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

Muhammad Akram Khan^*, Keith Miller, Kirstie Rawson, Yong Zhou

Biomedical Research Centre, Sheffield Hallam University, Sheffield, UK.

DOI: 10.4236/abc.2021.115017                

Abstract

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.

Keywords

Diarylthiosulfonates, Diaryldisulfides, Helicobacter pylori, Arylsulfenic Acids, Michael Reaction

1. 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 MIC₅₀ 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.

Figure 1. Enzymatic conversion of alliin into diallylthiosulfinate (allicin).

Figure 2. b-Elimination arylsulphoxides to produce sulphenic acids.

Staphylococcus aureus is a versatile pathogen that can cause a range of infectious diseases ranging from superficial skin infections to life-threatening septicaemia. The emergence of antibiotic-resistant strains of S. aureus including MRSA, VISA, LRSA and multi-drug resistant isolates has led to a great deal of interest in developing novel anti-staphylococcal agents [26] [27] [28].

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 Gram-negative bacterium, Escherichia coli.

2. Results and Discussion

2.1. Chemistry

Conjugate addition of thiolate anions to acrylonitrile furnished the arylsulfides 2a-i and 2k-m in excellent date yields (93% - 98%) with 2j obtained in 82% yield. Sodium metaperiodate is a specific oxidising agent for converting sulfides into sulfoxides. Thus oxidation of the sulfides 2a-m with one-molar equivalent of sodium metaperiodate in aqueous methanol yielded the corresponding sulfoxides 3a-m which were isolated in good yields (63% - 89%) and characterised spectroscopically. The FTIR spectra showed the >S=O absorbance at 1043 - 1093 cm⁻¹. Upon refluxing in toluene under nitrogen the sulfoxides 3a-m thermolized to furnish the disulfides 6a-m and diarythiosulfonates 7a-m as the only reaction products according to TLC analysis. The mixtures diaryldisulfides6a-m and diarylthiosulfonates 7a-m were separated and isolated by flash chromatography (Scheme 1).

Our disappointment in not obtaining the anticipated diarylthiosulfinates 5 as reaction products was shown by subsequent literature search that described diarylthiosulfinates 5 to be thermally unstable compounds due to the weak S-S bond (bond strength ~ 35 kcal) [31]. Thermolysis of the sulfoxides 3 initially generates arylsulfenic acids 4 which by virtue of their high reactivity condense to

Scheme 1. Synthesis of 1-arylsulphinyl-2-cyanoethanes 3 and their thermolysis to form products 6 and 7.

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].

2.2. 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 Gram-positive 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).

Scheme 2. The disproportionation and recombination of thiol and arylsulfinyl radicals to form the products 7 and 8.

Table 1. Antimicrobial activity of compounds 6a-f, 6h, 6j, 6k-m and 7b-e, 7g-h, 7j-l.

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 ortho- and 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.

3. Experimental

3.1. 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. ¹H NMR and ¹³C NMR spectra were recorded in CDCl₃ using a Bruker AC 250 spectrometer operating at 250 and 62.9 MHz, respectively. Chemical shifts (δ) are in ppm downfield from Me₄Si 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.

3.2. 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 MgSO₄ and evaporated to yield the crude sulfide 2 which according to TLC [1: 5 EtOAc-petrol] did not require any further purification. 2a [12], 2b: oil, 98% yield, IR ν 2248 (CN) cm⁻¹; ¹HNMR δ 2.43 (3H, s, CH₃), 2.56 (2H, t, J = 7.8 Hz, SCH₂-), 3.10 (2H, t, J = 7.8 Hz, -CH₂CN), 7.10 - 7.40 (4H, m, Ar); MS m/z 177 (M⁺, 73%), 137 (M-CH₂CN, 100%); 2c: oil, 93% yield, IR ν 2248 (CN) cm⁻¹; ¹HNMR δ 2.35 (3H, s, CH₃), 2.58 (2H, t, J = 7.8 Hz, SCH₂-), 3.08 (2H, t, J = 7.8 Hz, -CH₂CN), 7.03 - 7.30 (4H, m, Ar); EIMS: m/z 177 (M⁺, 64%), 137 (M-CH₂CN, 100%); 2d: oil, 93% yield, IR ν 2248 (CN) cm⁻¹; ¹HNMR δ 2.37 (3H, s, CH₃), 2.55 (2H, t, J = 7.8 Hz, SCH₂-), 3.10 (2H, t, J = 7.8 Hz, -CH₂CN), 7.15 (2H, d, J = 7.5 Hz, AB system Ar), 7.34 (2H, d, J = 7.5 Hz, AB system Ar); EIMS: m/z 177 (M⁺, 64%), 137 (M-CH₂CN, 100%); 2e: oil, 98% yield, IR ν 2248 (CN) cm⁻¹; ¹HNMR δ 2.58 (2H, t, J = 7.8 Hz, SCH₂-), 3.11 (2H, t, J = 7.8 Hz, -CH₂CN), 3.90 (3H, s, OCH₃), 6.83 - 6.94 (2H, m, Ar); 7.23 - 7.40 (2H, m, Ar); EIMS: m/z 193 (M⁺, 100%), 153 (M-CH₂CN, 69%); 2f: oil, 98% yield, IR ν 2248 (CN) cm⁻¹; ¹HNMR δ 2.63 (2H, t, J = 7.8 Hz, SCH₂-), 3.13 (2H, t, J = 7.8 Hz, -CH₂CN), 3.80 (3H, s, OCH₃), 6.80 (1H, d, J = 7.5 Hz, Ar H-4); 6.88 - 7.00 (2H, m, Ar); (1H, t, J = 7.5 Hz, Ar H-5); EIMS: m/z 193 (M⁺, 100%), 153 (M-CH₂CN, 68%), 140 (M-CH₂CH₂CN, 63%); 2g: oil, 93% yield, IR ν 2248 (CN) cm⁻¹; ¹HNMR δ 2.52 (2H, t, J = 7.8 Hz, SCH₂-), 2.98 (2H, t, J = 7.8 Hz, -CH₂CN), 3.80 (3H, s, OCH₃), 6.87 (2H, d, J = 7.5 Hz, AB system Ar H-3 and H-5), 7.40 (2H, d, J = 7.5 Hz, AB system Ar H-2 and H-6); EIMS: m/z 193 (M⁺, 100%), 153 (M-CH₂CN, 77%), 140 (M-CH₂CH₂CN, 70%); 2h: oil, 98% yield, IR ν 2252 (CN) cm⁻¹; ¹HNMR δ 2.64 (2H, t, J = 7.8 Hz, SCH₂-), 3.17 (2H, t, J = 7.8 Hz, -CH₂CN), 7.15 - 7.30 (2H, m, Ar); 7.35 - 7.45 (2H, m, Ar); EIMS: m/z 197.5 (M⁺, 52%), 157.5 (M-CH₂CN, 100%); 2i: oil, 98% yield, IR ν 2252 (CN) cm⁻¹; ¹HNMR δ 2.63 (2H, t, J = 7.8 Hz, SCH₂-), 3.12 (2H, t, J = 7.8 Hz, -CH₂CN), 7.17 - 7.30 (3H, m, Ar), 7.37 (1H, s, Ar); EIMS: m/z 197.5 (M⁺, 60%), 157.5 (M-CH₂CN, 100%); 2j: oil, 82% yield, IR ν 2243 (CN) cm⁻¹; ¹HNMR δ 2.58 (2H, t, J = 7.8 Hz, SCH₂-), 3.12 (2H, t, J = 7.8 Hz, -CH₂CN), 7.20 - 7.38 (4H, m, Ar); EIMS: m/z 197.5 (M⁺, 56%), 157.5 (M-CH₂CN, 100%); 2k: oil, 98% yield, IR ν 2248 (CN) cm⁻¹; ¹HNMR δ 2.65 (2H, t, J = 7.8 Hz, SCH₂-), 3.18 (2H, t, J = 7.8 Hz, -CH₂CN), 7.05 - 7.18 (1H, m, Ar); 7.20 - 7.50 (2H, m, Ar), 7.52 - 7.67 (1H, m, Ar); EIMS: m/z 242 (M⁺, 37%), 202 (M-CH₂CN, 100%); 2l: oil, 98% yield, IR ν 2243 (CN) cm⁻¹; ¹HNMR δ 2.57 (2H, t, J = 7.8 Hz, SCH₂-), 3.10 (2H, t, J = 7.8 Hz, -CH₂CN), 7.26 (2H, d, J = 7.5Hz, AB system Ar), 7.45 (2H, d, J = 7.5 Hz, AB system Ar); EIMS: m/z 242 (M⁺, 48%), 202 (M-CH₂CN, 51%); 2m: oil, 93% yield, IR ν 2248 (CN) cm⁻¹; ¹HNMR δ 2.60 (2H, t, J = 7.8 Hz, SCH₂-), 3.20 (2H, t, J = 7.8 Hz, -CH₂CN), 7.40 - 7.60 (3H, m, Ar); 7.72 - 7.95 (4H, m, Ar); EIMS: m/z 213 (M⁺, 49%), 202 (M-CH₂CN, 46%), 43 (100%).

Typical experimental procedure for the formation of 3-arylsulfinylpropanenitriles 3a-m:

To a vigorously stirred solution of sodium metaperiodate (0.0763 mol) in water (135 ml) at 0˚C was quickly added a solution of the sulfide 2 (0.0763 mol) in methanol (135 ml) and the mixture was stirred for 22h and allowed to come to RT. The precipitated inorganic solid was filtered at the pump and the mother liquor extracted with DCM (3 × 200 ml). After washing with water (100 ml) the organic layer was dried (MgSO₄) and evaporated to yield the crude sulfoxide 3 which was purified by flash chromatography [2:3 ethyl acetate-petrol followed by ethyl acetate]. 3a [12], 3b: 82% yield, IR ν 2246 (CN), 1035, 1068 (>S=O) cm⁻¹; ¹H NMR δ 2.30 (3H, s, CH₃), 2.40 - 2.60 (1H, m, -CHCN), 2.75 - 2.93 (2H, m, -SO-CH₂-), 3.05 - 3.22 (1H, m, -CHCN), 7.10 - 7.25 (1H, m, Ar), 7.30 - 7.45 (2H, m, Ar), 7.67 - 7.80 (1H, m, Ar); EIMS: m/z 193 (M⁺, 29%), 140 (M-CH₂=CH-CN, 46%), 139 (M-CH₂CH₂CN, 60%), 77 (100%); HRMS: Found 193.0565. Calcd. for C₁₀H₁₁NOS 193.0561; 3c: 85% yield, IR ν 2246 (CN), 1049, 1085 (>S=O) cm⁻¹; ¹H NMR δ 2.36 (3H, s, CH₃), 2.32 - 2.50 (1H, m, -CHCN), 2.72 - 2.95 (2H, m, -SO-CH₂-), 3.10 - 3.25 (1H, m, -CHCN), 7.20 - 7.40 (4H, m, Ar); EIMS: m/z 193 (M⁺, 14%), 140 (M-CH₂=CH-CN, 16%), 139 (M-CH₂CH₂CN, 67%), 66 (100%); HRMS: Found 193.0568. Calcd. for C₁₀H₁₁NOS 193.0561; 3d: 76% yield, IR ν 2246 (CN), 1045, 1085 (> S=O) cm⁻¹; ¹H NMR δ 2.33 (3H, s, CH₃), 2.27 - 2.50 (1H, m, -CHCN), 2.70 - 2.95 (2H, m, -SO-CH₂-), 3.05 - 3.20 (1H, m, -CHCN), 7.30 (2H, d, J = 7.5 Hz, AB system Ar), 7.43 (2H, d, J = 7.5 Hz, AB system Ar); EIMS: m/z 193 (M⁺, 13%), 140 (M-CH₂=CH-CN, 23%), 139 (M-CH₂CH₂CN, 100%), 91 (43%); HRMS: Found 193.0557. Calcd. for C₁₀H₁₁NOS 193.0561; 3e: 65% yield, IR ν 2246 (CN), 1039, 1068 (>S=O) cm⁻¹; ¹H NMR δ 2.25 - 2.45 (1H, m, -CHCN), 2.64 - 2.85 (1H, m, -SO-CH-), 2.97 - 3.13 (1H, m, -SOCH-), 3.15 - 3.34 (1H, m, -CHCN), 2.83 (3H, s, OCH₃), 6.86 (1H, d, J = 7.5 Hz, Ar), 7.10 (1H, t, J = 7.5 Hz, Ar), 7.43 (1H,t, J = 7.5 Hz, Ar), 7.60 (1H, d, J = 7.5Hz, Ar); EIMS: m/z 209 (M⁺, 18%), 156 (M-CH₂=CH-CN, 44%), 155 (M-CH₂CH₂CN, 100%); HRMS: Found 209.0517. Calcd. for C₁₀H₁₁NO₂S 209.0510; 3f: 89% yield, IR ν 2246 (CN), 1043 (>S=O) cm⁻¹; ¹H NMR δ 2.32 - 2.52 (1H, m, -CHCN), 2.67 - 2.94 (2H, m, -SO-CH₂-), 3.05 - 3.22 (1H, m, -CHCN), 2.75 (3H, s, OCH₃), 6.88 - 7.10 (3H, m, Ar), 7.28 - 7.43 (1H, t, J = 7.5 Hz, Ar); EIMS: m/z 209 (M⁺, 12%), 156 (M-CH₂=CH-CN, 18%), 155 (M-CH₂CH₂CN, 65%); HRMS: Found 209.0515. Calcd for C₁₀H₁₁NO₂S 209.0510; 3g: 89% yield, IR ν 2246 (CN), 1043, 1087 (>S=O) cm⁻¹; ¹H NMR δ 2.38 - 2.53 (1H, m, -CHCN), 2.69 - 2.95 (2H, m, -SO-CH₂-), 3.03 - 3.17 (1H, m, -CHCN), 3.77 (3H, s, OCH₃), 6.97 (2H, d, J = 7.5 Hz, AB system Ar), 7.47 (2H, d, J = 7.5 Hz, AB system Ar); EIMS: m/z 209 (M⁺, 16%), 156 (M-CH₂=CH-CN, 45%), 155 (M-CH₂CH₂CN, 100%); HRMS: Found 209.0509. Calcd for C₁₀H₁₁NO₂S 209.0510; 3h: 86% yield, IR ν 2246 (CN), 1043, 1087 (> S=O) cm⁻¹; ¹H NMR δ 2.38 - 2.40 (1H, m, -CHCN), 2.70 - 2.90 (1H, m, -SO-CH-), 3.00 - 3.18 (1H, m, -SO-CH-), 3.22 - 3.42 (1H, m, -CHCN), 7.30 - 7.60 (3H, m, Ar), 7.75 (1H, d, J = 7.5 Hz, Ar); EIMS: m/z 213.5 (M⁺, 38%), 160.5 (M-CH₂=CH-CN, 34%), 159.5 (M-CH₂-CH₂CN, 100%); HRMS: Found 213.00146. Calcd for C₉H₈NSCl 213.0015 (Cl³⁵); 3i: 73% yield, IR ν 2246 (CN), 1051 (>S=O) cm⁻¹; ¹H NMR δ 2.40 - 2.58 (1H, m, -CHCN), 2.70 - 2.98 (2H, m, -SO-CH₂-), 3.10 - 3.28 (1H, m, -CHCN), 7.33 - 7.47 (3H, m, Ar), 7.75 (1H, s, Ar); EIMS: m/z 213.5 (M⁺, 25%), 160.5 (M-CH₂=CH-CN, 30%), 159.5 (M-CH₂CH₂CN, 100%); HRMS: Found 213.0016. Calcd for C₉H₈NSCl 213.0015 (Cl³⁵); 3j: 66% yield, IR ν 2246 (CN), 1047, 1081 (>S=O) cm⁻¹; ¹H NMR δ 2.40 - 2.60 (1H, m, -CHCN), 2.74 - 2.98 (2H, m, -SO-CH₂-), 3.10 - 3.28 (1H, m, -CHCN), 7.43 - 7.60 (4H, m, Ar); EIMS: m/z 213.5 (M⁺, 15%), 160.5 (M-CH₂=CH-CN, 25%), 159.5 (M-CH₂CH₂CN, 100%); HRMS: Found 213.00146. Calcd for C₉H₈NSCl 213.0015 (Cl³⁵); 3k: 63% yield, IR ν 2246 (CN), 1056, 1093 (>S=O) cm⁻¹; ¹H NMR δ 2.40 - 2.60 (1H, m, -CHCN), 2.76 - 2.98 (1H, m, -SO-CH-), 3.08 - 3.25 (1H, m, -SO-CH-), 3.30 - 3.50 (1H, m, -CHCN), 7.42 (1H, t, J = 7.5 Hz, Ar), 7.50 - 7.65 (2H, m, Ar), 7.77 (1H, d, J = 7.5 Hz, Ar); EIMS: m/z 258 (M⁺, 20%), 205 (M-CH₂=CH-CN, 29%), 204 (M-CH₂CH₂CN, 100%); HRMS: Found 256.9516. Calcd for C₉H₈NSBr 256.9510 (Br⁷⁹); 3l: 83% yield, IR ν 2246 (CN), 1047, 1083 (>S=O) cm⁻¹; ¹H NMR δ 2.38 - 2.60 (1H, m, -CHCN), 2.74 - 2.97 (2H, m, -SO-CH₂-), 3.06 - 3.25 (1H, m, -CHCN), 7.44 (2H, d, J = 7.5 Hz, AB system Ar) 7.65 (2H, d, J = 7.5 Hz, AB system Ar); EIMS: m/z 258 (M⁺, 7%), 205 (M-CH₂=CH-CN, 58%), 204 (M-CH₂CH₂CN, 29%); HRMS: Found 256.9518. Calcd for C₉H₈NSBr 256.9510 (Br⁷⁹); 3m: 74% yield, IR ν 2246 (CN), 1040, 1068 (>S=O) cm⁻¹; ¹H NMR δ 2.35 - 2.53 (1H, m, -CHCN), 2.78 - 3.08 (2H, m, -SO-CH₂-), 3.10 - 3.35 (1H, m, -CHCN), 7.37 - 7.70 (3H, m, Ar), 77.70 - 8.05 (3H, m, Ar), 8.15 (1H, s, Ar); EIMS: m/z 229 (M⁺, 13%), 176 (M-CH₂=CH-CN, 35%), 175 (M-CH₂CH₂CN, 100%); HRMS: Found 229.0571 Calcd for C₁₃H₁₁NS 229.0561.

Typical experimental procedure for the formation of diaryldisulfides 6a-m and diarylthiosulfonates 7a-m:

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; ¹H NMR δ 2.50 (6H, s, 2x CH₃), 7.13 - 7.30 (6H, m, Ar), 7.55 - 7.65 (2H, d, J = 7.50 Hz, Ar); ¹³C NMR δ 20.85, 125.38, 126.05, 130.96, 133.32, 142.08; EIMS: m/z 246 (M⁺, 83%), 123 (M – SC₆H₄-Me, 100%); HRMS: Calcd for C₁₄H₁₄S₂ 246.0537; Found 246.0530; 7b: 30% yield, oil, IR ν 1315, 1145 cm⁻¹; ¹H NMR δ 2.15 (3H, s, CH₃), 2.69 (3H, s, CH₃), 7.02 - 7.50 (8H, m, Ar); ¹³C NMR δ 21.26, 21.64, 125.45, 126.10, 126.80, 128.25, 129.29, 131.24, 132.30, 133.20, 133.58, 137.65, 139.18, 142.10; EIMS: m/z 278 (M⁺, 50%), 123 (M – SC₆H₄-Me, 85%); HRMS: Calcd for C₁₄H₁₄O₂S₂ 278.04352; Found 278.04356; 6c: 30% yield, oil, IR ν 1079, 1141 cm⁻¹; ¹H NMR δ 2.34 (6H, s, 2x CH₃), 7.05 (2H, d, J = 7.80 Hz, Ar), 7.22 (2H, t, J = 7.80 Hz, Ar), 7.34 - 7.47 (4H, m, Ar); ¹³C NMR δ 21.02, 124.22, 125.78, 126.75, 136.05, 140.59; EIMS: m/z 246 (M⁺, 100%), 123 (M – SC₆H₄-Me, 77%); HRMS: Found 246.0526. Calcd for C₁₄H₁₄S₂ 246.0537 7c: 24% yield, oil, IR ν 1318(-SO₂), 1146 (S-O) cm⁻¹; ¹H NMR δ 2.30 (3H, s, CH₃), 2.35 (3H, s, CH₃), 7.15 (2H, s, Ar), 7.20 - 7.45 (6H, m, Ar); ¹³C NMR δ 21.42, 124.99, 128.28, 128.95, 129.49, 132.54, 133.89, 134.72, 13741, 137.42, 139.71, 143.42, 143.05; EIMS: m/z 278 (M⁺, 47%), 139 (SO₂C₆H₄-Me, 100%); HRMS: Calcd for C₁₄H₁₄O₂S₂ 278.04352; Found 278.0447; 6d: 35% yield, mp 45˚C - 47˚C, IR ν 1340, 1116 cm⁻¹; ¹H NMR δ 2.34 (6H, s, 2x CH₃), 7.13 (4H, d, J = 7.80 Hz, AB system Ar), 7.41 (4H, d, J = 7.80 Hz, AB system Ar), ¹³C NMR δ 21.48, 128.47, 129.70, 133.85, 137.33; EIMS: m/z 246 (M⁺, 100%), 123 (M – SC₆H₄-Me, 86%); HRMS: Calcd for C₁₄H₁₄S₂ 246.0537; Found 246.05424; 7d: 36% yield, mp 82˚C - 84˚C, IR ν 1321 (-SO₂), 1137 (S-O) cm⁻¹; ¹H NMR δ 2.38 (3H, s, CH₃), 2.43 (3H, s, CH₃), 7.07 - 7.30 (4H, m, Ar), 7.46 (2H, d, J = 7.80 Hz, Ar); ¹³C NMR δ 21.33, 21.51, 124.44, 127.43, 129.25, 130.08, 136.32, 140.30, 141.93, 144.51; EIMS: m/z 278 (M⁺, 48%), 139 (SO₂C₆H₄-Me, 90%); HRMS: Calcd for C₁₄H₁₄O₂S₂ 278.04352; Found 278.0447; 6e: 26% yield, mp 90˚C - 93˚C, IR ν 1238 cm⁻¹; ¹H NMR δ 3.91 (6H, s, 2x CH₃), 6.80 - 7.00 (4H, m, Ar), 7.13 - 7.30 (2H, m, Ar), 7.54 (2H, d, J = 7.60 Hz, Ar); ¹³C NMR δ 56.25, 110.92, 121.70, 128.14, 138.68, 156.99; EIMS: m/z 278 (M⁺, 82%), 139 (SC₆H₄-OMe, 100%); HRMS: Calcd. for C₁₄H₁₄O₂S₂ 278.0435; Found 278.0440; 7e: 35.5% yield, mp 113˚C - 115˚C, IR ν 1247 (-SO₂), 1095, 1153 (S-O) cm⁻¹; ¹H NMR δ 3.82 (3H, s, CH₃), 3.86 (3H, s, CH₃), 6.85 (4H, t, J = 7.80, Ar), 7.25 (2H, d, J = 7.80 Hz, Ar), 7.49 (2H, d, J = 7.80 Hz, Ar); ¹³C NMR δ 55.82, 56.06, 114.32, 114.72, 115.26, 115.46, 130.14, 130.57, 138.15, 138.60, 162.57, 163.92; EIMS: m/z 310 (M⁺, 44%), 177 (SO₂C₆H₄-OMe, 22%), 155 (SOC₆H₄OMe, 62%), 139 (SC₆H₄-OMe, 100%); HRMS: Calcd for C₁₄H₁₄O₄S₂ 310.0333; Found 310.0344; 6f: 31% yield, oil, IR ν 1249, 1085,1152 cm⁻¹; ¹H NMR δ 3.79 (6H, s, 2x CH₃), 6.78 (2H, d, J = 7.80 Hz, Ar), 7.05 - 7.15 (4H, m, Ar), 7.23 (2H, d, J = 7.8 Hz, Ar); ¹³C NMR δ 55.84, 111.09, 114.16, 119.47, 137.02, 161.00; EIMS: m/z 278 (M⁺, 41%), 139 (M-SC₆H₄-OMe, 100%); HRMS: Calcd for C₁₄H₁₄O₂S₂ 278.0435; Found 278.0439; 7f: 45% yield, oil, IR ν 1286(-SO₂), 1139,1183 (S-O) cm⁻¹; ¹H NMR δ 3.70 (6H, s, 2x OCH₃), 6.80 - 7.38 (8H, m, Ar); ¹³C NMR δ 55.85, 111.20, 112.19, 114.09, 119.36, 120.72, 121.63, 130.10, 130.62, 133.50, 139.64, 160.06, 161.00; EIMS: m/z 310 (M⁺, 73%), 155 (SC₆H₄-OMe, 100%); HRMS: Calcd for C₁₄H₁₄O₄S₂ 310.0333; Found 310.0340; 6g: 34% yield, oil, IR ν 1030, 1171 cm⁻¹; ¹H NMR δ 3.81 (6H, s, 2x OCH₃), 6.84 (4H, d, J = 7.75 Hz, AB system Ar), 7.41 (4H, d, J = 7.75 Hz, AB system Ar); ¹³C NMR δ 55.90, 114.50, 128.54, 130.21, 157.10; EIMS: m/z 278 (M⁺, 95%), 139 (SC₆H₄-OMe, 100%); HRMS: Calcd. for C₁₄H₁₄O₂S₂ 278.0435; Found 278.0435; 7g: 31% yield, oil, IR ν 1288 (-SO₂), 1170, 1032 (S-O) cm⁻¹; ¹H NMR δ 3.75 (6H, s, 2x OCH₃), 7.50 (4H, d, J = 7.80 Hz, AB system Ar), 7.65 (4H, d, J = 7.80 Hz, AB system Ar); ¹³C NMR δ 55.88, 114.64, 115.28, 124.90, 129.30, 130.37, 130.77, 130.81, 157.45, 165.72; EIMS: m/z 310 (M⁺, 60%), 155 (SC₆H₄-OMe, 100%); HRMS: Calcd. for C₁₄H₁₄O₄S₂ 310.0333; Found 310.0338; 6h: 41% yield, 88˚C - 90˚C, IR ν 1113, 1446 cm⁻¹; ¹H NMR δ 7.12 - 7.30 (4H, m, Ar), 7.38 (2H, d, J = 7.80 Hz, Ar), 7.56 (2H, d, J = 7.80 Hz, Ar), 7.18 - 7.47 (4H, m, Ar), 7.48 - 7.70 (4H, m, Ar); ¹³C NMR δ 127.17, 127.47, 127.73, 129.61, 131.80, 134.31; EIMS: m/z 286 (Cl³⁵) (M⁺, 90%), 143 (SC₆H₄-Cl³⁵, 85%); HRMS: Calcd. for C₁₂H₈ S₂Cl₂ 285.9444 (Cl³⁵); Found 285.9455; 7h: 37% yield, mp 157˚C - 160˚C, IR ν 1329(-SO₂), 1147 (S-O) cm⁻¹; ¹H NMR δ 7.08 - 7.30 (4H, m, Ar), 7.38 (2H, d, J = 7.80 Hz, Ar), 7.56 (2H, d, J = 7.80 Hz, Ar); ¹³C NMR δ 126.54, 127.60, 130.19, 130.91, 132.36, 133.10, 134.76, 139.72, 140.11, 140.21; EIMS: m/z 318 (Cl³⁵) (M⁺, 30%), 175 (SO₂C₆H₄-Cl³⁵, 35%), 143 (SC₆H₄-Cl³⁵, 68%), 159 (SOC₆H₄-Cl³⁵, 71%), 108 (100%); HRMS: Calcd. for C₁₂H₈O₂ S₂Cl₂ 317.9343 (Cl³⁵); Found 317.9330; 6i: 31% yield, oil, IR ν 1127, 1456 cm⁻¹; ¹H NMR δ 7.17 - 7.30 (4H, m, Ar), 7.30 - 7.42 (2H, m, Ar), 7.49 (2H, s, Ar); ¹³C NMR δ 125.33, 125.59, 129.15, 13.58, 133.50, 137.38; EIMS: m/z 286 (Cl³⁵) (M⁺, 100%), 143 (SC₆H₄-Cl³⁵, 78%); HRMS: Calcd. for C₁₂H₈ S₂Cl₂ 285.9444 (Cl³⁵); Found 285.9455; 7i: 32% yield, oil, IR ν 1294 (-SO₂), 1117 (S-O) cm⁻¹; ¹H NMR δ 7.16 - 7.65 (8H, m, Ar); ¹³C NMR δ 125.58, 126.39, 127.63, 127.61, 129.10, 130.71, 131.20, 133.61, 133.82, 133.93, 135.36, 140.10; EIMS: m/z 318 (Cl³⁵) (M⁺, 41%), 159 (SO₂C₆H₄-Cl³⁵, 100%), 143 (SC₆H₄-Cl³⁵, 64%); HRMS: Calcd. for C₁₂H₈O₂ S₂Cl₂ 317.9343 (Cl³⁵); Found 317.9343; 6j: 53% yield, mp 70 - 72˚C, IR ν 1151 cm⁻¹; ¹H NMR δ 7.28 (4H, d, J = 7.80 Hz, AB system Ar), 7.41 (4H, d, J = 7.80 Hz, AB system Ar), 7.28 (2H, t, J = 7.80 Hz, Ar), 7.48 - 7.64 (4H, m, Ar); ¹³C NMR δ 129.15, 129.79, 133.51, 135.04; EIMS: m/z 286 (Cl³⁵) (M⁺, 86%), 143 (M-SC₆H₄-Cl³⁵, 100%); HRMS: Calcd. for C₁₂H₈ S₂Cl₂ 285.9444 (Cl³⁵); Found 285.9451; 7j: 26%, mp 131˚C - 135˚C, IR ν 1322 (-SO₂), 1107 (S-O) cm⁻¹; ¹H NMR δ 7.25 - 7.39 (4H, m, Ar), 7.42 (2H, d, J = 7.80 Hz, AB system Ar), 7.52 (2H, d, J = 7.80 Hz, Ar); ¹³C NMR δ 129.25, 129.60, 130.22, 130.71, 137.98, 138.85, 140.87, 141.65; EIMS: m/z 318 (Cl³⁵) (M⁺, 50%), 175(M-SC₆H₄-Cl³⁵, 85%), 143 (SC₆H₄-Cl³⁵, 83%), 111 (100%); HRMS: Calcd. for C₁₂H₈O₂ S₂Cl₂ 317.9343 (Cl³⁵); Found 317.9345; 6k: 43% yield, 91˚C - 93˚C, IR ν 1357, 1151 cm⁻¹; ¹H NMR δ 7.09 (2H, t, J = 7.80 Hz, Ar), 7.28 (2H, t, J = 7.80 Hz, Ar), 7.54 (4H, d, J = 7.80 Hz, Ar); ¹³C NMR δ 121.00, 126.88, 127.83, 128.08, 132.80, 136.06; EIMS: m/z 378 (Br⁸¹) (M⁺, 28%), 374 (Br⁷⁹) (M⁺, 25%), 189 (SC₆H₄-Br⁸¹, 18%), 187 (SC₆H₄-Br⁷⁹, 17%); HRMS: Calcd. for C₁₂H₈ S₂Br₂ 373.8434 (Br⁷⁹); Found 373.8445; 7k: 35% yield, mp 138˚C - 140˚C, IR ν 1326 (-SO₂), 1151 (S-O) cm⁻¹; ¹H NMR δ 7.24 - 7.47 (4H, m, Ar), 7.57 (2H, t, J = 7.80 Hz, Ar), 7.71 (1H, d, J = 7.80 Hz, Ar), 7.80 (1H, d, J = 7.80 Hz, Ar); ¹³C NMR δ 121.07, 121.25, 127.18, 128.20, 128.95, 131.07, 131.22, 132.93, 133.52, 134.56, 135.95, 141.82; EIMS: m/z 410 (Br⁸¹) (M⁺, 17%), 406 (Br⁷⁹) (M⁺, 15%), 221 (SO₂C₆H₄-Br⁸¹, 18%), 219 (SO₂C₆H₄-Br⁷⁹, 19%), 205 (SOC₆H₄Br⁸¹, 64%), 203 (SOC₆H₄Br⁷⁹, 61%), 108 (100%); HRMS: Calcd. for C₁₂H₈ O₂S₂Br₂ 405.8332 (Br⁷⁹); Found 405.8344; 6l: 57% yield, 87˚C - 88˚C, IR ν 1149 cm⁻¹; ¹H NMR δ 7.34 (4H, d, J = 7.80 Hz, AB system Ar), 7.43 (4H, d, J = 7.80 Hz, AB system Ar); ¹³C NMR δ 121.43, 129.28, 132.09, 135.62; EIMS: m/z 378 (Br⁸¹) (M⁺, 52%), 374 (Br⁷⁹) (M⁺, 52%), 189 (SC₆H₄-Br⁸¹, 60%), 187 (SC₆H₄-Br⁷⁹, 57%); HRMS: Calcd for C₁₂H₈ S₂Br₂ 373.8434 (Br⁷⁹); Found 373.8439; 7l: 28% yield, mp 105˚C - 107˚C, IR ν 1323 (-SO₂), 1142 (S-O) cm⁻¹; ¹H NMR δ 7.24 (2H, d, J = 7.80 Hz, AB system, Ar), 7.44 (2H, d, J = 7.80 Hz, AB system, Ar), 7.52 (2H, d, J = 7.80 Hz, AB system Ar), 7.60 (2H, d, J = 7.80 Hz, AB system Ar); ¹³C NMR δ 126.49, 126.88, 128.82, 129.06, 132.10, 132.78, 137.70, 141.75; EIMS: m/z 410 (Br⁸¹) (M⁺, 38%), 406 (Br⁷⁹) (M⁺, 33%), 221 (SC₆H₄-Br⁸¹, 52%), 199 (SC₆H₄-Br⁷⁹, 51%), 108 (100%); HRMS: Calcd for C₁₂H₈ O₂S₂Br₂ 405.8332 (Br⁷⁹); Found 405.8321; 6m: 46% yield,; IR ν 1149 cm⁻¹; ¹H NMR δ 7.54 (2H, d, J = 7.80 Hz, H-3), 7.60 (4H, m, H-6, H-7), 7.86 (2H, s, H-1), 7.98 (4H, m, H-4, H-8), 8.06 (2H, d, J = 7.80 Hz, H-4); ¹³C NMR δ 126.58, 126.90, 128.09, 128.55, 129.30, 129.63, 129.68, 129.76; EIMS: m/z 318 (M⁺, 48%), 159 (SC₁₀H₇, 59%), 115 (100%); HRMS: Calcd. for C₂₀H₁₄S₂ 318.0537; Found 318.0536; 7m: 40% yield, IR ν 1323(-SO₂), 1124 (S-O) cm⁻¹; ¹H NMR δ 7.24 (1H, d, J = 7.80 Hz, H-3’), 7.60 (5H, m, H-1’, H-6, H-6’, H-7, H-7’), 7.85 (5H, m, H-4’, H-5, H-5’, H-8, H-8’), 8.15 (1H, d, J = 7.80 Hz, H-3), 8.38 (1H, d, J = 7.80 Hz, H-4), 8.85 (1H, s, H-1); ¹³C NMR δ 126.05, 126.60, 126.88, 127.06, 128.10, 128.71, 129.30, 129.35, 129.41, 129.69, 129.80, 129.90, 130.2; EIMS: m/z 350 (M⁺, 10%), 159 (SC₁₀H₇, 51%), 115 (100%); HRMS: Calcd for C₂₀H₁₄O₂S₂ 350.0435; Found 350.0436.

3.3. Microbiological Methods

Microorganisms: E. coli (JM 109) and S. aureus (SH1000) control strains were used to test antimicrobial activity. The series of compounds 6 and 7 were dissolved in DMSO to produce various concentrations which were refrigerated.

Inoculum: E. coli and S. aureus microorganisms were grown on Muller-Hinton Broth (MHB) for 24 hours at 37˚C. Starting innocula were prepared by diluting overnight cultures in fresh MHB to a culture density of 1 × 10⁶ cfu/ml. Minimum Inhibitory Concentrations (MIC) were determined according to the BSAC broth microdilution MIC methodology described by Andrews (2001).

4. Conclusion

We have shown how diarylthiosulfonates and diaryldisulfides, garlic-like organosulphur compounds can be conveniently synthesised in two steps in good yields. These 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. SAR analysis of the results has shown the presence of chloro- or bromo-groups in the 2-position of the 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.

Acknowledgements

The authors thank Smith, T.J., Biomedical Research Centre (BMRC), Sheffield Hallam University, for use of microbiological facilities. We thank David Kelly (University of Sheffield) for generous donations of the H. pylori strains used in these studies. We also thank Kevin Osborne and Daniel Kinsman for recording the large volume of NMR and MS data respectively. We express our thanks to Simon Thorpe, Chemistry Department, and the University of Sheffield for the measurement of accurate masses.

Conflicts of Interest

The authors declare no conflicts of interest regarding the publication of this paper.

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