Synthesis and Antimicrobial Activities Evaluation of New Spiro[4.5]dec-2-enes Containing 1,2,4-Triazole, Piperidine and Sulfonamide Moieties

A new series of 1,2,4,8-tetraazaspiro[4.5]dec-2-enes containing 1,2,4-triazole, piperidine and sulfonamide moieties have been synthesized from the reaction of 4-piperidone benzoylhydrazones having 1-methyl, 1-benzyl, and 1isopropyl groups with appropriate nitrilimines. The microanalysis and spectral data (IR, 1 H NMR, 13 C NMR and MS) of the synthesized compounds are in full agreement with their molecular structure. The microbial features of the synthesized compounds were studied by a known method. Some of titled compounds exhibited significant antimicrobial activity on several strains of microbes.


Introduction
The interest in the chemistry of hydrazonoyl halides is a consequence of the fact that they undergo a wide variety of reactions which provide routes to many of heterocyclic and spiro heterocyclic compounds [1][2][3][4]. Spiro heterocyclic compounds possess various pharmacological properties and hence their synthesis is of interest to organic chemists. Such compounds display pronounced antimicrobial [5], analgesic [6], anti-inflammatory [6], antimycobacterial [7], antifungal [8], antitumor [9,10] and antiviral [9,10] activities. Among these heterocycles, spiro azoles have been identified as privileged structures in medicinal chemistry and have attracted increasing interest in the recent years [11][12][13][14].
A survey of the literature has shown that compounds having azole derivatives to possess diverse biological activity and are widely used in a medicinal chemistry [15][16][17][18]. The 1,2,4-triazole derivatives has received a considerable attention in view of their diverse pharmacological activities [19][20][21][22] such as antimicrobial [23,24] sedative, anticonvulsant [25], anti-inflammatory properties [26] and anticancer agents. They are also use as intermediates in obtaining colour photosensitive materials as well as toners, inks and other photographic materials such as magenta coupler in a photosensitive emulsion layer [27][28][29][30]. The most developed procedure for construction of spirocompounds depends mainly on 1,3-dipolar cycloadditions to exocyclic double bonds [31]. Recently, we described a versatile and efficient one-pot synthesis of hexa and octaazadispiroheterocyclic compounds utilizing 1,4cyclohexanedione oxime or methyl hydrazones and nitrilimines, generated in situ from the corresponding hydrazonoyl halides by the action of a suitable base [32,33].

Apparatus and Chemicals
Melting points were determined using an electro thermal melting temperature apparatus and are uncorrected. The IR spectra were measured as KBr pellets using a Satellite 3000 Mid infrared spectrometer. 1 H NMR and 13 C NMR spectra were recorded on a Bruker AM 300 MHz spectrometer at r. t. in DMSO-d 6 solution u sing tetramethylsilane (TMS) as internal reference. Chemical shifts are expressed in δ (ppm) downfield from TMS and coupling constants are in Hertz (Hz). Electron impact (EI) mass spectra were run on a Shimadzu GCMS-QP1000 EX spectrometer at 70 eV. Elemental analysis were carried out at micro analytical laboratory, Cairo University, Cairo, Egypt. 1-Methyl, 1benzyl and 1-isopropyl-4-piperidone was purchased from Avocado Research Chemicals, England, and used without further purification. Hydrazonoyl chlorides 1 employed in this study, were prepared via direct coupling of the appropriate sulfa drug diazonium chloride with 3chloroacetylacetone or phenacyl chloride or αchloroacetoacetanilide or α-bromo-2-acetylfuran or α-bromo-2-acetylthiophene or ω-bromoacetonaphthone in ethanolic sodium acetate solution following standard procedures [41]. 1-substituted-4-piperidone benzoylhydrazones 3 were obtained by reacting the appropriate 1-substituted-4piperidone with benzolyhydrazine in excess following reported procedures [37,42].

Synthesis of 1,2,4,8-tetraazspiro[4.5]dec-2-enes (General Procedure)
To a stirred solution of hydrazonoyl halides 1 (5 mmol) and 1-substituted-4-piperidone hydrazones 3 (10 mmol) in 1,4-dioxane (50-70 ml), triethylamine (5 mmol) in 1,4dioxane (10 ml) dropwise added at room temperature. Stirring was continued to reaction completion for 12-16 hours (monitoring the reaction progress by TLC). The solvent was then removed under vacuum, and the residual solid was washed with water (100 ml) to get rid of the triethylamine salt. In some cases the residue was extracted with chloroform (3x30 ml) and the combined extracts were washed with water (50 ml), dried over anhydrous sodium sulfate. The solvent (CHC1 3 ) was evaporated in vacuum, and the crude product was triturated with ethanol (10-20 ml). The crude solid products were collected and recrystallized from appropriate solvents to afford the desired compounds 4a-u. The following compounds were prepared using this method:    13 [30]. The tested compounds were dissolved in dimethyl sulfoxide (DMSO). An inoculum of about 1.5 x 10 8 colony forming unit per spot was applied to the surfaces of Mueller-Hinton agar plates containing graded concentrations of the respective compound; plates were incubated at 37 o C for 18 h. The spot with the lowest concentration of compound showing no growth was defined as the minimum inhibitory concentration (MIC). All organisms used in this study were standard strains were obtained from the Microbiology laboratory (Al-Aqsa University) and included bacterial strain such as Enterococci, Escherichia coli, Staphylococcus aureus, Klebsiella spp, Proteus spp, and fungi strain such as Aspergillus niger, Candida albicans. The MIC of Tetracycline and fluconazole was determined concurrently as reference for antibacterial and antifungal activities, respectively (Table 1). Control DMSO was carried out with each experiment.

Results and Discussion
The nitrilimines 2 employed in this study was generated in situ from the corresponding hydrazonoyl halides 1 by the action of a suitable base as shown in Figure 1. The treatment of the resulting non-isolable nitrilimines 2, 1,3-dipole, with appropriate l-methyl, 1-isopropyl and 1-benzyl-4-piperidone benzoyl-hydrazones 3 in 1,4-dioxane or tetrahydrofuran in presence of triethylamine as a base, gave in each case a single product that proved to be the respective 1,3,4trisubstituted l,2,4-spirotriazole derivatives 4a-u, as cycloaddition products through the 1,3-dipolar cycloaddition of nitrilimines 2 to the exocyclic double bond (N=C) of benzoylhydrazone 3 instead of the spirotetrazine cyclocondensation products 5a-u ( Figure 1). The purity of obtained compounds was controlled by TLC and elemental analysis. Both the analytical and spectral data (IR, 1 H NMR, 13 C NMR and mass spectra) of the synthesized spirotriazoles were in full agreement with the proposed structures.

Spectroscopical Data of Spiro Compounds 4a-u
The characterization data of synthesized spiro compounds 4a-u are given in the experimental section. These compounds gave satisfactory analysis for the proposed structures which are confirmed on the bases of their spectroscopical data. The IR spectra showed the strong absorption band of CONH of the triazole ring in the region 3380-3360 cm -1 , in addition to, characteristic band of SO 2 NH in the region of 3340-3220 cm -1 , (Ar-C=O) at about 1680-1660 cm -1 and bands at 1350, 1150 cm -1 attributed to SO 2 of sulfonamide group. In the 1 H NMR spectra, a characteristic signal due to the PhCONH proton of the triazole ring appeared at 9.50-9.40 ppm. The NH of the tetrazine structures 5a-u if formed is expected to resonate at about 5-4 ppm [43]. The signals at 8.70, 6.60 ppm and 8.40, 6.80 ppm are attributed to thiazole and pyrimidine protons, respectively. Also, the spectra exhibit a characteristic singlet at 11.6-11.5 ppm and due to SO 2 NH proton. The structures of compounds 4a-u were further confirmed by 13 C NMR spectra, which account for the different carbons of these spirotriazoles. The signal at 89-91 ppm was attributed to the C-5 (spiro carbon) of the triazole