Enaminones as Building Blocks for the Synthesis of Substituted Pyrazoles with Antitumor and Antimicrobial Activities

Novel N-arylpyrazole-containing enaminones 2a,b were synthesized as key intermediates. Reactions of 2a,b with active methylene compounds in acetic acid in the presence of ammonium acetate afforded substituted pyridine derivatives 5a-d. Enaminones 2a,b also reacted with aliphatic amines such as hydrazine hydrate and hydroxylamine hydrochloride to give bipyrazoles 8a,b and pyrazolylisoxazoles 9a,b, respectively. On the other hand, treatment of 2a,b with a heterocyclic amine and its diazonium salt yielded the respective [1,2,4]triazolo[4,3-a]pyrimidines 12a,b and pyrazolylcarbonyl[1,2,4]triazolo-[3,4-c][1,2,4]triazines 14a,b. Moreover, 2-thioxo-2,3-dihydro-1H-pyrido[2,3-d]pyrimidin-4-one (17) was prepared via reaction of enaminone 2a with aminothiouracil (15). Cyclocondensation of 17 with the appropriate hydrazonoyl chlorides 18a-c gave the corresponding pyrido[2,3-d][1,2,4]triazolo[4,3-a]pyrimidin-5-ones 21a-c. The cytotoxic effects of compounds 2b, 14a and 17 against human breast cell line (MCF-7) and liver carcinoma cell line (HEPG2) were screened and in both lines they showed inhibition effects comparable to those of 5-fluorouracil, used as a standard. The antimicrobial activity of some products chosen as representative examples was also evaluated.


Introduction
The growing interest in bioactive N-arylpyrazoles has led to an increasing demand for efficient syntheses of this class of heterocyclic compounds. Several reports have found diverse applications for N-arylpyrazoles in medicine such as antitumor [1][2][3][4][5][6][7][8][9][10][11], antiviral [12], anti-inflammatory [13] agents, or OPEN ACCESS kinase inhibitors for the treatment of type 2 diabetes, hyperlipidemia, and obesity [14]. Moreover, these compounds have remarkable potential in nanomedicine applications against malignant gliomas [15]. 1-(4-Chlorophenyl)-4-hydroxy-3-substituted-1H-pyrazoles ( Figure 1) were reported by the U.S. National Cancer Institute (NCI) to have pronounced anticancer activity [16,17]. Structure modifications suggested in this work focused mainly on synthesis of polysubstituted pyrazole analogues to form A and B, having a variety of azoles and fused azoles at position 3. These substituents at position 3 are linked directly to the pyrazole ring or through the carbonyl group in order to improve the antitumor and antimicrobial activities of such compounds. This work is an extension of an ongoing research program devoted to the synthesis and characterization of different heterocyclic ring systems endowed with potential biological activities [18][19][20][21][22][23][24][25].
The structures of 2a,b were confirmed by their spectral data (IR, MS and 1 H-NMR) and elemental analyses. For example, the 1 H-NMR spectrum revealed two doublet signals at δ 5.88, 7.67 ppm with coupling constant J = 13 Hz assignable to olefinic protons (CH=CH) in a trans configuration [26,27] besides two singlet signals of the dimethylamino group at δ 2.8, 3.1 ppm.
Reactions of enaminones 2a,b with C-nucleophiles such as 2,4-pentanedione and ethyl 3-oxobutanoate were carried out in glacial acetic acid in the presence of ammonium acetate and led to formation of 6-(pyrazol-3-yl)-pyridine derivatives 5a-d via nucleophilic displacement of active methylene to the dimethylamino group followed by concurrent elimination of water molecule from non-isolable intermediates 4a-d (Scheme 2). The other possible isomeric structures 4-(pyrazol-3-yl)pyridines 7a-d were discarded based on 1 H-NMR data that revealed pyridyl hydrogens at C-4, C-5 as a pair of doublets at δ 7.5, 7.7 ppm, respectively, with J = 8 Hz assignable to 6-substituted-pyridines 5ad. The isomeric structures 7a-d should display pair of doublets corresponding to C-5, C-6 with a lower coupling constant (J = 2-3 Hz) [28]. Treatment of enaminones 2a,b with a N-nucleophile such as hydrazine hydrate in absolute ethanol under reflux afforded 1H,1'H-3,3'-bipyrazoles 8a,b. The structures of the products were substantiated by the 1 H-NMR spectra which displayed new pair of doublets at δ 7.53 and 7.58 ppm with (J = 7.5 Hz) corresponding to pyrazole protons at positions 4 and 5, respectively and another D 2 O exchangeable proton at δ 13 ppm assignable to the NH group. The products were formed via initial addition of the amino group in hydrazine to the enaminone double bond, followed by elimination of dimethylamine and water molecules to give the final isolable products 8a,b as previously mentioned [29] (Scheme 3). Similarly, enaminones 2a,b reacted with hydroxylamine hydrochloride in refluxing absolute ethanol in the presence of anhydrous potassium carbonate to yield products that may be formulated as pyrazolylisoxazoles 9a,b or its isomeric forms 10a,b. Structure 9 was assigned for the reaction products on the basis of the 1 H-NMR spectral data in which a resonance for H-4 and H-5 of isoxazole appeared typically at δ 6.78 and 8.50 ppm, respectively (see Experimental). The other isomeric structures 10a,b were ruled out as the isoxazole H-3 would be expected to resonate at a higher field of δ 8.0 ppm [30]. It is thus assumed that, the products 9a,b were formed via initial condensation of amino group of hydroxylamine with carbonyl group of enaminones 2a,b followed by elimination of dimethylamine (cf. Scheme 3). Next, the reactions of enaminones 2a,b with heterocyclic amines were investigated. Refluxing of enaminones 2a,b with 3-amino-1H- [1,2,4]triazole in glacial acetic acid gave the corresponding [1,2,4]triazolo[4,3-a]pyrimidines 12a,b via non-isolable intermediates 11a,b (Scheme 4).

Antitumor Screening Test
The cytotoxic effects of compounds 2b, 14a and 17 against human breast cell line (MCF-7) and liver carcinoma cell line (HEPG2) were evaluated using 5-fluorouracil as a standard sample in both lines.         The results of biological screening allow the following assumptions about the structure activity relationships (SAR) of these compounds: • The presence of nitrogenous fused heterocycles at position 3 of the main pyrazole moiety, linked directly or through carbonyl group, with multicenters for hydrogen accepting properties are essential for activity where it can intercalate within the DNA strands.

Antimicrobial Activity
The newly synthesized products 2a, 2b, 5b, 5c, 8b, 9b, 12b, 14a, 21a and 21b were tested for their antimicrobial activities using four species of fungi, namely Aspergillus fumigatus AF, Penicillium italicum PI, Syncephalastrum racemosum SR and Candida albicans CA, in addition to four bacterial species, namely Staphylococcus aureus SA, Pesudomonas aeruginosa PA, Bacillus subtilis BS and Escherichia coli EC. The organisms were tested against the activity of solutions of three different concentrations [5 mg/mL, 2.5 mg/mL, 1.25 mg/mL] of each compound and using inhibition zone diameter (IZD) in mm as criterion for the antimicrobial activity. The fungicide terbinafine and the bactericide chloramphenicol were used as references to evaluate the potency of the tested compounds under the same conditions. The results, depicted in Tables 1-3, revealed that compounds 2a and 5c exhibited high degree of inhibition against SA, and BS. Compounds 9b, 12b, 14a, 21a and 21b have high inhibition effects against AF, PI, SR and SA. These compounds also exhibited moderate inhibition effect against CA and BS. All the tested compounds were reflecting no inhibition of growth against PA and EC. Table 1. Antimicrobial activity of products 2a, 2b, 5b, and 5c.

General
All melting points were determined on an electrothermal Gallenkamp apparatus and are uncorrected. Solvents were generally distilled and dried by standard literature procedures prior to use. The IR spectra were measured on a Pye-Unicam SP300 instrument in potassium bromide discs. The 1 H NMR spectra were recorded on a Varian Mercury VXR-300 spectrometer (300 MHz) and the chemical shifts were related to that of the solvent DMSO-d 6 . The mass spectra were recorded on a GCMS-Q1000-EX Shimadzu and GCMS 5988-A HP spectrometers, the ionizing voltage was 70 eV. Elemental analyses were carried out by the Microanalytical Center of Cairo University, Giza, Egypt. Antitumor activity was evaluated by the National Institute of Cancer, Biology Department, Cairo University, Egypt. Antimicrobial activity was carried out at the Regional Center for Mycology and Biotechnology at Al-Azhar University, Cairo, Egypt. 3-Acetyl-4-(4-nitrophenyl)-1-aryl-1H-pyrazoles 1a,b [26] and hydrazonoyl halides [38][39][40][41][42][43] 18a-c were prepared following literature methods.

Agar diffusion well method to determine the antimicrobial activity
The microorganism inoculums were uniformly spread using sterile cotton swab on a sterile Petri dish Malt extract agar (for fungi) and nutrient agar (for bacteria). One hundred μL of each sample was added to each well (10 mm diameter holes cut in the agar gel, 20 mm apart from one another). The systems were incubated for 24-48 h at 37 °C (for bacteria) and at 28 °C (for fungi). After incubation, the microorganism's growth was observed. Inhibition of the bacterial and fungal growth were measured in mm. Tests were performed in triplicate [44].

Cytotoxic activity
The method applied is similar to that reported by Skehan et al. [45] using Sulfo-Rhodamine-B stain (SRB). Cells were plated in 96-multiwill plate (10 4 cells/well) for 24 h before treatment with the tested compounds to allow attachment of cell to the wall of the plate. Different concentrations of the compound under test (0, 2.5, 5, and 10 µg/mL) were added to the cell monolayer in triplicate wells individual dose, monolayer cells were incubated with the compounds for 48 h at 37 °C and in atmosphere of 5% CO 2 . After 48 h, cells were fixed, washed and stained with SRB stain, excess stain was washed with acetic acid and attached stain was recovered with tris-EDTA buffer, color intensity was measured in an ELISA reader. The relation between surviving fraction and drug concentration is plotted to get the survival curve of each tumor cell line after the specified compound. The response parameter calculated was the IC 50 value, which corresponds to the compound concentration causing 50% mortality in net cells (Figures 3-10).

Conclusions
In this study, synthetic routes to a wide variety of azoles, fused azoles, and azines at the 3-position of N-arylpyrazole ring were developed using novel enaminones as building blocks. Moreover, some of the newly synthesized products were tested as antitumor and antimicrobial agents and the results obtained were promising.