Synthesis, characterization and in vitro antimicrobial activity of novel fused pyrazolo[3,4-c]pyridazine, pyrazolo[3,4-d]pyrimidine, thieno[3,2-c]pyrazole and pyrazolo[3′,4′:4,5]thieno[2,3-d]pyrimidine derivatives

Background Some novel substituted pyrazolone, pyrazolo[3,4-c]pyridazine, pyrazolo[3,4-d]pyrimidine, pyrazolo[3,4-d]thiazolo[3,2-a]pyrimidinone, thieno[3,2-c]pyrazole and pyrazolo[3′,4′:4,5]thieno[2,3-d]pyrimidine derivatives have been reported to possess various pharmacological activities like antimicrobial, antitumor and anti-inflammatory. Results A novel series of azoles and azines were designed and prepared via reaction of 1,3-diphenyl-1H-pyrazol-5(4H)-one with some electrophilic and nucleophilic reagents. The structures of target compounds were confirmed by elemental analyses and spectral data. Conclusions The antimicrobial activity of the target synthesized compounds were tested against various microorganisms such as Escherichia coli; Bacillus megaterium; Bacillus subtilis (Bacterial species), Fusarium proliferatum; Trichoderma harzianum; Aspergillus niger (fungal species) by the disc diffusion method. In general, the novel synthesized compounds showed a good antimicrobial activity against the previously mentioned microorganisms.


Background
The compounds containing nitrogen are important category of heterocyclic compounds, which play a significant roles in modern pesticide industry (85% of pesticides with high activity and low toxicity contain nitrogen heterocyclic compound) [1]. Pyrazoles are important moieties as building blocks for many heterocyclic products and act as abinucleophile [2] with abroad spectrum of remarkable biological activities. Many derivatives containing pyrazole nucleus have been commercialized as herbicides, insecticides and fungicides for plant protection [3]. Heterocycles containing a pyrazole or pyrazolone nucleus have been reported to show abroad spectrum of biological activity including antimicrobial [4], anti-cyclooxygenase [5], anti-convulsant [6], antitubercular [7], antitumor [8], anti-inflammatory [9], analgesic [10], antidiabetic [11], antipshycotic [12][13][14]. In last few years, we have been involved in a program aimed at developing new efficient synthetic approaches for the synthesis of heterocyclic compounds of biological interest [15][16][17]. Since most of the pyrazole derivatives show anti-microbial activity, the synthesized compounds are also expected to show antimicrobial activity. Hence, our plan is to synthesize some substituted pyrazole derivatives and subsequently screen for their antimicrobial activity.
Pyrazol-5-one derivative 2 was exploited as a key intermediate for the synthesis of hitherto unknown fused pyrazole. Thus cyclocondensation of 2 with active methylene reagent such as malononitrile in ethanol under reflux in the presence catalytic amount of piperidine afforded indazole derivative 3 on the basis of analytical and spectral data (Scheme 1). The formation of 3 from the reaction of 2 with malononitrile is believed to be formed via initial condensation of malononitrile with the ring carbonyl and subsequent elimination of water followed by addition of methyl group on the triple bond system of cyano group. Also, compound 2 condensed with aryl aldehyde 4a in ethanol containing 10% sodium hydroxide to afford the condensation product 5 based on its elemental and spectral data (Scheme 1) [21]. Cyclization of 5 with ethyl cyanoacetate in ethanol in the presence of ammonium acetate at reflux temperature led to the formation of dihydropyridine derivative 6 (Scheme 1) [22][23][24][25]. The reactivity of methyl group in pyrazolone 2 toward aryl diazonium salts was also investigated aiming at preparation of new pyridazine derivatives. Thus, when 2 coupled with aryl diazoniuum salt 7a in ethanol in the presence of sodium acetate yielded hydrazone 8a on the basis on its spectral data. The 1 H-NMR spectrum of compound 8a recorded in DMSO-d 6 revealed a signal at δ = 12.00 ppm which could be attributed to hydrazone NH group. Similarly, pyrazolone 2 was coupled readily with aryl diazonium salts 7b in the same reaction conditions to give 8b as demonstrated in (Scheme 1).
Hydrazide 14 is used as a key precursor for many chemical transformations to synthesize a variety of important heterocycles. Thus, when compound 14 was allowed to react with triethylorthoformate in refluxing acetic anhydride afforded 5-amino-1,3-diphenyl-1H-thieno [3,2- Establishing structure of 16 was based on its elemental and spectral data. For example the infrared spectrum of thienopyrazole 16 revealed the absence of carbonyl group. The 1 H-NMR of the same product revealed absence of signals of ethyl fragment. The mass spectrum showed a very intense molecular ion peak at 361 (M + +2) and a number of fragments support the proposed structure Scheme 2 Synthessis of pyrazoles 10-13 [37]. Treatment of 14 with benzoyl chloride 17 afforded 5-amino-N′-benzoyl-1,3-diphenyl-1H-thieno[3,2-c]pyrazole-6-carbohydrazide 18 on the basis of its elemental analysis and spectral data. Moreover, the reaction of 18 with triethylorthoformate at reflux temperature afforded the fused pyrimidine derivative 19 (Scheme 3) [38].

Antimicrobial activity
The newly synthesized compounds and their derivatives have been screened for antibacterial activity against some gram negative bacteria (Escherichia coli) and some gram positive bacteria (Bacillus megaterium and Bacillus subtilis), and antifungal activity against Fusarium proliferatum, Trichoderma harzianum and Aspergillus niger, by the cup-plate method and agar diffusion disc method for determining MIC (minimum inhibitory concentration), ampicillin and colitrimazole were used as standards for comparison of antibacterial and antifungal activity, respectively.
The anti-bacterial activity of the synthesized compounds was tested against bacterial species (E. coli; B. megaterium; B. subtilis) and the antifungal activity was tested also against fungal species (F. proliferatum; T. harzianum; A. niger). Each compound was dissolved in DMF, About 100 mL of each compound will be pipetted and poured into the cups existed in nutrient agar plates containing medium which consisted of: peptic digest of animal tissue 5.00, sodium chloride 5.00, Beef extract 1.50, Yeast extract 1.50, Agar 15.00 all in gm/L, final pH at 25 °C; 7.4 ± 0.2) or Czapek's agar plates for fungi (sucrose 30.00, sodium nitrate 2.00, dipotassium phosphate 1.00, magnesium sulphate 0.50, potassium chloride 0.50, ferrous sulphate 0.01, Agar 15.00, all in gm/L, final pH at 25 °C; 7.3 ± 0.2), seeded with E. coli, B. megaterium and B. subtilis, F. proliferatum, T. harzianum and A. niger, respectively.
For determining minimum inhibitory concentration (MIC), serial dilutions of tested compounds (μg/ mL) as well as reference antibiotics were prepared using 10% DMF solution, paper discs of Whatman filter paper were prepared with standard size (8 mm), were cut and sterilized in an autoclave. The paper discs soaked in the desired compound solution were placed aseptically in the petri dishes containing agar media and microbial species. The petri dishes were incubated at 36-37 °C and the inhibition zones were recorded after 24 h of incubation in case of bacteria and after 5-7 days in case of fungi. Each treatment was replicated three times [40,41]. The antibacterial activity of a common standard antibiotic ampicillin and antifungal Clotrimazole was also recorded using the same procedure as above at the same concentration and solvents. The % activity index for the compound was calculated by the following formula.
Our results showed that most of checked compounds were active against most of micro-organisms used, while the discs which containing DMF solution (10%) alone were not exhibited any effect on the growing microorganisms (no inhibition zone around the discs). The results of antimicrobial and antifungal activity and its MIC are illustrated in Tables 1, 2. We found that compounds; 3, 13, 2, 12a and 20 showed promising broad spectrum antibacterial activities against E. coli. Compounds 14, 12b, 15, 2 and 24 showed maximum antimicrobial activity against B. megaterium, B. subtilis, F. proliferatum, T. harzianum and A. niger, respectively. Compounds; 9b, 8b, 6, 22a, 5a, 11b, 18 and 16 demonstrated moderate antimicrobial activity against gram positive, gram negative bacteria and fungi. On the other hand, 10a, 10b, 11a, 23a, 25 and 23b exhibited low antibacterial activity and moderate to low antifungal activity, whereas 25 and 23b showed high antibacterial activity against only B. subtilis. From Table 2, we observed that compounds; 13, 6, 3 and 14 showed the minimum inhibitory concentrations (MIC) for most tested bacteria and fungi, while compounds; 9b, 8b, 22a, 5a, 11b, 18 and 19 exhibited high concentrations of MIC as compared with standard antimicrobial agents used.

Chemistry
The melting points, the elemental analysis and the spectral data were recorded as reported in references [19].
Synthesis of 4-acetyl-1,3-diphenyl-1H-pyrazol-5(4H)-one (2). A mixture of pyrazolone 1 (0.01 mol) and acetyl chloride (0.01 mol) in acetic anhydride (10 mL) and sodium acetate (2 gm) was heated under reflux for 9 h. The reaction mixture was allowed to cool and poured into crushed ice then acidified with HCl. The separated solid was filtered, washed with water and crystallized from ethanol to give    (s, 1H, NH). 13 (8a-b). To a stirred cold solution of aryldiazonium chlorides 7a-b (0.01 mol), prepared by treating aniline derivatives (0.01 mol) with sodium nitrite (0.01 mol) in HCl, ethanol (30 mL) and catalytic amount of sodium acetate, the active methyl reagent 2 was added gradually. The stirring was continued for 2 h. The solid product so formed was filtered off, washed with water several times, dried and crystallized from the proper solvent to afford 8a-b.  General procedure for the synthesis of pyrazolopyridazinone derivatives (9a-b). A mixture of 8a-b (0.01 mol) and ammonium acetate (2.0 gm) was fused for 3.0 min in domestic microwave. The reaction mixture was left to stand, and then triturated with ethanol; the solid product so formed was collected by filtration and crystallized from the proper solvent to give 9a-b.