Synthesis and Biological Activity of Some New Pyrazoline and Pyrimidine Derivatives

Novas séries de pirazolinas, 3-aril-4,5-diidro-1H-pirazol-1-carbaldeídos (4-6), (aril-4,5-diidro1H-pirazol-1-il)etanonas (9-11) e 3-aril-4,5-diidro-1H-pirazóis (24 e 25) foram sintetizadas pela reação de chalconas (1-3) com hidrato de hidrazina em ácido fórmico, ácido acético ou etanol, respectivamente. Novos derivados de pirimidina 6-arilpirimidina-2-amina (32-34) também foram sintetizados a partir das mesmas chalconas de partida. As estruturas dos novos compostos sintetizados foram estabelecidas através do estudo dos espectros de IV, H RMN, C RMN e análise elementar. Todos os compostos foram avaliados quanto as suas atividades antibacteriana e antifúngica. Dentre estes compostos, três mostraram atividade relevante contra C. albicans e outros também apresentaram atividade contra E. coli.


Introduction
Chalcones are well known intermediates for the synthesis of various heterocyclic compounds. Compounds with the chalcone backbone have been informed to possess various biological activities. 1 Chalcones have been reported to possess antimicrobial, 2 anti-inflammatory, 2,3 antioxidant and anticancer properties. 4 They were also found to exhibit analgesic, 5 platelet antiaggregation, 6 antiulcerative, 7 antimalarial, 8 antiviral, 9 antileishmanial, 10 antitubercular 11 and antihyperglycemic properties, 12 as well as to inhibit the enzymes tyrosinase, 13 and aldose reductase. 9 Much attention has been paid to the synthesis of heterocyclic compounds bearing nitrogen-containing rings, like pyrazoline and pyrimidine systems, mainly due to their potential pharmacological activity. [14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31] Pyrazolines are well known and important nitrogencontaining 5-membered heterocycles, which were found to possess a broad spectrum of biological activities such as anti-inflammatory, 14 herbicidal, 15 antimicrobial, 16 antifungal, 17 antidepressant, 18 anticonvulsant, 19 antitumor, 20 antitubercular, 21 insecticidal, 22 antimycobacterial, 23 molluscicidal, 24 and antinociceptive. 25 A classical synthesis of 2-pyrazolines involves the base catalyzed 26 Claisen-Schmidt condensation of appropriate ketones with suitable aldehydes in the presence of potassium hydroxide in aqueous ethanolic solution at room temperature to give chalcones, 27 which undergo a subsequent cyclization reaction with hydrazines. 28 Several alternatives are available for this condensation, 29 including under acidic 28 or basic 30 conditions. On the other hand, pyrimidines have also been reported to show a variety of biological activities. 31 Based on the interest in the above biological activities exhibited by the pyrazoline and pyrimidine compounds, we report here the synthesis of a new series of pyrazoline and pyrimidine compounds.
Treatment of the methyl ketones 9 and 10 with benzaldehyde in alkaline medium at room temperature afforded the corresponding α,β-unsaturated ketones 12 and 13, respectively, the IR spectra of which showed the carbonyl group at 1650 and 1652 cm -1 . Their proton NMR spectra showed the disappearance of the CH 3 signals and exhibited pairs of signals at d 7.40, 8.67 and 7.28, 8.70 ppm, respectively, as doublets, due to the olefinic protons (H 1' , H 2' ).
The desired Schiff's bases 14-19 were prepared by heating the methyl ketones 9-11 with aryl hydrazines (phenyl hydrazine, p-nitrophenyl hydrazine) or hydrazine hydrate in ethanol. Their IR spectra showed a new absorption peak at 3285-3372 cm -1 due to the NH group, while their 1 H NMR spectra displayed the CH 3 protons as singlets in the range d 2.14-2.19 ppm. In addition, a broad singlet was observed in the range d 9.20-10.61 ppm, corresponding to the NH moiety. In case of compound 19, the signal of the NH 2 group appeared at d 7.22 ppm.
Heating the substituted 4,5-dihydro-1H-pyrazole derivative 14 with acetic anhydride afforded the corresponding tetrazole 20, which evidenced disappearance of NH signals in its IR and proton NMR spectra.
On the other hand, the reaction of chalcones 1-3 with an alcoholic solution of guanidine carbonate containing aqueous NaOH produced the corresponding 2-amino-4, 6-diarylpyrimidines 32-34 (Scheme 2). This transformation might proceed either by 1,4-addition or 1,2-addition of the guanidine to the chalcones, followed by cyclization of the intermediate, which undergoes proton shift and aromatization to yield the 2-aminopyrimidines. 32 The infrared spectra of the products showed two bands in the ranges 3054-3147 and 3323-3366 cm -1 corresponding to the NH 2 group. Furthermore, their 1

Evaluation of the biological activity
Four test organisms representing different groups of microorganisms were used to evaluate the bioactivity of the designed products. The inhibition zone and minimal inhibitory concentration results are given in Table 1.
From the data, it stems that compounds 1, 3, 7, 20, 23, 31 and 34 were the most active against E. coli, while compounds 18, 28 and 30 were found to be active against C. albicans. Some chlorinated compounds exhibited activity against C. albicans (28 and 30) and against E. coli (1, 7 and 20). In addition, some sulfur-containing compounds exhibited activity against C. albicans (28 and 30) and against E. coli (31). It was noticed that furan derivatives also showed activity against C. albicans (18), and against E. coli (3 and 34). No systematic variation was observed in the antibacterial and antifungal activities for the rest of the compounds. All tested compounds showed poor biological activity against P. aeruginosa and S. aureus.

Conclusions
In summary, new series of anthracenylpyrazolines and anthracenylpyrimidines were synthesized from 3-(anthracen-9-yl)-1-aryl-prop-2-en-1-one derivatives, and spectroscopically characterized. The b i o l o g i c a l activity of the compounds was evaluated against E. coli, P. aeruginosa, S. aureus and C. albicans by the agar diffusion method. The potency of compounds 18, 28 and 30 as antifungics against C. albicans is about 50% of that of Clotrimazole. On the other hand, the potency of compounds 1, 3, 7, 20, 23, 31 and 34 as antibacterials against E. coli is about 50% of that of Ampicillin.    Reagent quality solvents were used without purification. Melting points were obtained in open capillary tubes by using a MEL-Temp II melting point apparatus and are uncorrected. Infrared spectra (IR) were recorded on a Perkin-Elmer 1600 series Fourier transform instrument with the samples as KBr pellets. 1 H NMR and 13 C NMR spectra were recorded on JEOL 500 MHz spectrometers at ambient temperature using tetramethylsilane as an internal reference. Mass spectra were recorded on a JEOL JMS AX-500 spectrometer by using electron impact ionization at 70 eV. Elemental analyses were carried out by the University of Cairo Microanalytical Laboratories. The antimicrobial tests were carried out at the Pharmaceutical Department, Faculty of Pharmacy, Alexandria University. ChemDraw-Ultra-11.0 has been used for the nomenclature of the prepared compounds.

General procedure for the preparation of compounds 35 and 36
A mixture of 4-(anthracen-9-yl)-6-arylpyrimidin-2amine (32, 0.38 g, 0.001 mol or 33, 0.36 g, 0.001 mol) and acetic anhydride (10 mL) was heated on boiling water bath for 2 h. The reaction mixture was poured onto cold water and the precipitated product was filtered, washed with water, dried and recrystallized from ethanol/chloroform.

Measurement of the inhibition zone (IZ)
Compounds 1-36 were evaluated in vitro for antimicrobial activity against Escherichia coli ATCC8739 and Pseudomonas aeruginosa ATCC 9027 as gramnegative bacteria, Staphylococcus aureus ATCC 6538P as an example of gram-positive bacteria and Candida albicans ATCC 2091 as yeast-like fungus. The agar-diffusion method 33 was used for the determination of antibacterial and antifungal activity. From 1 mg mL -1 solutions of each of the test compounds in N,N-dimethylformamide (DMF), 75 µL was placed in a 6 mm diameter well in an agar plate seeded with the appropriate test organism in triplicates. Ampicillin trihydrate (10 µg per disc), Ciprofloxacin (5 µg per disc), Imipenem (10 µg per disc) and Clotrimazole (100 µg per disc) were used as standard antibacterial and antifungal agents, respectively. The plates were incubated at 37 °C for 24 h. The results were recorded for each tested compounds as the average diameter of inhibition zone of bacterial growth in mm (Table 1). DMF alone (control) showed no inhibition zone.

Minimal inhibitory concentration (MIC)
The microdilution susceptibility test in Muller-Hinton broth (oxoid) and Sabouraud liquid medium (oxoid) were used for the determination of antibacterial and antifungal activity with the same test organisms. The MIC measurements 34 were carried out for compounds that showed significant inhibition zones using the two-fold serial dilution technique with solutions in the concentration range 500-15.63 µg mL -1 . Suspensions of the microorganisms at 10 6 CFU mL -1 (Colony Forming Units mL -1 ) were used to inoculate the prepared test compounds in the above mentioned serial dilution broth. The culture tubes were incubated at 37 o C for 24-48 h. At the end of the incubation period the growth of bacteria was observed in the form of turbidity. The MIC is defined as the lowest concentration that showed no bacterial growth (Table 1).