Exocyclic enaminones as building blocks for synthesis of bioactive polyheterocyclic compounds

The reaction of exocyclic enaminones namely, 2-(dimethylaminomethylene)-3,4-dihydro-2H-naphthalen-1-one, 3(dimethylaminomethylene)-thiochroman-4-one and 2-(dimethyl-aminomethylene)-indane-1,3-dione, each with heterocyclic diazonium salts afforded the respective hydrazones which undergo either in situ dehydrative cyclization or cyclized by heating with acetic acid to give polycyclic compounds. The structure of all the newly synthesized products were confirmed by elemental and spectral (IR, I H NMR, Mass) data. Also, the biological activity of some of the prepared compounds was tested against some microorganisms and promising results were obtained.


INTRODUCTION
Literature reports indicate that enaminones constitute an important class of useful precursors in organic synthesis, in pharmaceutical development and in heterocyclic synthesis [1][2][3][4][5][6]. Since enaminones have two reactive sites, they are able to react with both electrophiles and nucleophiles [7][8][9][10][11] to give a variety of bioactive products. Several reports indicate that reaction of acyclic enaminones as nucleophiles with heterocyclic diazonium salts give the azo compounds which undergo in situ cyclization via elimination of dimethylamine to give the respective cyclized products [12,13] (Scheme 1). On the other hand, literature survey indicate that only one report [14] was found about reaction of exocyclic enaminones with heterocyclic diazonium salts.

Scheme 1
On the basis of these findings, we report herein a facile method for synthesizing tetra-and penta-heterocyclic ring systems via coupling reaction of a number of different exocyclic enaminones with heterocyclic diazonium salts. Our objective from this study is to investigate the reactivity of these exocyclic enaminones towards a variety of heterocyclic diazonium salts. Also, the activity of the products obtained from this reaction against a number of bacteria and fungi species was studied.

Experimental Materials
Melting points were determined on an electrothermal Gallenkamp apparatus and are uncorrected. The IR spectra were recorded in potassium bromide using Pye Unicam SP-1000 spectrophotometer. 1 HNMR spectra were recorded in DMSO-d6 using a varian Em-300 MHz Spectrometer and TMS as internal reference. Mass spectra were recorded on AEIMS30 mass spectrometer operating at 70ev. Elemental analyses were carried out by the Microanalytical Center of Cairo University, Giza, Egypt. Antimicrobial activity was carried out at the Regional Center for Mycology and Biotechnology (RCMB) at Al-Azhar University, Cairo, Egypt. The enaminones 4a,b and 5 were reported as described before [15][16][17].

Coupling of the enaminones 4a,b and 5 with heterocyclic amines
General procedure: To a stirred solution of the appropriate enaminones 4a,b or 5 (10 mmol) in ethanol (50 ml) was added sodium hydroxide (0.4 g, 10 mmol) and the mixture was cooled in an ice bath to 0-5 ºC. To the resulting solution, while being stirred, was added dropwise over a period of 20 min a solution of the appropriate heterocyclic diazonium salt, prepared as usual by diazotizing the respective heterocyclic amine (2-amino-[1,2,4]-triazole, 2-amino-benzimidazole or 5amino-3-phenyl-pyrazole) (10 mmol) in hydrochloric acid (6 M, 6 ml) or in nitric acid with sodium nitrite (1 M, 10 ml). The whole mixture was then left in a refrigerator overnight. The precipitated solid formed was collected, washed with water and finally crystallized from ethanol to give the respective hydrazone 8b, 12b, 14, 16, 18 or the cyclized compounds 9a, 11a,b and 12a, respectively.

Cyclization of hydrazones 8b, 12b, 14, 16 and 18
A solution of hydrazones 8b, 12b, 14, 16 or 18 (1 mmole) in glacial acetic acid (10 mL) was refluxed for 15 h. After cooling, the solution was then poured into ice and sodium acetate and the precipitate formed was filtered off and crystallized from the appropriate solvent to give compounds 9b, 13b, 15, 17 and 19, respectively.

5,6,7,12c-Tetraaza-indeno[2,1-c]fluoren-8-one (19)
Brown crystal, (65% yield), mp 152ºC (EtOH); IR (KBr) νmax 1720 (CO), 1534 (C=N). 1 (RCMB 010052). strains were used in this study. The microbial suspension equivalent to the turbidity of 0.5 McFarland (10 8 CFU/ml) standard was prepared from a fresh subculture of tested bacteria in Mueller Hinton Broth (MHB) and tested fungi in Sabouraud dextrose Broth (SDB) then this suspension was diluted to 10 6 CFU/ml using MHB for bacteria and Sabouraud dextrose Broth (SDB) for tested fungi. The adjusted microbial inoculum (100 μl) were added to each well of sterile 96-well flat-bottomed microtiter plate containing the tested concentration of tested samples (100 μl/well). As a result, last inoculum concentration of 5×10 5 CFU/ml was obtained in each well. Three wells containing microbial suspension with no sample using DMSO employed for dissolving the tested compound (Growth control) and two wells containing only media (background control) were included in this plate. Optical densities were measured after 24 hours at 37°C for bacteria and after 48 hours at 28°C for fungi using a multi-detection microplate reader at The Regional Center for Mycology and Biotechnology (Sun Rise -Tecan, USA) at 600 nm. Ampicillin, Gentamicin and Amphotericin B were used as standards for Gram positive bacteria, Gram negative bacteria and fungi respectively.
For the determination of MIC of tested samples by the micro-broth kinetic assay, the percentage of growth at each sample concentration was calculated with the following equation: % growth = [(OD600 of wells containing the sample/OD600 of the sample-free well) x 100] after substraction of background ODs (ODs of microorganism-free wells) [18].

Results and discussion
The starting exocyclic enaminones 4a,b and 5 were prepared in good yields as described before [15][16][17] via condensation of the corresponding cyclic ketones namely, tetralone, 4-thiochromanone or indane-1,3-dione, with dimethylformamidedimethylacetal (DMF-DMA) in dry xylene (or toluene) under reflux for 6hrs. The reaction of enaminones 4a,b each with a variety of diazonium salts of heterocyclic amines, namely, 3-amino-1,2,4-triazole, 2-aminobenzimidazole and 5-amino-3phenyl-1H-pyrazole, was then investigated. Thus, reaction of enaminones 4a,b, each with the diazonium salt of 3-amino-1,2,4-triazole at low temperature in ethanol and in the presence of sodium acetate afforded products 8b and 9a (Scheme 2). The structure assigned for the products 8b and 9a was confirmed based on elemental and spectral (IR, 1 HNMR and Mass) data (see Experimental). For example, the IR spectra for product 9a revealed the absence of both the carbonyl and the N-H stretching bands. Also, ¹H NMR displayed only singlet signal at δ 9.25 ppm and multiplet signals in the region δ 7.29-7.90 ppm due to the triazole and aromatic protons, respectively. The mass spectra of 8a and 9b revealed in each case a molecular ion peak which is in agreement with the assigned structure. Based on the foregoing data, we can conclude that product 9b were formed directly via in situ dehydrative cyclization of the respective hydrazone 8b (Scheme 2).

Scheme 2
Similarly, coupling reaction of 4a-b each with the diazonium salt of 3-amino-5-phenyl-pyrazole or 2-aminobenzimidazole under the same reaction conditions afforded the respective pentacyclic products 11a,b, 13a and the hydrazone 12b. The structure of the products was also identified on the basis of both elemental and spectral data (see Experimental). Treatment of the hydrazones 8b and 12b each with glacial acetic acid under reflux for 15hrs, gave the respective polycyclic compounds 9b and 13b (Scheme 2 and 3).
The structure assigned for the cyclized products 9b and 13b was established through the data obtained from both elemental and spectral analysis. For example, the IR spectrum of products revealed the absence of the characteristic bands due to the N-H groups. Also, the 1 Table 1).The products 12b and 16 have no activity against Candida albicans (CA). Also, the latter products together with products 8b and 11b have no activity against Pseudomonas aeruginosa (PA). Compound 14 showed higher potency against the fungus, namely, Syncephalastrum racemosum (SR) than the standard fungicide Amphotericin B (see Table 1) S e p t e m b e r 1 9 , 2 0 1 4 The test was done using the diffusion agar technique, well diameter: 6.0 mm (100 µl was tested), RCMB: Regional Center for Mycology and Bio-technology Antimicrobial unit test organisms NA: No activity, data are expressed in the form of mean± SD.

Minimum inhibition concentration MIC:
The minimum inhibition concentration and IC50 of four of the newly synthesized products 8b, 11b, 14 and 18 was examined against all the employed fungi and bacteria and the results obtained are depicted in the following Tables 2-5. These results indicated that the most reactive derivative is compound 14.

Conclusion:
In the present paper, we described a facile method for synthesis of tetra-and pentacyclic compounds via coupling reaction of a number of exocyclic enaminones with some heterocyclic diazonium salts. The tetra-and pentacyclic compounds were obtained either by in situ dehydrative cyclization of the initially formed hydrazones or by thermal treatment of the respective hydrazones with glacial acetic acid. Also, some of the newly synthesized products were tested against some fungi and bacteria and the results obtained indicate that all the tested compounds have high activity against most of the employed microorganisms. Moreover, compound 14 showed promising results since it has a higher potency against the fungus Syncephalastrum racemosum than the standard employed fungicide Amphotericin B.