An Eco-Friendly Synthesis of Heterocyclic Moieties Condensed with Pyrazole System under Green Conditions and Their Biological Activity

The efficient, facile and green synthesis of 4-bromo pyrazolone by using N-bromo saccharine as valuable green reagent encouraged us to prepare some new fused heterocycles as furopyrazole, pyranopyrazole, imidazopyrazole, pyrazolothiazole, pyrazol thiazolopyrimidine, pyrazolothiazine, oxathinopyrazole, pyrazolobenzooxazine, and pyrazoloquinoxaline. The synthesis was carried out by a basic condensation of bromo pyrazolone 2 and a suitable reagent in a one-pot reaction using chitosan as a green basic catalyst. The reactions were carried out by microwave irradiation technique as a green source of energy as well as the conventional heating. The antioxidant activity of the prepared compounds was studied using 1,1-phenyl-2-picrylhydrazyl (DPPH) assay and their antibacterial activity against Gram-positive, Gram-negative bacteria and antifungal activity was evaluated.


Introduction
The goals of green chemistry are to reduce and prohibit the pollution of nature, ensure perpetual life on earth and minimize the use and production of hazardous materials [1]- [3].
The chemistry of pyrazole system has attracted much attention and many methods for synthesis have been extended [4].Pyrazoles exist in many compounds that are used as pharmaceuticals and agrochemicals [5].Fused pyrazoles have fungicidal [6], herbicidal [7], veridical [8] and insecticidal activity [9] [10] and have been used for the treatment of rheumatoid arthritis [11] [12].Beside the pharmaceutical importance pyrazoles are very important class of heterocycles due to their commercial uses in dyestuffs and food coloring agents [13].
In continuation to our program aiming at the synthesis of fused heterocyclic moities of anticipated biological activity, we report herein the eco-friendly synthesis of fused pyrazole derivatives [14]- [16].

Chemistry
Melting points were recorded on a Gallenkamp melting point apparatus and uncorrected.The infrared spectra were recorded on Perkin-Elmer FTIR 1430 spectrophotometer using the KBr disk technique.The 1 H NMR and 13 C NMR spectra were recorded on a Bruker AC spectrometer (300 MHz) at 25˚C in DMSO-d 6 with TMS as internal standard and chemical shifts are reported in ppm as δ values.Reactions were conducted under microwave irradiation in closed vessels under magnetic stirring in a Synthos 3000 (Anton Paar) microwave with dual magnetrons system and with maximum power of 1000 W. Mass spectra were measured on a Finnigan MAT 8222 EX mass spectrometer at 70 eV.Microanalyses were performed on Perkin-Elemer 2400 Elemental Analyzer at microanalytical center at Cairo University.Reaction progress was monitored by thin layer chromatography (TLC) using benzene/acetone (2/1 by volume) as eluent.The strains for the biological activity were obtained from the Culture Collection of Bacteriology Laboratory, Microbiology Unit, Faculty of Science, Tanta University.

Cyclization of Compound 2 with β-Mercapto Derivatives (12-14)
A mixture of compound 2 (0.6 g, 20 mmol), appropriate reagent cysteine, ethyl mercapto acetate and/or 2-mercapto ethanol (20 mmole), and chitosan (0.5 g) in dry dioxane (10 ml), was refluxed for 4 h.The reaction mixture was cooled, filtered and the filtrate evaporated under reduced pressure; the residue was triturated with pet.ether 40˚C -60˚C.The obtained products were refluxed in glacial acetic acid (10 ml) for 2 h.The reaction mixture was cooled and poured into ice water to give compounds (12-14).A mixture of compound 2 (0.6 g, 20 mmol), o-aminophenol and/or o-phenylene diamine (20 mmole) and chitosan (0.5 g) in dry dioxane (20 ml) was heated under reflux for 6 h.The reaction mixture was cooled and filtered.The filtrate evaporated under reduced pressure and the residue was triturated with pet.ether 40˚C -60˚C.The obtained products were refluxed in 10 ml glacial acetic acid for 2 h.The reaction mixture was cooled and poured into ice water to give compounds 15, 16.

Antimicrobial Assay
An aliquot of 0.1 ml of each bacterial strain was inoculated and spread on nutrient agar while 0.1 ml of the yeast was spread on sabaroud agar slopes.Antimicrobial activity of the synthesized compounds was tested in vitro against different types of bacteria and one fungal strain by the cut plug method [17].The assay plates were inoculated with 100 ml containing the diluted inoculums (107 CFU/ml) of each tested organism that were spread on the corresponding media.After solidification, the wells were made and 10 mg of the synthesized chemicals were dissolved in 1 ml DMSO and inserted in the wells.Nutrient agar plated was incubated at 37˚C for 24 h while plates were incubated at 25˚C for 48 h.The zones of inhibition around the wells were measured and the average based on three replies was recorded.

DPPH Radical Scavenging Assay
The antioxidant activities of the tested compounds were measured by using DPPH radical scavenging assay with L-ascorbic acid as drug reference [18].Each tested sample and L-ascorbic acid (50 μg) was dissolved in 1 ml DMSO.The dissolved sample (250 ml) was added to 1 ml DPPH/DMSO solution (6 μg/50ml) and the total volume was adjusted to 3 ml with DMSO.An equal amount of DMSO was used as a control.After vortexing the mixture was incubated for 30 min in dark at room temperature.Absorbance was measured using a spectrophotometer at 517 nm).DPPH radical scavenging % = 1/(A sample/A control) × 100.Serial dilutions (5 -50 μg/ml) of each compound were measured by the same assay to obtain the IC50 according to Brand-Williams et al. [19].

Chemistry
The synthetic routes for the synthesis of compounds 2-16 are outlined in (Schemes 1-5).
The key intermediate 2 was easily prepared from 1-phenyl-3-pyridyl-5-pyrazolone 1 [23] by bromination with N-bromosaccharine (sodium salt of saccharine with potassium bromide and oxone in water at room temperature) in 90% yield (Scheme 1).It is worthy mentioning that the classical bromination methods (bromine and/or Nbromo-succinamide in chloroform) the yield did not exceed 68%.
The I.R spectrum of compound 2 shows a strong absorption band at ν 1558 cm −1 (C=O) and a band at ν 748 cm −1 (C-Br).The 1 H NMR and mass spectrum of the prepared compound confirm the structure.
The structure of compounds 3-5 was proved on the basis of analytical and spectral data.Thus IR spectrum for compound 3 shows bands at ν 3345 cm −1 (NH 2 ), ν 2219 cm −1 (CN) and the disappearance of ν C=O band.The 1 H NMR spectrum for compound 3 showed the presence of a singlet for NH 2 protons at δ 4.47 ppm which disappeared by mixing with D 2 O.The IR spectrum for compound 4 shows bands at ν 3324 cm −1 (NH 2 ), ν 3350 cm −1 (NH 2amide ) and ν 1680 cm −1 (C=O).The 1 H NMR spectrum of compound 4 shows the presence of a singlet for NH 2 protons at δ 5.27 ppm.All spectroscopic and analytical data are given in experimental part.
The IR spectrum for compound 5 shows bands at ν 3415 cm −1 (NH 2 ), ν 1710 cm −1 (C=O ester).The 1 H NMR spectrum of compound 5 showed triplet for CH 3 at δ 1.23 ppm, quartet for CH 2 at δ 4.23 ppm for ester protons and a singlet for NH 2 protons at δ 4.65 ppm.All spectroscopic and analytical data are given in experimental part.
The structure of compound 6 was proved by spectral data, IR spectrum showed bands at ν 3414 cm −1 (NH 2 ), ν 2954 cm −1 (CH 3aliph ) and ν 2362 cm −1 (CN).The 1 H NMR spectrum of compound 6 showed a singlet for OCH 3 at δ 3.75 ppm, a singlet for NH 2 protons at δ 4.61 ppm and a singlet proton of pyrane ring at δ 4.82 ppm.All spectroscopic and analytical data are given in experimental part.The reaction of compound 2 with urea, guanidine hydrochloride and thiourea in dioxane in the presence of chitosan gave the corresponding imidazopyrazoles 7 and 8 or pyrazolothiazole 9 respectively (Scheme 3).
The structure of compounds 7-9 was confirmed by analytical and spectral data.The IR spectram showed bands at ν 3329 -3354 cm −1 (OH, NH 2 ) and disappearance of ν C-Br and ν C=O bands. 1 H NMR spectram for compounds 7 and 8 showed a singlet NH proton of imidazole moiety at δ 11.23 ppm.All spectroscopic and analytical data are given in experimental part.On the other hand, the reaction of compound 9 with some ketoesters named ethyl acetoacetate and ethyl benzoylacetate in dioxane in the presence of chitosan gave the corresponding 5-substituted pyrazol [4,3-b] thiazolo[3,2-a]pyrimidine-7-ones 10 and 11 respectively (Scheme 4).
The structure of compounds 12-14 was proved on the basis analytical and spectral data.The IR spectrum of compound 12 showed bands at ν 3342 cm −1 (OH), ν 1710 cm −1 (C=O) of carboxylic group and ν 675 cm −1 (C-S).Also, 1 H NMR spectrum showed doublet and triplet for CH 2 and CH of thiazine ring protons at δ 3.44 and 4.20 ppm respectively and a singlet of carboxylic proton at δ 11.23 ppm.The IR spectram of compounds 13 and 14 showed bands at ν 2891, 2884 cm −1 (CH 2 aliph. of oxazine ring) respectively.Also, 1 H NMR spectrum of compound 13 showed doublet and triplet CH 2 and CH of oxazine ring protons at δ 3.44 and 4.20 ppm respectively, while, The 1 H NMR spectrum of compound 14 showed two triplet signals for two CH 2 protons of oxazine ring at δ 3.36 and 4.15 ppm respectively.All spectroscopic and analytical data are given in experimental part.The structure of compounds 15 and 16 was proved by analytical and spectral data.The IR spectrum showed bands at ν 3346, 3352 cm −1 (NH) and disappearance of ν C=O and ν C-Br bands.The 1 H NMR spectrum showed multiplet at δ 6.50 -8.71 and 7.32 -8.56 ppm respectively corresponding to aromatic and heteroaromatic protons.All spectroscopic and analytical data are given in experimental part.Scheme 6 Preparation of pyrazolo [3,4-b]benzo(e) [1,4] The microwave irradiation technique as a source of energy was used.Under this technique interesting results were obtained in which the reaction time was reduced from 4-8 hours to only few minutes (15 -25 min.)and the yields were increased from 50% -82% to 80% -92%.Also, the products obtained are more pure than that obtained by conventional heating procedure (Table 1).

Antimicrobial Evaluation
Throughout history, there has been a continual battle between humans and the multitude of microorganisms that cause infection and disease.Diseases caused by microbial infection are a serious menace to the health of human beings and often have connection to some other diseases whenever the body system gets debilitated.During the 20 th century, vaccines for bacterial toxins and many other common acute viral infections were developed and made widely available.
The antimicrobial properties of the synthesized compounds 7-16 were tested against Gram-negative bacteria (Klibsella, E. coli, Serratia and Citrobacter), Gram-positive bacteria (Bacillus subtilis, Bacillus cereus, Pseudomonas vulgarus and Staphylococcus aureus) along with the non-filamentous fungus (Candida albicans) along with the non-filamentous fungus (Candida albicans) as pathogenic bacterial strains.Three different broadly used antibiotics (Amoxycilline, Chlormphenicol and Tetracycline) were used as references.
Reviewing the antimicrobial activity data (Table 2), it is concluded that compound 9 and compound 16 is the most active among all the synthesized compounds against most of the tested organisms, while compounds 7, 8 and 12-15 were found to have slight or moderate activity.It is worthy mentioning that minor change in molecular configuration of these compounds profoundly influences the biological activities.
Minimum inhibitory concentration (MIC) is important in diagnostic laboratories to confirm resistance microorganisms to an antimicrobial agent and also to monitor the activity of new antimicrobial agents.An MIC is generally regarded as the most basic measurement of the activity of an antimicrobial agent against organism.The present data shows that the most sensitive organism to the tested compound 9 is Candida albicans, Staphylo coccusaures, Citrobacter, Bacillus cereus and E. coli which showed (MIC) value of 250, 180, 250, 250 and 250 respectively.While, the most sensitive organism to the tested compound 16 is Candida albicans, Bacillus subtilis and Pseudonas vulgarus which showed (MIC) value of 250, 180 and 250 respectively compared with other (MIC) for the other organisms (Table 3).The sensitivity of microorganisms to the tested compounds is identified in the following manner * : Highly sensitive = Inhibition zone: 15 -20 mm; moderately sensitive = Inhibition zone: 10 -15 mm; slightly sensitive = Inhibition zone: 1 -10 mm; Not sensitive = Inhibition zone: 0 mm; * each result represents the average of triplicate readings.

Antioxidant Screening
Since antioxidants are gaining attention as a potential means of treating a large number of life style diseases like cancer, it is immense significance via a convenient synthetic methodology.The DPPH radical has been widely used to the ability of compounds to behave as been radical scavengers or hydrogen donors.Briefly the assay measures the decrease in absorbance of the DPPH radicals at a characteristic wave length after 60 min incubation of the DPPH radical with different concentrations (from 5 μg/ml to 50 μg/ml) of the antioxidant compounds according to the method of Brand-Williams et al. [19].The absorbance of the reaction mixture was recorded by bcc using a UV visible spectrometer (Genway 6305).L-ascorbic acid (vitamin C) was used as standard antioxidant (positive control).Results are expressed as the percentage of the DPPH free radical scavenging at (five concentrations), each value is expressed as the average of three experiments per concentration ±SD.Radical of the tested measured and the results were depicted in (Table 4) and (Figure 1).From DPPH method the range of IC 50 for compounds (2-16) is from 3.2 -6.6 µg/ml.The highest IC 50 (6.6µg/ml) and lowest activity showed in compound 2. The fused furane, pyrane, imidazole, oxathiine and benzooxine rings with pyrazole ring showed decrease in IC 50 (4.1 -5.5) and increase in antioxidant activity.The fused pyrazole ring with thiazole, thiazolopyrimidine and quinoxaline rings showed more decrease in IC 50 (3.3 -3.6) and more increase in antioxidant activity.Thiazine carboxylic acid fused with pyrazole ring 12 showed much closed antioxidant reactivity IC 50 (3.2µg/ml) compared with standard ascorbic acid due to the free carboxylic group in thiazine ring.

Conclusion
In the present work we could manage to report a new, simple eco-friendly technique for the synthesis of heterocyclic moieties fused with pyrazole system.The antimicrobial screening and antioxidant activity of some synthesized compounds have shown promising activities.

Figure 1 .
Figure 1.Scavenging antioxidant percentage of the tested compounds.

Table 1 .
Experimental data for the synthesis of compounds 2-8 by traditional methods and microwave assisted methods.

Table 2 .
Diameters of inhibition zones (mm) of newly synthesized compounds against different test bacteria and fungi on nutrient agar at 30˚C after 24.

Table 3 .
Minimal inhibitory concentration (MIC) of the provided samples against test microorganisms (MIC) μg/ml.All the dilutions of both samples and standards were performed by double fold dilution.

Table 4 .
Percentage of free radical scavenging activity (DPPH radical) obtained for the tested compounds.