Synthesis and Evaluation of In Vitro Antibacterial and Antitumor Activities of Novel N,N-Disubstituted Schiff Bases

To get inside the properties of N,N-disubstituted Schiff bases, we synthesized three high-yielding benzaldehyde Schiff bases. We used the reaction between salicylaldehyde and different diamine compounds, including diamine, ethanediamine, and o-phenylenediamine, determining the structure of obtained molecules by nuclear magnetic resonance spectroscopy and electrospray ionization mass spectroscopy. We thus evaluated the microbicidal and antitumor activity of these compounds, showing that salicylaldehyde-hydrazine hydrate Schiff base (compound 1a) significantly inhibited the growth of S. aureus; salicylaldehyde-o-phenylenediamine Schiff base (compound 1c) displayed a strong capability to inhibit the proliferation of leukemia cell lines K562 and HEL. Moreover, we observed that the antibacterial action of 1a might be associated with the regulation of the expression of key virulence genes in S. aureus. Compound 1c resulted in a strong apoptotic activity against leukemia cells, also affecting the cell cycle distribution. Overall, our novel N,N-disubstituted Schiff bases possess unique antibacterial or antitumor activities that exhibit the potent application prospect in prophylactic or therapeutic interventions, providing new insights for developing new antibacterial and anticancer chemical agents.


Introduction
Recently, drug resistance of clinical bacteria and their pathogenicity as the major reasons caused the increasing rate of death in infectious diseases and tumor in humans because of the lack of effective drugs and methods for medical prevention and treatment [1][2][3]. Therefore, developing novel efficient antibacterial and antitumor agents is urgently needed based on new chemical compositions that have new structure with the natural products [4]. Schiff bases, discovered and named by the chemist Hugo Schiff, as a powerful candidate, exhibited various biological and physicochemical treated activities, antibacteria, anti-inflammation, and antitumor [5]. From chemical structure, we know that the carbonyl group (C=O) in some ketone or aldehyde-compounds is replaced by special functional groups, such as azomethine and/or imine group, to form series of special Schiff bases which are produced by the reaction of aldehydes or ketones with primary amines in the specific conditions [6,7]. Previous studies have established the synthesis method and the biological activity assayed results showed that the presence of special functional groups (imine or azomethine subunits) is critical to their biological activities in various nonnatural, natural, and natural-derived compounds [8].
There are evidences confirming that salicylaldehyde Schiff bases, obtained from the condensation of the salicylaldehyde and its derivatives in alkaline grind solution [8,9], showed a better carrying oxygen ability and catalysis of mimic enzymes due to their structure similar to the porphyrin and phthalocyanine rings, which displayed a great anticancer, anti-inflammatory, antibacterial, and antiviral activity. N,N-Disubstituted Schiff bases are a series of easy flowed electronic bridge structures, which can chelate with metal ions to form a flat rigid conjugate structure with fluorescence 2 Biochemistry Research International characteristics when aromatic rings were introduced to the salicylaldehyde moiety [9][10][11]. However, a diamine can react with salicylaldehyde by condensation reaction in alkaline grind solution to produce N,N-disubstituted Schiff bases [8]; their biological activity has been attributed to the presence of the (-CONHN=CH-) moiety. Some hydrazide-hydrazone derivatives possessed a broad range of biological activities in vivo and in vitro including analgesic, anticonvulsant [12], antidepressant [13], antimicrobial [14,15], antitumor [16,17], and anti-inflammatory [18] activities, while little available research reported the biological activity and mechanism of N,N-disubstituted Schiff bases. In this paper, we synthesized and characterized three N,N-disubstituted hydrazone Schiff bases, thus assessing their antibacterial and antitumor activities in vitro. Results will validate the antibacterial and antitumor efficacy and large differences of structure-activity relationships of N,N-disubstituted hydrazone Schiff bases in vitro and also provided the chance for developing the potential antibacterial agents from the Schiff bases.

Materials and Methods
2.1. General. All equipment, spectrometer and column chromatography, and chemical or biological agents used in this study were the same as previously published paper [19]. Cell lines PC3, MDA, WM9, BPH1, K562, and HEL were a gift obtained from the Sunnybrook Research Center in Canada.

Synthesis Procedure for N,N-Disubstituted Schiff Bases.
Compounds a, b, and c were synthesized according to the report of Przybylski et al. [11]. In brief, salicylaldehyde (0.01 mol, 2 eq) was injected in 50 mL anhydrous ethanol in a round bottom flask and then added to 85% hydrazine hydrate (0.005 mol, 1 eq). The reaction mixture was then refluxed for 7 h at 80 ∘ C under Ar 2 protection and detected by thin layer chromatography (TLC) assay. After cooling, the obtained product was filtered and then washed with cold ethanol and dried. Recrystallization was done using ethanol. Compounds b and c were prepared by adding ethanediamine and ophenylenediamine.

Antibacterial Activity
Assay. The antibacterial activity in vitro of the compounds was assessed in vitro by turbidimetric assays [19,20]. The minimum inhibitory concentration (MIC) value was determined with broth microdilution method [21].

In Vitro Gene Expression.
The methods were the same as described previously [19,21].

Antitumor Activity Assay.
Antitumor activity was evaluated by performing the MTT assay [17]. Briefly, PC3, MDA, WM9, BPH1, K562, and HEL cells were seeded in 96-well microculture plates at the density of 5 × 10 3 cells/well and incubated for 24 h to allow cell adhesion. Cells were then treated with various concentrations of assayed compounds for 48 h and then observed with an inverted fluorescence microscope (Nikon, Japan). MTT (20 L of 5 mg/mL solution) was added to each well and incubated at 37 ∘ C for additional 4 h. All medium was then removed and added 200 L Tris-DMSO solution. Plates were lightly shaken up for dissolving the mixture to measure the absorbance at 570 nm using an ELISA plate reader.

Cell Apoptosis Assay.
Cells apoptosis was also evaluated using flow cytometer based on the reported methods [16,17]. Apoptotic cells were defined as annexin V positive control. The treated cell was trypsinized, washed using PBS solution, transferred to microcentrifuge tubes for centrifugation at 1000 rpm for 5 minutes, and resuspended in binding buffer. Propidium iodide (Becton Dickinson Pharmingen, Franklin Lakes, NJ, USA) was added to the cells to a 20 g/mL of final concentration. The mixture was transferred to a 96-well plate to analyze induced apoptosis by flow cytometer (Becton Dickinson Biosciences, Franklin Lakes, NJ, USA). resuspended using PBS solution containing 1 g/mL RNase and incubated at 37 ∘ C for 30 minutes and added to a final concentration of 20 g/mL propidium iodide. The mixture was transferred into a 96-well plate to analyze the propidium iodide signal intensity using flow cytometer with FACSArray (BD Biosciences, Franklin Lakes, NJ, USA). The signal intensity was determined by the percentage of cells at G 0 , G 1 , and S phases.

Statistical
Analysis. SPSS 18.0 software was used for analyzing the data and reported results indicated the mean ± SD of three experiments. For all the experiments, the statistical significance of difference between each group was determined by one-way ANOVA followed by Student's t-test. The statistical significance of difference between every two groups was investigated with LSD method. < 0.05 was defined as significant and < 0.01 was considered extremely significant. Dates were presented as the mean ± SEM of three assays.

Chemistry.
Three N,N-disubstituted Schiff bases ( a, b, and c) were produced according to the condensation reaction between salicylaldehyde and different diamine compounds, including diamine (Scheme 1), ethanediamine (Scheme 2), and o-phenylenediamine (Scheme 3), in the presence of an alkali [11]. Three diamine compounds (1 eq) and salicylaldehyde (1 eq) were added to anhydrous ethanol with stirring and refluxing to produce the three target compounds. All reactions were determined by TLC assay. The structures of compounds a-c were determined with ESI-MS data and NMR.

Antibacterial Activity of N,N-Disubstituted Schiff Bases.
A preliminary evaluation of 100 mol/L purified N,Ndisubstituted Schiff bases was assayed to determine the antibacterial activity by assessing the antigrowth capability to several clinical pathogenic bacteria. Results obtained are summarized in Table 1. We observed that the growth of S. aureus was inhibited significantly by compound a, with an extent similar to the positive control. Moreover, compounds a and b both exhibited slight inhibitory activity against E. coli (inhibition < 50% bacterial cell growth). We also observed that the three analyzed compounds could selectively suppress the growth of A. baumannii, K. pneumonia, and P. aeruginosa in a very slight extent. However, we decided to further investigate the antibacterial properties of compound a that exhibited more than 50% bacterial cell growth inhibition at the concentration of 100 mol/L. We thus confirmed the antibacterial activity by determining the minimum inhibitory concentration (MIC) values.
To this aim, the bacteria were cultured at 37 ∘ C for 8 hours in LB medium containing different concentrations of compound a in order to investigate whether the synthesis compounds demonstrated antibacterial activity at a concentration < 100 mol/L. The MIC value of each obtained compound for the bacterial growth was defined as the lowest concentration of the compound that reduced the growth by 1% compared to the control [19]. Obtained results indicated that salicylaldehyde-hydrazine hydrate Schiff base (compound a) displayed an activity for inhibiting the growth of S. aureus in a concentration-dependent manner (Figure 1), with a MIC value of 9.75 ± 1.02 mol/L, which was similar to the positive control Streptomycin.

Effect of the Compound 1a on Expression of Virulence
Genes in S. aureus. Salicylaldehyde-hydrazine hydrate Schiff base ( a) showed the better inhibition to the growth of S. aureus. To further investigate the action of this compound and its effect on the expression of associated virulence factors, we cultured S. aureus ATCC 25923 with the treatment of a sublethal dose of the compound (50 mol/L) for 8 h; the expressional level in mRNA (transcript abundance) of the key virulence factors hla, sbi, saeR, and mecA was determined Values are mean ± standard deviation of three independent experiments. The bacteria were seeded in 96-well microplates at concentration of 1 × (gyrB housekeeping gene was used as control) by RT-PCR (Figure 2(a)) and semiquantitative RT-PCR methods (Figure 2(b)). Compound a resulted in a significant ( < 0.01) induction of the expression of saeR and mecA genes, with 12-fold and 6-fold higher expression than the negative control, respectively. Besides, compound a downregulated the expression of sbi gene ( < 0.01), about 5-fold lower than the negative control. Finally, no changes were observed in the expression of hla genes.  Figure 4(c)) cell lines was increased with the increase of assayed concentrations of the compounds, indicating the dose-dependent trend of the inhibitory response. However, the same trend was not observed in the inhibitory activity of the compounds on the WM9 cell (Figure 4(b)). All cells treated with 5 mol/L of each compound for 48 h were also analyzed using a flow cytometer, in order to observe apoptosis ( Figure 5). Compared to untreated cells ( Figure 5(c)), K562 and HEL cells treated with compounds b and c showed apoptosis rates significantly increased (Figure 5(a)). Furthermore, compound c was showed to be able to induce high levels of apoptosis in two leukemia cell lines. However, very low apoptosis rates were observed in PC3 and BPH1 cells (Figure 5(b)).

Effects of N,N-Disubstituted Schiff Bases on Cell Cycle.
Effects of active compounds on cell cycle distribution in leukemia (Figure 6(a)) and prostate (Figure 6(b)) cells were evaluated using flow cytometer. After the incubation with  20 mol/L of the compounds for 48 hours, cells were harvested and analyzed. Results showed that cells distribution in G1 and S phases was affected in the two K562 and HEL leukemia cells treated with compound c ( < 0.01, Figure 6(c)). However, compound b did not induce changes in the two cell lines. Compound a was found to significantly increase the G1 phase of prostate PC3 ( < 0.01) and BPH1 cells ( < 0.05) along with a reduction of the number of cells in S phase (Figure 6(d)). Compound b could only slightly change the G1 and S phases of PC3 cell ( < 0.05) but has no significant effect on BPH1 cells. Finally, the compound c induced no significant changes in cell cycle profile of the PC3 and BPH1 cells.

Discussion
In the present study, we synthesized three novel N,Ndisubstituted Schiff bases and evaluated their properties as antibacterial and antitumor agents. Overall, we found    of the regulation of genes associated with virulence factors [19]. Transcript of saeR gene, a key member of the virulence regulatory system saeR/S that plays an important role in the development of staphylococcal skin lesions in mice [22], was upregulated about 12-fold in S. aureus following the incubation with a with respect to the control. Besides, we have also analyzed the expression of other virulence genes sbi, hla, and mecA at the transcriptional level. Gene hla encodes the -hemolysin, which is essential for S. aureus and causes skin infections diseases in both animal and human [23]; gene sbi encodes for a crucial immunomodulatory protein in the complement evasion [24]; gene mecA encodes for the altered penicillin-binding protein 2a conferring resistance to -lactam antibiotic [25].
Our results demonstrate that the transcriptional expression of several virulence genes was upregulated by compound a. The reduction of transcript levels of other virulence genes of S. aureus involved in the saeR/S virulence regulatory system indicates that compound a may regulate in an intricate manner a grown number of S. aureus virulence genes, supporting the hypothesis that the antigrowth activity of Schiff bases against S. aureus may associate with the up-or downregulation on the expression of related virulence gene. Our results also indicate that only the compound c possesses a slight inhibitory activity against prostate cells along with a strong capability to inhibit leukemia cell proliferation, thus representing a novel powerful candidate as antitumor agent.