Synthesis and In Vitro Anti-Helicobacter and Anti-Staphylococcal Activities of Novel Diaryldisulfides and Diarylthiosulfonates

Arylthiols were reacted with acrylonitrile under basic conditions to form the corresponding aryl sulfides which were oxidised with sodium metaperiodate in aqueous methanol to yield 3-arylsulphinylpropanenitriles that upon thermolysis in refluxing toluene produced a mixture of diarylthiosulfonates and diaryldisulfides. The mixture of the two products was easily separated by flash chromatography and characterized spectroscopically. The diarylthiosulfonates and diaryldisulfides, garlic-like organosulfur compounds, were tested for their antimicrobial properties against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Helicobacter pylori and had been found to have good activity against S. aureas and H. pylori with no activity against the other two organisms.


Introduction
The medicinal properties of crushed garlic and onions are well recognized [1] [2]. The natural products in garlic responsible for the therapeutic actions are identified as the thiosulfinate esters alliin (S-allyl-L-cysteine sulfoxide) and allicin (diallylthiosulfinate) ( Figure 1) which have been reported to have a broad range of biological activities such as for example anti-inflammatory [1] and antibacterial [3] [4] actions.
In the laboratory synthetic versions of thiosulfinate esters have usually been made by either the oxidation of diaryldisulfides [5] [6] [7] [8] with peracids or by condensation of a sulfinyl chloride with a thiol in the presence of a tertiary amine [8] [9] [10]. A third method which has been little used for making symmetrical diarylthiosulfinates involves the condensation of arylsulfenic acids generated in situ by thermolysis of arylsulfoxides possessing β-hydrogens. The β-elimination of sulfoxides to yield sulfenic acids and the condensation of sulfenic acids to form thiosulfinate esters are well documented in the literature [11] ( Figure  2).
In our previously reported synthesis of phenylsulfinyl alkene derivatives by the Markovnikov addition of benzenesulfenic acid to terminal alkynes, generated in situ by thermolysis of 3-phenylsulfinylpropanenitrile [12] the diphenylthiosulfinate produced as a by-product was always disposed of. In this article, we report this synthetic strategy of making diarylthiosulfinates by condensation of arylsulfenic acids in the absence of any terminal alkynes as trapping agents.
The antimicrobial activity of garlic is mainly due to the thiosulfinate ester called allicin and is reported to be three times more effective on Gram-positive bacteria than Gram-negative ones [13]. The antibacterial activity of aqueous garlic extract against 16 H. pylori strains has been assessed and MIC 50 concentrations in the range 2 -5 mg ml −1 were able to inhibit the strains. In most cases, the inhibitory concentrations [4] were also bactericidal.
Helicobacter pylori and its clinical association with the development of peptic ulcers has led to the development of various chemotherapeutic agents to eliminate infection caused by this pathogen [14] [15] [16] [17] [18]. H. pylori is also implicated in the development of acute and chronic gastritis, gastric adenocarcinoma and gastric lymphoma (MALT), and has been classified as a class I carcinogen in humans and is a major contributing factor in the development of gastric cancer [19]. Infection with H. pylori is typically treated with a combination of clartithromycin, ampicillin and a proton pump inhibitor, but this triple therapy approach is costly [20]. The infection is eradicated in up to 90% of patients but side effects, poor compliance and the development of antimicrobial resistance are common causes of treatment failure [21]. H. pylori infection has been implicated with increased COX-2 expression in gastric antral mucosa for both NSAID users and non-users [22] [23] [24]. We have been engaged with the search for compounds with anti-H. pylori activity and have recently reported anti-H. pylori activity of novel quinoline-derived propionic acid esters [25]. In this paper, we report the synthesis and antimicrobial activities of a set of diaryl disulfides 6 and diaryl thiosulfonates 7.  Escherichia coli is a common commensal organism in the human gastrointestinal tract but it is also a significant pathogen that can cause a range of human infections including diarrheal diseases and urinary tract infections through wound infections and life-threatening ulcerative colitis and septicaemia [29] [30].
Due to the structural similarity of our synthetic compounds 6 and 7 to thiosulfinate esters found in garlic, we decided to investigate their preliminary antimicrobial activity against Helicobacter pylori and a Gram-positive bacterium, Staphylococcus aureus (including resistant strains of this species), and the Gramnegative bacterium, Escherichia coli.

Chemistry
Conjugate addition of thiolate anions to acrylonitrile furnished the arylsulfides form diarylthiosulfinates 5 and water. Compounds of the formula RS(O)SR tend to be unstable and usually cannot be isolated. At the high temperature of the reaction the weak sulfinyl-sulfide S-S bond cleaves homolytically to generate radical pairs [32]. The recombination of arylsulfinyl radicals then produces the isolated symmetrical product 6 (Scheme 2).
The recombination of arylsulfinyl radicals did not produce any of the anticipated α,α'-diaryldisulfoxide 8 as reaction product [33] [34] but instead produced the thiosulfonate 7 as the isolated reaction product. The only evidence to date for the existence of stable α,α-disulfoxides has been provided by bridged bicyclic compound 9 [35].

Microbiological Results and Discussion
An increase in resistance of bacteria and fungi towards currently available antibacterial and antifungal agents has resulted in a huge demand for the identification of novel antimicrobial agents. This is due to the rapid development of antimicrobial-resistant bacterial and fungal strains as well as a lack of new antimicrobial drugs that are effective against these resistant strains. Antimicrobial screening assays provide a robust method for the discovery of potential inhibitors of microbial growth. Due to the structural similarity of our synthetic compounds 6 and 7 to thiosulfinate esters found in garlic we decided to investigate their preliminary antimicrobial activity against Helicobacter pylori and a Grampositive bacterium, Staphylococcus aureus (including resistant strains of this species, and the Gram-negative bacterium, Escherichia coli. The diarylthiosulfonates 7d, 7h, 7j, 7k, 7l and 7m and diaryldisulfides 6d, 6h, 6j, 6k, 6l and 6m were dissolved in 50% DMSO and tested for their antimicrobial activities using the broth microdilution method (Table 1). Although there was no antimicrobial activity of any of the compounds against the Gram-negative pathogen E. coli there was some very promising effect on the common Gram-positive pathogen S. aureus. Compounds 6 and 7 were significantly inactive in antimicrobial activity against E. coli showing activity only at > 256 μg/mL but displayed modest activity against the other two organisms H. pylori 3339 and S. aureus. All the derivatives 6e, 6j, 6l, 7b, 7c, 7g and 6g as a mixture, 7j and 7k have shown modest antimicrobial activity against H. pylori 3339 when compared with the standard anti-Helicobacter agents shown in Table 1.
There is a noticeable common Structure-Activity Relationship (SAR) observed in inhibitory activity for the two different sets of compounds 6 and 7 on the H. pylori strain 3339. It was seen that when the aromatic rings in compounds 6 and 7 are orthoand para-substituted with -OMe, -Cl and -Br groups the compounds tend to have the lowest concentration inhibitory effects overall. On the other hand some of the compounds 6 and 7 have shown modest to good antimicrobial activity against S. aureus In particular the diaryldisulfides 6h, 6k and diarylthiosulfonates 7h and 7k gave the lowest inhibitory responses that are either equal or better than ampicillin. Thus, it is interesting to note that diarylthiosulfonate 7k is the most active at 2 μg/mL whilst its counterpart diaryldisulfide 6k inhibits at a somewhat higher concentration of 8 μg/mL. Similarly the 2-chloro derivatives of 6 and 7 showed good inhibitory action against S. aureus at 4 μg/mL.
Thus, a noticeable structure-activity feature of the most promising four active compounds 6h, 6k, 7h and 7k is the presence of chloro-or bromo-groups in the 2-position of the aromatic rings. We conclude that for good inhibitory activity against S. aureus compounds 6 and 7 require of an ortho-substituted chlorine or bromine atoms as best candidates for further antibacterial studies.

General Methods
Melting points are uncorrected and were determined on Stuart Scientific SMP3 apparatus. Infrared spectra were recorded with an ATI Mattson Genesis series FTIR spectrophotometer. 1 H NMR and 13 C NMR spectra were recorded in CDCl 3 using a Bruker AC 250 spectrometer operating at 250 and 62.9 MHz, respectively. Chemical shifts (δ) are in ppm downfield from Me 4 Si as internal standard and J values are given in Hz. Mass spectra were recorded with EI-VG 7070E mass spectrometer. Accurate masses were determined on VG Autospec, EI mass spectrometer with magnetic sector instrument.

Typical Experimental Procedure for the Formation of Sulfides 2 a-m
To a magnetically stirred solution of thiol 1 (0.10 mol) and acrylonitrile (20 ml, an excess) in THF (40 ml) at 0˚C was added a solution of tetrabutylammonium fluoride in THF (2 ml) and the mixture was allowed to stir and come to room temperature overnight. The solvent was rotary evaporated and the residue extracted with DCM (160 ml). The organic solution was washed with 2M NaOH solution (2 × 40 ml) and water (50 ml) before being dried over MgSO 4 and evaporated to yield the crude sulfide 2 which according to TLC     The sulfoxide 3 (0.03 mol) in dry toluene (80 ml) was heated at reflux under nitrogen for 2h after which the solvent was evaporated and the residue showing two spots by TLC [1:5 ethyl acetate-petrol] was separated by flash chromatography to yield firstly 6 followed by 7. 6a [12] and 7a [12]. 6b: 32% yield, oil, IR ν 1461, 1041, 1149 cm −1 ; 1 H NMR δ 2.50 (6H, s, 2x CH 3 ), 7.13 -7.30 (6H, m, Ar), aromatic rings to be crucial for antimicrobial activities. By SAR analysis the most promising four active compounds were 6h, 6k, 7h and 7k against Gram-positive organisms H. pylori and S. aureus. These compounds offer the promising prospects for development into clinical candidates by further studies.