Synthesis , Chemical Reactivity and Fungicidal Activity of Pyrido [ 1 , 2-b ] [ 1 , 2 , 4 ] triazine Derivatives

A síntese de alguns novos derivados de pirido[1,2-b][1,2,4]triazinas (2-12) foi obtida através da ciclocondensação de 4-aril-1,6-diamino-2-oxo-1,2-diidropiridina-3,5-dicarbonitrilas (1a,b) com compostos α,β-bifuncionais. Foram também preparadas pirido[1,2:2 ́,3 ́]triazino[5 ́,6 ́-f] triazinas (13-14). O comportamento de 1a,b frente às interações com indol-2,3-diona e seu análogo N-acetil foi estudado em diferentes condições de reação. As estruturas dos novos produtos foram deduzidas a partir de análise elementar e de dados espectroscópicos (UV, IR, H RMN, C RMN e espectrometria de massas). Os novos compostos sintetizados foram testados quanto à atividade antifungos.


Introduction
Polyfunctional pyridines are highly reactive intermediates that have been extensively used in heterocyclic synthesis. 1-Diamines are very active substrates for building of various heterocyclic systems 2 and are largely used in formation of complexes.3 In symmetrical diamines, the product will be the same irrespective of which amine participates first in the reaction.In the case of unsymmetrical diamines, the substituents influence the initial participation of a particular amino group in the reaction, resulting in chemoselective products. Te electron withdrawing/ donating nature of substituents in diamine influences the nucleophilicity of the amino group.On the other hand, 1,2,4-triazine derivatives exhibit marked biological and pharmacological effects and are use for the building fused, condensed and isolated heterobicyclic systems. 4On the basis of these observations, the objective of this work is the study of the chemical reactivity of 4-aryl-1,6diamino-2-oxo-1,2-dihydropyridine-3,5-dicarbonitriles (1a,b) and their use for preparation of nitrogen bridgehead pyrido [1,2-b]  [1,2,4]triazines in view of their antifungal activity.
Heterocyclization of diaminopyridones 1a,b with phenacyl bromide 7  KOH (Scheme 1).The IR spectra of 4a and 4b showed absorption bands at 3445 and 3320 cm -1 assigned to NH groups, respectively, while the IR spectrum of 5 showed two absorption bands at 3327 and 3243 cm -1 for two NH groups.
Some new 8-aryl-2,6-dioxo-1,2-dihydropyrido[1,2-b] [1,2,4]triazine-7,9-dicarbonitriles (6a-g) have been synthesized by cyclocondensation of compounds 1a,b with α-oxocarboxylic acids, namely pyruvic, α-oxobutyric, 4-chlorostyrylglyoxalic and phenoxypyruvic acids in refluxing glacial acetic acid (Scheme 2).It should be noted that this reaction occurred preferentially between the N 1 -amino group (N-NH 2 ) and the α-keto function of the electrophile to form a hydrazone intermediate, which underwent a cyclodehydration reaction between the other amino group at C 6 (C-NH 2 ) and the hydroxyl group of the acid function affording the target pyridotriazine derivatives 6a-g. 13C NMR spectra gave good evidence for the formation of compounds 6a-g.For examples, the 13 C NMR spectrum of compound 6a showed a new signal at 18.60 ppm characteristic for a methyl group in position 3.In the case of compound 6f the vinyl carbons were observed in the spectrum in their expected positions at 122.75 and 135.70 ppm for C α and C β , respectively.
Cyclic 1,2-bioxygen compounds were also used for building various fused heterocyclic systems. 8Thus, compounds 7a,b were prepared from refluxing compound 1a,b with diethyl oxalate in dry dioxane and/or with oxalyl chloride in warm DMF (Scheme 2).Some new pyridotriazines were obtained from cyclocondensation of 1,6-diaminopyridones 1a,b with α-dicarbonyl compounds.Thus, treatment of 1a with butane-2,3-dione in glacial acetic acid afforded 2,3-dimethylpyrido [ The course of the reactions of cyclic 1,2-bi-oxygen heterocyclic compounds with aromatic heterocyclic o-diamines was shown to depend on the reaction conditions, type of solvent and also the substituents in the diamino compounds. 10Thus, reaction of compounds 1a,b with indole-2,3-dione (isatine) in different media can yield different products.Treating 1a with indole-2,3dione in absolute ethanol and few drops of piperidine produced the Schiff base condensate, 6-amino-4-(3,4,5trimethoxyphenyl)-2-oxo-1-[2-oxo-1,2-dihydro-3-indolo-3-ylidine)amino]-1,2-dihydropyridine-3,5-dicarbonitrile (15).Alternatively, warming indole-2,3-dione with alcoholic NaOH solution yielded a 2-aminophenylglyoxalic acid that adds to 1,6-diaminopyridinone 1b to give the condensation product 16.Indolotriazinopyridines 17a,b were produced from ring closure reaction of compounds 15 and/or 16 in boiling glacial acetic acid in the presence of freshly fused sodium acetate.Acetylation of compound 17a by refluxing with acetic anhydride afforded the N-acetyl derivative 18 (Scheme 4).IR spectrum of 18 indicated that NH group disappeared and a new characteristic band at 1734 cm -1 appeared for the C=O of the acetyl group.The mass spectrum revealed the parent peak at m/z 494 which is coincident with the formula weight in agreement with the postulated structure.
N-acetylisatine showed a different behavior. 11Reaction of 1a with N-acetylisatine in absolute ethanol in the presence of few drops of piperidine led to 8- 9-dicarbonitrile (20) and not to the isomeric product 21 (Scheme 4).This reaction can be explained by an increase in the positive charge on the α-carbon atom in comparison to isatine itself due to the electron withdrawing acetyl group which facilitates the nucleophilic attack of more nucleophilic amino group (N-NH 2 ) at this position with concomitant opening of five membered ring.Apparently, the reaction can be claimed to proceed via intermediate 19, as also observed by previous workers in reaction with other diamines. 12However, this type of intermediate was reported to be unstable and not isolated.Vol. 20, No. 7, 2009

Fungicidal activity
Several new synthesized compounds were screened for their antifungal activities against two species of fungi, namely Alternaria alternata and Aspergillus niger using the disc diffusion method. 13The tested compounds were dissolved in DMF (which has no inhibitory activity) to get concentrations of 1 mg mL -1 solution.The antibiotic fluconazole was used as standard antifungal reference.The inhibition zones of microbial growth surrounding the filter paper disc (2.5 mm) were measured in millimeters at the end of an incubation period at 30 ○ C for 3 days.Inhibition of the organisms was evidenced by a clear zone surrounding each disk (Table 1).
All the tested compounds showed variable activities toward the two species of fungi, some of them comparable to standard fluconazole.The most active triazines were 2d, 3, 9b and 10.
From the results obtained, it is clear that increasing the percentage of nitrogen in the tested compounds led to higher effects toward the tested fungi.The antifungal effects decrease in the order of: 10 > 9b > 3 > 2d for higher activity and 14 > 6b > 4 > 8b > 5b >1a for moderate activity.The lower activity was observed by compound 17a (Table 1).

Experimental
Melting points are uncorrected and were recorded in open capillary tubes on a Stuart SMP3 melting point apparatus.Infrared spectra were recorded on FT-IR Bruker Vector 22 spectrophotometer using KBr wafer technique.UV absorption spectra (DMF) were recorded on a Jasco model (V-550) UV spectrophotometer. 1 H NMR and 13 C NMR spectra were measured on Gemini (200 MHz) spectrometer and Bruker (250 MHz) AC spectrometer using DMSO-d 6 as solvent and TMS (chemical shift in ppm) as an internal standard.Mass spectra were obtained using a Shimadzu GCMS qp 1000 ex instrument mass spectrometer (70 eV).

Compound 6f
Crystallized from ethanol as white crystals, yield 85%, mp > 300 ºC. 1  A mixture of 1a or 1b (0.01 mol) and diethyl oxalate (0.01 mol) in dry dioxane (50 mL) was refluxed for 4 h, after cooling the reaction mixture was concentrated.The solid obtained was filtered and crystallized to give 7a,b.

Method 2
Compound 1a or 1b (10 mmol) was dissolved in DMF (50 mL), oxalyl chloride (10 mmol) was added dropwise within 15 min.The reaction mixture was refluxed for 4 h, after cooling the reaction mixture was poured into ice.The solid obtained was filtered and crystallized to give 7a,b.

8-Aryl
A mixture of 1a or 1b (5 mmol) and benzoin (5 mmol) in glacial acetic acid (50 mL) and anhydrous sodium acetate (1 g) was refluxed for 8 h, after cooling the reaction mixture was poured onto ice.The solid obtained was filtered, washed several times with water and crystallized to give 10a,b.

Method 2
Compounds 10a or 10b (5 mmol) was dissolved in methanol (50 mL), ferric chloride (10%, 20 mL) in methanol (30 mL) was added and refluxed for 3 h, after cooling the reaction mixture was concentrated.The solid obtained was filtered and crystallized to give 9a,b.Melting point and mixed melting point showed no depression with 9a,b obtained from the above experiment.

Scheme 3 Table 1 .
Fungicidal activity of some of the new compounds 1