Tandem Reactions Using Nitrile Imines : Synthesis of Some Novel Heterocyclic Compounds with Expected Biological Activity

New functionalized 7,9-dimethylpyrimido[4,5-d][1,2,4]triazolo[4,3-a]pyrimidine5,6,8(1H,7H,9H)-trione derivatives were synthesized via reaction of the hydrazonoyl halides with 7,8-dihydro-1,3-dimethyl-7-thioxopyrimido[4,5-d]pyrimidine-2,4,5(1H,3H,6H)trione. The biological activity of the products has been evaluated. The mechanism and the regioselectivity of the studied reactions have been discussed.


INTROdUCTION
Chemists are constantly working to discover new and improved reactions.One of the primary motivating goals of this research is the development of cleaner, more efficient transformations to shorten syntheses and save money on chemicals.The strategy of using reactions in tandem is also aimed at shortening syntheses.Tandem reactions are commonly referred to under the nebulous phrase "multistep one-pot reactions". 1 On cost and amounts of reagents, solvents, and reduce the amount of waste that is generated.So, our aim is to use such idea of tandem reactions of organic compounds to synthesize new heterocycles with expected biological activity in short steps by using nitrile imines via 1,3-dipolar cycloaddition.basically a nitrile imine 1 is a flexible system of three atoms over which four pi-electrons are distributed.Although one can write seven possible resonance structures for such a system, the 1,3dipolar sextet structure 1A with its complementary nucleophilic and electrophilic centers will be used throughout this article, although theoretical calculations have indicated that all the octet zwitterionic structure 1B is the most stable contributor to the resonance hybrid. 2

R -C
As very authorative review of the chemistry of the precursors of nitriliminesas well as generation of nitrile imines was reported in 2010 by Shawali. 2 Pyridopyrimidines have good biological importance. 3ecently some fused heterocyclic compounds containing nitrogen atom show a wide range of pharmacological activities.Pyrimidopyrimidines are annelated to uracils that have considerable interest in recent years. 4,5Derivatives of pyrimidopyrimidine display potent inhibitory properties regarding tyrosine kinase domain of epidermalgrowth factor receptor. 6yrimido [4,5-d]pyrimidine fused system represent attractive pharmacological applications such as antitumor, 7 antiviral, 8 antioxidant, 9 antifungal 10 and hepatoprotective activities. 11Pyrimidopyrimidines have a ring system that can be found marine derived natural products such as crambescidin 23 alkaloid.Keeping this importance in mind herein the reactions of 7-thioxopyrimdo [4,5-d]pyrimidin-2,4,5-trione with hydrazonoyl halides will be reported.

Reactions of 4 with active chloromethylene compounds (10b,d,f)
To a solution of 4 (2.4 g, 0.01 mol) in chloroform was added triethylamine (1.4 ml, 0.01 mol) and the mixture was stirred for 10 min at room temperature.To the resulting clear solution was added active chloromethylene derivative (10) (0.01 mol) dropwise while stirring the reaction mixture.After complete addition, the reaction mixture was stirred for further 24 h at room temperature.The solid that precipitated was filtered off, washed with water, dried and finally crystallized from dioxane to give pure 11.The compounds 11b,d,fprepared are listed below together with their physical constants.

Synthesis of the thiohydrazonates (7)
To a solution of each of 11b, 11d and 11f (10 mmol) in ethanol (40 ml) was added sodium acetate trihydrate (1.38 g, 10 mmol) and the mixture was cooled to 0-5 %C in an ice bath.To the resulting cold solution was added portionwise a cold solution of benzenediazonium chloride, prepared by diazotizing aniline (10 mmol) dissolved in hydrochloric acid (6 M, 6 ml) with a solution of sodium nitrite (0.7 g, 10 mmol) in water (10 ml).After complete addition of the diazonium salt, the reaction mixture was stirred for further 12 h at room temperature.The solid precipitated was filtered off, washed with water, dried and crystallized from dimethyformamide/EtOH (1:1 v:v) to give the respective pure 7b, 7d and 7f.

Method B (for 5b, 5d and 5f)
To a stirred ethanolic sodium ethoxide solution, prepared from sodium metal (0.23 g, 10 mg atom) and absolute ethanol (20 ml), was added each of the compound 7b, 7d and 7f (10 mmol) and the reaction mixture was stirred at room temperature for 15 h, during which the starting reactants 7 dissolved and the crude product preciptated.The latter was filtered, washed with water, dried and finally crystallized from the proper solvent to give a product identified as 5b, 5d and 5f, respectively.The latter products proved to be identical in all respects (mp, mixed mp, IR) with that obtained from 4 and the respective hydrazonoyl halides 1.Their yields were 60-70%.[4,5-d

Antimicrobial assay
Cultures of four fungal species namely Aspergillusfumigatus AF, Penicilliumitalicum pI,Syncephalastrumracemosum SR and Candida albicans.CA as well as four bacterial species namely Staphylococcus aureus SA, Pseudomonas aeruginosa pA, Bacillus subtilis BS and Escherichia coli EC were used to investigate the antimicrobial activity of the compounds 5a-g.The antimicrobial activity was assayed biologically using the diffusion plate technique.The latter technique was carried out by pouring a spore suspension of the fungal species (one ml of sterile water contains approximately 108 conidia) or spreading bacterial suspension over a solidified malt agar medium.The layer is allowed to set for 30 min.A solution of the test compound 5a-g (1.0ug/ml) in dimethylformamide was placed onto sterile 5mm filter paper discs and allowed to dry, then the discs were placed on the centre of the malt agar plate and incubated at optimum incubation temperature 28 ± 2 0 C. Test organism growth may be affected by the inhibitory action of the test compound, so a clear zone around the disc appears as an indication of the inhibition of test organism growth.The size of the clearing zone is proportional to the inhibitory action of the compound.The fungicide Terbinafin and the bactericide Chloramphenicol were used as standards under the same conditions.Measurements were considered after 72 h for fungi and 24 h for bacteria.The results are summarized in table 1.
The formation of compounds 5 from the thione 2 and hydrazonoyl halides 1 could beaccounted for by one of the two pathways indicated in scheme 2. As depicted in this scheme,it is suggested that the studied reactions started with the hydrazonoylation of 2 to give therespective thiohydrazonate esters 7.This is followed by Smiles type rearrangement 17,18 of 7 to form the respective thiohydrazides 8, which in turn underwent cyclization to give 5 as end products (route a, Scheme 2).It seems that both intermediates 7 and 8 are consumed,under the employed reaction conditions as soon as they are formed since all attempts to isolate them failed.Alternatively, reaction of the thione 2 with hydrazonoyl halides 1 starts with the formation of the amidrazone intermediates 9 which cyclize to give 5 (route b, Scheme 1).0][21][22] Furthermore the suggested route a and the involvement of 7 and 8 as intermediates in the formation of 4 by this route were evidenced by alternate synthesis of 5b, 5d and 5f (Scheme 3).Thus, treatment of 2 with each of 3-chloro-2,4-pentanedione (10b), ethyl a-chloroacetoacetate (10d) and á-chloroacetoacetanilide(10f) in chloroform in the presence of triethylamine afforded the respective substituted products 11b, 11d and 11f.Coupling of each of these compounds with benzenediazonium chloride in ethanol in thepresence of sodium acetate yielded the thiohydrazonates 7b, 7d and 7f, respectively (Scheme 2) via Japp-Klingemann cleavage of the acetyl group. 22Treatment of the products 7 with sodium ethoxide in ethanol afforded the respective compounds 5b, 5d and 5f identical in all respects with those obtained from reactions of 2 with each of 1b, 1d and 1f, respectively.This finding indicates that 7 and 8 are intermediates in the studied reactionsof 1 with 3 and they are consumed as soon as they are formed under the employed reaction conditions.Finally, the suggestion that the site of cyclization of the thiohydrazide intermediates 8 involves N-3 rather than N-1 to give 5is consistent with literature reports.[25] In conclusion, the studied reactions of hydrazonoyl halides 1 with the thione 2 are both site and regioselective and lead to the title ring system.

Antimicrobial activity
The compounds 5a-g were tested for their antimicrobial activities against four fungal speciesnamely Aspergillusfumigatus AF, Penicilliumitalicum pI, Syncephalastrumracemosum SR and Candida albicans CA as well as four bacteria species namely Staphylococcus aureus SA, Pseudomonas aeruginosa pA, Bacillus subtilis BS and Escherichia coli EC.The organisms were tested against the activity of solutions of concentration of 1.0mg/ml of each compoundand using inhibition zone diameter in cm (IZD) as criterion for the antimicrobial activity.Terbinafin as an antifungal agent and Chloramphenicol as an antibacterial agent were used asreferences to evaluate the potency of the tested compounds under the same  a Chloramphenicol as standard antibacterial agent, bTerbinfin as standard antifungal agent, ++ inhibition value 0.6-1.0cm; +, inhibition value 0.1-0.5 cm beyond control; 0, no inhibition detected.
conditions.Theresults are depicted in table 1.The results reveal that compounds 5a, 5b, 5d and 5g exhibited thehighest degree of inhibition against the tested organisms AF and SA, respectively, their activity is similarto that of the standard antifungal and antibacterial agents used.All other compounds eitherexhibit no activity or being less active against the tested species.

Chart 1 :
13 C comparison between different triazole derivatives