Microwave-Assisted Synthesis of some Novel Azoles and Azolopyrimidines as Antimicrobial Agents

In this study, new derivatives of pyrazole, isoxazole, pyrazolylthiazole, and azolopyrimidine having a thiophene ring were synthesized under microwave irradiation. Their pharmacological activity toward bacteria and fungi inhibition was screened and compared to the references Chloramphenicol and Trimethoprim/sulphamethoxazole. The antimicrobial results of the investigated compounds revealed promising results and some derivatives have activities similar to the references used.


Introduction
Five-membered heterocyclic ring systems are very significant class of compounds, not only due to their abundance in nature, but also for their chemical and biological value. Thiophene derivatives have been fully-known for their therapeutic applications. They possess antihypertensive [1], antimicrobial [2], diabetes mellitus [3], antiviral [4], analgesic and anti-inflammatory [5], and antitumor activities [6,7]. Pyrazoles and thiazoles exist in many naturally occurring substances and representing an interesting array of azole compounds. They have a wide range of biological activities as for example, anti-inflammatory [8,9], antimicrobial [10][11][12][13], Akt kinase inhibitive [14], anticonvulsant [15], and antitumor activities [16]. On the other hand, microwave-assisted organic synthesis is a tool by which we can achieve goals in a few minutes with high yield as compared to conventional heating [17][18][19][20][21]. Motivated by these findings, and in continuation of our ongoing research program dealing with the synthesis of bioactive heterocyclic ring systems [22][23][24][25][26], we were encouraged to synthesize heterocyclic having thiophene incorporated pyrazole, thiazole, and/or pyrimidine derivatives under microwave irradiation to investigate their antimicrobial activity.

Synthesis
1,3-Di(thiophen-2-yl)prop-2-en-1-one 1 was cyclized with different types of nitrogen nucleophiles, namely, thiosemicarbazide, hydrazine derivatives 3a-c, and hydroxylamine hydrochloride which afforded pyrazole derivatives 2, 4a-c and isoxazole derivative 5, respectively (Scheme 1). The previous reactions were carried out under conventional heating and under microwave irradiation as shown in Table 1. The heating under microwave was more efficient than thermal heating as it reduced the reaction time and increased the product yields in all cases.
It was reported that pyrazolylthiazole derivatives have a wide range of biological activities such as antimicrobial [27], anti-inflammatory [27], hypotensive [28], and antitumor activities [29]. So we became interested in synthesizing the pyrazolylthiazole derivatives from the reaction of 1-thiocarbamoyl-3,5-di-(2-thienyl)-2-pyrazoline 2 with hydrazonoyl chlorides. Thus, conventional heating or microwave irradiation of mixture of carbothioic acid amide derivative 2 and 2-oxo-Narylpropanehydrazonoyl chloride 6a-e in dioxane in the existence of a base catalyst yielded in each case only one isolated product (Scheme 2). The spectroscopic information confirmed the reaction products 8a-e. For example, the mass spectra of the isolated products 8a-e displayed the expected molecular ion. Also, all derivatives 8a-e showed in their 1 H-NMR spectra the characteristic signals for CH 3 , H-5, and CH 2 (see experimental part). The structure of products 8 was further supported by an alternative synthesis. Thus, reaction of compound 1 with 2-hydrazinyl-4-methyl-5-(phenyldiazenyl)thiazole 9 under reflux in ethanol led to the formation of product 8a (Scheme 2). reactions were carried out under conventional heating and under microwave irradiation as shown in Table 1. The heating under microwave was more efficient than thermal heating as it reduced the reaction time and increased the product yields in all cases. It was reported that pyrazolylthiazole derivatives have a wide range of biological activities such as antimicrobial [27], anti-inflammatory [27], hypotensive [28], and antitumor activities [29]. So we became interested in synthesizing the pyrazolylthiazole derivatives from the reaction of 1-thiocarbamoyl-3,5-di-(2-thienyl)-2-pyrazoline 2 with hydrazonoyl chlorides. Thus, conventional heating or microwave irradiation of mixture of carbothioic acid amide derivative 2 and 2-oxo-Narylpropanehydrazonoyl chloride 6a-e in dioxane in the existence of a base catalyst yielded in each case only one isolated product (Scheme 2). The spectroscopic information confirmed the reaction products 8a-e. For example, the mass spectra of the isolated products 8a-e displayed the expected molecular ion. Also, all derivatives 8a-e showed in their 1 H-NMR spectra the characteristic signals for CH3, H-5, and CH2 (see experimental part). The structure of products 8 was further supported by an alternative synthesis. Thus, reaction of compound 1 with 2-hydrazinyl-4-methyl-5-(phenyldiazenyl)thiazole 9 under reflux in ethanol led to the formation of product 8a (Scheme 2). Scheme 1. Synthesis of pyrazoline derivatives 2, 4a-c, and 5.

Antimicrobial Activity
In vitro antimicrobial screening of compounds 2, 4a-c, 5, 8a-e, 11a,b, 13,and 15 prepared in the study was carried out using cultures of two fungal strains Aspergillus niger (ATCC) (ASP) and Candida albicans (ATCC10231) (CA), as well as three bacteria species, namely, Gram positive bacteria, Staphylococcus aureus (ATCC 29213) (SA), and Bacillus subtilus (ATCC 6051) (BS) and the Gram negative bacteria is Escherichia coli (ATCC 25922) (EC). Chloramphenicol and Trimethoprim/sulphamethoxazole antibacterial agents were used as references to evaluate the potency of the examined compounds under the same conditions. The activity was investigated by measuring the diameter of inhibition zone (IZD) in mm ± standard deviation beyond well diameter (6 mm) generated on a range of environmental and clinically pathogenic microorganisms (gram-positive and gram-negative bacteria and fungi) utilizing (0.1 g/mL) concentration of tested samples and the outcomes are portrayed in Table 2. For the antifungal activity: All tested compounds were inactive against Aspergillus niger (ATCC) (ASP) while, compounds 4c, 8c, and 11b have excellent activity against Candida albicans (ATCC 10231) (CA) with inhibition zones 23, 24, and 25 respectively. For the antibacterial activity: it was found that Gram positive bacteria are more sensitive to the tested compounds especially SA rather than BS as five compounds 2, 4c, 8b, 8d, and 15 have potent activity against SA while for BS only compounds 4a and 4c showed good activity. In the case of Gram negative activity with EC, two derivatives 2 and 8c revealed higher activity. The used solvent DMSO concentration did not exhibit any influence on bacteria or fungi. bacteria is Escherichia coli (ATCC 25922) (EC). Chloramphenicol and Trimethoprim/sulphamethoxazole antibacterial agents were used as references to evaluate the potency of the examined compounds under the same conditions. The activity was investigated by measuring the diameter of inhibition zone (IZD) in mm ± standard deviation beyond well diameter (6 mm) generated on a range of environmental and clinically pathogenic microorganisms (gram-positive and gram-negative bacteria and fungi) utilizing (0.1 g/mL) concentration of tested samples and the outcomes are portrayed in Table 2. For the antifungal activity: All tested compounds were inactive against Aspergillus niger (ATCC) (ASP) while, compounds 4c, 8c, and 11b have excellent activity against Candida albicans (ATCC 10231) (CA) with inhibition zones 23, 24, and 25 respectively. For the antibacterial activity: it was found that Gram positive bacteria are more sensitive to the tested compounds especially SA rather than BS as five compounds 2, 4c, 8b, 8d, and 15 have potent activity against SA while for BS only compounds 4a and 4c showed good activity. In the case of Gram negative activity with EC, two derivatives 2 and 8c revealed higher activity. The used solvent DMSO concentration did not exhibit any influence on bacteria or fungi.

General Experimental Procedures
Melting points were measured with an IA 9000-series digital melting-point apparatus (Bibby Sci. Lim. Stone, Staffordshire, UK). Solvents were generally distilled and dried by standard literature procedures prior to use. IR spectra were recorded in potassium bromide discs on FTIR 8101 PC infrared spectrophotometers (Shimadzu, Tokyo, Japan). NMR spectra were recorded on a Mercury VX-300 NMR spectrometer (Varian, Inc., Karlsruhe, Germany) operating at 300 MHz ( 1 H-NMR) and run in deuterated dimethylsulfoxide (DMSO-d6). Chemical shifts were related to that of the solvent. Mass spectra were recorded on a Shimadzu GCeMS-QP1000 EX mass spectrometer (Tokyo, Japan) at 70 eV. Microwave reactions were performed with a Millstone Organic Synthesis Unit with a touch control terminal (MicroSYNTH, Giza, Egypt) and a continuous focused microwave power delivery system in a pressure glass vessel (10 mL) sealed with a septum under magnetic stirring. The

Moderate activity
Molecules 2017, 22, 346 4 of 9 bacteria is Escherichia coli (ATCC 25922) (EC). Chloramphenicol and Trimethoprim/sulphamethoxazole antibacterial agents were used as references to evaluate the potency of the examined compounds under the same conditions. The activity was investigated by measuring the diameter of inhibition zone (IZD) in mm ± standard deviation beyond well diameter (6 mm) generated on a range of environmental and clinically pathogenic microorganisms (gram-positive and gram-negative bacteria and fungi) utilizing (0.1 g/mL) concentration of tested samples and the outcomes are portrayed in Table 2. For the antifungal activity: All tested compounds were inactive against Aspergillus niger (ATCC) (ASP) while, compounds 4c, 8c, and 11b have excellent activity against Candida albicans (ATCC 10231) (CA) with inhibition zones 23, 24, and 25 respectively. For the antibacterial activity: it was found that Gram positive bacteria are more sensitive to the tested compounds especially SA rather than BS as five compounds 2, 4c, 8b, 8d, and 15 have potent activity against SA while for BS only compounds 4a and 4c showed good activity. In the case of Gram negative activity with EC, two derivatives 2 and 8c revealed higher activity. The used solvent DMSO concentration did not exhibit any influence on bacteria or fungi.

General Experimental Procedures
Melting points were measured with an IA 9000-series digital melting-point apparatus (Bibby Sci. Lim. Stone, Staffordshire, UK). Solvents were generally distilled and dried by standard literature procedures prior to use. IR spectra were recorded in potassium bromide discs on FTIR 8101 PC infrared spectrophotometers (Shimadzu, Tokyo, Japan). NMR spectra were recorded on a Mercury VX-300 NMR spectrometer (Varian, Inc., Karlsruhe, Germany) operating at 300 MHz ( 1 H-NMR) and run in deuterated dimethylsulfoxide (DMSO-d6). Chemical shifts were related to that of the solvent. Mass spectra were recorded on a Shimadzu GCeMS-QP1000 EX mass spectrometer (Tokyo, Japan) at 70 eV. Microwave reactions were performed with a Millstone Organic Synthesis Unit with a touch control terminal (MicroSYNTH, Giza, Egypt) and a continuous focused microwave power delivery system in a pressure glass vessel (10 mL) sealed with a septum under magnetic stirring. The

Low activity
Molecules 2017, 22, 346 4 of 9 bacteria is Escherichia coli (ATCC 25922) (EC). Chloramphenicol and Trimethoprim/sulphamethoxazole antibacterial agents were used as references to evaluate the potency of the examined compounds under the same conditions. The activity was investigated by measuring the diameter of inhibition zone (IZD) in mm ± standard deviation beyond well diameter (6 mm) generated on a range of environmental and clinically pathogenic microorganisms (gram-positive and gram-negative bacteria and fungi) utilizing (0.1 g/mL) concentration of tested samples and the outcomes are portrayed in Table 2. For the antifungal activity: All tested compounds were inactive against Aspergillus niger (ATCC) (ASP) while, compounds 4c, 8c, and 11b have excellent activity against Candida albicans (ATCC 10231) (CA) with inhibition zones 23, 24, and 25 respectively. For the antibacterial activity: it was found that Gram positive bacteria are more sensitive to the tested compounds especially SA rather than BS as five compounds 2, 4c, 8b, 8d, and 15 have potent activity against SA while for BS only compounds 4a and 4c showed good activity. In the case of Gram negative activity with EC, two derivatives 2 and 8c revealed higher activity. The used solvent DMSO concentration did not exhibit any influence on bacteria or fungi.

General Experimental Procedures
Melting points were measured with an IA 9000-series digital melting-point apparatus (Bibby Sci. Lim. Stone, Staffordshire, UK). Solvents were generally distilled and dried by standard literature procedures prior to use. IR spectra were recorded in potassium bromide discs on FTIR 8101 PC infrared spectrophotometers (Shimadzu, Tokyo, Japan). NMR spectra were recorded on a Mercury VX-300 NMR spectrometer (Varian, Inc., Karlsruhe, Germany) operating at 300 MHz ( 1 H-NMR) and run in deuterated dimethylsulfoxide (DMSO-d6). Chemical shifts were related to that of the solvent. Mass spectra were recorded on a Shimadzu GCeMS-QP1000 EX mass spectrometer (Tokyo, Japan) at 70 eV. Microwave reactions were performed with a Millstone Organic Synthesis Unit with a touch control terminal (MicroSYNTH, Giza, Egypt) and a continuous focused microwave power delivery system in a pressure glass vessel (10 mL) sealed with a septum under magnetic stirring. The bacteria is Escherichia coli (ATCC 25922) (EC). Chloramphenicol and Trimethoprim/sulphamethoxazole antibacterial agents were used as references to evaluate the potency of the examined compounds under the same conditions. The activity was investigated by measuring the diameter of inhibition zone (IZD) in mm ± standard deviation beyond well diameter (6 mm) generated on a range of environmental and clinically pathogenic microorganisms (gram-positive and gram-negative bacteria and fungi) utilizing (0.1 g/mL) concentration of tested samples and the outcomes are portrayed in Table 2. For the antifungal activity: All tested compounds were inactive against Aspergillus niger (ATCC) (ASP) while, compounds 4c, 8c, and 11b have excellent activity against Candida albicans (ATCC 10231) (CA) with inhibition zones 23, 24, and 25 respectively. For the antibacterial activity: it was found that Gram positive bacteria are more sensitive to the tested compounds especially SA rather than BS as five compounds 2, 4c, 8b, 8d, and 15 have potent activity against SA while for BS only compounds 4a and 4c showed good activity. In the case of Gram negative activity with EC, two derivatives 2 and 8c revealed higher activity. The used solvent DMSO concentration did not exhibit any influence on bacteria or fungi.

General Experimental Procedures
Melting points were measured with an IA 9000-series digital melting-point apparatus (Bibby Sci. Lim. Stone, Staffordshire, UK). Solvents were generally distilled and dried by standard literature procedures prior to use. IR spectra were recorded in potassium bromide discs on FTIR 8101 PC infrared spectrophotometers (Shimadzu, Tokyo, Japan). NMR spectra were recorded on a Mercury VX-300 NMR spectrometer (Varian, Inc., Karlsruhe, Germany) operating at 300 MHz ( 1 H-NMR) and run in deuterated dimethylsulfoxide (DMSO-d6). Chemical shifts were related to that of the solvent. Mass spectra were recorded on a Shimadzu GCeMS-QP1000 EX mass spectrometer (Tokyo, Japan) at 70 eV. Microwave reactions were performed with a Millstone Organic Synthesis Unit with a touch control terminal (MicroSYNTH, Giza, Egypt) and a continuous focused microwave power delivery system in a pressure glass vessel (10 mL) sealed with a septum under magnetic stirring. The

General Experimental Procedures
Melting points were measured with an IA 9000-series digital melting-point apparatus (Bibby Sci. Lim. Stone, Staffordshire, UK). Solvents were generally distilled and dried by standard literature procedures prior to use. IR spectra were recorded in potassium bromide discs on FTIR 8101 PC infrared spectrophotometers (Shimadzu, Tokyo, Japan). NMR spectra were recorded on a Mercury VX-300 NMR spectrometer (Varian, Inc., Karlsruhe, Germany) operating at 300 MHz ( 1 H-NMR) and run in deuterated dimethylsulfoxide (DMSO-d 6 ). Chemical shifts were related to that of the solvent. Mass spectra were recorded on a Shimadzu GCeMS-QP1000 EX mass spectrometer (Tokyo, Japan) at 70 eV. Microwave reactions were performed with a Millstone Organic Synthesis Unit with a touch control terminal (MicroSYNTH, Giza, Egypt) and a continuous focused microwave power delivery system in a pressure glass vessel (10 mL) sealed with a septum under magnetic stirring. The temperature of the reaction mixture was monitored using a calibrated infrared temperature control under the reaction vessel, and control of the pressure was performed with a pressure sensor connected to the septum of the vessel. Elemental analyses were carried out at the Microanalytical Centre of Cairo University, Giza, Egypt. Compounds 10a,b, 12, and 14 were purchased from Sigma-Aldrich and utilized as it is without previous treatments. Compounds 1, 2, 6a-e, and 9 were prepared as previously reported in the respective literature [30][31][32].

Synthesis of Pyrazoline Derivatives 4a-c
Method A: A mixture of chalcone 1 (0.220 g, 1 mmol) and hydrazine derivative (1 mmol) in ethanol (20 mL) in the presence of catalytic drops of acetic acid was refluxed for 3-5 h (monitored by TLC). The reaction mixture was poured into water and the solid product was collected by filtration followed by washing with ethanol. The crude products were then recrystallized from ethanol to give pure pyrazolines 4a-c, respectively.
Method B: Repetition of the same reactions of method A with heating in a microwave oven at 500 W and 120 • C for a period of time. The reaction mixture was treated similar to method A to obtain compounds 4a-c. Compounds 4a-c with their physical constants and spectral data are depicted as shown below: Method A: A mixture of chalcone 1 (0.220 g, 1 mmol), hydroxylamine. HCl (0.069 g, 1 mmol), and anhydrous sodium acetate (0.3 g) in acetic acid (20 mL) was stirred at room temperature for 6 h. The formed solid was filtered, washed with water, and crystallized from dioxane to give isoxazoline derivative 5.
Method B: The above reaction of chalcone 1 and hydroxylamine with the same quantity in method A were heated under microwave irradiation at 500 W and 150 • C for 10 min. The reaction mixture was treated similarly to method A to obtain compounds 5 as yellow solid; m.p.