Bioactive 2-(Methyldithio)Pyridine-3-Carbonitrile from Persian Shallot (Allium stipitatum Regel.) Exerts Broad-Spectrum Antimicrobial Activity

Antibiotic resistance is a problem that continues to challenge the healthcare sector, especially in clinically significant pathogens like methicillin-resistant Staphylococcus aureus (MRSA). Herein is described the isolation and structure elucidation of a bioactive compound from Allium stipitatum with antimicrobial activity. Crude Allium stipitatum dichloromethane extract (ASDE) was subjected to systematic purification by chromatographic procedures to afford various bioactive fractions. A fraction that exhibited anti-MRSA activity (4 µg·mL−1) was further characterized to determine the structure. The structure of the compound was elucidated as 2-(methyldithio)pyridine-3-carbonitrile (2-Medpy-3-CN). The 2-Medpy-3-CN compound, which was screened for antimicrobial activity, exhibited minimum inhibitory concentrations (MICs) in the range of 0.5 to >64 µg·mL−1 for tested bacterial species and 0.25 to 2 µg·mL−1 for Candida spp. Further studies are important to confirm the drug target and mechanism of action.


Introduction
The sustained increase in life-threatening infectious diseases and emerging infectious diseases (EID) due to the multidrug-resistant (MDR) pathogens is an immense and serious global challenge. Infections associated with MDR Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA) and vancomycin-resistant S. aureus (VRSA) [1], and with vancomycin-resistant

Bioassay-Guided Fractionation
A total of 80 fractions were obtained through silica gel column chromatography (CC) and all the fractions were subjected for thin-layer chromatography (TLC) analysis. Fractions that displayed similar TLC patters were pooled together and divided into six major fractions (D 1 -D 6 ). Screening for antibacterial activity of fractions D 1 -D 6 showed fraction D 3 to have strong anti-MRSA activity with a minimum inhibitory concentration (MIC) of 32 µg·mL −1 . The MICs of fractions D 2 and D 4 were 128 µg·mL −1 and 256 µg·mL −1 , respectively. However, fractions D 1 , D 5 , and D 6 did not show any activity (Table 1). Fraction D 3 which was subjected to further purification by preparative TLC (PTLC) showed eight major fractions (D 3/1 -D 3/8 ). Screening for antibacterial activity of fractions D 3/1 -D 3/8 showed fraction D 3/6 to have strong anti-MRSA activity with an MIC of 16 µg·mL −1 . The MICs of fractions D 3/5 and D 3/6 were 16 µg·mL −1 , while the MICs of fractions D 3/4 and D 3/7 were 32 µg·mL −1 . Fractions D 3/1 -D 3/3 and D 3/8 did not show any activity (Table 2). Fraction D 3/6 was separated into 120 subfractions by CC, which were pooled into six subfractions (D 3/6a -D 3/6f ) based on the TLC patterns. Screening for antibacterial activity of subfractions D 3/6a -D 3/6f showed D 3/6e to have strong anti-MRSA activity with an MIC of 4 µg·mL −1 . Subfraction D 3/6d inhibited the growth of MRSA at 32 µg·mL −1 , while subfractions D 3/6a-c and D 3/6f did not show any activity (Table 3).

Time-to-Kill Assay
Studies on the bacterial killing kinetics of 2-Medpy-3-CN on MRSA showed that the growth control in brain heart infusion(BHI) without any antibiotics (non-treated) maintained viability for 24 h, while MRSA treated with the test antibacterial compound at 1×, 2×, and 4× MIC showed significant reduction in growth, indicating that 2-Medpy-3-CN is strongly bactericidal in killing >90% of the cells at 2 h post-treatment. The compound did not exhibit a concentration-dependent activity, and no significant difference in log 10 colony-forming units (CFU)·mL −1 reduction was observed. It was observed that, with an initial inoculum of~10 6 CFU·mL −1 , 2-Medpy-3-CN (1×, 2×, and 4× MIC) declined the growth of MRSA as the incubation time increased from 0 to 2 h. The average reductions in CFUs were found to be 1.8-log 10 , 2.1-log 10 , and 1.8-log 10 decreases in CFU·mL −1 at 0.5, 1, and 2 h post-treatment, respectively ( Figure 2).

Time-to-Kill Assay
Studies on the bacterial killing kinetics of 2-Medpy-3-CN on MRSA showed that the growth control in brain heart infusion(BHI) without any antibiotics (non-treated) maintained viability for 24 h, while MRSA treated with the test antibacterial compound at 1×, 2×, and 4× MIC showed significant reduction in growth, indicating that 2-Medpy-3-CN is strongly bactericidal in killing >90% of the cells at 2 h post-treatment. The compound did not exhibit a concentration-dependent activity, and no significant difference in log10 colony-forming units (CFU)·mL −1 reduction was observed. It was observed that, with an initial inoculum of ~10 6 CFU·mL −1 , 2-Medpy-3-CN (1×, 2×, and 4× MIC) declined the growth of MRSA as the incubation time increased from 0 to 2 h. The average reductions in CFUs were found to be 1.8-log10, 2.1-log10, and 1.8-log10 decreases in CFU·mL −1 at 0.5, 1, and 2 h post-treatment, respectively ( Figure 2).

Discussion
The 2-Medpy-3-CN compound was isolated as a yellow-colored oil (later formed as yellow crystals). The LC-MS data displayed a molecular mass of 183.00471 m/z, signifying a molecular formula of C7H6N2S2, and the spectrum also gave the isotype motif for two sulfur atoms. The 1 H-NMR spectrum (Table 5; Figure S1, Supplementary Materials) showed a deshielded methyl singlet at δ2.60 and the presence of four hydrogens represented a characteristic ABCD aromatic structure/scheme. The 13 C-NMR and DEPT-135 spectra (Table 5; Figures S2 and S8, Supplementary Materials) showed the presence of three methine aromatic carbons at δ152.18-120.58, a deshielded quaternary carbon at δ120.58S, and a methyl carbon at δ23. 23. This deshielded nature of the hydrogen atom (δ8.77, H-6) of 2-Medpy-3-CN points out the phenomenon of a typical coupling of an alpha hydrogen to the nitrogen of a pyridine structure [27]. Hence, this pattern of coupling and correlation strongly underscores the possibility that 2-Medpy-3-CN could be a pyridine ring-containing natural compound.
Correlation spectroscopy (COSY) revealed the correlations of the aromatic hydrogens of 2-Medpy-3-CN ( Figure S6, Supplementary Materials). In particular, the double-doublet of H-3 (δ8.77) was coupled to a H-4 triplet (δ7.89), which finally coupled to αH-6 doublet (δ7.26). At δ2.60, the presence of a methyl group appeared as a singlet featuring without couplings, indicating that it belongs to the aromatic nucleus. Its selected appearance in the 1 H spectrum at 2.60 ppm determined that it was deshielded slightly and was attached to one of the sulfur atoms (apparently as a heteroatom). Heteronuclear multiple bond correlation spectroscopy (HMBC) showed the weak

Discussion
The 2-Medpy-3-CN compound was isolated as a yellow-colored oil (later formed as yellow crystals). The LC-MS data displayed a molecular mass of 183.00471 m/z, signifying a molecular formula of C 7 H 6 N 2 S 2 , and the spectrum also gave the isotype motif for two sulfur atoms. The 1 H-NMR spectrum (Table 5; Figure S1, Supplementary Materials) showed a deshielded methyl singlet at δ 2.60 and the presence of four hydrogens represented a characteristic ABCD aromatic structure/scheme. The 13 C-NMR and DEPT-135 spectra (Table 5; Figures S2 and S8, Supplementary Materials) showed the presence of three methine aromatic carbons at δ 152.18-120.58, a deshielded quaternary carbon at δ 120.58S, and a methyl carbon at δ 23.23. This deshielded nature of the hydrogen atom (δ 8.77, H-6) of 2-Medpy-3-CN points out the phenomenon of a typical coupling of an alpha hydrogen to the nitrogen of a pyridine structure [27]. Hence, this pattern of coupling and correlation strongly underscores the possibility that 2-Medpy-3-CN could be a pyridine ring-containing natural compound.
Correlation spectroscopy (COSY) revealed the correlations of the aromatic hydrogens of 2-Medpy-3-CN ( Figure S6, Supplementary Materials). In particular, the double-doublet of H-3 (δ 8.77) was coupled to a H-4 triplet (δ 7.89), which finally coupled to αH-6 doublet (δ 7.26). At δ 2.60, the presence of a methyl group appeared as a singlet featuring without couplings, indicating that it belongs to the aromatic nucleus. Its selected appearance in the 1 H spectrum at 2.60 ppm determined that it was deshielded slightly and was attached to one of the sulfur atoms (apparently as a heteroatom). Heteronuclear multiple bond correlation spectroscopy (HMBC) showed the weak correlation ( 4 J) between the methyl hydrogens and the C-2 quaternary carbon (HSQC), which suggested its terminal position on a disulfide side-chain α to the nitrogen ( Figures S3 and S4, Supplementary Materials). Stereochemistry on the nuclear Overhauser spectroscopy (NOESY) effect of methyl hydrogens to H-3 was also determined ( Figure S5, Supplementary Materials).
The promising antibacterial activities of pyridine and thiopyridines derivatives ere adequately reported [29][30][31]. The 2-Medpy-3-CN compound demonstrated outstanding potency with low MIC values ranging from 0.5 to >64 µg·mL −1 . The MICs of 2-Medpy-3-CN are consistent with the earlier data on pyridine based compounds from other research groups [25,26,28]. O'Donnell and his colleagues were the first to isolate and report pyridine-N-oxide alkaloids with disulfide functional groups from natural sources [26]. Three compounds named 2-(methyldithio)pyridine-N-oxide, 2[(methylthiomethyl)dithio]pyridine-N-oxide, and 2,2'-dithio-bis-pyridine-N-oxide (dipyrithione) were reported. Of the three compounds, 2-(methyldithio)pyridine-N-oxide and 2[(methylthiomethyl)dithio]pyridine-N-oxide were shown to possess strong antibacterial activity against fast-growing Mycobacterium sp., MRSA, and MDR variants of S. aureus with MICs of 0.5-8 µg·mL −1 . These results were in accordance with the present study results where an MIC of 4 µg·mL −1 was observed for MSSA and MRSA strains. Following O'Donnell's discovery of pyridine N-oxide compounds, Krejčová and his colleagues reported the anti-inflammatory and neuroprotective effects of five naturally occurring sulfur-containing pyridine-N-oxides from A. stipitatum [28]. The compound of the present study, 2-Medpy-3-CN, harbors nitrile as a functional group, which is another likely perception for its strong antimicrobial properties. Carbonitrile-containing semisynthetic derivatives of pyridine molecules exhibit strong antibacterial activity [32][33][34]. In addition to antibacterial activity, 2-Medpy-3-CN also exhibited very strong anticandidal activity, attributed to a broad-spectrum antimicrobial activity at a very low MIC level of 2 µg·mL −1 for C. tropicalis. Semisynthetic pyrimidine derivatives with carbonitrile as the functional group at the fifth position were reported to have strong antibacterial and antifungal activities at 12.5 µg·mL −1 [33,35]. The extreme potency of 2-Medpy-3-CN foreshadows its possibility in clinical use.
Time kill study was used to explore the bactericidal activity of 2-Medpy-3-CN using MRSA American Type Culture Collection (ATCC) 43300. The killing kinetics of 2-Medpy-3-CN demonstrated time-dependent killing instead of a dose-dependent pattern. At 1× MIC (4 µg·mL −1 ), 2-Medpy-3-CN effected a time-dependent reduction in the viability of the tested bacterial strain. Increasing the concentration of 2-Medpy-3-CN did not show any change in the bactericidal action. The killing rates were very much similar at 2× and 4× MIC, with 1.8-log 10 , 2.1-log 10 , and 1.8-log 10 reductions in the number of CFU·mL −1 after 0.5, 1, and 2 h of incubation, respectively. For the first time, our study reports the killing kinetics or the rate of killing of pyridine compounds on MRSA. A recent study on novel 2-thiopyridines against actively growing and dormant cells of M. tuberculosis for seven days at 10 µg·mL −1 showed more than 2-log-unit killing effect [31]. Remarkably, the tested concentration and the CFU reductions were similar to the results of the present study. Testing 2-Medpy-3-CN on a diverse panel of antibiotic-resistant S. aureus isolates at various MICs disclosed the consistency of anti-MRSA activity.
Absorption, distribution, metabolism, and excretion (ADME) properties were used to determine the "drug-like" characteristics of the ligand molecule. ADME predictions can be used to focus lead optimization efforts in enhancing the desired properties of a given compound. The expected ADME property of the tested compound was evaluated with the QikProp module of Schrodinger. Almost all the predicted properties of the tested compound were in the range as predicted by QikProp for 100% (>80% is high) of known oral drugs, and the compound also satisfies Lipinski's rule of five to be considered as having drug-like potential.

Plant Material and Preparation of Extracts
The source and the preparation of the extract were described earlier [22]. Briefly, dried bulbs (5 kg) of A. stipitatum were ground into fine powder and extracted with 10 L of dichloromethane for 72 h (for each solvent) by maceration at room temperature. The extract was filtered through Whatman No.1 paper to remove solid plant materials, and the filtrate was dried under vacuum (BÜCHI Rotovapor R-200, Flawil, Switzerland) at 40 • C. Upon filtration and solvent volatilization, the dichloromethane extract yielded 164 g (3.28%) of residue.

General Experimental Procedures
Analytical-grade solvents, including hexane and dichloromethane used for extraction and isolation, were purchased from (Merck KGaA, Darmstadt, Germany). Column chromatography was carried out using silica gel Merck 7734 (70-230 mesh ASTM) and Merck 9385 (230-400 mesh ASTM). Thin-layer chromatography (TLC) analyses were carried out on Merck silica gel DC-plastikfolien 60 F254 plastic sheets and TLC spots were visualized using a UV lamp at 254 and 366 nm, respectively. Melting point was recorded using an electrothermal digital melting point apparatus. Ultraviolet (UV) and IR spectra were recorded on a Varian UV-visible light (UV-Vis) 50 and Perkin-Elmer 100 Fourier-transform infrared (FTIR) spectrophotometers, respectively. The isolated compound was dissolved in CDCl 3 (deuterated chloroform), and the NMR spectra (1D and 2D NMR) were recorded on a Bruker AVANCE 500 Ultrashield NMR spectrometer. The molecular weight of the isolated compound was determined on an Agilent 1290 Infinity II LC system (Agilent Technologies, Santa Carla, CA, USA) coupled to a 6520 Q-TOF tandem mass spectrometer for separation. The chemical shifts (δ) were determined from the residual solvent peaks.

Isolation of Potential Antimicrobial Compounds
TLC analysis of the Allium stipitatum dichloromethane (ASDE) extract showed the presence of large, clear spots when visualized under a UV chamber and iodine staining. The crude dichloromethane extract of A. stipitatum bulbs (164 g) was subjected to silica gel column chromatography (CC) using CH 2 Cl 2 as the mobile phase. A total of 80 fractions (50 mL in volume) were collected in 100-mL conical flasks. All fractions were subjected to TLC analysis. Fractions that showed similar TLC patterns were pooled together which yielded six major fractions (D 1 -D 6 ). The six fractions (D 1 -D 6 ) were tested for their antibacterial activity against MRSA. Fraction D 3 (1.354 g) which exerted remarkable antibacterial activity, was subjected to preparative thin-layer chromatography (PTLC) using CH 2 Cl 2 as the mobile phase to yield eight subfractions (D 3/1 -D 3/8 ). Subfraction D 3/6 (302 mg) was further subjected to column chromatography (CC) using CH 2 Cl 2 as the mobile phase to yield 120 fractions (10 mL in volume). Fractions were pooled together based on TLC patterns. Subfraction D 3/6e (31 mg) was identified as a pure fraction and subjected to NMR and mass analysis. The purification scheme of fraction D 3/6e is illustrated in Scheme 1. At each purification step, the fractions and subfractions were tested for their antibacterial activity using the micro-broth dilution method.

Test Microorganisms
MRSA ATCC 43300 was used as model organism to screen for the antimicrobial activity. For the anti-MRSA activity, the confirmed compound was further screened for broad-spectrum activity against other pathogenic bacteria including Acinetobacter baumannii, A. iwoffii, Enterobacter sp., Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella typhi, Shigella dysenteriae, and Stenotrophomonas maltophilia through MIC (minimum inhibitory concentration) and MBC (minimum bactericidal concentration) determinations. The compound was also tested for antifungal activity against five different pathogenic forms of Candida species, namely C. albicans, C. glabrata, C. krusei, C. parapsilosis, and C. tropicalis, for MIC and MFC (minimum fungicidal concentration).

MIC, MBC, and MFC Measurements
For the determination of MIC, MBC, and MFC, the compound was dissolved in 10% dimethyl sulfoxide (DMSO) with a graded concentration in the range of 64-0.125 µg·mL −1 with MHB for bacteria and Roswell Memorial Park Institute (RPMI-1640) medium for Candida strains. The MIC of the compound was determined using microbroth dilution method as described for bacteria and yeast [36,37]. Fluconazole was used as an antifungal standard and its MIC was determined using the Etest method. Appropriate antibiotic controls were included as positive controls based on the recommendations by the Clinical Laboratory Standards Insititute CLSI (2012). DMSO (10%) was used as a negative control and broth cultures without antibiotics served as growth controls.

Time-To-Kill Assay for Detecting the Bactericidal Effect of the Compound for MRSA
The killing kinetics of the compound on MRSA ATCC 43300 at 0×, 1× (4 µg·mL −1 ), 2× (8 µg·mL −1 ), and 4× (16 µg·mL −1 ) MIC was determined according to the method described previously [38] with some modifications. Bacterial and yeast suspensions were diluted to 1 × 10 6 CFU·mL −1 . The compound concentrations were adjusted to 1× MIC, 2× MIC, and 4× MIC. Cultures treated with the varying concentrations of the compound were incubated at 37 • C for 0, 0.5, 1, 2, 4, 8, and 12 h. Aliquots of 100 µL were pipetted out from each tube at each time point, serially diluted in phosphate-buffered saline (PBS) and spread onto Mueller Hinton Agar MHA plates. Tubes without the compound served as growth controls (0×). The plates were incubated at 37 • C for 24 h followed by the enumeration of the colonies. Killing curves were constructed by plotting the log 10 CFU·mL −1 versus time over a 24 h time period. A decline in bacterial/fungal growth to ≥3log 10 CFU·mL −1 from the initial inoculum was considered to be bactericidal/fungicidal.

Prediction of ADME Properties
Schrodinger's QikProp module is an accurate, quick, and easy-to-use absorption, distribution, metabolism, and excretion (ADME) prediction program that was designed to produce certain ADME-related descriptors. QikProp consists of two modes: normal mode and fast mode. In the present study, the QikProp program, version 3.4 was run in normal processing mode with default options [39]. To set up the calculation, a pose viewer file (generated after docking with Glide) was used to consider the receptor and source of ligands. The program was run (with default options) and reasonable descriptors were produced.

Statistical Analysis
All experiments were performed in triplicate except for the time-to-kill assay. Differences between the treated and untreated (control) groups were analyzed using GraphPad Prism 5.0. One-way ANOVA was performed and a Dunnett's post hoc test was used for the comparison of multiple means. Significance was set at a p-value of <0.05.

Conclusions
The present study isolated a novel broad-spectrum antimicrobial compound 2-(methyldithio)pyridine-3-carbonitrile. The 2-Medpy-3-CN compound showed excellent antimicrobial activity against a panel of bacterial and fungal pathogens. The compound inhibited the growth of Gram-positive and Gram-negative pathogens along with Candida species at MICs ranging from 0.5 to >64 µg·mL −1 . The 2-Medpy-3-CN compound was strongly bactericidal against MRSA in less than 2 h post-treatment. Further studies are vital to identify the drug target and mechanism of action for this highly potent antimicrobial compound.
Supplementary Materials: The following are available online at http://www.mdpi.com/1420-3049/24/6/1003/ s1, 1 H and 13 C-NMR data and other related spectra for 2-Medpy-3-CN within this article, along with ADME toxicity data, can be found in the Supplementary Materials.