Azoniaallene salts as versatile building blocks in the synthesis of antibacterial and antifungal heterocyclic compounds

Substituted 2-azoniaallene salts 1 are strong bifunctional electrophiles, undergo cyclization reactions furnish many series of heterocyclic compounds, where reacted with p-tolyl urea, phenyl thiourea and thiosemicarbazone derivatives to afford triazinium salts, and converted to corresponding free bases 3, 5, 7 under treatment with Na2CO3. While triazole derivatives 8 and 9 were obtained by the reaction 2-azoniaallene salts 1 with benzohydrazide and phenyl hydrazine, respectively. Benzoxazinium salts 10 and 11 were acquired when asymmetric 2-azoniaallene salt reacted in (1:1) ratio with p-cresol and 3-methyl1-phenyl-5-pyrazolone, respectively. The reaction of 2-azoniaallene salt with malononitrile furnished the primidinium salt 12 which underwent neutralization with Na2CO3 followed by heterocyclization with hydrazine hydrate afforded the bicyclic compound 3-aminopyrazolo[3,4-d]pyrimidine 14, which is highly reactive for nucleophilic addition to phenyl isothiocyanate to furnish thiourea derivative 15. Moreover, 14 undergo condensation with aldehydes to give imine derivatives 16a,b. All free base compounds were screened for their antimicrobial activities.


Chemistry
All solvents were dried by standard methods.All experiments for the synthesis of hexachloroantimonate salts were carried out with exclusion of moisture.The melting points were determined with Electrothermal (Barstead 9100) apparatus and are uncorrected.IR spectra were recorded with Shimadzu (IR Prestige-21) FT-IR spectrometer. 1 H and 13 C NMR spectra were determined with Jeol ( 1 H NMR, 13 C NMR: 400 MHz), the chemical shifts in ppm are expressed on the δ scale using tetramethylsilane as internal standard.Mass spectra were measured with GC-MSQP 1000Ex Shimaduz.Microanalyses were performed with EuroEA Elemental Analyzer.

Antimicrobial Activity
The free base Compounds (3, 5, 7a, 7b, 9, 13, 14, 15, 16a and 16b) were screened for their in-vitro antibacterial activity against garm(+) bacteria; [Staphlococcus aurous, Staphylococcus epidormidis, Stroptococcus faecalis] and gram(−) bacteria; [Escherichi coli, Klebsiella pneumonia, Proteus mirabilis, Pseudomonus aeuroginosa] employing well-diffusion method at the concentration of 100 µg/mL and 150 µg/mL in Müller-Hinton agar media and also for in-vitro antifungal activity against Candida albicans and Saccharomyces cervisiae by welldiffusion method at the concentration of 100 µg/mL and 150 µg/mL using sabouraud-dextrose agar.DMF was used as a solvent control for antimicrobial activity.(CN10) Gentamicin, (CTX30) Cefotaxim, (FOX30) Cefoxitix, (E15) Erythromycin, (TE30) Tetracyclin, (MXF5) Moxifloxacin, (VA30) Vancomycin and (CIP5) Ciprofloxacin were used as reference antibacterial drugs where (CLT10) Clotrimazole and (FCA25) Fluconazole as reference antifungal drugs.The area of inhibition of zone measured in mm.The results are listed in Table 1.New derivatives of triazoles and oxazines were synthesized through cycloaddition of 2-azoniaallene salts.Triazole salt 8 was obtained in simple reaction of azoniaallene salt 1 and benzohydrazide under stirring conditions for 30 minutes and its structure was secured by the presence of NH band at 3329 cm -1 and disappearance the (C=O) band of benzohydrazide.The asymmetric 2-azoniaallene salt reacted with phenyl hydrazine under reflux to afford the triazole salt which failed to be crystallized so treated directly with sodium carbonate to furnish the triazole derivative 9.The mass spectrum of 9 confirmed the presence of molecular ion peak at m/z = 387 for the molecular formula C 23 H 21 N 3 O 3 .Cyclization of azoniaallene to afford the benzoxazine salt 10 was acquired when p-cresol and 2-azoniaallene salt reacted in (1:1) ratio.On the reaction of 3-methyl-1-phenyl-5-pyrazolone with azoniaallene salts, a good yield of 11a,b was obtained (Scheme 2).

RESULTS
Primidinium salt 12, formed under reaction of malononitrile with azoniaallene salt 1, was confirmed by IR which showed sharp band for nitrile group at 2264 cm -1 , and 3209 cm -1 for NH group.Treatment of salt 12 with sodium carbonate solution afforded the corresponding free pyrimidine base 13 with the same sharp band for nitrile group at 2229 cm -1 .The bicyclic system 3-aminopyrazolo [3,4-d]pyrimidine 14 was obtained by reflux 13 and hydrazine hydrate in ethanol.The chemical structure of 14 was based on its spectral data and elemental analysis.The amino group of 14 is highly versatile; it is highly reactive for nucleophilic addition to phenyl isothiocy-anate in hot pyridine to furnish the corresponding thiourea derivative 15.Moreover, compound 14 undergo condensation with aromatic aldehyde e.g.p-anisaldehyde and p-toulaldehyde to give the corresponding imine derivatives 16a,b (Scheme 3).

CONCLUSION
In the present research studies, our efforts are to synthesize some new heterocyclic compounds triazine, triazole, oxazine, and primidine based on the bifunctional electrophile azoniaallene salts.These synthesized compounds are characterized by various elemental and spectral analyses.The antimicrobial data showed that the new compounds in general have moderate to good activity compared to the reference antibacterial & antifungal drugs.Looking at the structure activity relationship (SAR) for compounds (7a, 7b) observed that the change in chemical structure has no significant effect on growth inhibition of bacteria and fungi.Where for (16a, 16b) there are some differences especially in the diameter of inhibition zone (mm) of Gram(−) bacteria and fungi.Work is underway in our laboratories to increase the efficacy and specificity of the titled scaffolds as antimicrobial by structural refinements and modulation.

Substituted 2 -
azoniaallene salts 1 with stabilizing groups R 1 , R 2 have found considerable preparative application.Azoniaallene salts are strong bifunctional electrophiles, which should undergo cyclization reactions with molecules with two nucleophilic centers.Here we repot such cyclizations.2-Azoniaallene salts are versatile building blocks in furnishes many series of heterocyclic compounds, as triazine derivatives (Scheme 1).p-Tolyl urea was reacted with symmetric and asymmetric 2-azoniaallene salts 1 in dichloroethane to afford the corresponding oxotriazinium salts 2a,b.The free base triazine compound 3 was achieved by treatment of hexachloroantimonate salt 2a with Na 2 CO 3 solution.The chemical structure of 2 and 3 were secured by their elemental analyses and spectral data.The IR spectrum of 2a displayed bands at NH band at 3282 and 1739 cm -1 due to NH and C=O groups, respectively.The 1 H NMR spectrum of 2a showed singlet signal at δ = 2.26 ppm for the methyl protons, multiplet in the range δ = 7.27 -8.4 for the aromatic protons and singlet at δ = 11.33 for the (NH) proton.Under similar conditions, phenyl thiourea reacted with 2-azoniaallene salt 1 to furnish the thiotriazinium salt 4. The IR spectrum of this salt showed a sharp and intensive peak for NH at 3240 cm -1 .The 1 H NMR showed multiplet signal of aromatic protons and singlet signal a of (NH) proton at 7.53 -8.57and 10.82, respectively.The free thiotriazine compound 5 was obtained by treatment the corresponding salt 4 with solution of Na 2 CO 3 .The mass spectrum of compound 5 showed the molecular ion peak at m/z = 411 corresponding to the formula C 21 H 13 Cl 2 N 3 S. Aminothiazole with a good leaving group was reacted with 2-azoniaallene salts 1 to afford the bicyclic thiazolo[3,2-a][1,3,5]triazinium salts 6a,b which seems to represent a new ring system.The NMR spectrum of 6a showed a doublet signal at δ = 6.46 for C 2 -thiazole proton, a multiplet signal in the region δ = 7.13 -7.67 for the aromatic protons and a doublet signal at δ = 8.11 for C 3 -thiazole proton.Moreover, the reaction of thiosemicarbazone derivatives with symmetric azoniaallene salt 1 (R 1 = R 2 = Ph) afforded the corresponding triazinium salts, which underwent neutralization by Na 2 CO 3 solution furnished the corresponding triazine derivatives 7a,b (Scheme 1).