Microwave-assisted synthesis of some hybrid molecules containing penicillanic acid or cephalosporanic acid moieties and investigation of their biological activities

Ethyl 4-amino-2-fluorophenylpiperazin-1-carboxylates containing a 1,3-oxazol(idin)e, 5-thioxo-1,2,4-triazole, 1,3,4-thiadiazole, 5-thioxo-1,3,4-oxadiazole, or 1,3-thiazole nucleus were obtained starting from ethyl piperazine-1-carboxylate (1) by several steps. The treatment of amine, 3 or hydrazide, 9 with several aromatic aldehydes generated the corresponding arylmethyleneamino (3a–f) or arylidenehydrazino (12a–c) compounds. The Mannich reaction between the 1,2,4-triazole or 1,3,4-oxadiazole compounds and 7-aca produced cephalosporanic acid derivatives. Penicillanic acid derivatives were obtained when 6-apa was used in the Mannich reactions. The synthesized compounds were screened for their antimicrobial, antilipase, and antiurease activities. Some of them were found to possess good-moderate antimicrobial activity against the test microorganisms. Two compounds exhibited antiurease activity, and four of them displayed antilipase activity.


Introduction
The limitations of the existing antibacterial drugs caused by various reasons including drug resistance, the serious side effects, and/or lack of efficacy made infectious diseases a vicious cycle. In addition, the treatment of resistant strains requires a prolonged therapy containing the use of more toxic drugs and increases the financial burden. The rising prevalence of multi-drug resistant bacteria continues to serve medicinal chemists to search and discove novel antimicrobial agents effective against pathogenic microorganisms resistant to current treatment.
Among the strategies addressed to the synthesis of compounds possessing antimicrobial activity, the syntheses of hybrid molecules incorporating different heterocyclic moieties have been attracting widespread attention (Mallikarjuna et al., 2009).
A number of N-containing heterocyclic compounds constitute important building blocks in organic and medicinal chemistry. For example, triazoles have been shown to possess a number of desirable activities in the context of medicinal chemistry. Ribavirin (antiviral), rizatriptan (antimigraine), alprazolam (psychotropic), fluconazole, and itraconazole (antifungal) are the best examples for potent drugs possessing triazole nucleus Walczak et al., 2004;Jones et al., 1965;Ashok et al., 2007). Tazobactam, a b-lactamase inhibitor is the other best known example of triazole containing structures with the broad spectrum antibiotic piperacillin (Kategaonkar et al., 2010).
Substituted piperazines constitute another class of important pharmacophores, which are found in many marketed drugs, such as the HIV protease inhibitor, Crixivan (Chaudhary et al., 2006). Ciprofloxacin, norfloxacin, pefloxacine, ofloxacin, and enoxacin are fluoroquinolone class antibacterial drugs characterized by having a piperazine moiety at C-7 of quinolone skeleton, and they have been used for the treatment of bacterial infections (Foroumadi et al., 2005).
Mannich bases, which are known to be physiologically reactive since their basic function rendering the molecule soluble in aqueous solvents when it is transformed into aminium salt, have been reported as potential biological agents (Karthikeyan et al., 2006). N-Mannich bases have been used successfully to obtain prodrugs of amine as well as amide-containing drugs (Zhao et al., 2009). Some Mannich bases derived from 1,2,4-triazole nucleus have been reported to possess protozocidal and antibacterial activity (Ashok et al., 2007;Almajan et al., 2009;Bayrak et al., 2009Bayrak et al., , 2010Demirbas et al., 2009;Bektas et al., 2010;Patole et al., 2006).
Schiff bases have gained importance in medicinal and pharmaceutical fields due to their most versatile properties as organic synthetic intermediates and also possessing a broad range of biological activities, such as antituberculosis, anticancer, analgesic and anti-inflammatory, anticonvulsant, antibacterial, and antifungal activities (Patole et al., 2006, Hearn andCynamon, 2004;Ren et al., 2002;Demirbas et al., 2002;Lohray et al., 2006).
We envisage that hybrid compound incorporating a 4-(2-fluorophenylene)-piperazine core with several heterocyclic moieties responsible for biological activity in a single molecular frame could lead to the novel potent antimicrobial and antiurease agents. Highly substituted piperazines can be expected to increase antimicrobial activity probably by enhancing lipophilicity of molecule.
In continuation of our research program on the synthesis of hybrid molecules containing various heterocyclic moieties, we planned the synthesis of 4-(2-fluorophenyl)piperazine derivatives along with their antimicrobial and antiurease activities.

Results and discussion
The main aim of the present study is the synthesis and antimicrobial activity evaluation of new piperazine derivatives incorporating several heterocyclic moieties including 1,3-oxadiazole, 1,2,4-triazole, 1,3-oxa(thia)zole, penicillanic acid, and/or cephalosporanic acid. Synthesis of the intermediate and target compounds was performed according to the reactions outlined in Schemes 1, 2, and 3. The starting compound ethyl 1-piperazinecarboxylate (1) was provided commercially.
Ethyl 4-(4-amino-2-fluorophenyl)piperazine-1-carboxylate (3), that was obtained starting from compound 1 by two steps, was converted to the corresponding arylmethylenamino derivatives (4a-f) by the treatment with several aromatic aldehydes. In the FT-IR and 1 H NMR spectra of these compounds, no signal pointing the -NH 2 group was seen. Instead, additional signals derived from aldehyde moiety were recorded at the related chemical shift values in the 1 H NMR spectra.
The cyclocondensation of compound 5, that was obtained from the reaction of 4 with benzylisocyanate, with ethyl bromoacetate or 4-chlorophenacyl bromide produced the corresponding hybrid molecules incorporating a 4-oxo-1,3-oxazolidine (6) or 4-chlorophenyl)-1,3-oxazole (7) nucleus in the 2-fluorophenylpiperazine-1-carboxylate skeleton. The 1 H and 13 C NMR spectra of compound 7 exhibited additional signals at aromatic region originated from 4-chlorophenyl nucleus as a result of condensation. Moreover, the elemental analyses and mass spectral data of derivatives 6 and 7 were compatible with the suggested structures.
The treatment of compound 3 with ethyl bromoacetate at room temperature in the presence of triethylamine resulted in the formation of compound 8. When compound 8 was converted to the corresponding hydrazide (9) by refluxing with hydrazine hydrate, the signals originated from ester function was disappeared in the 1 H and 13 C NMR spectra. Instead, new signals due to -NHNH 2 protons were seen at 5.93 and 9.09 ppm. Meanwhile, the stretching frequency band of this group was recorded at 3,313 cm -1 as a wide signal characteristic for the hydrazide structure. Compounds 6 and 7 gave mass fragmentation confirming the proposed structures.
The synthesis of compounds 10 and 11 was carried out by the treatment of compound 7 with the corresponding isothiocanates. These compounds displayed spectroscopic data and elemental analysis results consistent with the assigned structures.
The intramolecular cyclization of compound 10 generated the corresponding 1,3,4-thiazole compound (12) in acidic media. On the other hand, the basic treatment of compounds 10 and 11 caused to the cyclization of the (arylamino)carbonothioylhydrazino side change leading to the formation of 5-thioxo -4,5-dihydro-1H-1,2,4-triazol derivatives (13 and 14). With the conversion of compounds 10 and 11 to compounds 12-14, two of NH signals were disappeared in the 1 H NMR spectra. It is well-known that type of compounds can stay in thioxo or mercapto tautomeric form. In the present study, compounds 13 and 14 are present predominately in the thioxo form as it was shown by the C=S band at 1,244-1,250 cm -1 in the FT-IR spectra of these compounds. Furthermore, the 1 H NMR spectra of compounds 13 and 14 revealed clearly the absence of the signal originated from SH proton, instead of that, two signals due to NH proton on 1,2,4-triazol ring was recorded at 10.45 (for 13) or 11.27 (for 14), that is characteristic for 4,5-dihydro-1H-1,2,4-triazoles.
The synthesis of Mannich bases (15-17) was performed by the reaction of compounds 13 and 14 with 6-aminopenicillanic acid, 6-apa (for 17) or 7-aminocephalosporanic acid, 7-aca (for 15 and 16) in tetrahydrofuran at room temperature in the presence of triethylamine and formaldehyde. The occurrence of the alkylaminomethylation was provided by the disappearance of signal for the proton at the N-1 nitrogen of the 1,2,4-triazole ring. Moreover, in 1 H and 13 C NMR spectra, additional signal corresponding to the 6-apa or 7-aca-ammonium salt was recorded at the related chemical shift value.
The synthesis of ethyl arylidenehydrazino-piperazine-1carboxylate derivatives (19a-c) was performed by microwave irradiation of compound 9 with several aromatic aldehydes namely 3-hydroxy-4-methoxybenzaldehyde, pyridine-4-carbaldehyde, and 2-hydroxybenzaldehyde. In the FT-IR spectra of these arylidenehydrazino compounds, absorption bands characteristic for NH groups were visible in  the ranges of 3,357-3,181 cm -1 . Another piece of evidence for condensation was the appearance of a signal as singlet integrating for one proton in the 1 H NMR spectra, which corresponds to the N=CH proton of azomethyne group. Moreover, these compounds gave mass fragmentation and elemental analysis confirming the proposed structures.  Ethyl 4-(2-fluoro-4-{[(5-thioxo-4,5-dihydro-1,3,4-oxadiazol-2-yl)methyl]amino} phenyl)piperazine-1-carboxylate (20) was prepared from the reaction of compound 9 with CS 2 in the basic media. The attempts for aminoalkylations of compound (20) by Mannich reaction allowed the isolation of the corresponding products (21 and 22) after 4 (for 21) or 6 h (for 22) at room temperature. This idea originated from the intent to introduce the penicillanic acid or cephalosporanic acid nucleus to (piperazin-1-yl)-2-thioxo-1,3,4-oxadiazole skeleton. As different from 20, the NMR spectra of the obtained Mannich bases (21 and 22) displayed additional signals derived from penicillanic-or cephalosporanic-acid moiety and -CH 2 -linkage at the related shift and integral values as D 2 O nonexchangeable signals.
Among the synthesized compounds ethyl 4-(2-fluoro-4-nitrophenyl)piperazine-1-carboxylate (2) exhibited activity on Bacillus cereus (Bc), that is Gram positive spore bacillus. With the reduction of nitro group of 2 to amine (compound 3), additional activities towards Staphylococcus aureus (Sa), that is Gram positive coccus, Candida albicans (Ca), and Saccharomyces cerevisiae (Sc), which are yeast like fungi. For the imine compounds (4a-f), the highest activity was observed against Mycobacterium smegmatis (Ms) that is an atypical tuberculosis factor leading mortality, with the inhibition zone varying between 10 and 25 mm. The compounds containing 1,2,4-triazole and cephalosporanic-or penicillanic-acid moiety (compounds 15-17) displayed goodmoderate activity on some of the test microorganisms. The highest activity was observed for compound 17 on Bc with the inhibition zone of 16 mm. This result is better than standard drug ampicillin. Other compounds containing penicillanic acid or cephalosporanic acid core The synthesized compounds were assayed for their in vitro urease inhibitory activity against Jack bean urease. Two of those compounds showed perfect urease inhibition. No inhibitory effect was detected for other compounds. Thiourea with IC 50 value 54.56 ± 4.17 lg mL -1 was used as standard inhibitor. Among tested compounds, compound 15 was found to be the best inhibitory effect against urease with an IC 50 value of 4.67 ± 0.53 lg mL -1 . At the various final concentrations the compound 15 showed more inhibitory effect than standard urease inhibitor thiourea. Also, compound 17 has the highest inhibitory activity than thiourea. These compounds might be considered as potential antibiotics to treat infections.
All compounds were evaluated with regard to pancreatic lipase activity and compounds 12, 13, 14, and 15, which are 1,3,4-thiadizole or 1,2,4-triazole derivatives including also 4-fluorophenylpiperazine nucleus, showed moderate anti-lipase activities at final concentration of 6.25 lg mL -1 . No inhibitory effect was detected for other compounds. Orlistat, known pancreatic lipase inhibitor used as anti-obesity drug, showed inhibitory effect by 99 % at the same concentration.
Among the synthesized compounds, the compounds containing 1,2,4-triazole and cephalosporanic-or penicillanic-acid moiety (15-17) displayed good-moderate activity on some of the test microorganisms. The highest activity was observed for compound 17 on Bc with the inhibition zone of 16 mm. This result is better than standard drug ampicillin. Moreover, compounds 15 and 17 exhibited an inhibitory effect against urease. Other compounds containing penicillanic acid or cephalosporanic acid core (21 and 22) displayed good-moderate activity against the test microorganisms. Furthermore, compounds 12, 13, 14, and 15, which are 1,3,4-thiadizole or 1,2,4triazole derivatives including also 4-fluorophenylpiperazine nucleus, showed moderate anti-lipase activities at final concentration of 6.25 lg mL -1 .

General information for chemicals
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 aluminum sheets. The mobile phase was ethyl acetate:diethyl ether, 1:1, and detection was made using UV light. FT-IR spectra were recorded as potassium bromide pellets using a Perkin Elmer 1600 series FT-IR spectrometer. 1 H NMR and 13 C NMR spectra were registered in DMSO-d 6 on a BRUKER AVANCE II 400 MHz NMR Spectrometer (400.13 MHz for 1 H and 100.62 MHz for 13 C). The chemical shifts are given in ppm relative to Me 4 Si 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.