cyclooxygenases ( cox ) or prostaglandin endoperoxide synthases are the key enzymes in the synthesis of prostaglandins , the main mediators of inflammation , pain , and increased body temperature ( hyperpyrexia ) . the cox-1 is responsible for formation of important biological mediators such as prostanoids , including prostaglandins , prostacyclin , and thromboxane , and involved in pain causing , blood clotting , and protecting the stomach , whereas cox-2 is involved in the pain by inflammation and plays a major role in prostaglandin biosynthesis in inflammatory cells and central nervous system . when cox-1 is inhibited , inflammation is reduced , but the protection of the lining of the stomach is also lost . this can cause stomach upset as well as ulceration and bleeding from the stomach and even the intestines . whereas cox-2 is usually specific to inflamed tissue , there is much less gastric irritation associated with cox-2 inhibition together with the decreased risk of peptic ulceration . therefore , selective cox-2 inhibitors such as celecoxib and rofecoxib had been developed for ease of inflammation associated with cox . the use of coxib drugs such as rofecoxib ( vioxx ) and valdecoxib ( bextra ) was withdrawn from the market in 2004 and 2005 , respectively , because of increased risk of heart attacks and strokes with long - term use . at present , celecoxib ( celebrex ) is the only cox-2 inhibitor available in the united states . the isatin ( indole-2,3-dione ) pharmacophore has attracted and still attracts much attention from medicinal chemists because of great importance in their biological as well as synthetic approach of medicinal chemistry . the various derivatives of isatin are known to possess a range of biological properties including anticancer , anticonvulsant , antidepressants , antimicrobial , antiviral , anxiolytics activities , molecular docking studies of isatin derivatives as egfr inhibitors , and anti - inflammatory , and the potent anti - inflammatory effect of indomethacin , etodolac , and tenidap encouraged us to further explore the pharmacological properties of the indole nucleus . it has been reported that the nature of substituents at the 2- or 3-position of the indole nucleus plays an important role in modulating their anti - inflammatory properties [ 1418 ] . amide - containing compounds have been shown to possess a wide range of biological activities , including anti - inflammatory properties . interestingly , the replacement of the carboxylic groups by amide groups in nsaid drugs indomethacin , meclofenamic acid , and ketoprofen conferred the compounds greater selectivity for cox-2 over the cox-1 enzyme . in addition , many thiazoline derivatives exhibit a wide variety of biological activities such as antimicrobial , anti - inflammatory , antihistaminic , antihypertensive , hypnotic , and anticonvulsant . keeping in view of biological importance of the two molecular moieties , namely , isatins and 2-hydroxybenzohydrazide , to study the condensation of isatins with 2-hydroxybenzohydrazide ( ii ) the synthesized compounds were screened for in vivo anti - inflammatory activity and molecular docking studies . these results were supported to design novel specific inhibitors of cox-2 by the comparative modelling of cox-1 and cox-2 enzymes with the available pharmacophore . we hope that these molecules may be further explored as powerful and novel nonulcerogenic anti - inflammatory with selective inhibition of cox-2 . all the chemicals used were of analytical grade and obtained from himedia and sd fine chemicals . the completion of the reaction , the purity of the compounds checked by tlc using e - merck 0.25  mm silica gel plates , and visualization were accomplished with uv light . h nmr spectra were recorded on an avance-300  mhz in cdcl3 solvent using tms as an internal standard ( chemical shifts in  , ppm ) . the ir spectra were recorded on rkin elmer spectrum bx-1 spectrometer by using kbr disc . male adult albino wister rats ( 150350  g ) were acclimatized for a period of 14 days prior to performing the experiments and maintained under standard husbandry conditions and had free access to food and water ad libitum . the animals were divided into different groups ; each consists of six animals which were fasted overnight prior to the study . the protocol of the present study was reviewed and approved by the institutional animal ethical committee . in a 5  lit . rb flask , chloral hydrate ( 0.54  mol ) and 1200  ml of water were placed . to this solution , crystallized sodium sulphate ( 1300  g ) was then added followed by a solution of an appropriate aromatic amine ( i ) in 300  ml of water and concentrated hydrochloric acid ( 0.52  mol ) . finally , a solution of hydroxylamine hcl ( 1.58  mol ) in 500  ml of water was added . the contents of the flask were heated over a wire - gauge by a mecker burner so that vigorous boiling begins in about 45 minutes . after 1 to 2 minutes of vigorous boiling the reaction was completed . during the heating period itself it was filtered under suction , air dried , and purified by recrystallization from suitable solvent(s ) . sulphuric acid ( 600  g , d , 1.84 , 326  ml ) were warmed at 50c in a one liter rb flask fitted with an efficient mechanical stirrer and to this , finely powdered appropriate isonitrosoacetanilide ( ii , 0.46  mol ) was added at such a rate so as to maintain the temperature between 60c and 70c but not higher . external cooling was applied at this stage so that the reaction could be carried out more rapidly . after the addition of isonitroso compound was completed the temperature of the solution was raised to 80c and maintained at that temperature for 10 minutes , to complete the reaction . then the reaction mixture was cooled to room temperature and poured on crushed ice ( 2.5  kg ) while stirring . after standing for about half - an - hour , the product separated was filtered , washed several times with small portions of cold water , and dried . purification of the compound was affected by the recrystallization from methanol . in a 500  ml of rb flask , 10  g of methyl salicylate ( v ) and 50  ml of distilled alcohol were placed and the reaction mixture was shaken for 5 minutes . to this add 20  ml of hydrazine hydrate ( 99% ) and the contents of the flask were refluxed for 3 hours , the completion of the reaction monitored by tlc . the resultant white crystalline solid was filtered and washed repeatedly , with small portions of cold alcohol . the product was dried and purified by recrystallization from methanol , yield 90% , m.p . a mixture of an appropriate indole-2,3-dione ( 0.01  mol ) and 2-hydroxybenzohydrazide ( 0.01  mol ) was taken into methanol ( 50  ml ) in presence of glacial acetic acid which was heated under reflux on water bath for 6 - 7 hours . the coloured compounds were thus obtained upon cooling , were filtered , were washed with small portions of cold methanol , and were dried . spectral data of the synthesized compounds 
 
 ( 1 ) 2-hydroxybenzohydrazide ( 
 vi 
 ) . ir ( kbr ) cm : 3320.78 ( nh ) , 1623 ( c = o ) , 1479 ( c - n ) , 830500 ( ar ) ; ms : m / z 152 ( m ) . 1735.97 ( c = o ) , 1663.97 ( c = o ) , 1527.60 ( c = n ) ; h nmr ( 300  mhz , dmso - cdcl3 ) :   [ ppm ] , 6.857.85 ( m , 8h , ar - h ) , 10.9 ( s , 1h , conh ) , 11.6 ( s , 1h , indole nh ) , 14.4 ( s , 1h , oh phenolic ) ; ms : m / z 281 ( m ) . ir ( kbr ) cm : 3170 ( nh ) , 3060 ( c - h ) , 1719 ( c = h - nmr ( dmso - d6 ) :   [ ppm ] , 2.6 ( s , 3h , ch3 ) , 7.2 ( s , 1h , ar - h ) , 7.4 ( d , 1h , ar - h ) 7.6 ( t , 2h , ar - h ) , 7.7 ( d , 1h , ar - h ) , 7.92 ( d , 2h , ar - h ) , 11.6 ( s , 1h , indole nh ) , 11 ( s , 1h , conh ) , 14.1 ( s , 1h , oh phenolic ) . ms : m / z 295 ( m ) . ir ( kbr ) cm : 3170.58 ( nh ) , 1724.26 ( c = o ) , 1675.74 ( c = o ) , 1528.08 ( c = h - nmr ( dmso - d6 ) :   [ ppm ] : 6.92 ( s , 1h , ar - h ) , 7.21 ( d , 1h , ar - h ) , 7.52 ( t , 2h , ar - h ) , 7.81 ( d , 1h , ar - h ) , 7.94 ( d , 2h , ar - h ) , 10.4 ( s , 1h , conh ) , 12.6 ( s , 1h , indole ) , 14.6 ( s , 1h , oh phenolic ) . ms : m / z 315 ( m ) , 317 ( m ) . 
 ( 5 ) 2-hydroxy - n-(5-bromo-2-oxoindolin-3-ylidene)benzohydrazide ( viid ) . ir ( kbr ) cm : 3236.95 ( nh ) , 1676.20 ( c = o ) , 1628.43 ( c = o ) , ] : 7.02 ( s , 1h , ar - h ) , 7.42 ( d , 1h , ar - h ) , 7.62 ( t , 2h , ar - h ) , 7.84 ( d , 1h , ar - h ) , 7.89 ( d , 2h , ar - h ) , 11.4 ( s , 1h , conh ) , 12.2 ( s , 1h , indole nh ) , 14.1 ( s , 1h , oh phenolic ) . ms : m / z 360 ( m ) . 
 ( 6 ) 3-(2-(2-hydroxybenzoyl)hydrazono)-2-oxoindoline-5-carboxylic acid ( 
 viie 
 ) . ir ( kbr ) cm : 3289.98 ( nh ) , 1702.00 ( c = o ) , 1676.94 ( c = o ) , h - nmr ( dmso - d6 ) : 7.12 ( s , 1h , ar - h ) , 7.32 ( d , 1h , ar - h ) , 7.42 ( t , 2h , arh ) , 7.91 ( d , 1h , arh ) , 7.94 ( d , 2h , arh ) , 10.1 ( s , 1h , conh ) , 12.6 ( s , 1h , indole nh ) , 14.0 ( s , 1h , oh phenolic ) , 14.8 ( s , 1h , carboxylic ) . ms : m / z 326 ( m ) . ir ( kbr ) cm : 3160 ( nh ) , 3122 ( c - h ) , 1731.90 ( c = o ) , 1665.9 ( c = o ) , 1542.60 ( c = h - nmr ( dmso - d6 ) :   [ ppm ] , 3.8 ( s , 3h , ch3 ) , 6.957.82 ( m , 8h , ar - h ) , 11.9 ( s , 1h , conh ) , 13.8 ( s , 1h , oh phenolic ) . ms : m / z 296 ( m ) . ir ( kbr ) cm : 3260 ( nh ) , 3068 ( c - h ) , 1791.90 ( c = o ) , 1685.47 ( c = o ) , 1542.60 ( c h nmr ( dmso - d6 ) :   [ ppm ] , 4.7 ( s , 3h , ch3 ) , 6.827.80 ( m , 8h , ar - h ) , 11.5 ( s , 1h , conh ) , 13.4 ( s , 1h , oh phenolic ) . ms : m / z 323 ( m ) . ir ( kbr ) cm : 3219.98 ( nh ) , 1722 ( c = o ) , 1677.94 ( c = o ) , 15249.67 ( c = n ) ; h nmr ( 300  mhz , dmso - cdcl3 ) : 7.02 ( d , 1h , ar - h ) , 7.22 ( d , 1h , ar - h ) 7.4 ( t , 2h , ar - h ) , 7.87 ( t , 1h , arh ) , 7.9 ( d , 2h , arh ) , 11.1 ( s , 1h , conh ) , 12.0 ( s , 1h , indole nh ) , 14.2 ( s , 1h , oh phenolic ) , 14.5 ( s , 1h , carboxylic ) ; ms : m / z 326 ( m ) . ir ( kbr ) cm : 3170.58 ( nh ) , 1724.26 ( c = o ) , 1675.74 ( c = o ) , 1528.08 ( c = n ) , 762 ( c - cl ) ; h - nmr ( 300  mhz , dmso - cdcl3 ) :   [ ppm ] : 6.92 ( d , 1h , arh ) , 7.21 ( d , 1h , arh ) , 7.52 ( t , 2h , arh ) , 7.81 ( t , 1h , arh ) , 7.94 ( d , 2h , arh ) , 10.4 ( s , 1h , conh ) , 12.6 ( s , 1h , indole nh ) , 14.6 ( s , 1h , oh phenolic ) ; ms : m / z 315 ( m ) , 317 ( m ) . 
 ( 11 ) 2-hydroxy - n-(7-bromo-2-oxoindolin-3-ylidene)benzohydrazide ( viij ) . ir ( kbr ) cm : 3236 ( nh ) , 1674 ( c = o ) , 1628 ( c = o ) , 1542 , ( c = n ) ; h - nmr ( 300  mhz , dmso ) :   [ ppm ] : 7.20 ( d , 1h , arh ) , 7.40 ( d , 1h , arh ) , 7.60 ( t , 2h , arh ) , 7.84 ( m , 1h , arh ) , 7.89 ( d , 2h , arh ) , 11.4 ( s , 1h , conh ) , 12.8 ( s , 1h , indole nh ) , 14.12 ( s , 1h , oh phenolic ) ; ms : m / z 345 ( m ) . the study was conducted in accordance with oecd guidelines ( testing of chemical number 423 ) . the test compounds , suspended in normal saline , were administered intraperitoneally , in doses of 10  mg to 1000  mg per kg ( b.w . ) . the animals were observed for 48 hours from the time of administration of test compound , in a dose of 1000  mg / kg produce mortality ( ld50 ) . soon , tenth of the ld50 , that is , 100  mg / kg ( ed50 ) , was selected as a dose for an anti - inflammatory . in carrageenan model albino rats of all groups were treated with subcutaneous injection of 0.1  ml of 1% w / v solution of carrageenan into the subplantar region of the right hind paw . the paw was marked with permanent marker at the planter region where the paw volume was to be measured . the indomethacin 10  mg / kg and test compounds 100  mg / kg were suspended in 0.3% sodium carboxymethyl cellulose . one hour after the injection of carrageenan in subplantar region of right paw . mean normal paw volume was measured 30 min prior to carrageenan injection by using plethysmometer . mean increase in the paw volume for control group ( after carrageenan injection ) and test group was measured at 1  hr , 2  hr , 3  hr , and 4  hr . the mean percent inhibition of indomethacin and tested compounds at 10  mg  kg concentrations was compared with control using repeated measures anova with dunnet 's test . the data obtained is expressed as mean standard error of mean ( sem ) : ( 1)%  inhibition = mean edema of control group  mean edema of testmean edema of control group100 . grip docking employs the plp scoring function in a novel way for fast and accurate capturing of ligand receptor interactions in the active site of proteins . for this purpose , vlife mds 4.3 ( http://www.vlifesciences.com ) incorporates the piecewise linear pairwise potential ( plp ) [ 30 , 31 ] scoring function in grip docking method that includes interactions like hydrogen bonding , steric interactions , vanderwaal 's interactions , hydrophobic interactions , and electrostatic interactions . the plp score is designed to enable flexible docking of ligands to perform a full conformational and positional search within a rigid binding site . the functional form of the ligand - protein interaction energy in plp scoring function is shown in figure 1 . protein potential are as follows : for steric interactions , a = 3.4 , b = 3.6 , c = 4.5 , d = 5.5 , e = 0.4 , and f = 20.0 ; for hydrogen bond interactions , a = 2.3 , b = 2.6 , c = 3.1 , d = 3.4 , e = 2.0 , and f = 20.0 . the units of a , b , c , and d are angstrom ; for e and f the units are arbitrary energy units . the x - ray crystal structure of cox-1 and cox-2 ( pdb codes : 3n8y and 3ln1 , resp . ) was downloaded from the protein data bank protein complexed with indomethacin and celecoxib , respectively . water molecules were removed and hydrogen was added to the crystal structure of protein , refined by completing the incomplete residues and missing residues . the side chain hydrogen was then added to the crystal structure and its positions were optimized up to the rms gradient 1 by aggregating the other part of the receptor . the 2d structure of test compounds built and then converted into the 3d with the help of vlife mds 4.3 software . the 3d structures were then energetically minimized up to the rms gradient of 0.01 using merck molecular force field ( mmff ) . all optimized ligands were docked into active site of cox proteins ; the active site with the largest surface area is selected for docking . the parameters fixed for docking simulation were like this number of placements : 30 , rotation angle : 30 , exhaustive method , scoring function : plp score . by rotation angle , , the method will check all the 30 possible placements into the active site pocket and results in few best placements out of 30 . for each ligand , all the conformers with their best placements and their dock scores will be saved in output folder . the method also highlights the best placement of best conformer of one particular ligand which is having best ( minimum ) plp score . after completion of the docking process , the minimum interaction energy between each ligand and cox proteins for the best ligand pose inside the receptor cavity was obtained as the plp score . some of the new isatin derivatives were obtained by condensation of 2-hydroxybenzohydrazide with various isatins in presence of glacial acetic acid depicted in scheme 2 . all synthesized compounds were evaluated for in vivo anti - inflammatory activity at the dose of 100  mg / kg by carrageenan induced paw edema method . from the data it was revealed that all tested compounds significantly reduced carrageenan induced edema and the results ( r1 = 5-cl ) and viid ( r1 = 5-br ) may be considered as potent anti - inflammatory agents and comparable with standard anti - inflammatory drug , indomethacin . interestingly , derivatives with halo substitution at position c-5 and position c-7 of the isatin , compounds viic ( r1 = 5-cl ) , viid ( r1 = 5-br ) , viii ( r1 = 7-cl ) , and viij ( r1= 7-br ) exhibited substantial activity with 65% , 63% , 62% , and 60% edema reduction . on the other hand , compounds with alkyl and acetyl substitution at position n-1 of the isatin and compounds viif ( r2 = ch3 ) , viig ( r2 = coch3 ) are next in the order of activity with 57.58% of edema reduction , whereas compounds viib ( r1 = 5-ch3 ) , viia ( r1 = r2 = h ) , viie , and viih ( 5&7 cooh ) exhibited 52% , 54% , 55% , and 49% of oedema reduction , respectively . compounds having substitution with electron withdrawing groups at c-5 and c-7 position of isatin moieties showed more anti - inflammatory activity whereas the other substitutions on various positions of isatin nucleus are next in the order of activity . the docking study was performed using the vlife mds 4.3 . all the ten 2-hydroxy - n-(2-oxo-1,2-dihydro-3h - indol-3-ylidene)benzohydrazide derivatives were docked into the active site of the enzymes cox-1 ( pdb i d : 3n8y ) and cox-2 ( pdb i d : 3ln1 ) which showed better docking scores than the reference compounds indomethacin and celecoxib and the results were presented in table 3 . compounds viie and viih showed good docking scores 68.73 , 43.30 with cox-2 enzyme even though they were not considered good anti - inflammatory agents because they are also having good dock scores 26.79 , 27.28 with cox-1 enzymes so these may be the causes of gastric irritation . the compounds viic , viid , and viif are considered as good anti - inflammatory agents because of good binding scores 57.27 , 62.02 , and 58.18 into the active site of cox-2 and least dock scores 8.03 , 9.17 , and 8.94 on cox-1 enzymes . compounds viic and viid showed hydrogen bond interaction with gln-275 and aromatic interaction with his-193 and compound viif showed hydrophobic interactions with lys197 , asn217 , his218 , gly221 , gly274 , glu222 , and gln275 with cox-2 enzyme whereas the reference compounds showed hydrophobic , van der waals interactions presented in table 4 , figures 27 . we described herein the synthesis and characterization of a novel 2-hydroxy - n-(2-oxo-1,2-dihydro-3h - indol-3-ylidene)benzohydrazide derivatives . all the synthesized compounds are subjected for the evaluation of in vivo anti - inflammatory activity at dose 100  mg / kg . among all , the compounds viic and viid had good anti - inflammatory activity . electron withdrawing groups seemed to be necessary factors in providing higher anti - inflammatory activity . the molecular docking studies further help in understanding the various interactions between the ligands and enzyme active sites in detail and thereby help to design novel potent inhibitor . the docking experiments were carried out for all the synthesized compounds on cox-1 and cox-2 enzymes and compared the docking score with reference compounds indomethacin and celecoxib . the compounds viic , viid , and viif showed higher binding score , which are further attributed to the in vivo activity of these compounds .
a novel synthesis of 2-hydroxy - n-(2-oxoindolin-3-ylidene ) benzohydrazide derivatives was synthesized by the condensation of 2-hydroxybenzohydrazide with substituted isatins . 
 the synthesized compounds were characterized by ft - ir , 1h - nmr , and mass spectral data . 
 further , the compounds were screened for in vivo anti - inflammatory activity by carrageenan induced paw edema method . 
 the tested compounds have shown mild - to - moderate anti - inflammatory activity . 
 the compounds viic and viid exhibited 65% and 63% of paw edema reduction , respectively . 
 the molecular docking studies were also carried out into the active site of cox-1 and cox-2 enzymes ( pdb i d : 3n8y , 3ln1 , resp . ) using vlife mds 4.3 . 
 the compounds viic , viid , and viif exhibited good docking scores of 57.27 , 62.02 , and 58.18 onto the active site of cox-2 and least dock scores of 8.03 , 9.17 , and 8.94 on cox-1 enzymes and were comparable with standard cox-2 inhibitor celecoxib . 
 a significant correlation was observed between the in silico and the in vivo studies . 
 the anti - inflammatory and docking results highlight the fact that the synthesized compounds viic , viid , and viif could be considered as possible hit as therapeutic agents .