positive control , a column with all solutions with the exception of the test compound , and a column with all solutions with the exception of the bacterial solution , adding 10  l of nutrient broth instead .
the plates were prepared in triplicate and placed in an incubator set at 37c for 1824  h. the colour change was then assessed visually .
the average of three values was calculated and that was the mic for the test material and bacterial or fungal strain .  
the new ligand and their metal complexes were evaluated for their in vitro antibacterial activity against e. coli and s. aureus strains and in vitro antifungal activity against c. albicans and a. niger strains by using serial dilution method .
the minimum inhibitory concentration ( mic ) measurement determined for compounds showed significant growth inhibition zones using serial dilution method .
the results mentioned in figure 1 indicate that most of the tested compounds displayed variable inhibitory effects on growth of tested against bacterial strain and antifungal strain .
the metal complex 4d showed excellent antibacterial activity against e. coli and s. aureus strains but metal - complexes 4a4c showed moderate activity against tested antibacterial strains .
the inhibitory growth of metal - complexes 4a4d are almost double than novel synthesized ligand 5-(diethylamino)-2-(5-nitro-1h - benzimidazol-2-yl)phenol ( 3 ) , and the compound 4b is less active against s. aureus antibacterial strain . regarding the structure - activity relationship of the novel compound 5-(diethylamino)-2-(5-nitro-1h - benzimidazol-2-yl)phenol complex with cobalt(ii ) metal showed better activity than fe , ni , and cu metals .  
the results mentioned in figure 1 showed that ligand as well as metal complexes show good inhibitory growth in case c. albicans as well as a. niger strains .
these results indicate that after coordination of biologically important transition , metal with 5-(diethylamino)-2-(5-nitro-1h - benzimidazol-2-yl)phenol has substantial effect on the antimicrobial activity against tested microorganism .
in general , most of the tested compounds revealed better activity against the antibacterial strain ( e. coli , s. aureus ) and antifungal strain ( c. albicans , a. niger ) .
it was also noticed that ligand ( 3 ) and metal complexes 4a4d showed stronger antibacterial activity than antifungal one .   in order to examine the thermal stability of these complexes , thermal gravimetric ( tg ) and differential scanning colorimeter ( dsc ) analysis
the tg results indicate that the skeleton of the synthesized ligand and its transition metal complexes are stable up to 250c . above 250c ,
the comparisons of the td ( decomposition temperature ) showed that the thermal stability of the 3 and 4a4d decreases in the order 4b > 4c > 4d > 4a > 3 .
metal complexes and ligand do not decompose completely even up to temperature 600c . in the dsc curve
metal complexes 4a4d shows exothermic peak 310 , 395 , 290 , and 385 , respectively , as shown in figure 3 .
the electrochemical behavior of all the complexes and ligand were studied by using cyclic voltametry ( cv ) in dimethyl methyl sulphoxide ( 0.1  m net4clo4 ) in the potential range 1.6 to + 1.2  v by using platinum auxiliary electrode and pt disc - working electrode at ambient temperature ( 300  k ) with no trace of decomposition as reflected in smooth curve .
cyclic voltammetric studies of the ligand 3 and complexe 4a4d in dimethyl formamide solution under nitrogen atmosphere are irreversible .
the result of cyclic voltammetry of ligand closely resembles with that of metal complexes compounds , which serve as further evidences for similar structural and electronic properties figure 4 .
the uv - vis absorption and emission spectra of ligand 3 and its metal complexes 4a4d were recorded in dmf at room temperature , and the compound concentrations are 1  10  m. the max ( absorbance ) values of ligand 3 is 294 ( 0.484 ) , and metal complexes 4a4d were obtained as 387 ( 0.629 ) , 366 ( 1.00 ) , 354 ( 0.818 ) , and 339 ( 1.043 ) nm , respectively . as can be seen , the absorption characteristics of metal complexes 4a4d are nearly same , while ligand absorbs in blue region as compared to complexes .
in conclusion , we have synthesized new ligand 5-(diethylamino)-2-(5-nitro-1h - benzimidazol-2-yl)phenol and their metal complexes . these novel compounds were evaluated for in vitro antibacterial activity against e. coli and s. aureus strains as well as for antifungal activity against c. albicans and a. niger strains using serial dilution technique .
all synthesized compounds are confirmed by ft - ir , h - nmr , and atomic absorption spectroscopy .
the reaction was monitored by tlc using on 0.25  mm e - merck silica gel 60 f254 precoated plates , which were visualized with uv light .
melting points were measured on standard melting point apparatus from sunder industrial product mumbai and are uncorrected .
the ft - ir spectra were recorded on perkins - elmer 257 spectrometer using kbr discs .
h - nmr spectra were recorded on vxr 300-mhz instrument using tms as an internal standard .
phosphorous oxychloride ( pocl3 ) ( 2.75  ml , 0.03 mole ) was slowly added to dimethylformamide ( dmf ) ( 3.65  ml , 0.05 mole ) at 510c under the stirring . to this cooled reagent
, 3-(diethyl amino)phenol ( 0.01  mole ) was added by dissolving it into dmf ( 6  ml ) under the stirring , and the resulting mixture was heated at 75c for 4  hrs .
the reaction mixture was cooled to room temperature and then poured into ice water ( 60  ml ) .
reaction mass was neutralised with sodium carbonate , brown colored solid separated out , filtered the separated product washed with cold water , dried , and crystallised from ethanol to get pure product ( m.p .
phosphorus trichloride ( 0.33  mol ) was added dropwise to a solution of the p - n , n - diethyl salicylaldehyde ( 0.33  mol ) n , n - diethyl m - amino phenol ( 0.33  mol ) in ethanol ( 50  ml ) , maintaining the temperature at 4045c .
the mixture was heated at 60c for 4  h , after the completion of reaction ( monitored by tlc ) cooled the reaction mass at room temperature and made alkaline to ph 8 with aqueous sodium bicarbonate solution ( 20%  w / v ) .
reaction mass was concentrated under vacuum , and the solid which obtained was collected and crystallized from isopropyl alcohol .
ir ( kbr , cm ) : 2991 , 1620 , 1520 , 1338 , 1149 , 946 , 817 , 733 . 

h - nmr ( 300  mhz ) :  1.21 ( t , 6h , ch3 ) , 3.42 ( q , 4h , ch2 ) , 6.27 ( d , 1h , aromatic h ) , 7.26 ( d , 1h , aromatic h ) , 7.29 ( m , 2h , aromatic h ) , 7.88 ( d , 1h , aromatic h ) , 7.85 ( d , 1h , aromatic h ) , 8.17 ( s , 1h , nh ) , 12.43 ( s , 1h , oh ) .
4a : ir ( kbr , cm ) : 2972 , 1606 , 1498 , 1262 , 1144 , 1074 , 821 , 726 . 

4b : ir ( kbr , cm ) : 2974 , 1608 , 1498 , 1336 , 1258 , 1148 , 1063 , 1019 , 818 . 

4c : ir ( kbr , cm ) : 2973 , 1607 , 1497 , 1336 , 1258 , 1149 , 1019 , 820 . 

4c : ir ( kbr , cm ) : 2974 , 1607 , 1498 , 1339 , 1261 , 1069 , 827 , 732 . to a solution of ligand , 5-(diethylamino)-2-(5-nitro-1h - benzimidazol-2-yl)phenol ( 3 ) ( 0.1 mole ) in methanol
( 15  ml ) was added a few drops of triethyl amine and solution of metal salts ( 0.05 mole ) in methanol ( 2  ml ) .
the product , thus , separated was filtered , washed with water followed by methanol , and dried to give 4a4d .
atomic absorption spectra were recorded using atomic absorption spectrometer model gbc 932 ( gbc scientific equipment , australia ) .
exactly weighed dye samples were dissolved in 20  ml of dimethyl sulphoxide and diluted to 100  ml with distilled water and analyzed by gbc 932 plus atomic absorption spectrometer ( aas ) .
acetylene was used as fuel , and air was used as carrier gas . certified 1000  mg / l standard solution of iron ( merck , mumbai ) was used to perform calibration using hallow cathode lamp for iron at 248.3  nm wavelength .
the samples were prepared in such a manner that they will result in 2  mg / l solution containing 1  :  2 complexes .
table 1 compares the experimental results of aas analysis and with one calculated on the theoretical basis .
the results of aas analysis are in well agreement with the predicted results within the limitations of the experimental error , which confirms the proposed 1  :  2 metal complex stoichiometric between metal and ligand .
 synthesis and antimicrobial activities of new metal [ co(ii ) , cu(ii ) , ni(ii ) , and fe(ii ) ] complexes from 5-(diethylamino)-2-(5-nitro-1h - benzimidazol-2-yl)phenol are described . 
 the newly synthesized ligands were characterized by ir , 1h nmr , and lc - ms analysis , and metal - ligand complex formations were confirmed by using atomic absorption spectroscopy and elemental analysis . 
 all complexes show significant in vitro antibacterial activities against e. coli and s. aureus strains and in vitro antifungal activity against c. albicans and a. niger strains by using serial dilution method . 
 the antibacterial activities were expressed as the minimum inhibitory concentration ( mic ) in g / ml . thermal properties and electrochemical behavior of novel transition metal complexes have been studied . 