cancer is a leading cause of death worldwide , accounting for 7.6 million deaths ( around 13% of all deaths ) in 2008 . deaths from cancer worldwide are projected to continue rising , with an estimated 13.1 million deaths in 2030 . cancer causes 1 in 8 deaths worldwide and is rapidly becoming a global pandemic . according to the international agency for research on cancer , there were 12.7 million new cancer cases in 2008 . if rates do nt change , the global cancer burden is expected to nearly double to 21.4 million cases and 13.5 million deaths by 2030 . urease is an enzyme which produces numerous amounts of ammonia in the helicobacter pylori by catalysis the hydrolyzing of urea into carbon dioxide and ammonia . the produced ammonia makes the growth and survival of bacteria in highly acidic media of gastric wall , by increasing the ph of environment ( 1 ) . hence , urease activity plays an important role in gastric diseases , formation of kidney stones ( 2 ) and development of urolithiasis , pyelonephritis and hepatic encephalopathy ( 3 ) . discovering a new agent for inhibition of urease and potent anti - ulcer drugs are of special demand because h. pylori has become resistant to many antibiotics ( 4 ) . pyridopyrimidine derivatives have received ample attention in pharmaceutical and agrochemical sectors ( 5 ) due to their prominent biological applications such as antibacterial ( 6 ) , antiallergic ( 7 ) , cns stimulants ( 8) , inhibitors of adenosine kinase enzyme ( 9 ) or dihydrofolate reductase inhibitors ( 10 ) . preparation of compounds with promising drug like activity without tedious and time consuming purification is in great interest . recently using new catalysts opened exciting opportunities in the field of synthesizing medicinal compounds or green chemistry by minimizing waste , cost , and time ( 11 ) . the catalytic systems offer the opportunity of synthesizing molecules via novel routes , which may be difficult or impossible with traditional methods and also increase the yield of the resultant compounds .   as a part of our program to develop catalytic and environmentally friendly methods in the synthesis of biologically active heterocycles ( 12 , 13 ) , we explored novel synthetic strategies for the synthesis of pyridopyrimidine derivatives ( 14 ) as urease inhibitor by using biacidic carbon catalyst . gc - mass analysis was performed on a gc - mass model : 5973 network mass selective detector , gc 6890 agilent . ir spectra were recorded from kbr disk using a ft - ir bruker tensor 27 instrument . melting points were measured using the capillary tube method with an electro thermal 9200 apparatus . the h nmr ( 400 mhz ) was run on a bruker dpx , 250 mhz . synthesis of the catalyst 
  the biacidic carbon catalyst is prepared as the following procedure ( 15 ) . the mixture of glucose ( 10 g ) , citric acid ( 5 g ) , hydroxyethylsulfonic acid ( 3 g ) , and deionized water ( 80 ml ) was placed in a 100 ml teflon - lined stainless steel autoclave , which was heated in an oven at 180 c for 4 h. the resulting products were filtered , washed with water and methanol , and dried in a vacuum oven at 100 c for 4 h to give biacidic carbonaceous material ( 2.3 g ) as a black powder . 
 general procedure for preparation of spiro pyridopyrimidine - indolines 
 4a - e 
  a mixture of 1,3-indandione ( 1 mmol ) , 6-aminouracil ( 1 mmol ) , isatin derivatives ( 1 mmol ) and catalytic amount of biacidic carbon ( 20 mg ) in ethanol ( 5 ml ) was refluxed for appropriate time ( table 2 ) . after the completion of reaction as indicated by tlc , the catalyst was recovered by filtering and washing with acetone , and then dried in an oven at 80 c for about 1 h followed by crystallization using ethanol and water . 5'-chloro-1h - spiro[indeno[1,2-b ] pyrido [ 2,3-d]pyrimidine-5,3'-indoline]-2,2',4,6 ( 3'h,10'h)-tetraone ( 4d ) 
  orange powder , mp 315 - 319 c , ir ( kbr ) ( max /cm ) : 3549 , 2800 , 1720 , 1701 , 1666 , 1617 . ms , m / z : 418 ( m ) , 341 , 293 , 271 , 255 , 243 , 229 , 195 , 161 , 75 , 51 . h nmr ( 400 mhz , dmso - d6 ) : h ( ppm ) 6.77 - 7.96 ( 7h , m , h - ar ) , 10.55 - 10.58 ( 3h , s , 3nh ) , 10.99 ( 1h , s , nh ) . 8',8'-dimethyl-8',9'-dihydro-1'h - spiro [ indoline-3,5'-pyrimido[4,5-b]quinoline]-2,2',4 ' , 6'(3'h,7'h,10'h)-tetraone ( 7a ) 
  pale orange powder , mp 310 - 312 c , ir ( kbr ) ( max /cm ) : 3402 , 3126 , 2955 , 1720 , 1673 , 1613 , 1400 . ms , m / z : 378 ( m ) , 320 , 308 , 307 , 292 . h nmr ( 400 mhz , dmso - d6 ) : h ( ppm ) 1.06 ( 6h , 2ch3 ) , 6.72 - 7.44 ( 4h , m , h - ar ) , 8.88 ( 1h , s , nh ) , 10.11 ( 2h , s , 2nh ) , 10.29 ( 1h , s , nh ) . 1'h - spiro[indoline-3,5'-pyrido[2,3-d:6,5-d']dipyrimidine-2,2',4',6',8'(3'h,7'h,9'h,10'h)-pentaone ( 7b ) 
  pale orange powder , mp 333 - 334 c , ir ( kbr ) ( max /cm ) : 3435 , 3200 , 2806 , 1732 , 1649 , 1546 . ms , m / z : 366 ( m ) , 352 , 295 , 283 , 252 , 43 . h nmr ( 400 mhz , dmso - d6 ) : h ( ppm ) 6.18 - 7.17 ( 4h , dd , h - ar ) , 7.95 ( 1h , s , nh ) , 9.09 ( 1h , s , nh ) , 10.08 ( 1h , s , nh ) , 10.60 ( 1h , s , nh ) . 1,3-dimethyl-1'h - spiro[indoline-3,5'-pyrido[2,3-d:6,5-d']dipyrimidine- 2,2',4',6',8 ' ( 3'h,7'h,9'h,10'h)-pentaone ( 7c ) 
  pale orange powder , mp 327 - 330 c , ir ( kbr ) ( max /cm ) : 3489 , 3184 , 1772 , 1702 , 1664 , 1501 . ms , m / z : 394 ( m ) , 367 , 352 , 284 . h nmr ( 400 mhz , dmso - d6 ) : h ( ppm ) 3.09 ( 3h , s , ch3 ) , 3.37 ( 3h , s , ch3 ) , 6.82 - 7.28 ( 4h , dd , h - ar ) , 9.32 ( 1h , s , nh ) , 10.46 ( 1h , s , nh ) , 11.06 ( 1h , s , nh ) , 11.95 ( 1h , s , nh ) . urease inhibition assay 
  in this work , the total released ammonia from urease activity has been determined by the berthelot colorimetric method ( 16 ) . in brief , solution composed of 800 l urea as substrate and 15 l enzyme and inhibitors with various concentrations were incubated in water bath at 37 c for 30 minutes . fresh phenol solution ( solution a ) was prepared by dissolving 1000 mg of phenol and 7 mg of sodium nitroprusside in 100 ml of deionized water and fresh solution b was prepared by dissolving 500 mg sodium hydroxide pellets and adding 840 l of sodium hypochlorite solution ( 11 - 14% ) to 100 ml of water . 100 l sample from incubated solution was transferred to microplate well and followed by adding 500 l of solution a and b. the mixture was stirred and kept at 37 c for 30 minutes . after that , the plate was read at 625 nm using the biotech synergy h1 micropalte reader . docking simulation procedure 
  all of the processes include of preparation of input files ( receptor and ligand preparation ) and docking parameters were similar to our previous study ( 17 ) . briefly , to validate observed activity , molecular docking was performed using auto dock 4.2 using lamarckian genetic algorithm ( 18 ) on pdb structure 3la4 ( http://rcsb.org ) with resolution of 2  using default docking parameters values . the graphical user interface auto dock tools 1.4.5 ( 19 ) was employed to set up the enzyme and ligand input files as reported previously ( 20 ) . ligand scout 3.0 program was used for demonstration ligand - enzyme interaction ( 21 ) . the titled compounds were synthesized in a mild , convenient and simple procedure using a three - component reaction ( scheme 1 ) . in this method indandione 1 , various isatin 2a - e and 6-aminouracil 3 were mixed and refluxed in ethanol for a short periods of time ( 10 - 30 min , table 1 ) in presence of nano biacidic carbon catalyst yielding pyridopyrimidine derivatives 4a - e ( table 2 ) in high yields ( figure 1 , table 2 ) . synthesis of spiro indeno[1,2-b]pyrido[2,3-d ] pyrimidine-5,3-indolines using biacidic carbon as catalyst biacidic carbon catalyzed the synthesis of spiro pyridopyrimidine to optimize the reaction procedure , different conditions were applied as shown in table 1 . the best results were obtained under a solvent - free condition at 130 c ( table 1 , entry 5 ) in etoh as a solvent ( table 1 , entry 1 ) and used for preparation of other derivatives . optimization of the reaction conditions in the synthesis of 4a using biacidic carbon as catalyst . with these optimized conditions , the scope of the reaction was inspected using various isatin as the acceptors . as can be seen from the data reported in table 2 , the use of 5-chloroisatin , resulted in improved condensation yield ( 95% ) but the yield could be decrease by using other isatin derivatives such as n - benzylisatin , 5-bromoistain and n - methylisatin ( 70 - 76% ) . it s noteworthy that in the all following cases the reaction is carried out in a very short reaction time ( 10 - 30 min ) . synthesis of 4 catalyzed by biacidic carbon the mechanism of the reaction and formation of compound 4a can be explained by the condensation , addition , cyclization and dehydration reactions which illustrated in scheme 2 . first , according to acidic property of the catalyst , the carbonyl group of isatin 2a was protonated , which then condenses with enamine group of 6-aminouracil 3 to afford 5 . the proposed reaction mechanism this method also works well for the other -diketone compounds 6a - c instead of indandione under the similar reaction condition ( scheme 3 ) . the desired products 7a - c were obtained in good to excellent yields ( 50 - 90% ) and at the moderate reaction times ( 3 - 4 h ) as shown in table 3 . extension of this method using different -diketones in the optimized conditions exploration of the reaction scope in relation to the -diketone components using biacidic carbon as catalyst 
 biological activity 
  all synthetic compounds were tested against urease inhibitory activity ( table 4 ) and shows good urease inhibitory potency in the range of ic50= 1.94 - 14.46 m in comparison to hydroxyurea as a standard inhibitor ( m ) . to confirm obtained results and extract a preliminary structure activity relationships various parameters such as log p , mr , clog p and cmr were calculated ( table 4 ) using chemoffice packages ( www . on the other hand , the docking studies were applied for all synthesis compounds ( 4a-7c ) . correlation between urease inhibitory activity and physiochemical properties of tested compounds reveals that compounds with lipophilicity values in the range of log p = - 0.44 to 0.61 , shows good urease inhibitory potency ( table 4 ) . based on this deduction , compound 7b with lipophilic character was less active than 4a ( table 4 ) . on the other hand , molar refractivity ( mr ) which mentioned in table 4 , has a prominent influence on the activity whereas the compound 4c with mr= 131.16 hasnt any inhibition activity because it could nt provide a good interaction with the active site . according to our previous studies , the compounds with methyl bearing substitute on the barbituric acid ring not only havent inhibitory activity but also activate the urease enzyme ( 22 ) . this could be explained by existence of two barbiturate rings in its structure , which one of barbiturate acts as an activator and the other is inhibitor thus results inactivation of compound . meanwhile , the compound 7b which hasnt any substitution on the barbiturate ring shows the inhibitory potency with ic50= 14.46 m . in compound 7a compares to 4a the indenone ring is replaced by 5,5-dimethylcyclohex-2-enone and this replacement hasnt any significant changes to the inhibitory activity ( ic50   4a/7a is 1.94/2.81 ) . changing the substitution on the oxindole ring also causes considerable alteration in the inhibitory activity since the replacement of h in 4a with halogen ( 5-cl and 5-br , 4d and 4e , respectively ) decreases the inhibitory activity as a result of the steric hindrance or increase in lipophilicity due to size of halogen groups . urease inhbitory activity of spiro pyridopyrimidine compounds our results reveal that changing the substitution on nitrogen atom of oxindole ring alters the inhibition value for instance conversion of nh to n - me ( 4b ) causes to decrease activity and conversion of nh to n - bn ( 4c ) causes the totally abolishing of the inhibition . it seems that hydrogen binding ability of nitrogen of isatin rings is necessary for higher activity and increasing the sizes of substituted groups will decreases binding of molecule to active site . herein , the molecular docking was used in order to predict the orientation of spiro indeno[1,2-b]pyrido[2,3-d]pyrimidine-5,3-indolines family in the urease binding pocket . in this way the planner structure of this ligand lets it to sit deeply inside of active site cleft . moreover , the suitable positions of chemical groups led to favorable interactions with the receptor . the other carbonyl moiety comprises a hba with his545 alongside a weak ionic interaction with the ni atom . the two nitrogen atoms in the barbituric ring donate their hydrogen atoms in hydrogen bonds with his492 and asp633 residues . thus , the barbituric cycle which is included in all synthesized compounds is responsible for four hydrogen bonds with urease protein . besides , the hydrogen of nitrogen atom in 9 position of the ligand is involved a hbd toward asp494 . the last hydrogen bond is seen between cme592 and carbonyl group connected to the cyclopentadiene moiety . in addition , 4a can be more stabilized due to presence of - interaction with his593 . optimal binding model for compound 4a into active site of jack - bean urease enzyme docked by autodock 4.2 ( a ) , interaction of ligand with key residues are shown in 3d and 2d models ( b , c ) . the yellow sphere indicates hydrophobic region , the red line shows hydrogen bond acceptor , the green line point out hydrogen bond donor features and purple cycle is - interaction as a conclusion the spiro indenopyridopyrimidine derivatives are valuable compounds which show inhibition potency through urease and offer the opportunity of synthesizing the new molecules of this group . in the continuous of other studies by this investigation the key role of barbiturate ring in preparation of inhibitory potent compounds was either confirmed .
new series of spiro indeno[1,2-b]pyrido[2,3-d]pyrimidine-5,3-indolines as new urease inhibitors were synthesized by the catalytic procedure in high yield and short reaction time . in this method , 
 biacidic carbon was prepared as a novel heterogeneous acid and was subsequently used as an efficient catalyst . 
 the inhibitory activities of synthesized compounds were tested against jack bean urease using berthelot colorimetric assay and docking simulation using autodock 4.2 . 
 the compound 4a with ic50 = 1.94 m has the most inhibitor activity in this study . 
 other derivatives such as 4b , 4d , 4e and 7a were found to be more potent urease inhibitors than the standard inhibitor hydroxyurea , yielding ic50 values of 4.35 , 5.557 , 7.44 , 2.81 and 14.46 m , respectively ( ic50 of hydroxyurea = 100 m ) .