Synthesis and Biological Evaluation of N- Pyrazolyl Derivatives and Pyrazolopyrimidine Bearing a Biologically Active Sulfonamide Moiety as Potential Antimicrobial Agent

A series of novel pyrazole-5-carboxylate containing N-triazole derivatives 3,4; different heterocyclic amines 7a–b and 10a–b; pyrazolo[4,3-d]pyrimidine containing sulfa drugs 14a,b; and oxypyrazolo[4,3-d]pyrimidine derivatives 17, 19, 21 has been synthesized. The structure of the newly synthesized compounds was elucidated on the basis of analytical and spectral analyses. All compounds have been screened for their in vitro antimicrobial activity against three gram-positive and gram-negative bacteria as well as three fungi. The results revealed that compounds 14b and 17 had more potent antibacterial activity against all bacterial strains than reference drug Cefotaxime. Moreover compounds 4, 7b, and 12b showed excellent antifungal activities against Aspergillus niger and Candida albicans in low inhibitory concentrations but slightly less than the reference drug miconazole against Aspergillus flavus.


Introduction
Antimicrobial resistance has become a serious health problem, so the increased rate of microbial infections and resistance to antimicrobial agents [1] prompted us to identify a novel structure that may be used in designing new, potent, and broad spectrum antimicrobial agents. Pyrazoles are one of the most common pharmaceutically active compounds, and have attracted much attention due to their broad spectrum of biological activities such as anti-inflammatory [2,3], COX inhibitory [4], hypoglycemic [5], CDK2/Cyclin A [6], p38 MAP kinase [7], and antidepressive activities [8], and have been widely used in biopharmaceutical and pesticides. Pyrazole plays a unique role in drug discovery programs. Pyrazole derivatives are an important class of heterocyclic compounds showing a wide range of biological activities such as antimicrobial [9][10][11] (Figure 1).
Pyrazolopyrimidines have important biological functions including herbicidal [12] and antitumor activity [13]; pyrazolopyrimidine derivatives have been found as purine analogs [14] and have significant properties as antimetabolites in purine biochemical reactions such as neuropeptide Y1 receptor antagonists [15]. Also, the pyrazolo [4,3-d]pyrimidinone class of compounds is very important in the treatment of impotence [16], used as an PDE5 inhibitor. PP30 possesses a good kinase selectivity profile used as ATP-competitive mTORC1/mTORC2 inhibitors [17,18] (Figure 1). In view of these observations and as a continuation of our previous work on heterocyclic chemistry, we report herein the synthesis of some new heterocyclic-containing pyrazolo [3,2-d]pyrimidine moieties and the study of their antimicrobial activities in comparison to Cefotaxime and miconazole as reference drugs.

Compd. No
Treatment of compounds (12a,b) with hydrazine hydrate in refluxing ethanol afforded the cyclic N-amino compounds (13a,b). The formation of N-amino derivatives (13a,b) proceeded via loss of 1 mol of H2S, followed by intramolecular cyclization to give (13a,b), respectively. IR spectra of compounds (13a,b) revealed the presence of characteristic bands of NH2, C=O and SO2 groups. Thioureido derivatives (14a,b) were obtained via a reaction of compounds (13a,b) with phenyl isothiocyanate (Scheme 3).
Treatment of compounds (12a,b) with hydrazine hydrate in refluxing ethanol afforded the cyclic N-amino compounds (13a,b). The formation of N-amino derivatives (13a,b) proceeded via loss of 1 mol of H 2 S, followed by intramolecular cyclization to give (13a,b), respectively. IR spectra of compounds (13a,b) revealed the presence of characteristic bands of NH 2 , C=O and SO 2 groups. Thioureido derivatives (14a,b) were obtained via a reaction of compounds (13a,b) with phenyl isothiocyanate (Scheme 3).

Antimicrobial Evaluation
The molar refractivity and the values of the MIC against the tested microorganisms are reported in Tables 1-3. Among the 24 newly synthesized compounds, compounds 14b with MIC value ranging from (6-8 µg·mL −1 ) and 17 with MIC (4-10 µg·mL −1 ) were found to have more potent antibacterial activity against all strains than the reference drug Cefotaxime with MIC value (6-13 µg·mL −1 ). It was found that lipophilicity plays a major role in determining where drugs are distributed within the body after adsorption and, as a consequence, how rapidly they are metabolized and excreted. In the biological system drug disposition depends on the ability to cross membranes, so there is a strong relationship with measures of lipophilicity [23]. So the strong lipophilic character of the molecule

Antimicrobial Evaluation
The molar refractivity and the values of the MIC against the tested microorganisms are reported in Tables 1-3. Among the 24 newly synthesized compounds, compounds 14b with MIC value ranging from (6-8 µg·mL −1 ) and 17 with MIC (4-10 µg·mL −1 ) were found to have more potent antibacterial activity against all strains than the reference drug Cefotaxime with MIC value (6-13 µg·mL −1 ). It was found that lipophilicity plays a major role in determining where drugs are distributed within the body after adsorption and, as a consequence, how rapidly they are metabolized and excreted. In the biological system drug disposition depends on the ability to cross membranes, so there is a strong relationship with measures of lipophilicity [23]. So the strong lipophilic character of the molecule

Antimicrobial Evaluation
The molar refractivity and the values of the MIC against the tested microorganisms are reported in Tables 1-3. Among the 24 newly synthesized compounds, compounds 14b with MIC value ranging from (6-8 µg·mL −1 ) and 17 with MIC (4-10 µg·mL −1 ) were found to have more potent antibacterial activity against all strains than the reference drug Cefotaxime with MIC value (6-13 µg·mL −1 ). It was found that lipophilicity plays a major role in determining where drugs are distributed within the body after adsorption and, as a consequence, how rapidly they are metabolized and excreted. In the biological system drug disposition depends on the ability to cross membranes, so there is a strong relationship with measures of lipophilicity [23]. So the strong lipophilic character of the molecule plays a major role in producing the antimicrobial effect. In this context the presence of the hydrophobic moiety would be important for such activity. The lipophilicity of the compounds, expressed as log P, explains the main predictor for the activity. The octanol/water partition coefficient C log P was calculated using the software ACD/log P 1.0 and the results are shown in Table 1. The molar refractivity (MR) of the newly synthesized compounds was also calculated using the software ACD/log P 1.0 to explain the activity behavior of the synthesized compounds. From Tables 2 and 3 it can be inferred that the higher value of molar refractivity favors the activity ratio.   Pyrazole containing N-methyl piperazine 7b with MIC (6-12 µg·mL −1 ) displayed not only comparable antibacterial activity against all the tested bacterial strains, but also excellent antifungal activity against Aspergillus niger and Candida albicans in low inhibitory concentrations MIC (6 µg·mL −1 ), but moderate activity against Aspergillus flavus with MIC (10 µg·mL −1 ). Furthermore compounds 6, 9, and 10d, and pyrazole-containing sulphonamide derivatives 12a,b and 14a with MIC (8-16 µg·mL −1 ) exhibited good antibacterial activity slightly lower than the reference drug cefotaxime, but 12c,d and 13a,b exhibited moderate antibacterial activity. Also compounds 2-4, 19, and 21 with MIC (10-16 µg·mL −1 ) exhibited moderate inhibitory efficiency against all the tested bacterial strains.
Antifungal evaluation in vitro revealed that most of the newly prepared compounds exhibited completely different results in comparison with their antibacterial activities. Pyrazoles 2-4 with an oxiran-2-yl-methyl group and derivatives of triazolyl moieties at the N-1 position showed good inhibitory efficiency against all the tested fungal strains, which gave comparable or superior inhibitory potency to the first-line antifungal drug miconazole. Compound 7b exhibited equipotent activity against Aspergillus niger and Candida albicans, but good activity against Aspergillus flavus compared to miconazole. Their activity may be attributed to the presence of N-butylmethylpiprazine. Compound 12b, which has sulphonamide derivatives, exhibited equipotent activity against Aspergillus niger and Candida albicans but good activity against Aspergillus flavus; pyrazolopyrimidine sulphonamide derivatives (13a) exhibited good antifungal activities with MIC value (8-10 µg·mL −1 ). Most of the newly synthesized compounds (6, 7(a,c,d), 9, 10(a,c,d), 12(a,c,d), 13b, 17, 19, and 21 exhibited moderate antifungal activity against some of the fungal strains, while compounds 10b and 14a,b exhibited low antifungal activity compared to the reference drug miconazole.

General Information
All melting points were measured on an Electrothermal 9100 series digital melting point apparatus (Shimadzu, Tokyo, Japan). Microanalytical data were gathered with a Vario Elementar apparatus (Shimadzu). Elemental analyses of all compounds were within ±0.4% of the theoretical values. The IR spectra (KBr) were recorded on a Perkin Elmer 1650 spectrometer (Shelton, CT, USA). 1 H-NMR and 13 C-NMR spectra were recorded on a JEOL EX-300 and JEOL ECA-500 (Shimadzu, Tokyo, Japan).
Chemical shifts were expressed in ppm relative to SiMe 4 as internal standard in DMSO-d 6 as a solvent. Mass spectra were recorded on a 70 eV Finnigan SSQ 7000 spectrometer (Thermo-Instrument System Incorporation, Columbia, MD, USA). The purity of the compounds was checked on aluminum plates coated with silica gel (Merck, Darmstadt, Germany). Chemicals and solvents (Analar ≥ 99%) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Ethyl 4-amino-3-(4-chlorophenyl)-1-[2-hydroxy-3-(1H-1,2,4-triazol-1-yl)-propyl]-1H-pyrazole-5-carboxylate (3)
To a stirred suspension of potassium carbonate (1.51 g, 12 mmol) in ethanol was added 1,2,4-triazole (6.91 g, 10 mmol). The mixture was stirred at 60 • C for 1 h. The reaction was cooled to room temperature, compound 2 (3.22 g, 10 mmol) was added at the room temperature and stirred for 2 h under reflux. After the reaction came to an end, the solvent was evaporated and the residue was extracted with chloroform. The combined organic extracts were dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was purified by column chromatography To a stirred solution of sodium hydroxide (3%, 20 mL) was added compound 3 the mixture was stirred at 100 • C for 3 h. After the reaction was completed, the mixture was treated with formic acid to adjust the pH to 7, and then the suspension was filtered and washed with water three times to give

General Procedure for the Synthesis of 6 and 9
A mixture of compound 1 (2.65 g, 10 mmol) and 1,4-dibromobutane 5 (2.16 g, 10 mmol) and /or (E)-1,4-dibromobut-2-ene 8 (2.14 g, 10 mmol) in dry DMF (10 ml) along with anhydrous potassium carbonate (1.88 g, 15 mmol) was stirred at room temperature for about five hours. The reaction mixture was then poured into ice cold water. The contents were then extracted with diethyl ether and the ether layer was washed with a brine solution and dried over anhydrous sodium sulfate. The solvent was removed and the crude product so obtained was crystallized from ethanol.

General Procedure for Synthesis of 7a-d and 10a-d
N-Nucleophile (10 mmol) was dissolved in DMF (20 mL) along with anhydrous potassium carbonate (15 mmol) The bromo compound (6 and 9) (10 mmol) was added to the solution, and the mixture was stirred at room temperature for 3 h. After completion of the reaction, the mixture was poured into ice cold water. The precipitate obtained was washed with water, dried under a vacuum, and crystallized from ethanol to afford 7a-d and from dioxane to afford 10a-d in good yield.

General Procedure for Synthesis of 19 and 21
A mixture of 15 (2.60 g, 10 mmol), was stirred with potassium carbonate (1.38 g, 10 mmol) in dry DMF (20 mL) for 1 h, followed by the addition of the appropriate alkenyl halide (10 mmol) in portions. The reaction mixture was stirred at room temperature overnight, then refluxed for 4 h. After cooling it was then filtered to remove insoluble materials, and the filtrate was poured into ice water to give the crude product as a precipitate, which in turn was filtered off and dried. The crude product obtained in all cases was purified by recrystallization from ethanol.