1. Introduction

The goal of the anti-infective therapy is to rid the host of the pathogens, whereas the therapy of other diseases takes aim at direct the host [1]. Although significant progress has been made for the treatment and control of microbial infections by introducing new strategies and combinatorial therapy, antimicrobial resistance continues to be one of major concerns to the public health and scientific communities worldwide. Because, infections caused by resistant pathogens fail to response to treatment resulting in prolonged illness and greater risk of death. The history of antimicrobial chemotherapy has clearly demonstrated that the drugs used for the treatment of microbial infections are also responsible for making these diseases more difficult to treat in future due to the increasing antimicrobial resistance [1, 2]. In order to overcome the antimicrobial resistance, several strategies have been developed; one of which contains the development of hybrid molecules synthesized by combination of several pharmacophores that are selected on the basis of their known bio profiles [3–6].

Nitrogen-containing heterocycles constitute an extraordinary class of synthetic organic chemistry, natural products, drugs and food industry [7]. Among them, piperazine nucleus locates in the structures of a number of important drugs such as ciprofloxacin, norfloxacin, pefloxacin, ofloxacin, lomefloxacin, which are fluoroquinolone class antibacterials used for the treatment of various infections caused by Gram negative bacteria (Fig. S1 in Supporting information) [8–10].

The science of organic synthesis is constantly enriched by the improvement of synthetic methodologies. Microwave assisted techniques were reported to be more effective in perspective of environment, reaction time, high yields, ease of work-up and isolation of products. Moreover, solvents which are often expensive, toxic, difficult to remove in the case of aprotic dipolar solvents with high boiling point, and are environmentally polluting agents, are not necessary most of the microwave assisted synthesis [11]. Recently, multicomponent reactions (MCRs) have received considerable attention by synthetic organic and medicinal chemists for the construction of complex molecules having biological activity. When compared with conventional organic reactions, MCRs have some superior properties including high conversion rate, minimal reaction time and structural complexity. Thus, MCRs are also considered as green chemical processes [12]. Among these, Mannich reaction, a one pot three-component condensation reaction, provide synthetically and biologically important β-aminoalkylated compounds, which are important intermediates for the construction of various nitrogen-containing natural products and pharmaceuticals [13].

Most of free radicals are unstable species and capable of abstracting electrons from other molecules. Hydrogen peroxide (H₂O₂), the hydroxyl radical (HO), the superoxide anion radical (HOA), which are the predominant reactive oxygen species generated by cell metabolism or by exogenous factors have essential roles in cell signaling, apoptosis and gene expression. The damages of DNA, proteins and lipids caused by excessive free radical attacks can resulted in some diseases such as cancer, neurological degeneration and arthritis, as well as the process of aging. The discovery of new agents with antioxidant properties has become another extraordinary active area of preventive medicinal chemistry [14–16].

In light of these considerations, as the continuation of our ongoing efforts endowed with the discovery of hybrid compounds, we reported here the synthesis and biological activities of new hybrid molecules containing several heterocyclic nuclei with biological activity by ecofriendly and traditional methods (Fig. S2 in Supporting information). Docking some of the synthesized compounds into the active sites of the α-glucosidase and urease enzymes was performed as well to predict the binding affinities and non-covalent interactions between enzyme-ligand at molecular level.

2. Results and discussion 2.1. Chemistry

In the present study, the ecofriendly synthesis, antimicrobial, enzyme inhibition and antioxidant activity screening, and molecular docking studies of new hybrid molecules was intended. The structures of newly synthesized compounds were established on the basis of physicochemical, elemental analysis and spectral data (FT IR, ¹H NMR, ¹³C NMR and EI-MS).

Compounds 2, 5a and 6a were synthesized following the procedure reported by us earlier [2]. The cyclocondensation of the hydrazide (2) with CS₂ in ethanol-water in basic media generated the compound, {[4-(2-methoxyphenyl)piperazin-1-yl]methyl}-1,3,4-oxadiazol-2-thiol (3) under reflux, and also microwave irradiated conditions (Scheme 1). This idea originated from the aim to obtain an active hydrogen compound with biological activity that can be used as an intermediate for further condensation reactions leading to the formation of new bioactive products. In order to optimize the method, microwave (MW) irradiation was applied at different power values of 80, 100, 150 and 200 W and the progress of reaction was monitored by TLC. The complete conversion of the starting hydrazide (2) was observed after microwave irradiation at 200 W for 4 min. It is noteworthy to underline that shorter reaction time or lower microwave energy power caused to lower conversion rate, while increasing reaction time or MW power resulted in decomposition of the target product as revealed by TLC analysis. Assignment of the structure of 3 was based on correct elemental analysis results. FT IR spectrum of compound 3 revealed the presence of -SH group at 2832 cm^-1. A singlet characteristic for the -SH group was recorded at 14.35 ppm in the ¹H NMR spectra of compound 3. In the mass spectrum, the molecular ion peak (m/z=307.36, M+1) is present corresponding to the molecular weight of the assigned for compound 3.

The nucleophilic addition of compound 2 to alkyliso(thio) cyanates afforded the corresponding carbo(thio)amides (5a–d) (Scheme 1). The reaction conditions were examined in DCM under reflux conditions as well as under solvent free microwave irradiation conditions with a view to maximizing the yield of the product. To optimize reaction conditions, the synthesis of compound 5a was selected as model reaction and microwave (MW) irradiation was applied at different power values of 100, 150, 200, 250 and 300 W (the progress of reaction was monitored by TLC). The complete conversion of the starting hydrazide (2) was observed after microwave irradiation at 100 W for 10 min. Compared with conventional thermal heating, microwave irradiation decreased the reaction time from 20 h to 10 min. It is noteworthy to underline that shorter reaction time or lower microwave energy power caused to lower conversion rate, while increasing reaction time or MW power resulted in decomposition of the target product as revealed by TLC analysis. FT IR spectra of compounds 5a, 5b and 5d revealed the presence of -C=S group 1235–1244 cm^-1. On the other hand, the FT-IR spectrum of compound 5c exhibited -C=O stretching band at 1658 cm^-1. In the ¹³C NMR spectra of these compounds (5a–d), -C=S or -C=O function resonated between 162.44–181.25 ppm. Another evidence for the formation of carbo(thio)amides was the presence of three -NH-signals at 7.16–9.22 ppm in the ¹H NMR spectra as D₂O exchangeable singlet. The elemental analysis data of carbon, hydrogen and nitrogen were formed to be satisfactory and within the permissible limit error.

The synthesis of triazoles (6a–d) was carried out by the treatment of NaOH in ethanol-water under reflux condition in order to introduce the 1,2,4-triazole nucleus to piperazine skeleton, because it is well known that more efficacious bioactive compounds can be designed by joining two or more biologically active heterocyclic systems together in a single molecular framework [2, 14–16] (Scheme 1). In addition, type of compounds 6a–d can be considered as useful tolls having active NH group for further one pot three component aminoalkylation reactions leading to the formation new bioactive compounds. Then, the scope of these reactions was investigated under microwave irradiation. Compared with conventional thermal heating, microwave irradiation decreased the reaction time from 4–13 h to 5–13 min and increased the yields from 80%–88% to 92%–100% (Table 1).

It is well known that 1,2,4-triazol-3-(thi)ol derivatives can exist as mercapto-thioxo (or enol-keto) tautomeric forms. The -SH proton due to mercapto form resonates at about 13–14 ppm, while NH signal originated from thioxo tautomer appears at 9–12 ppm as D2O exchangeable signals [2, 17–20]. In the FT-IR spectra of compounds 6a and 6d, the presence of stretching bands at 2829 (for 6a) and 2822 cm^-1 (for 6d) originated from -SH function supported the mercapto form for compounds 6a and 6d. The -SH proton resonated at 13.85 and 13.84 ppm as D₂O exchangeable peaks, respectively, in the ¹H NMR spectra of these compounds. On the other hand, the signal due to thioxo (for 6b) or keto (for 6c) group was recorded at 1238 or 1707 cm^-1 as expected, while no signal due to -SH (for 6b) or -OH (for 6c) function was observed. Moreover, the D₂O exchangeable signal belonging to NH group pointing the thioxo or keto form for compounds 6b and 6d appeared at 11.62 and 11.72 ppm, respectively. The additional support for the formation of the targeted compounds, 6a-d was obtained by the appearance of [M+1], [M+2], [M+Na] and/or [M+K] ion peaks at corresponding m/z values confirming their molecular masses; and these compounds have given elemental analysis results consistent with the proposed structures.

The one-pot, three-component Mannich type reaction of compounds 3 and 6a-d with several amines namely 6-aminopencillanic acid, 7-aminocephalosporanic acid, norfloxacin and ciprofloxacin in the presence of formaldehyde generated the aminoalkylated products (4a, 4b; 7a–h) (Scheme 1). This reaction proceeds via the formation of immonium salt which subsequently attacks the N-1 of triazole or oxadiazole N-3 giving rise to the corresponding Mannich bases. Two methods were applied for this treatment including conventional and microwave assisted techniques. Time and yield data for the synthesis of compounds 4 and 7a-h by microwave and conventional methods were given in Table 1.

In comparison with the long refluxing time, microwave irradiation provided more efficient and green way for one pot Mannich type condensation with relatively higher product yield. The number of signals and their chemical shifts are in accordance with the assigned structures for the Mannich bases. In the ¹H NMR and ¹³C NMR spectra, additional signals corresponding to the 6-aminopenicillanic acid, norfloxacin or ciprofloxacin, were recorded at the related chemical shift values, while the spectra of these compounds showed the disappearance of the characteristic bands of triazole (or oxadiazole) -NH or -SH. Moreover, the preparation of Mannich bases was verified by registration of their mass spectrums which were in accordance with their molecular masses and the elemental analysis data (carbon, hydrogen and nitrogen) were±0.4% of the theoretical values.

The synthesis of new analogues (10a-i and 13a-c) of triazole class antifungals was carried out starting from the corresponding 3H-1,2,4-triazole compounds (6a-d) via three steps, because in recent years, serious fungal infections have emerged as an important complication and a major cause of disease and mortality [21, 22]. The structures of these compounds were confirmed on the basis of spectroscopic methods and elemental analysis (Schemes 2 and 3).

2.2. Biological activity 2.2.1. Antimicrobial activity

All the newly synthesized compounds were screened for their antimicrobial activity and the results obtained were presented in Table 2. Among these, compound 3, that contains a 1,3,4-oxadiazole nucleus linked to piperazine skeleton via a methylene linkage, was found to be active on Escherichia coli (Ec), a Gramnegative, facultatively anaerobic bacterium, Yersinia pseudotuberculosis (Yp), a Gram-negative bacterium, Staphylococcus aureus (Sa) and Enterococcus faecalis (Ef), which are Gram positive cocci, and Mycobacterium smegmatis (Ms), a nonpigmented rapidly growing mycobacterium with the mic values varying 31.25–250 µg/mL. The conversion of compound 3 to the corresponding Mannich bases (4a-d) containing a β-lactame or fluoroquinolone unit resulted in an increase in the antimicrobial activity. Among these, compounds 4a and 4b, which contain a fluoroquinolone nucleus attached to piperazinyl-oxadiazole skeleton displayed excellent antimicrobial activities. In fact, the activities of these compounds (4a and 4b) is better than standard drug ampicillin with the MIC values < 0.24 µg/mL. Other mannich bases derived from compound 3 (4c and 4d), having a β-lactam nucleus instead of fluoroquinolone, exhibited good-moderate activity on some of the test microorganisms. Among these, the MIC values of compound 4c on Ec and Ms varying 7.81 and 3.91 µg/mL are better than standard drugs, ampicillin and streptomycin.

Among the carbon(thio)amides, compounds 5b-d were found to have good-moderate activity against the test microorganisms with the MIC values between 7.8–125 µg/mL except Pa and Ef. Carbothioamide 5a containing a N-phenyl nucleus linked to thioamide carbon exhibited activity towards none of the test microorganisms. Among the 1,2,4-triazole derivatives (6a-d), the best activity was observed for 6d, that includes a benzyl group at the position 4 of 4H-1,2,4-triazole-3-thiol skeleton, on Ec and Ms with the MIC values 7.81 µg/mL and 3.91 µg/mL, respectively. On the other hand, excellent activities with the MIC values varing < 0.12–1.95 mg/mL were observed for compounds 7a-h, the synthesis of which was carried out by the aminoalkylation of the corresponding compounds 6a-d with norfloxacine or ciprofloxacine, concequently, these compounds can be regarded as fluorquinolone-triazole-phenylpiperazine hydrids. On the other hand, these compounds are inactive against Candida albicans (Ca) and Saccharomyces cerevisiae (Sc), yeast like fungy. Only slight activity was observed for 7d and 7h on Ca and Sc with the MIC values 500 µg/mL.

Table 2 revealed that compounds 10a, 10i and 13a-c, which can be considered as new analogues of miconazole, econazole or sulconazole (Fig. 1) containing triazole nucleus instead of imidazole exhibited good antifungal activity on Saccharomyces cerevisiae (Sc) and Candida albicans (Ca) with the MIC values 62.5 or 78 µg/mL as expected.

2.2.2. Principal component analysis (PCA)

The analyzed results for comparing the microwave irradiation method (MIM)and conventional method (CM) byPCAareshownin Fig. 1a. PC of Mannich bases listed in Table 1 (4a-d and 7a-h) explained 78.43% of total variation, where PC1 accounts for 61.59% of the variance and PC2 for 16.85%. PCA indicated that compounds 4b, 4a and 7e at the right lower plan and compounds 7f-h at the right upper plan on PC1 were mostly closely associated and strong correlated with the CM-yield, and MIM-yield (r=0.958, 0.878, 0.897, P < 0.05), while MIM-time and CM-time did not correlate with any of them. In contrast, compounds 4c, 7d and 4d at the right lower plan on PC2 were closely associated with CM-time-Acat and negatively strong correlated with MIM-yield (r=-0.948), CM-yield (r=-0.972).

Table 3 shows antioxidant capacity (µmol TE/g) values of 35 synthesized novel compounds measured by DPPH, FRAP and CUPRAC assays. The AC values given in the table significantly (P < 0.05) differed within and among assayed tests. The highest AC values were obtained from compound 4b (832.91), following 4a (556.05) as fluoroquinolone for DPPH, 5d (4744.08mmol/TEg) following 1 (3094.08) for FRAP and 4b (1192.38) following 7g (894.22 µmol) as fluoroquinolone for CUPRAC. The table includes compound 1, hydrazide (2), carbothioamide (5d) and carboxamide (5c) each, five compounds in triazole (6a-d), three compounds in ethanone (8a, b, d), four compounds in ethanol (9a-d), ten compounds in conazole (10a-i and 13c) and nine compounds in fluoroquinolone (4a, 4b, 7a, b, d, e, f, h, g) groups.

The Fig. 1b represents the antioxidant capacity (AC) data of the synthesized 35 novel compounds assayed using the three assays applied to PCA. The PCA of the compounds' AC values explained 91.03% of total variation, where PC1 accounts for 58.56% of the variance and PC2 for 32.47%. PC1 separated DPPH and CUPRAC from the other AC assay, FRAP. First, DPPH, following CUPRAC having a positive loading along the axis on PC1, was associated and positively high correlated (r=0.729, P < 0.05) with higher AC of compounds, 4b, 7d, 7h, 5c and 6c, which are three fluoroquinolones, one carboxamide and one triazole derivatives, respectively. FRAP was associated more with AC of compounds 4a, 7a, 7g and 7b as fluoroquinolone, 6a, 6b and 6d as triazole and 2 as hydrazide derivatives and did not correlate with other two AC tests (r=0.062). In contrast, the remaining 22 compounds did not lead to a complete separation for AC values depending on the AC assay types. They were situated on the negative and positive axes on PC2, contributing more or less equal numbers of compounds at the left lower and upper plans of the principal component.

The average AC values of hydrazide (2), carbo(thio)amide (5a-d) triazole (6a-d), ethanone (8a, b, d), ethanol (9a-d), conazole (10a-i and 13c), fluoroquinolone (4a, 4b, 7a, b, d, e, f, h, g) derivatives were also applied to PCA (Fig. 1c). PC of AC values of the compounds at nine derivative levels explained 90.80% of total variation, where PC1 accounts for 51.60% of the variance and PC2 for 39.20%. PCA indicated that carboxamide derivative with CUPRAC and fluoroquinolone, triazole and hydrazide with DPPH at the right lower and upper plan on PC1 and carbothioamide, benzylethanone and phenylyethanone with FRAP at the left upper plan were most closely significantly associated and correlated with 18 compounds in these groups. However, the remaining five derivative groups with 17 compounds situated at the left lower plan on PC2 were associated and correlated within alone, but not the three AC assay tests (Fig. 1c).

2.2.3. Urease inhibitory activity

The newly prepared compounds were assayed for their in vitro urease inhibitory activity against Jack bean urease with the inhibition rate 59% and 57%, respectively. Two of those compounds (10d and 13a), which are new analogues of triazole class antifungals, showed promising antiurease activity. The remaining compounds displayed no inhibitory effect (Table 4).

2.2.4. α-Glucosidase inhibitory activity

All compounds were evaluated with regard to α-glucosidase inhibition. Compounds 10a, 13a and 13b, which may be regarded as new analoques of triazole class antifungals, exhibited good inhibitory effect at various concentrations with inhibitory rates of 80%, 91% and 99% of 100 µmol/L, respectively (Table 4). Acarbose, an α-glucosidase inhibitor used as an anti-diabetic drug, exhibited an inhibitory effect of 83% at the same concentration. No significant inhibitory effect was detected for other compounds.

2.3. Molecular docking results

In order to predict the binding mode and affinities of compounds to target enzymes saccharomyces cerevisiae α-glucosidase and H. pylori urease, molecular docking was carried out. For comparison, an active and an inactive compound were selected for the corresponding enzymes according to the experimental results on their inhibitory effects.

The most energetically profitable poses of the active compound 13b (99±5% inhibition) and inactive compound 9b (5±1% inhibition) in the active site of saccharomyces cerevisiae α-glucosidase enzyme were presented in Figs. 2 and S3 (in supporting information), respectively in both three–dimensional (3D) and two-dimensional (2D) spaces.

For the docking of the compounds 13b and 9b to α-glucosidase, binding affinity values of -8.2 and -7.9 kcal mol^-1 were predicted, correspondingly. Weak hydrogen bonds of the C-H…O, C-H…π and O-H…π were the characteristic interactions for the active compound 13b (Fig. 2b). Three C-H…O interactions were obtained between the different methylene groups of 13b and both Pro312 and Asp242 amino acids in the distance range of 3.16– 3.30 Å. Aromatic dichlorobenzene ring of 13b was constituted two C-H…π interactions with Pro243 residue at 3.98 and 4.38 Å distance. Another C-H…π interaction was formed between Tyr158 and 13b at a distance of 3.54 Å. In addition, the presence of water molecules in the binding site of α-glucosidase caused an O-H…π interaction with anisole moiety of 13b (3.96 Å). In case of inactive compound 9b, it was formed a π…π interaction of His280 and triazole ring at 3.91 Å and a C-H…π interaction between the endmost methyl group of compound and Tyr158 amino acid at 4.11 Å distance (Fig. S3b in Supporting information). Smaller molecular structure of 9b with less functional group resulted with fewer non-covalent interactions and lower binding affinity comparison to 13b.

Active compound 10d (59±3% inhibition) and inactive compound 10g (4±1% inhibition) were given as an example of interaction with H. pylori urease in Figs. S4 and S5, respectively.

Cys321 and piperazine ring of 10d formed S–H…N hydrogen bond interaction at 3.45 Å distance. 10d was interacted with Met317 amino acid via C-H…S (at 3.70 Å) and arene-H (3.81 Å) interactions. The other arene-H contacts were obtained for the compound and His322, His323 and Ala169 residues (Fig. S4b in Supporting information). In contrary to large non-covalent interactions network between 10d and urease, only a C-H…π interaction was formed among inactive compound 10g and His322 residue of the enzyme. A noticeable difference in the energy of binding with urease was also predicted for two compounds: -9.6 kcal mol^-1 for the active compound 10d and 7.3 kcal/mol for the inactive 10g. Lower binding affinity value of 10g can be attributed to the weak interaction with the enzyme urease.

3. Conclusions

This study reports the successful synthesis of some new hybrid compounds or conazole derivatives starting from methoxyphenyl piperazine. The effect of MW irradiation on the progress of the reactions was investigated. The antimicrobial, antioxidant and enzyme inhibitory activity studies were also performed in the study. The antimicrobial screening suggested that the compounds containing norfloxacin or ciprofloxacin nucleus displayed excellent antimicrobial activity. Some of them displayed inhibition properties on S. cerevisiae better or equivalent to fluconazole, while, most of the synthesized compounds exhibited good antioxidant activity. Moreover, two compounds showed promising antiurease activity and three of them displayed good anti α-glucosidase activity.

Docking some of the synthesized compounds into the active sites of the α-glucosidase and urease was carried out and the obtained results complemented well the experimental results on α-glucosidase and urease inhibitory effects of the compounds. Higher binding affinities and much more interaction networks were observed for active compounds in contrary to inactive ones. It was predicted with the docking studies that triazole and anisole moieties in the structure of the synthesized compounds contributed to the stabilization of corresponding enzymes through noncovalent interactions.

4. Experimental 4.1. General

All the chemicals were purchased from Fluka Chemie AG Buchs (Switzerland) and used without further purification. Melting points of the synthesized compounds were determined in open capillaries on a Büchi B-540 melting point apparatus and are uncorrected. Reactions were monitored by thin-layer chromatography (TLC) on silica gel 60 F254 aluminium sheets. The mobile phase was ethyl acetate:diethyl ether (1:1), and detection was made using UV light. FT-IR spectra were recorded using a Perkin Elmer 1600 series FTIR spectrometer. ¹H NMR and ¹³C NMR spectra were registered in DMSO-d₆ on a BRUKER AVENE Ⅱ 400 MHz NMR spectrometer (400.13 MHz for ¹H and 100.62 MHz for ¹³C). The chemical shifts are given in ppm relative to Me₄Si as an internal reference, J values are given in Hz. The elemental analysis was performed on a Costech Elemental Combustion System CHNS-O elemental analyzer. All the compounds gave C, H and N analysis within ±0.4% of the theoretical values. The mass spectra were obtained on a Quattro LC-MS (70 eV) Instrument. Microwave irradiated reactions were performed in a CEM Discovery monomode synthesis reactor. Compounds 2, 5a and 6a were reported by us earlier [10].

4.1.1. {[4-(2-Methoxyphenyl)piperazin-1-yl]methyl}-1,3,4-oxadiazole-2-thiol (3)

Method 1: Compound 2 (10 mmol) and CS₂ (0.60 mL, 10 mmol) were added to a solution of KOH (0.56 g, 10 mol) in 50 mL H₂O and 50 mL ethanol and the reaction mixture was refluxed for 7 h. Then, the reaction content was acidified with conc. HCl to pH 6. The precipitate formed was filtered off, washed with H₂O and recrystallized from ethanol to afford the desired compound. Yield: 89%.

Method 2: The solution of compound 2 (1 mmol) and CS₂ (0.60 mL, 1 mmol) in H₂O+ethanol (5 mL+5 mL) containing KOH (1 mmol) was irradiated in monomode microwave reactor in closed vessel with the pressure control at 100℃, 200 W maximum power for 4 min (hold time). After the completion of the reaction, (monitored by TLC), the mixture was acidified with conc. HCl to pH 6. The precipitate formed was filtered off, washed with H₂O and recrystallized from ethanol to afford the desired compound. Yield: 94%.

4.1.2. General method for the synthesis of compounds 5a-d

Method 1: A mixture of compound 2 (10 mmol) and the corresponding iso(thio)cyanate (10 mmol) in dichloromethane was stirred at room temperature for 20 h (monitored by TLC). After evaporating the solvent under reduced pressure, a solid obtained. The crude product was purified by crystallisation from ethanol (for 5a and 5c) or ethyl acetate (for 5b).

Method 2: A mixture of compound 2 (1 mmol) and the corresponding iso(thio)cyanate (1 mmol) was irradiated in closed vessels with the pressure control at 100℃, 100 W (the progress of reaction was monitored by TLC) for 10 min. Then, the mixture was poured into ice water and a solid formed. The crude product was collected by filtration and purified by crystallization from dimethyl sulfoxide.

4.1.3. General method for the synthesis of compounds 6a-d

Method 1: A solution of corresponding carbo(thio)amide 5a-d (10 mmol) in ethanol/water (1:1) was refluxed in the presence of 2 mol/L NaOH (20 mmol) for 4 h (for 6a), 13 h (for 6b) or 9 h (for 6c) and 8 h (for 6d) (the progress of the reaction was monitored by TLC). Then, the resulting solution was cooled to room temperature and acidified to pH 5 with 37% HCl. The precipitate formed was filtered off, washed with water, and recrystallized from ethanol to give the target compound.

Method 2: The mixture of compound 5a-d (1 mmol) and 2 mol/L NaOH (2 mmol) in ethanol (10 mL) was irradiated in monomode microwave reactor in open vessel (physical parameters were given in Table 1). Then the resulting solution was cooled to room temperature and acidified to pH 5 with 37% HCl. The precipitate formed was filtered off, wash with water, and recrystallized from ethyl acetate (for 6a and 6c), ethanol (for 6d) and methanol (for 6b) to give the target compounds.

4.1.4. General method for the synthesis of compounds 4a-d and 7a-h

Method 1: The solution of 7-aminocephalosporanic acid (for 4d), 6-aminopenicillanic acid (for 4c), ciprofloxacin (for 4b, 7b, 7d, 7f, 7h) or norfloxacin (for 4a, 7a, 7e, 7g), (10 mmol) in dimethyl formamide was stirred at room temperature in the presence of formaldehyde (37%, 30 mmol) for 15 min. Then, the corresponding compound 3 or 6a-d (10 mmol) was added into it and stirred for additional 20–24 h. The reaction mixture was poured to ice-water and a solid obtained. This crude product was recrystallized from an appropriate solvent to give the desired compound.

Method 2: The mixture of 7-aminocephalosporanic acid (for 4d), 6-aminopenicillanic acid (for 4c), ciprofloxacin (for 4b, 7b, 7d, 7f, 7h), norfloxacin (for 4a, 7a, 7e, 7g), (1 mmol), formaldehyde (37%, 3 mmol) and the corresponding compound 3 or 6 (1 mmol) in dimethyl formamide (10 mL) was irradiated in monomode microwave reactor in closed vessel at 100℃ (physical parameters were given Table 1). The solid formed after the mixture was poured to ice-water was filtered off and purified by crystallisation from an appropriate solvent to give the desired compound.

4.1.5. General method for the synthesis of compounds 8a, 8b, 8d and 11

Method 1: The solution of compounds 6a, 6b and 6d (10 mmol) in ethanol was stirred in the presence of sodium ethoxide (10 mmol) at room temperature for 15 h (the progress of the reaction was monitored by TLC). Then, 2-bromo-1-(4-chlorophenyl)etanone (30 mmol) was added into it and stirred for additional 12 h. After evaporating the solvent under reduced pressure, a solid obtained. The crude product was recrystallized from an appropriate solvent to afford the desired product.

Method 2: The solution of compounds 6a, 6b and 6d (1 mmol) and sodium ethoxide (1 mmol) in ethanol (10 mL) was irradiated in monomode microwave reactor in closed vessels at 90℃, 100 W, for 1.5 h (for 8a, 8b and 8d) or 2 h (for 11) (the progress of the reaction was monitored by TLC). Then, 2-bromo-1-(4-chlorophenyl)etanone (3 mmol) was added into it and irradiated for additional 1 h. The mixture was poured into ice-water and a solid obtained. This crude product was collected by filtration and recrystallized from an appropriate solvent to afford the desired product.

4.1.6. General method for the synthesis of compounds 9a, 9b, 9d and 12

Method 1: NaBH₄ (50 mmol) was added the solution of the corresponding compound 8a, 8b, 8d and 11 (10 mmol) in ethanol and the mixture was stirred at room temperature for 24 h (the progress of the reaction was monitored by TLC). After evaporating the solvent under reduced pressure, a solid obtained. The crude product was recrystallized from an appropriate solvent to afford the desired product.

Method 2: The solution of compound 8a, 8b, 8d and 11 (1 mmol) in ethanol (10 mL) was irradiated in monomode microwave reactor in closed vessel in the presence of NaBH₄ (3 mmol) with pressure control (physical parameters were given in Table 1). Then, the mixture was poured into ice-water and a solid obtained. This crude product was collected by filtration and recrystallized from an appropriate solvent to afford the desired product.

4.1.7. General method for the synthesis of compounds 10a-i and 13a-c

Method 1: NaH (10 mmol) was added the solution of the corresponding compound 9 or 12 (10 mmol) in THF and the mixture was refluxed for 5 h. Then, the corresponding substituted benzylchloride was added into it and the refluxed for additional 9 h (for 10a-i) or 12 h (for 13a-c). After the completion of the reaction, solvent was evaporated under reduced pressure, and the obtained oily product was extracted with 15 mL of ethyl acetate three times in the presence of K₂CO₃. The organic layer was dried on Na₂SO₄. After the removal of solvents at a reduced pressure, an oily product formed which was purified by column chromatography (n-hexane/ ethyl acetate) on silica gel.

Method 2: NaH (1 mmol) was added the solution of the corresponding compound 9 or 12 (1 mmol) in THF (10 mL) and the mixture was irradiated in monomode microwave reactor in closed vessel with pressure control at 100℃ and 100 W, for 10 min. Then, the corresponding substituted benzylchloride (3 mmol) was added into it and irradiation was continued for 45 minute (for 10a-i) or 50 min (for 13a-c) at 125℃, 150 Watt. Solvent was evaporated under reduced pressure, and the obtained oily product was extracted with 15 mL of ethyl acetate three times in the presence of K₂CO₃. The organic layer was dried on Na₂SO₄. After the removal of solvents at a reduced pressure, an oily product formed which was purified by column chromatography (n-hexane/ethyl acetate) on silica gel.

4.2. Antimicrobial activity

The test microorganisms were obtained from the Hifzissihha Institute of Refik Saydam (Ankara, Turkey) and were as follows: Escherichia coli (E. coli) ATCC35218, Yersinia pseudotuberculosis Y. pseudotuberculosis) ATCC911, Pseudomonas aeruginosa (P. aeruginosa) ATCC43288, Enterococcus faecalis (E. faecalis) ATCC29212, Staphylococcus aureus (S. aureus) ATCC25923, Bacillus cereus (B. cereus) 709 Roma, Mycobacterium smegmatis (M. smegmatis) ATCC607, Candida albicans (C. albicans) ATCC60193 and Saccharomyces cerevisiae (S. cerevisia) RSKK 251. All the newly synthesized compounds were weighed and dissolved in hexane to prepare extract stock solution of 20.000 microgram/milliliter (µg/mL).

The antimicrobial effects of the substances were tested quantitatively in respective broth media by using double microdilution and the minimal inhibition concentration (MIC) values (µg/mL) were determined. The antibacterial and antifungal assays were performed in Mueller-Hinton broth (MH) (Difco, Detroit, MI) at pH.7.3 and buffered Yeast Nitrogen Base (Difco, Detroit, MI) at pH 7.0, respectively. The micro dilution test plates were incubated for 18–24 h at 35℃. Brain Heart Infusion broth (BHI) (Difco, Detriot, MI) was used for M. smegmatis, and incubated for 48–72 h at 35℃ [23]. Ampicillin (10 µg) and fluconazole (5 µg) were used as standard antibacterial and antifungal drugs, respectively. Dimethylsulphoxide with dilution of 1:10 was used as solvent control. Only positive results were presented in Table 5.

4.3. Antioxidant activity 4.3.1. Antioxidant activity studies

DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging activity: The scavenging activity of different chemicals was determined using the free radical DPPH (2,2-diphenyl-1-picrylhydrazyl), as describedby Blois[24]. A 100-µL: chemicalsolutionwas mixed with 1 mL of freshly prepared methanolic DPPH solution. The reaction mixture was incubated for 30 min at room temperature in the dark and was then measured at 520nm. The activity was expressed as µmol Trolox equivalent.

FRAP (the ferric reducing ability of plasma): FRAP was measured using the method described by Benzie & Strain [25] with some modification. To 100 µL of each sample was added 2.9 mL freshly prepared FRAP reagent containing 300 mmol/L acetate buffer (pH 3.6), 10 mmol/L TPTZ (2,4,6-tripyridyle-striazine) and 20 mmol/L FeCl.6H₂O in proportions of 10:1:1 (v/v/v). The mixture was incubated for 30 min at 37℃ and measured at 593 nm. The values were expressed as µmol of Trolox/g.

CUPRAC (cupric ion reducing antioxidant capacity): CUPRAC was measured following the procedure described by Apak et al. [26] with some modification. Briefly, 100 µL of each chemical solution was mixed with 900 µL bi-distilled water, 1 mL acetate buffer solution (1 mmol/L, pH: 7.0), 1 mL CuCl₂ (10 mmol/L) and 1 mL 7.5 mmol/L neocuproine to a final volume of 4 mL. The reaction mixture was then incubated in the dark for 30 min at room temperature, and the absorbance of the reaction mixture was measured at 450 nm against a water blank. Trolox was used as the standard calibration curves, and the results were expressed as µmol Trolox equivalent per g.

4.4. α-Glucosidase inhibition assay [27]

α-Glucosidase inhibition assay was performed spectrophotometrically. α-Glucosidase from Saccharomyces cerevisiae (Sigma-Aldrich) was dissolved in phosphate buffer (pH 6.8, 50 mmol/L). Test compounds were dissolved in DMSO. To 96-well microtiter plates was added 20 µL of test sample, 20 µL of enzyme (20 mU/mL) and 135µL of buffer This was then incubated for 15 minutes at 37℃. After incubation, 25 µL of p-nitrophenyl-α-Dglucopyranoside (2 mmol/L, Sigma Aldrich) was added and change in absorbance was monitored for 20 minutes at 400 nm. The test compound was replaced by DMSO (10% final) as the control. Acarbose (Sigma-Aldrich) was used as a standard inhibitor. The assays were performed in triplicate. The IC₅₀ value was determined as the concentration of compound giving 50% inhibition of maximal activity.

4.5. Urease inhibition assay [28]

Reaction mixtures comprising 25 µL of Jack Bean urease, 55 µL of buffer (0.01 mol/L K₂HPO₄, 1 mmol/L EDTA and 0.01 mol/L LiCl, pH 8.2) and 10 mmol/L urea were incubated with 5 µL of the test compounds at room temperature for 15 min in microtiter plates. The production of ammonia was measured following the indophenol method and was used to determine the urease inhibitory activity. The phenol reagent (45 µL, 1% w/v phenol and 0.005% w/v sodium nitroprusside) and alkali reagent (70µL, 0.5% w/v sodium hydroxide and 0.1% v/v NaOCl) were added to each well. Increasing absorbance at 625 nm was measured after 20 min, using a microplate reader (SpectraMax M5, Molecular Device, USA). The percentage inhibition was calculated from the formula 100–(OD_testwell/OD_control)×100. Thiourea was used as the standard inhibitor. In order to calculate IC₅₀ values, different concentrations of synthesized compounds and standard were assayed under the same reaction conditions.

4.6. Molecular docking

Before the docking, initial structures of the compounds were built and optimized by the GAUSSIAN 09 program [29]. Geometry optimizations were performed using Density Functional Theory (DFT) at the B3LYP (Becke–3 parameter–Lee-Yang-Parr)/6-31G (d, p) level [30], [31] with dual Xeon workstation (2.3 GHz, RAM 64 GB).

The crystal structures of the saccharomyces cerevisiae α-glucosidase and H. pyloriurease enzymes were obtained from RCSB Protein Data Bank (http://www.rcsb.org/pdb/), under the accession codes, 3A4A [32] and 1E9Y [33], respectively. Molecular Operating Environment (MOE) software [34] was used for molecular docking studies. Enzyme-ligand complexes were energy minimized to a gradient of 0.01 kcal/(mol Å), and protonated by means of the force field AMBER99. Charges on the enzyme and ligands were assigned using the force field AMBER99 and force field MMF94X, correspondingly. The active sites of enzymes were identified by the site finder application in MOE. Triangle Matcher Algorithm and two rescoring functions, London dG and GBVI/WSA dG were used to produce 20 poses of each ligand. All poses generated with docking were analyzed and the best-scored pose for each compound was selected for further investigation of interactions with the corresponding enzyme.

4.7. Statistical analysis

All measurements were performed in triplicate (n=3), and the data were presented as mean±pooled standard deviation (mean±SD). The data were analyzed using a one-way ANOVA and Duncan's multiple range test for comparison among means at significance levels of P < 0.01 or 0.05 (IBM SPSS Statistics V22.0). A statistical software package (XLSTAT version 2014.6) using ADDINSOFT (Damremont, Paris, France) was used to perform principal component analysis (PCA).

Acknowledgement

The support was provided by Scientific and Technological Research Council of Turkey (TUBITAK, No. 113Z181).

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.cclet.2016.12.012.