Antimicrobial and antiurease activities of newly synthesized morpholine derivatives containing an azole nucleus

2-[6-(Morpholin-4-yl)pyridin-3-ylamino]acetohydrazide (4) was obtained starting from 6-morpholin-4-ylpyridin-3-amine (2) via the formation of ester (3) and then converted to the corresponding Schiff bases (5, 6) with the reaction with aromatic aldehydes. The carbothioamide (9), obtained from the reaction of hydrazide with phenylisothiocyanate, was converted to the corresponding 1,2,4-triazole (11) and 1,3,4-thiadiazole (12) derivatives by the treatment with NaOH or H2SO4, respectively. The cyclocondenzation of 9 with 4-chlorophenacyl bromide or ethyl bromoacetate produced the corresponding 1,3-thiazole (10) or 1,3-thiazolidine derivatives (13), respectively. Antimicrobial and antiurease activities of newly synthesized compounds were investigated. Some of them were found to be active on M. smegmatis, and they displayed activity toward C. albicans and S. cerevisiae in high concentration. Compound 10 proved to be the most potent showing an enzyme inhibition activity with an IC50 = 2.37 ± 0.19 μM.


Introduction
Urea amidohydrolases (ureases) have been known as a class of large heteropolymeric enzymes with the active site containing two nickel (II) atoms and to accelerate hydrolysis of urea to ammonia gas with the reaction rate at least 10 14 over the spontaneous reaction. Ureases are widely distributed in nature and are found in a variety of plants, algae, fungi, and bacteria (Kot et al., 2010). Medically, bacterial ureases have been reported as important virulence factors implicated in the pathogenesis of many clinical conditions such as pyelonephritis, hepatic coma, peptic ulceration, and the formation of injection-induced urinary stones and stomach cancer. The catalytic mechanism of their action has been believed to be the same of all urease inhibitors in which the amino acid sequences of the active site are principally conserved (Xiao et al., 2010). The active site of the native enzyme binds three water molecules and a hydroxide ion bridged between two nickel ions (Bachmeier et al., 2002). In the course of enzymatic reaction, urea replaces these three water molecules and bridges the two metal ions. The surrounding by a hydrogen-bonding network strongly activates the inert urea molecule; it is subsequently attacked by the hydroxide ion, forming a tetrahedral transition state. As a result, ammonia is released from the active site followed by the negatively charged carbamate (Adil et al., 2011). The latter decomposes rapidly and spontaneously, yielding a second molecule of ammonia. The ammonia generated may cause disruption to several metabolic functions in a large number of animal tissues and organs (Adil et al., 2011).
Urease is also indispensable for colonization of human gastric mucosa by Helicobacter pylori. The ammonia produced has been shown to be toxic for various gastric cell lines. Furthermore, urease activity was proposed to damage the gastric epithelium via its interaction with the immune system by stimulating an oxidative burst in human neutrophils (Ito et al., 1998). H 2 O 2 generated in this oxidative burst probably reacts with ammonia and chloride to yield the toxic monochloramine (Kot et al., 2010). Finally, the ammonia may reach the serum and contribute to symptoms of hepatic encephalopathy in patients suffering from cirrhosis. Apart from ammonia, the carbon dioxide generated by urea hydrolysis may play a significant role for survival of H. pylori in the gastric mucosa (Cobena et al., 2008;Miroslawa et al., 2010;Xiao et al., 2010;Khan et al., 2010a, b;Ito et al., 1998;Keri et al., 2002;Ashiralieva and Kleiner, 2003).
Moreover, urea constitutes the predominant source of nitrogen containing fertilizers used in agriculture, accounting for 50 % of the total world fertilizer nitrogen consumption. However, the efficiency of urea is decreased by its hydrolysis with the enzyme urease to ammonia gas in soil. Besides the economic impact for farmers, NH 3 lost to the atmosphere from applied urea causes eutrophication and acidification of natural ecosystems on a regional scale (Cobena et al., 2008).
Several classes of compounds have been reported as the agents having antiurease activity; among them hydroxamicacids are the best recognized urease inhibitors (Adil et al., 2011;Krajewska, 2009;Muri et al., 2003). Phosphoramidates, another class of antiurease agents, have been reported as the most potent compounds (Amtul et al., 2002;Kot et al., 2001). However, the teratogenicity of hydroxamicacid in rats and degradation of phosphoramidates at low pH (Adil et al., 2011, Domínguez et al., 2008Kreybig et al., 1968) restrict their use as a drug in vivo. Another class of compounds showing enzyme's inhibitory activity is polyphenols such as gallocatechin that is a polyphenol extracted from green tea and quercetin, a naturally occurring flavonoid having anti-H. pylori activity (Matsubara et al., 2003;Shin et al., 2005).
During the recent decades, the human population being afflicted with life-threatening infectious diseases caused by multidrug-resistant Gram-positive and Gram-negative pathogen bacteria has been increasing at an alarming lscale around the world as a result of antimicrobial resistance. In spite of the wide range of antimicrobial drugs with different mechanisms of action used for the treatment of microbial infections either alone or in combination and also the existence of many compounds used in different phases of clinical trials, microbial infections have been posing a worldwide problem. There is already evidence that antimicrobial resistance is associated with an increase in mortality (Bayrak et al., 2010a(Bayrak et al., , b, 2009aDemirbas et al., 2009). The growing number of reports of antibiotic resistance worldwide has led to fears that some lethal human pathogens, such as Mycobacterium tuberculosis, will soon become untreatable (Dye and Williams, 2009;Dye and Phill, 2006;Koca et al., 2005;Zalavadiya et al., 2009). Tuberculosis (TB) causes the death of approximately three million patients in the world every year. These numbers make TB one of the leading infectious causes of death, eclipsed only by AIDS. Synthetic drugs for treating TB have been available for over half a century, but incidences of the disease continue to be on the rise worldwide. The causative organism, Mycobacterium tuberculosis, is a tremendously successful colonizer of the human host and is estimated to have latently infected approximately one-third of humanity. A growing number of immunocompromised patients are attributed to cancer chemotherapy, organ transplantation, and HIV infection, which are the major factors contributing to this increase. Therefore, it is necessary to search for and synthesize new classes of antimicrobial compounds that are effective against pathogenic microorganisms that have developed resistance to the antibiotics (Dye and Williams, 2009;Dye and Phill, 2006;Koca et al., 2005;Zalavadiya et al., 2009;Bayrak et al., 2010a, b).
In the field of medicinal chemistry, azoles belong to a class of antimicrobial agents that are widely used and studied because of their safety profile and high therapeutic index. Ribavirin, rizatriptan, alprazolam, vorozole, letrozole, and anastrozole are the best examples of drugs containing 1,2,4-triazole moiety (Ashok et al., 2007;Rao et al., 2006;Hancu et al., 2007;Cai et al., 2007). Among azole-based drugs, conazoles, such as itraconazole, fluconazole, voriconazole, and ravuconazole constitute a major class being used for the treatment of fungal infections (Yu et al., 2007;Gupta et al., 2007;Schiller and Fung, 2007).
Another important pharmacophore group is the morpholine nucleus incorporated in a wide variety of therapeutically important drugs, one of which is linezolid which belongs to the oxazolidinone class of antibiotics and is used for the treatment of infections caused by gram-positive bacteria (Wyrzykiewicz et al., 2006;Dixit et al., 2005;Raparti et al., 2009;Bektas et al., 2010Bektas et al., , 2012Bayrak et al., 2009a, b). In addition, 4-phenylmorpholine derivatives have been reported to possess antimicrobial, antiinflammatory, and central nervous system activities (Dixit et al., 2006), Oxazolidinones are a relatively new class of synthetic antibacterial agents, having a new mechanism of action that involves early inhibition of bacterial protein synthesis. This class of compounds is particularly active against gram-positive organisms. Oxazolidinones are thought not to be cross-resistant with other types of antibiotics because of their different action mechanisms, which include interaction with the bacterial ribosome to inhibit bacteria. (Zheng et al., 2010;Giera et al., 2006;Das et al., 2005;Gage et al., 2000;Cui et al., 2005). Hence, oxazolidinone class of antibacterial compounds attracted considerable attention of a number of research groups during the last decade to get more efficacious and less toxic drug (Srivastava et al., 2008).
Thiazolidinone derivatives have been further reported to possess diverse pharmacological properties, such as antibacterial, antifungal, anticonvulsant, anticancer, antituberculosis, and antihuman immunodeficiency virus type 1 (HIV-1) activities. Thiazolidinones are novel inhibitors of the bacterial enzyme MurB, a precursor acting during the biosynthesis of peptidoglycan as an essential component of the cell wall of both gram-positive and gramnegative bacteria. (Bonde and Gaikwad, 2004;Aridoss et al., 2007;Küçükgüzel et al., 2002;Capan et al., 1999;Barreca et al., 2001;Andres et al., 2000;El-Gaby et al., 2009) The identification and synthesis of combinational chemotherapeutic drugs with different mechanisms of action and with few side effects are an important part of the efforts to overcome antimicrobial resistance (Bayrak et al., 2010a, b). A recent survey of novel small-molecule therapeutics has revealed that the majority of the drugs results from an analog-based approach and that their market share represents two-thirds of all drug sales (Vicini et al., 2008).
In the present study, as a part of our ongoing study on the synthesis of bioactive hybrid molecules, we aimed to obtain the far derivatives of linezolid. It was reported that SAR studies of linezolid demonstrated a high tolerance for structural variation at the 4-position of the phenyl ring (Weidner-Wells et al., 2002). In the structures of the newly synthesized compounds, the phenyl ring substituted by pyridine and oxazolidinone scaffold by other azole rings such as 1,3-thiazole, 1,3-thiazolidinone, 1,2,4-triazole, 1,3,4-thiadiazole, and 1,3,4-oxadiazole nucleus.

Results and discussion
The synthetic route for the newly synthesized compounds (3-13) is illustrated and outlined in Schemes 1 and 2.
The synthesis of compound 3 was performed from the reaction of ethyl bromoacetate with compound 2 that is available commercially. Then, compound 3 was converted to the corresponding hydrazide (4)  hydrazide function, whereas the signals due to ester group disappeared in the NMR spectrum. The treatment of hydrazide, 4 with aromatic aldehydes, namely, 4-bromobenzaldehyde and cinnamaldehyde produced the corresponding Schiff bases, compounds 5 and 6. In the 1 H NMR spectra of these compounds, the signal derived from NH 2 group disappeared; instead, new signals originated from aldehyde moiety were recorded at the related chemical shift values in the 1 H NMR and 13 C NMR spectra. Moreover, these compounds (5 and 6) exhibited EI-MS and elemental analysis data consistent with the proposed structures.
The basic treatment of intermediate 9 afforded 5-[(6morpholin-4-ylpyridin-3-yl)methyl]-4-phenyl-4H-1,2,4triazole-3-thiol (11), while the cyclization of 9 in acidic media yielded 5-[(6-morpholin-4-ylpyridin-3-yl)methyl]-N-phenyl-1,3,4-thiadiazol-2-amine (12). In the 1 H NMR spectrum of compound 11, the signal due to SH group was recorded at 13.91 ppm as an evidence of intramolecular cyclization. This group was seen at 2,857 cm -1 in the FT-IR spectrum of compound 11. Two NH signals were recorded at 6.04 and 10.23 ppm as D 2 O exchangeable peaks in the 1 H NMR spectrum of compound 12. In the 13 C NMR spectra of compounds 11 and 12, other signals belonging to 1,2,4-triazole or 1,3,4-thiadiazole nuclei resonated at the chemical shift values consistent with the literature (Bektas et al., 2010(Bektas et al., , 2012. Furthermore, [M] ? ion peaks were observed at the related m/z values supporting the proposed structures. In addition, these compounds gave reasonable elemental analysis data. The newly synthesized compounds 3-13 were evaluated in vitro for their antimicrobial activities. The results are presented in the Table 1. Among the compounds tested, compound 8, which contains different heterocyclic moieties such as morpholine, pyridine, piperazine, and 1,3,4oxadiazole important antimicrobial activity, was found to be active against all the microorganisms. All compounds except compounds 6, 7, 10, and 13 exhibited activity toward Mycobacterium smegmatis (Ms), a nonpigmented, rapidly growing mycobacterium and an atypical tuberculosis factor leading to morbidity and mortality. The highest Ms activity with the MICvalue 15.6 lg/mL was observed for compound 12 that is a 1,2,4-triazole derivative containing morpholine and pyridine nuclei as well. All the tested compounds were found to be active on yeast like fungi, Candida albicans (Ca) and Saccharomyces cerevisiae (Sc), in high concentrations with the MIC values of 500 or 1,000 lg/mL, whereas all compounds, except compound 8, displayed no activity against gram-negative bacterial strain. In contrast to other compounds, compound 12 demonstrated a low activity against Pseudomonas aeruginosa (Pa), a gram-negative bacillus.
Almost all the compounds showed moderate-to-good urease inhibitory activity ( Table 2). The inhibition was increased with increasing compound concentration. Potent compound have their activities in the range of 2.37-13.23 lM. Lower IC 50 values indicate higher enzyme inhibitor activity. Compound 10 proved to be the most potent showing an enzyme inhibition activity with an IC 50 = 2.37 ± 0.19 lM. The least active compound 3 had an IC 50 = 13.23 ± 2.25 lM.

Conclusion
In this study, the synthesis of some morpholine derivatives (3-13) were performed, some of which contain an azole moiety, and their structures were confirmed by IR, 1 H NMR, 13 C NMR, Mass spectroscopic, and elemental analysis techniques. In addition, the newly synthesized compounds were screened for their antimicrobial and antiurease activities. Some of them were found to possess activity on M. smegmatis, C. albicans ATCC, and S. cerevisiae. Furthermore, all the compounds exhibited moderate-to-good antiurease activity

General information for chemicals
All the chemicals were purchased from Fluka Chemie AG Buchs (Switzerland) and used without further purification.

Synthesis of compound 3
Ethylbromoacetate (10 mmol) was added to the mixture of compound 2 (10 mmol), and triethylamine (10 mmol) was added dropwise in dry tetrahydrofurane at 0-5°C. Then, the reaction content was allowed to reach to room temperature and stirred for 11 h (the progress of the reaction was monitored by TLC). The precipitated triethylammonium salt was removed by filtration. After evaporating the solvent under reduced pressure, a brown solid appeared. This crude product was recrystallized from ethanol-water (1:2) to afford the desired product.

Synthesis of compound 4
Hydrazide hydrate (25 mmol) was added to the solution of compound 2 (10 mmol) in absolute ethanol, and the mixture was allowed to reflux for 7 h. On cooling the reaction mixture to room temperature, a white solid appeared. The crude product was filtered off and recrystallized from ethanol to give the desired compound 4.

Syntheses of compounds 5 and 6
The solution of compound 4 (10 mmol) in absolute ethanol was refluxed with appropriate aldehyde (10 mmol) for 6 h. Then, the reaction content was allowed to cool to room temperature, and a solid appeared. This crude product was filtered off and recrystallized from ethanol to obtain the desired compound.

Synthesis of compound 8
To the solution of corresponding compound 7 (10 mmol) in dichloromethane, formaldehyde (37 %, 1.55 mL) and phenyl piperazine (10 mmol) were added, and the mixture was stirred at room temperature for 3 h. After removing the solvent under reduced pressure, a solid was obtained. This crude product was treated with water, filtered off, and recrystallized from ethyl acetate/petroleum ether (1:2) to yield the desired compound.

Synthesis of compound 9
The mixture of compound 4 (10 mmol) and phenylisothiocyanate (10 mmol) in absolute ethanol was refluxed for 6 h. On allowing the reaction content to be cooled to room temperature, a white solid was formed. This crude product was filtered off and recrystallized from ethylacetate to afford the desired compound.    ,4.84;N,16.13,S,6.15. Found: C,59.85;H,4.78;N,16.22; S, 6.18.

Synthesis of compound 11
A solution of compound 9 (10 mmol) in ethanol:water (1:1) was refluxed in the presence of 2N NaOH for 3 h, then, the resulting solution was cooled to room temperature, and acidified to pH 4 with 37 % HCl. The precipitate formed was filtered off, washed with water, and recrystallized from ethyl acetate to afford the desired compound.

Synthesis of compound 12
Concentrated sulfuric acid (64 mmol) was added into compound 9 (10 mmol) drop by drop under stirring, and the reaction content was stirred in an ice bath for 15 min. The mixture was allowed to reach to room temperature and stirred for an additional 3 h. Then, the resulting solution was poured into ice-cold water and made alkaline to pH 8 with ammonia. The precipitated product was filtered, washed with water, and recrystallized from ethanol to afford the desired product.