Abstract
Six series of benzoheterocyclic sulfoxide derivatives were designed and synthesised as Pseudomonas aeruginosa (P. aeruginosa) quorum sensing inhibitors in this paper. We experimentally demonstrated that 6b significantly inhibited the formation of P. aeruginosa PAO1 biofilm without affecting the growth. Further mechanistic studies showed that 6b affected the luminescence of quorum sensing reported strain PAO1-lasB-gfp and the production of P. aeruginosa PAO1 elastase virulence factor which was regulated by las system. These experimental results indicate that 6b acts as a quorum sensing inhibitor mainly through the las system. Furthermore, silico molecular docking studies demonstrated that 6b and the P. aeruginosa quorum sensing receptor LasR were molecularly bound via hydrogen bonding interactions. Preliminary structure-activity relationship and docking studies illustrated that 6b shows great promise as anti-biofilm compounds for further studies in order to solve the problem of microbial resistance in future.
Introduction
With the excessive and indiscriminate abuse of antibiotics, the emergence of multiple drug resistant (MDR) bacterial strains has been bred. The abuse of antibiotics directly results in the death of 16 million human beings annually due to infections.Citation1–2 It is necessary to emphasise that approximately 65% of infectious diseases are related to the proliferation of bacterial communities by forming biofilm.Citation3 The bacteria in biofilms show more significant resistance to antibiotics and to the host immune responses than their plankton counterparts.Citation4 In modern clinical microbiology, formation of biofilm is generally considered a pathogenicity characteristic of chronic infection.Citation5–6 Biofilm formation is regulated by a phenomenon commonly known as quorum sensing (QS), in which bacteria release small signalling molecules, produce virulence factors, and form biofilms in a cell density-based manner.Citation2,Citation7,Citation8 Therefore, inhibiting the formation of bacterial biofilms by interfering with bacterial quorum sensing is an efficient strategy to develop alternative therapies for the control and prevention of bacterial infections.
Thus far, the QS network of P. aeruginosa is one of the most classical networks of quorum sensing and has been widely used in clinical studies.Citation9 The QS network is mainly composed of three subsystems, las, rhl and pqs, which are interrelated and mutually regulated, leading to bacterial infection in the host ().Citation10 The las and rhl systems rely on two different N-acyl-L-homoserine lactone (AHL) type signal molecules, N-3-oxo-dodecanoyl homoserine lactone (3-oxo-C12-HSL, OdDHL) and N-butanoyl homoserine lactone (C4-HSL, BHL). The third system, pqs, employs 2-alkyl-4-quinolones (3, 2-hepyl-4-hydroxyquinoline (HHQ) or 2-heptyl-3- hydroxy-4-quinolone (PQS)) as the quorum sensing signal molecule (QSSMs).Citation11–13 QS signals control about 6% of the P. aeruginosa genome through its complex but finely tuned mechanism.Citation14
Figure 1. The main three QS signalling networks in P. aeruginosa.
By analysing the quorum sensing signal system of P. aeruginosa, the receptor proteins LasR, RhlR, and PqsR, that specifically bind to signal molecules have been identified, which are closely related to the pathogenicity of P. aeruginosa.Citation15–18 Among these, the LasR transcription activator protein controls the production and expression of numerous P. aeruginosa exoproducts, which is a hot topic in this field.Citation19 Reports show that after the quorum sensing system of P. aeruginosa is blocked, the secretion of virulence factors and biofilm formation ability of P. aeruginosa are significantly reduced.Citation10,Citation20,Citation21 Therefore, quorum sensing inhibitors (QSIs) are expected to become a novel drug against P. aeruginosa infections.
Studies have revealed that crude garlic extract had significant inhibitory effect on P. aeruginosa QS. The sulfur-containing compound ajoene was identified as QSIs in garlic extract by further bioassays.Citation22 Givskov and Yang et al.Citation23 first reported the synthesis of compounds with disulphide bond framework as QSIs in addition to the natural ajoene, which showed excellent quorum-sensing activity against P. aeruginosa and could reduce the production of QS-regulated virulence factors. They confirmed that disulphide derivatives with benzothiazole skeletons were pivotal functional groups for quorum sensing activity, but monosubstituted thioethers with benzothiazole skeletons did not show QS activity. It is well known that sulfoxides have a wide range of biological activities, as well as benzoheterocyclic skeletons,Citation24–25 and both allicin and ajoene have sulfoxide structures. We hypothesised that benzoheterocyclic monosulfide may have quorum sensing activity after being oxidised to sulfoxides. We attempted to replace the disulphide bond by sulfoxide in hope of finding new lead compound to inhibit the quorum sensing of P. aeruginosa (). Hence, we designed and synthesised a series of benzoheterocyclic sulfoxide compounds and evaluated the quorum sensing bioactivity against P. aeruginosa.
Figure 2. The design of the title compounds.
In this study, we report a series of benzoheterocyclic sulfoxide derivatives against biofilm inhibitory activity of P. aeruginosa. (A part of structures have been reported without bioactive resultsCitation26). According to the expression of green fluorescent protein (GFP) reporter strains (PAO1-lasB-gfp, PAO1-rhlA-gfp, PAO1-pqsA-gfp), 6b showed excellent biofilm inhibitory activities and significantly inhibited the expression of PAO1-lasB-gfp strains. Furthermore, we analysed the experimental results of P. aeruginosa PAO1 virulence factors including elastase, pyocyanin and rhamnolipid, as well as the silico molecular docking results with LasR receptor protein in quorum sensing. In summary, it was described that 6b could reduce biofilm formation and virulence factor production by hydrogen bond interaction antagonistically binding to P. aeruginosa QS receptor protein.
Results and discussion
Chemistry
To verify the above hypothesis, we referred to 2-mercaptobenzothiazole disulphide with the excellent P. aeruginosa quorum-sensing activity to design the new candidate compounds.Citation23 We retained the core part of the benzothiazole and replaced the disulphide bond with sulfoxide to synthesise a series of benzoheterocyclic sulfoxide derivatives. The synthetic routes of titled compounds were shown in Scheme 1. The key intermediates 3 (the different heterocyclic thioethers) were synthesised from benzyl bromides 1 and heterocyclic thiols 2 in the presence of triethylamine in acetonitrile. Benzyl bromide 1 was added dropwise into the mixture at room temperature.Citation26 Then intermediates 3 were oxidised by meta-chloroperoxybenzoic acid in dichloromethane to obtain the different types of compounds. We first synthesised 4a-4l (containing benzo thiazole), 5a-5l (containing benzoxazole), the preliminary screening of P. aeruginosa biofilm activity showed that compounds with benzoxazole have good inhibitory effects. Furthermore, we considered chloro-substitution as an important halogen group for QS activity, a series of 6a-6l (containing 5-chlorobenzoxazole) and 7a-7f (containing 6-chlorobenzooxazole) were synthesised. To investigate whether the chloro-substitution on the benzoxazole ring was the key active site, we synthesised the series 8a-8f (containing 5-methylbenzoxazole) and 9a-9f (containing 5-methoxybenzoxazole).
Scheme 1. The synthetic route of the title compounds.
Biological evaluation
Evaluation of inhibition of P. aeruginosa biofilm and SAR studies
We tested the inhibitory activity of 4, 5, and 6 against P. aeruginosa PAO1 biofilm, benzimidazole used as positive control.Citation27–28 As shown in , the 4a-4l series compounds showed little, or no biofilm inhibitory effect compared to other groups. We also tested 5-chloro-substituted benzothiazole derivatives during the experiments, however, they showed little biofilm inhibitory activity (insignificant and not shown in results). The anti-biofilm results showed that 5b and 6b were more active than 4b, while 5d and 6d were more active than 4d. Moreover, 5e and 6e were more active than 4e. All of above indicates that compounds containing benzoheterocyclic oxazole had better inhibitory activities of P. aeruginos PAO1 biofilm than benzoheterocyclic thiazole, and the benzoxazole ring might be the active functional group that plays a key role in biofilm activity. When the heterocyclic rings were benzoxazole structure, 5c, 5d, 6c, and 6d showed relatively weaker activity than 5b and 6b. It indicated that the Rʺ might lead to better inhibitory activity when it was substituted at para-position. By analysing compounds with various substituents on the Rʺ phenyl ring, including those with electron-withdrawing groups (5e, 5f), or with electronic donating groups (6a, 6 g), they showed only a slight impact on the anti-biofilm activity. Moreover, compound 6b with chloro-substitution had the best anti-biofilm effect, with an inhibition rate of 46.13 ± 0.79%. The inhibition activity of biofilm was better than fluoro- (6e, 38.64 ± 0.32%) and bromo- (6f, 14.60 ± 1.23%) substitutions at the para-position. We also tested the biofilm inhibitory activity of all compounds against Pseudomonas aeruginosa at 100 μM, and the SAR was basically consistent with the concentration of 50 μM.
Table 1. Biofilm inhibition rates of derivatives against the P. aeruginosa PAO1.
Next, to further explore the effect of Rʺ as substitution on the anti-biofilm activity, compounds 7, 8, and 9 were synthesised. Antibiofilm activity was enhanced when chloro-substitution on aromatic ring involved. When 5- or 6-position of benzoxazole ring was substituted by chloro, 6b and 7b showed good inhibitory activities (). Especially, the inhibition rate of 7b was 43.64 ± 2.49%, which meant that there was no difference between the chloro-substitutions at the 5- and 6-position on benzoxazole ring. However, when we changed the chloro-substitution on 5-position of benzoxazole ring to methyl or methoxy group, the inhibition rate of 8b and 9b were 19.93 ± 0.57% and 29.44 ± 0.39%. 8 and 9 with various substitutions had no particularly excellent inhibition effects except for 9b and 9f. A comparison with the serials data of 6 indicated that both the benzoxazole ring and the chloro-substitution were the key active functional group. Preliminary structure-activity relationships indicated that the benzoxazole heterocyclic ring was critical for optimal activity. In addition, the para-position on aromatic ring of benzyl group was an excellent choice for inhibitors design.
Table 2. Biofilm inhibition rates of derivatives against the P. aeruginosa PAO1.
Previous reports have confirmed that disulphide bonds are bioactive, while, without the disulphide bonds in garlic analogues will render the QSI ineffective.Citation21 The above experimental results verified our speculation that although the monosubstituted thioethers of the benzothiazole skeleton have no QS activity, they can inhibit the formation of P. aeruginosa PAO1 biofilm after oxidation to sulfoxide. Therefore, the sulfoxide bond is also an important framework for inhibitory biofilm and the sulfoxide derivatives also show quorum sensing activity, which has never been reported before. The structure-activity relationships indicated that the benzoxazole heterocycle and the sulfoxide bonds were the keys to the optimal activity, and the para-position of the benzene ring was the best choice for designing inhibitors.
Effect of 6b on biofilm growth and formation
Based on the above results, compounds 5b, 5h, 6b, 7b and 9b with better biofilm inhibitory activities were selected for further study (). All compounds were cultured for 24 h and the OD value at 600 nm was evaluated before biofilm experiment. The normal growth of P. aeruginosa PAO1 was unaffected at concentration of 50 μM. 6b was chosen as an example and tested at concentrations of 50 μM, 25 μM, 12.5 μM, 5 μM and 2.5 μM. An equal amount of dimethyl sulfoxide (DMSO) was added to the control group. The effect of 6b on P. aeruginosa PAO1 growth was assessed hourly by monitoring OD600 culture (). As a result, 6b had no effect on the growth of P. aeruginosa PAO1. Cytotoxicity of 6b was also evaluated (see in Supporting information Figure S-1). In addition, it was observed that 6b impeded biofilm formation in the images taken by confocal laser scanning microscopy (CLSM) (). The density of the biofilm formed by 6b at 50 μM was lower than that of the control group, and the biofilm formation was also inhibited at 25 μM.
Figure 3. Effects of 6b on P. aeruginosa PAO1 biofilm growth and formation. (A) Biofilm inhibition at 50 μM of 5b, 5h, 6d, 7b, 9b for 24 h in microtiter plate. (B) Growth at different concentrations of 6b (50, 25, 12.5, 5, 2.5 μM) for 16 h. (C) CLSM images of biofilm formed for 24 h with 50 μM and 25 μM of 6b, and benzimidazole used as positive control (An equal amount of dimethyl sulfoxide solvent was set as control group). Data represents the average of three-independent determinations of triplicate samples.
Effect of 6b on QS system report strains
According to the regulation process of the QS system of P. aeruginosa, Givskov et al.Citation21,Citation29–31 fused their respective promoters in the las, rhl and pqs pathways with unstable green fluorescent protein (GFP) to construct three reporter strains PAO1-lasB-gfp, PAO1-rhlA-gfp and PAO1-pqsA-gfp, and a screening system for detecting small molecule QSI based on the growth of bacteria was established. LasB encodes the virulence factor elastase, which has been shown to be transcriptionally controlled by LasR.Citation32 In rhl system, rhlA is the first gene of rhlA and rhlB operons, encoding rhamnotransferase required for the synthesis of rhamnolipid; while pqsA is the operation required by the first gene of pqsABCDE to produce pqs signal, and the pqs system can enhance the pyocyanin expression of P. aeruginosa.Citation33–34
To further verify the QS activity of 5b, 5h, 6b, 7b and 9b, the PAO1-lasB-gfp, PAO1-rhlA-gfp and PAO1-pqsA-gfp strains were introduced for screening. As shown in , 6b and 7b could significantly inhibit the fluorescence expression of PAO1-lasB-gfp strain at 20 μM. 5b, 5h, 6b, 7b, 9b also had a little inhibitory effect on PAO1-rhlA-gfp and PAO1-pqsA-gfp strains, but the effect was not as significant as PAO1-lasB-gfp. Furthermore, 6b was selected as the key research object. Under the premise of function-permitting growth of the reporter strain (), 6b showed a significant dose-dependence on PAO1-lasB-gfp strain at different concentrations of 20 μM, 10 μM, 5 μM, 2.5 μM and 1.25 μM (). From the obtained dose-response curve, the IC50 value was estimated to be 2.08 ± 0.25 μM ().
Figure 4. The effects of 5b, 5h, 6d, 7b, 9b on QS system report strain (A) PAO1-lasB-gfp, (B) PAO1-rhlA-gfp, and (C) PAO1-pqsA-gfp. The experiments were done triplicate.
Figure 5. (A) Dose-dependent inhibition curves of 6b incubated with the QS monitors PAO1-lasB-gfp. (B) Growth at different concentrations of 6b (20, 10, 5, 2.5, 1.25 μM). (C) IC50 values calculations were based on three biological replicates and performed by nonlinear fitting, using Graphpad Prism 6 software. The IC50 values of 6b was 2.08 ± 0.25 μM for PAO1-lasB-gfp. The experiments were also done in triplicate.
Effect of 6b on virulence factors
As it is mentioned above, the QS system of P. aeruginosa controls the production of various virulence factors and biofilm formation. To further verify the active mechanism of 6b inhibiting biofilm to las system, we measured the effects of 6b on the production of three virulence factors, elastase, pyocyanin, and rhamnolipid. The results were showed that 6b significantly reduced the production of elastase at 50 μM, 25 μM and 12.5 μM in a concentration-dependent manner (). 6b only inhibited the production of pyocyanin at high concentrations, while 6b has almost no effect on the production of rhamnolipid. Las, rhl and pqs systems control elastase, rhamnolipid and pyocyanin respectively. These results suggested that the presence of 6b inhibited the QS lasB gene and reduced the production of the virulence factor elastase. It was further demonstrated that 6b inhibited biofilm information through the las pathway.
Figure 6. Effects of 6b on the production of (A) elastase (B) pyocyanin (C) rhamnolipid at different concentrations (50, 25, 12.5 and 0 μM). Error bars are means ± SDs. * = p < 0.05 versus the control, ** = p < 0.01 versus the control, *** = p < 0.001 versus the control.
Molecular docking study
The las QS system uses OdDHL as autoinducer, which is synthesised by the lasI protein. Upon binding to the autoinducer OdDHL, the receptor protein LasR gets activated and forms a complex. The LasR-OdDHL complex bind to conserve las-rhl cassettes located in target genes promoters, thereby activating their transcriptional expression.Citation10 In order to further explore the molecular mechanism of 6b regulating QS system, silico molecular docking was performed to predict the binding model of 6b with its homologous signal receptor protein LasR. The lowest binding energy for the docked conformations was chosen from 30 docking conformations as modes for the corresponding compound. As shown in , 6b was well positioned by the active pocket of LasR, the diaromatic character occupying several cavities in the LasR protein. Specifically, chlorine and benzene part of benzoxazole 6b formed hydrogen bond and π-H interactions with the residues Leu 110 and Trp 88, while oxygen and nitrogen in OdDHL were observed to form hydrogen bond interactions with Trp 60, Trp 88, Asp 73, Tyr 56 and Ser 129, respectively. It indicated the importance of substituted benzoxazole with chlorine for P. aeruginosa biofilm activity. Besides, the benzene substituted with chlorine in 6b occupied the same cavities in the LasR protein as the long alkyl chain in OdDHL. Docking models revealed that 6b interacted with the amino acid residues of LasR, which explained the observed SARs.
Figure 7. Predicted binding model of compounds (OdDHL, 6b) and LasR (PDB code: 2uv0). (A) The pocket surface view of interactions between OdDHL, 6b with receptor protein lasR; (B) Superposition of native docking of OdDHL and OdDHL in LasR X-ray crystal structure, showing as OdDHL in green, OdDHL in LasR X-ray crystal structure in orange; (C) Details of 6b binding. 6b was shown in blue. The Residues involved in interactions with compounds are depicted as sticks in black and named in red. The hydrogen bonds are shown as aqua dashed lines.
Conclusion
To solve the drug resistance problem of P. aeruginosa mediated by biofilm, medicinal chemists have proposed an efficient strategy that could reduce the production of pathogen extracellular virulence factors without killing the pathogen, which can also alleviate the drug resistance problem a certain extent. According to the excellent QSIs performance of sulfur-containing compounds, we designed and synthesised a series of benzoheterocyclic sulfoxides and evaluated their ability to inhibit QS in vitro. The results showed that 6b significantly inhibited the biofilm formation of P. aeruginosa, with an inhibition rate of 46.13 ± 0.72%. The results of mechanism study confirmed that 6b could effectively inhibit las system in a dose-dependent manner, and the IC50 value of inhibitory concentration against PAO1-lasB-gfp strain was 2.08 ± 0.25 μM. In addition, 6b attenuated the production of elastase, a virulence factor of P. aeruginosa PAO1. Molecular docking analysis showed that 6b and LasR receptor protein inhibited the expression of las system gene by forming hydrogen bonds, thus, inhibiting the production of virulence factors and P. aeruginosa biofilm production. Our study provides a new approach for designing quorum sensing inhibitors as new antimicrobial resistance agents. Sulfoxide derivatives have been proposed as a new QSIs model, which will provide a reference for the medical community to solve the global drug resistance problem. In future studies, we will optimise more efficient and practical QSIs based on 6b to develop new anti-infection drugs.
Experimental section
Chemistry
Materials and methods
The solvents and reagents used in this experiment were obtained commercially without further purification. All compounds were structurally identified by 1H NMR and 13 C NMR and high-resolution mass spectrometry (ESI-HRMS). Unless otherwise stated, 1H and 13C NMR spectra were recorded on Bruker Avance III 400 at 600 and 150 MHz or 400 and 100 MHz spectrometer. Chemical shifts are recorded as δ in units of parts per million (ppm), while tetramethylsilane (TMS) was used as an internal standard. Compounds were dissolved in CDCl3. High resolution mass spectra were obtained on an SCIEX series X500B QTOF mass spectrometer. Thin-layer chromatography (TLC) was performed using Huanghai GF254 Silica gel plates. Column chromatography was performed using silica gel (200–300 mesh, Beijing, China) with a linear solvent gradient.
General synthesis of compounds 4a–9f
Benzyl bromide 1 (1.2 mmol) was added dropwise to a solution of heterocyclic thiol 2 (1.0 mmol) and Et3N (1.5 mmol) in MeCN (10 mL) at room temperature. The reaction solution was quenched with 6 M HCl aqueous solution, and then extracted with EtOAc (3 × 20 mL). The combined organic phase was dried with anhydrous Na2SO4, concentrated in vacuum and purified by column chromatography (PE/EA = 50/1 ∼ 30/1 ratio) to obtain intermediates thioether 3. Then intermediates 3 (1.0 mmol) and meta-chloroperoxybenzoic acid (1.0 mmol) are added in dichloromethane to react and extracted with saturated sodium thiosulphate solution, dried and purified by column chromatography to obtain the different title compounds 4a-4l, 5a-5l, 6a-6l, 7a-7f, 8a-8f, 9a-9f.
2-(benzylsulfinyl) benzo[d]thiazole (4a) white solid, yield 85%. 1H NMR (400 MHz, Chloroform-d) δ 8.13 − 8.09 (m, 1H), 7.94 (dd, J = 8.0, 1.1 Hz, 1H), 7.61 − 7.55 (m, 1H), 7.49 (ddd, J = 8.4, 7.2, 1.1 Hz, 1H), 7.29 (ddd, J = 9.5, 6.2, 4.3 Hz, 3H), 7.19 − 7.15 (m, 2H), 4.59 − 4.28 (m, 2H). 13C NMR (101 MHz, CDCl3) δ 176.86, 153.69, 135.99, 130.45, 128.74, 128.69, 128.30, 126.92, 126.15, 123.89, 122.24, 62.77. ESI-HRMS: calcd for C14H11NOS2 [M + H]+, 274.0360; found, 274.0361.
2-((4-chlorobenzyl) sulfinyl) benzo[d]thiazole (4b) white solid, yield 81%. 1H NMR (400 MHz, Chloroform-d) δ 8.13 − 8.08 (m, 1H), 7.97 − 7.94 (m, 1H), 7.62 − 7.55 (m, 1H), 7.53 − 7.47 (m, 1H), 7.23 (d, J = 8.4 Hz, 2H), 7.11 − 7.04 (m, 2H), 4.62 − 4.11 (m, 2H). 13C NMR (101 MHz, CDCl3) δ 176.30, 153.57, 135.86, 134.88, 131.61, 128.75, 126.91, 126.52, 126.15, 123.79, 122.20, 61.47. ESI-HRMS: calcd for C14H10ClNOS2 [M + H]+, 307.9971; found, 307.9969.
2-((3-chlorobenzyl) sulfinyl) benzo[d]thiazole (4c) white solid, yield 89%. 1H NMR (600 MHz, Chloroform-d) δ 8.11 (dt, J = 8.3, 0.9 Hz, 1H), 7.96 (ddd, J = 8.1, 1.2, 0.6 Hz, 1H), 7.59 (ddd, J = 8.3, 7.2, 1.2 Hz, 1H), 7.50 (ddd, J = 8.3, 7.2, 1.2 Hz, 1H), 7.28 (ddd, J = 8.1, 2.1, 1.1 Hz, 1H), 7.22 − 7.15 (m, 2H), 7.06 (dt, J = 7.6, 1.3 Hz, 1H), 4.39 (dd, J = 107.1, 13.2 Hz, 2H). 13C NMR (151 MHz, CDCl3) δ 176.48, 153.69, 135.95, 134.54, 130.45, 130.29, 129.84, 128.91, 128.56, 127.02, 126.27, 123.95, 122.26, 62.10. ESI-HRMS: calcd for C14H10ClNOS2 [M + H]+, 307.9971; found, 307.9973.
2-((2-chlorobenzyl) sulfinyl) benzo[d]thiazole (4d) white solid, yield 90%. 1H NMR (600 MHz, Chloroform-d) δ 8.23 − 7.90 (m, 2H), 7.58 (ddd, J = 8.3, 7.2, 1.2 Hz, 1H), 7.50 (ddd, J = 8.2, 7.2, 1.2 Hz, 1H), 7.41 (dd, J = 8.0, 1.2 Hz, 1H), 7.32 − 7.26 (m, 2H), 7.22 (td, J = 7.4, 1.3 Hz, 1H), 4.62 (dd, J = 175.6, 12.9 Hz, 2H). 13C NMR (151 MHz, CDCl3) δ 176.60, 153.70, 136.01, 135.15, 132.91, 130.35, 129.81, 127.07, 127.01, 126.97, 126.31, 124.13, 122.26, 61.11. ESI-HRMS: calcd for C14H10ClNOS2 [M + H]+, 307.9971; found, 307.9976.
2-((4-fluorobenzyl) sulfinyl) benzo[d]thiazole (4e) white solid, yield 86%. 1H NMR (600 MHz, Chloroform-d) δ 8.10 (dt, J = 8.3, 0.9 Hz, 1H), 7.94 (dt, J = 8.0, 0.9 Hz, 1H), 7.58 (ddd, J = 8.3, 7.2, 1.2 Hz, 1H), 7.49 (ddd, J = 8.3, 7.2, 1.2 Hz, 1H), 7.14 − 7.10 (m, 2H), 6.97 − 6.92 (m, 2H), 4.41 (dd, J = 101.5, 13.4 Hz, 2H). 13C NMR (151 MHz, CDCl3) δ 176.60, 163.92, 162.28, 153.71, 135.99, 132.22, 132.16, 127.00, 126.23, 123.91, 122.29, 115.79, 115.64, 61.57. ESI-HRMS: calcd for C14H10FNOS2 [M + H]+, 292.0266; found, 292.0272.
2-((4-bromobenzyl) sulfinyl) benzo[d]thiazole (4f) white solid, yield 85%. 1H NMR (600 MHz, Chloroform-d) δ 8.10 (dt, J = 8.3, 0.9 Hz, 1H), 7.95 (dt, J = 8.1, 0.9 Hz, 1H), 7.58 (ddd, J = 8.3, 7.2, 1.2 Hz, 1H), 7.49 (ddd, J = 8.2, 7.2, 1.1 Hz, 1H), 7.42 − 7.36 (m, 2H), 7.03 − 6.99 (m, 2H), 4.38 (dd, J = 101.8, 13.3 Hz, 2H). 13C NMR (151 MHz, CDCl3) δ 176.45, 153.69, 135.98, 131.99, 131.79, 127.18, 126.99, 126.23, 123.90, 123.21, 122.30, 61.67. ESI-HRMS: calcd for C14H10BrNOS2 [M + H]+, 351.9465; found, 351.9469.
2-((4-methylbenzyl) sulfinyl) benzo[d]thiazole (4 g) white solid, yield 84%. 1H NMR (400 MHz, Chloroform-d) δ 8.10 (d, J = 7.6 Hz, 1H), 7.96 (dd, J = 8.1, 1.1 Hz, 1H), 7.57 (dt, J = 8.2, 1.0 Hz, 1H), 7.49 (tt, J = 7.2, 0.9 Hz, 1H), 7.08 (d, J = 1.7 Hz, 4H), 4.58 − 4.20 (m, 2H), 2.31 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 177.02, 153.71, 138.71, 135.99, 130.35, 129.45, 126.90, 126.12, 125.18, 123.89, 122.26, 62.66, 21.23. ESI-HRMS: calcd for C15H13NOS2 [M + H]+, 288.0517; found, 288.0522.
2-((naphthalen-2-ylmethyl) sulfinyl) benzo[d]thiazole (4h) white solid, yield 79%. 1H NMR (400 MHz, Chloroform-d) δ 8.11 (dd, J = 8.4, 1.0 Hz, 1H), 7.90 (dd, J = 8.1, 1.0 Hz, 1H), 7.81 − 7.68 (m, 4H), 7.58 (ddd, J = 8.4, 7.2, 1.2 Hz, 1H), 7.50 − 7.41 (m, 3H), 7.25 − 7.22 (m, 1H), 4.73 − 4.46 (m, 2H). 13C NMR (101 MHz, CDCl3) δ 176.90, 153.70, 135.98, 133.12, 130.25, 128.46, 127.97, 127.66, 127.44, 126.93, 126.54, 126.39, 126.15, 125.80, 123.88, 122.26, 63.24. ESI-HRMS: calcd for C18H13NOS2 [M + H]+, 324.0517; found, 324.0519.
2-((4-nitrobenzyl) sulfinyl) benzo[d]thiazole (4i) yellow solid, yield 84% 1H NMR (400 MHz, Chloroform-d) δ 8.09 (dd, J = 8.9, 2.3 Hz, 3H), 7.93 (dd, J = 8.1, 1.1 Hz, 1H), 7.63 − 7.58 (m, 1H), 7.53 − 7.48 (m, 1H), 7.31 − 7.28 (m, 2H), 4.68 − 4.43 (m, 2H). 13C NMR (101 MHz, CDCl3) δ 175.53, 153.61, 148.00, 135.88, 135.30, 131.37, 127.21, 126.49, 123.94, 123.56, 122.35, 60.92. ESI-HRMS: calcd for C14H10N2O3S2 [M + H]+, 319.0211; found, 319.0198.
2-((4-(trifluoromethyl) benzyl) sulfinyl) benzo[d]thiazole (4j) white solid, yield 78%. 1H NMR (400 MHz, Chloroform-d) δ 8.11 (d, J = 8.2 Hz, 1H), 7.95 (d, J = 8.1 Hz, 1H), 7.61 (dd, J = 7.3, 1.0 Hz, 1H), 7.55 − 7.47 (m, 4H), 7.28 (s, 1H), 4.58 (d, J = 13.2 Hz, 1H), 4.41 (d, J = 13.2 Hz, 1H). 13C NMR (101 MHz, CDCl3) δ 176.15, 153.68, 135.99, 132.24, 130.83, 127.11, 126.37, 125.57, 125.53, 123.95, 122.32, 61.70. ESI-HRMS: calcd for C15H10F3NOS2 [M + H]+, 342.0234; found, 342.0232.
2-((3-methoxybenzyl) sulfinyl) benzo[d]thiazole (4k) white solid, yield 75%. 1H NMR (400 MHz, Chloroform-d) δ 8.10 (dt, J = 8.3, 0.9 Hz, 1H), 7.95 (dt, J = 8.1, 0.9 Hz, 1H), 7.57 (ddd, J = 8.3, 7.2, 1.3 Hz, 1H), 7.48 (ddd, J = 8.3, 7.2, 1.2 Hz, 1H), 7.19 (dd, J = 8.3, 7.5 Hz, 1H), 6.85 (ddd, J = 8.4, 2.6, 0.9 Hz, 1H), 6.79 (dt, J = 7.5, 1.2 Hz, 1H), 6.66 (dd, J = 2.5, 1.6 Hz, 1H), 4.40 (dd, J = 71.2, 13.1 Hz, 2H), 3.60 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 177.00, 159.69, 153.71, 136.04, 129.71, 126.96, 126.20, 123.89, 122.85, 122.29, 115.14, 115.11, 63.03, 55.08. ESI-HRMS: calcd for C15H13NO2S2 [M + H]+, 304.0466; found, 304.0463.
2-((4-methoxybenzyl) sulfinyl) benzo[d]thiazole (4 l) white solid, yield 78%. 1H NMR (400 MHz, Chloroform-d) δ 8.10 (dt, J = 8.3, 0.9 Hz, 1H), 7.95 (dt, J = 8.0, 0.9 Hz, 1H), 7.58 (ddd, J = 8.3, 7.2, 1.3 Hz, 1H), 7.48 (ddd, J = 8.3, 7.2, 1.2 Hz, 1H), 7.10 − 7.07 (m, 2H), 6.81 − 6.77 (m, 2H), 4.53 − 4.26 (m, 2H), 3.77 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 177.04, 159.97, 153.69, 135.96, 131.65, 126.85, 126.05, 123.83, 122.22, 120.08, 114.11, 62.24, 55.21. ESI-HRMS: calcd for C15H13NO2S2 [M + Na]+, 326.0285; found, 326.0258.
2-(benzylsulfinyl) benzo[d]oxazole (5a) white solid, yield 74%. 1H NMR (600 MHz, Chloroform-d) δ 7.83 − 7.79 (m, 1H), 7.61 (dt, J = 8.2, 0.8 Hz, 1H), 7.49 − 7.42 (m, 2H), 7.34 − 7.27 (m, 3H), 7.24 (dt, J = 6.7, 1.6 Hz, 2H), 4.64 − 4.52 (m, 2H). 13C NMR (151 MHz, CDCl3) δ 163.73, 151.64, 140.40, 130.23, 129.02, 128.26, 127.09, 125.60, 123.98, 122.63, 121.26, 111.46, 110.14, 109.84, 60.80. ESI-HRMS: calcd for C14H11NO2S [M + H]+, 258.0589; found, 258.0590.
2-((4-chlorobenzyl) sulfinyl) benzo[d]oxazole (5b) yellow solid, yield 77%. 1H NMR (600 MHz, Chloroform-d) δ 7.85 − 7.79 (m, 1H), 7.61 (d, J = 7.6 Hz, 2H), 7.46 (ddd, J = 12.1, 7.7, 1.1 Hz, 2H), 7.27 (d, J = 3.1 Hz, 1H), 7.17 (d, J = 8.4 Hz, 2H), 4.65 − 4.48 (m, 2H). 13C NMR (151 MHz, CDCl3) δ 163.37, 151.66, 140.33, 135.31, 131.55, 129.21, 127.20, 126.71, 125.71, 121.27, 111.48, 110.12, 109.91, 59.76. ESI-HRMS: calcd for C14H10ClNO2S [M + H]+, 292.0199; found, 292.0199.
2-((3-chlorobenzyl) sulfinyl) benzo[d]oxazole (5c) white solid, yield 84%. 1H NMR (600 MHz, Chloroform-d) δ 7.87 − 7.84 (m, 1H), 7.64 (dd, J = 7.8, 1.2 Hz, 1H), 7.52 − 7.46 (m, 2H), 7.33 − 7.31 (m, 1H), 7.26 − 7.23 (m, 1H), 7.18 − 7.14 (m, 2H), 4.63 − 4.52 (m, 2H). 13C NMR (151 MHz, CDCl3) δ 134.86, 130.30, 130.20, 129.25, 128.43, 127.24, 125.73, 124.06, 122.68, 121.30, 111.49, 110.16, 109.93, 60.05. ESI-HRMS: calcd for C14H10ClNO2S [M + H]+, 292.0199; found, 292.0199.
2-((2-chlorobenzyl) sulfinyl) benzo[d]oxazole (5d) white solid, yield 80%. 1H NMR (600 MHz, Chloroform-d) δ 7.83 (dt, J = 7.9, 0.9 Hz, 1H), 7.66 − 7.63 (m, 1H), 7.50 (td, J = 7.8, 1.3 Hz, 1H), 7.47 − 7.41 (m, 2H), 7.30 (ddd, J = 7.1, 4.3, 2.5 Hz, 2H), 7.21 (td, J = 7.5, 1.2 Hz, 1H), 4.80 (dd, J = 119.7, 12.8 Hz, 2H). 13C NMR (151 MHz, CDCl3) δ 163.83, 151.69, 140.39, 134.99, 132.84, 130.60, 129.95, 127.35, 127.26, 126.66, 125.65, 121.40, 111.52, 58.77. ESI-HRMS: calcd for C14H10ClNO2S [M + H]+, 292.0199; found, 292.0199.
2-((4-fluorobenzyl) sulfinyl) benzo[d]oxazole (5e) yellow solid, yield 68%. 1H NMR (600 MHz, Chloroform-d) δ 7.82 (d, J = 7.4 Hz, 1H), 7.62 − 7.58 (m, 1H), 7.49 − 7.42 (m, 2H), 7.21 (dd, J = 8.6, 5.3 Hz, 2H), 6.97 (t, J = 8.6 Hz, 2H), 4.63 − 4.49 (m, 2H). 13C NMR (151 MHz, CDCl3) δ 163.48, 151.65, 140.35, 132.09, 132.03, 127.16, 125.68, 124.11, 121.24, 116.13, 115.99, 111.46, 109.97, 59.68. ESI-HRMS: calcd for C14H10FNO2S [M + H]+, 276.0495; found, 276.0503.
2-((4-bromobenzyl) sulfinyl) benzo[d]oxazole (5f) white solid, yield 79%. 1H NMR (600 MHz, Chloroform-d) δ 7.84 − 7.80 (m, 1H), 7.64 − 7.60 (m, 1H), 7.50 − 7.40 (m, 4H), 7.13 − 7.09 (m, 2H), 4.62 − 4.38 (m, 2H). 13C NMR (151 MHz, CDCl3) δ 163.37, 151.64, 140.33, 132.15, 131.82, 127.24, 127.18, 125.70, 123.51, 121.26, 111.47, 59.80. ESI-HRMS: calcd for C14H10BrNO2S [M + Na]+, 357.9513; found, 357.9493.
2-((4-methylbenzyl) sulfinyl) benzo[d]oxazole (5 g) white solid, yield 70%. 1H NMR (600 MHz, Chloroform-d) δ 7.84 − 7.80 (m, 1H), 7.63 − 7.59 (m, 1H), 7.49 − 7.43 (m, 2H), 7.12 − 7.08 (m, 4H), 4.64 − 4.45 (m, 2H), 2.30 (s, 3H). 13C NMR (151 MHz, CDCl3) δ 163.87, 151.65, 140.44, 139.04, 130.13, 129.74, 127.05, 125.58, 125.05, 121.26, 111.47, 60.56, 21.21. ESI-HRMS: calcd for C15H13NO2S [M + H]+, 272.0745; found, 272.0745.
2-((naphthalen-2-ylmethyl) sulfinyl) benzo[d]oxazole (5h) yellow solid, yield 81%. 1H NMR (600 MHz, Chloroform-d) δ 7.82 − 7.76 (m, 2H), 7.75 − 7.70 (m, 2H), 7.58 (dd, J = 7.5, 1.7 Hz, 1H), 7.49 − 7.43 (m, 5H), 7.32 − 7.29 (m, 1H), 4.81 − 4.66 (m, 2H). 13C NMR (151 MHz, CDCl3) δ 163.83, 151.64, 140.41, 133.24, 133.23, 130.12, 128.85, 127.93, 127.68, 127.08, 126.70, 126.52, 125.67, 125.59, 121.23, 111.44, 61.13. ESI-HRMS: calcd for C18H13NO2S [M + Na]+, 330.0564; found, 308.0559.
2-((4-nitrobenzyl) sulfinyl) benzo[d]oxazole (5i) yellow solid, yield 87%. 1H NMR (600 MHz, Chloroform-d) δ 8.16 − 8.13 (m, 2H), 7.84 − 7.81 (m, 1H), 7.62 − 7.59 (m, 1H), 7.51 − 7.45 (m, 2H), 7.44 − 7.42 (m, 2H), 4.73 − 4.64 (m, 2H). 13C NMR (151 MHz, CDCl3) δ 162.83, 151.71, 148.27, 140.25, 135.56, 131.31, 127.43, 125.91, 123.96, 121.32, 111.51, 59.34. ESI-HRMS: calcd for C14H10N2O4S [M + H]+, 303.0440; found, 303.0440.
2-((4-(trifluoromethyl) benzyl) sulfinyl) benzo[d]oxazole (5j) white solid, yield 78%. 1H NMR (600 MHz, Chloroform-d) δ 7.84 (dd, J = 7.6, 1.5 Hz, 1H), 7.64 − 7.61 (m, 1H), 7.58 (d, J = 8.1 Hz, 2H), 7.52 − 7.46 (m, 2H), 7.39 (d, J = 8.0 Hz, 2H), 4.70 − 4.61 (m, 2H). 13C NMR (151 MHz, CDCl3) δ 163.18, 151.69, 140.32, 132.39, 131.29, 131.07, 130.68, 127.29, 125.90, 125.78, 123.98, 121.28, 111.49, 59.84. ESI-HRMS: calcd for C15H10F3NO2S [M + H]+, 326.0463; found, 326.0461.
2-((3-methoxybenzyl) sulfinyl) benzo[d]oxazole (5k) yellow solid, yield 69%. 1H NMR (600 MHz, Chloroform-d) δ 7.86 − 7.82 (m, 1H), 7.64 (dd, J = 7.9, 1.2 Hz, 1H), 7.48 (ddd, J = 12.9, 7.7, 1.3 Hz, 2H), 7.22 (d, J = 7.9 Hz, 1H), 6.88 − 6.83 (m, 2H), 6.75 (t, J = 2.1 Hz, 1H), 4.64 − 4.54 (m, 2H), 3.67 (s, 3H). 13C NMR (151 MHz, CDCl3) δ 163.81, 159.90, 151.64, 140.43, 130.05, 129.58, 127.11, 125.63, 122.50, 121.26, 115.18, 115.15, 111.48, 60.96, 55.12. ESI-HRMS: calcd for C15H13NO3S [M + H]+, 288.0694; found, 288.0695.
2-((4-methoxybenzyl) sulfinyl) benzo[d]oxazole (5 l) yellow solid, yield 63%. 1H NMR (600 MHz, Chloroform-d) δ 7.81 (dd, J = 7.6, 1.5 Hz, 1H), 7.63 − 7.59 (m, 1H), 7.45 (dtd, J = 19.3, 7.5, 1.3 Hz, 2H), 7.16 − 7.13 (m, 2H), 6.83 − 6.78 (m, 2H), 4.60 − 4.49 (m, 2H), 3.76 (s, 3H). 13C NMR (151 MHz, CDCl3) δ 163.88, 160.20, 151.64, 140.44, 131.49, 127.01, 125.56, 121.22, 119.96, 114.48, 111.45, 60.25, 55.27. ESI-HRMS: calcd for C15H13NO3S [M + Na]+, 310.0513; found, 310.0491.
2-(benzylsulfinyl)-5-chlorobenzo[d]oxazole (6a) white solid, yield 80%. 1H NMR (600 MHz, Chloroform-d) δ 7.80 (s, 1H), 7.55 − 7.50 (m, 1H), 7.44 (d, J = 8.8 Hz, 1H), 7.32 − 7.27 (m, 2H), 7.21 (d, J = 8.2 Hz, 2H), 7.11 − 7.06 (m, 1H), 4.87 − 4.37 (m, 2H). 13C NMR (151 MHz, CDCl3) δ 165.20, 150.21, 141.38, 131.29, 130.19, 129.15, 129.06, 127.97, 127.56, 121.15, 112.24, 60.81. ESI-HRMS: calcd for C14H10ClNO2S [M + H]+, 292.0199; found, 292.0198.
5-chloro-2-((4-chlorobenzyl) sulfinyl) benzo[d]oxazole (6b) white solid, yield 85%. 1H NMR (600 MHz, Chloroform-d) δ 7.80 (t, J = 1.5 Hz, 1H), 7.53 (dd, J = 8.8, 1.1 Hz, 1H), 7.45 (dt, J = 8.8, 1.6 Hz, 1H), 7.26 (s, 2H), 7.18 − 7.11 (m, 2H), 4.61 − 4.47 (m, 2H). 13C NMR (151 MHz, CDCl3) δ 164.95, 150.21, 141.35, 135.44, 131.51, 131.38, 129.25, 127.65, 126.52, 121.15, 112.27, 59.80. ESI-HRMS: calcd for C14H9Cl2NO2S [M + H]+, 325.9810; found, 325.9817.
5-chloro-2-((3-chlorobenzyl) sulfinyl) benzo[d]oxazole (6c) white solid, yield 85%. 1H NMR (600 MHz, Chloroform-d) δ 7.82 − 7.80 (m, 1H), 7.54 (d, J = 8.8 Hz, 1H), 7.45 (dd, J = 8.8, 2.1 Hz, 1H), 7.31 (ddd, J = 8.1, 2.0, 1.1 Hz, 1H), 7.25 − 7.22 (m, 2H), 7.12 (dt, J = 7.7, 1.3 Hz, 1H), 4.59 − 4.49 (m, 2H). 13C NMR (151 MHz, CDCl3) δ 164.93, 150.22, 141.33, 134.90, 131.39, 130.28, 130.24, 130.05, 129.34, 128.39, 127.69, 121.17, 112.27, 60.07. ESI-HRMS: calcd for C14H9Cl2NO2S [M + H]+, 325.9810; found, 325.9814.
5-chloro-2-((2-chlorobenzyl) sulfinyl) benzo[d]oxazole (6d) white solid, yield 82%. 1H NMR (600 MHz, Chloroform-d) δ 7.88 − 7.79 (m, 1H), 7.57 (dd, J = 8.8, 0.5 Hz, 1H), 7.46 (dd, J = 8.7, 2.1 Hz, 1H), 7.41 (dd, J = 7.9, 1.2 Hz, 1H), 7.32 − 7.30 (m, 1H), 7.22 (td, J = 7.5, 1.3 Hz, 1H), 7.10 − 7.08 (m, 1H), 4.80 (dd, J = 111.6, 12.8 Hz, 2H). 13C NMR (151 MHz, CDCl3) δ 165.30, 150.29, 134.98, 132.84, 131.36, 130.73, 130.00, 127.76, 127.39, 126.36, 122.55, 121.30, 112.30, 58.78. ESI-HRMS: calcd for C14H9Cl2NO2S [M + H]+, 325.9810; found, 325.9822.
5-chloro-2-((4-fluorobenzyl) sulfinyl) benzo[d]oxazole (6e) yellow solid, yield 79%. 1H NMR (600 MHz, Chloroform-d) δ 7.80 (t, J = 1.6 Hz, 1H), 7.53 (d, J = 8.7 Hz, 1H), 7.44 (ddd, J = 8.5, 2.1, 0.9 Hz, 1H), 7.23 − 7.16 (m, 2H), 7.04 − 6.94 (m, 2H), 4.63 − 4.42 (m, 2H). 13C NMR (151 MHz, CDCl3) δ 165.03, 164.08, 162.43, 150.21, 141.36, 132.00, 131.36, 127.61, 123.88, 121.13, 116.20, 116.05, 112.25, 59.73. ESI-HRMS: calcd for C14H9ClFNO2S [M + H]+, 310.0105; found, 310.0113.
2-((4-bromobenzyl) sulfinyl)-5-chlorobenzo[d]oxazole (6f) white solid, yield 81%. 1H NMR (600 MHz, Chloroform-d) δ 7.80 (d, J = 2.0 Hz, 1H), 7.54 (d, J = 8.8 Hz, 1H), 7.46 − 7.41 (m, 3H), 7.09 (d, J = 8.5 Hz, 2H), 4.63 − 4.37 (m, 2H). 13C NMR (151 MHz, CDCl3) δ 164.85, 150.21, 141.31, 132.20, 131.78, 131.39, 127.66, 126.95, 123.66, 121.15, 112.27, 59.81. ESI-HRMS: calcd for C14H9BrClNO2S [M + H]+, 369.9304; found, 369.9313.
5-chloro-2-((4-methylbenzyl) sulfinyl) benzo[d]oxazole (6 g) yellow solid, yield 80%. 1H NMR (600 MHz, Chloroform-d) δ 7.80 (d, J = 2.0 Hz, 1H), 7.53 (d, J = 8.7 Hz, 1H), 7.44 (dd, J = 8.7, 2.0 Hz, 1H), 7.09 (s, 4H), 4.61 − 4.49 (m, 2H), 2.30 (s, 3H). 13C NMR (151 MHz, CDCl3) δ 165.35, 150.19, 141.42, 139.17, 131.23, 130.08, 129.29, 127.50, 124.75, 121.14, 112.23, 60.57, 21.21. ESI-HRMS: calcd for C15H12ClNO2S [M + H]+, 306.0356; found, 306.0359.
5-chloro-2-((naphthalen-2-ylmethyl) sulfinyl) benzo[d]oxazole (6h) yellow solid, yield 87%. 1H NMR (600 MHz, Chloroform-d) δ 7.79 (q, J = 2.7, 2.3 Hz, 2H), 7.74 (d, J = 8.9 Hz, 3H), 7.52 − 7.45 (m, 3H), 7.43 (dd, J = 8.8, 2.1 Hz, 1H), 7.27 (dd, J = 8.4, 1.9 Hz, 1H), 4.83 − 4.66 (m, 2H). 13C NMR (151 MHz, CDCl3) δ 165.33, 150.21, 141.41, 133.28, 133.22, 131.28, 130.16, 128.93, 127.93, 127.70, 127.54, 126.97, 126.81, 126.63, 125.35, 121.13, 112.23, 61.18. ESI-HRMS: calcd for C18H12ClNO2S [M + Na]+, 364.0175; found, 364.0179.
5-chloro-2-((4-nitrobenzyl) sulfinyl) benzo[d]oxazole (6i) yellow solid, yield 72%. 1H NMR (600 MHz, Chloroform-d) δ 8.19 − 8.12 (m, 2H), 7.81 (d, J = 2.0 Hz, 1H), 7.53 (d, J = 8.8 Hz, 1H), 7.48 − 7.40 (m, 3H), 4.76 − 4.59 (m, 2H). 13C NMR (151 MHz, CDCl3) δ 164.38, 150.26, 148.33, 141.24, 135.33, 131.60, 131.30, 130.47, 127.89, 124.00, 121.21, 112.31, 59.34. ESI-HRMS: calcd for C14H9ClN2O4S [M + Na]+, 358.9869; found, 359.0133.
5-chloro-2-((4-(trifluoromethyl) benzyl) sulfinyl) benzo[d]oxazole (6j) white solid, yield 81%. 1H NMR (600 MHz, Chloroform-d) δ 7.81 (d, J = 2.0 Hz, 1H), 7.56 (d, J = 8.1 Hz, 2H), 7.52 (d, J = 8.7 Hz, 1H), 7.45 (dd, J = 8.8, 2.1 Hz, 1H), 7.36 (d, J = 8.0 Hz, 2H), 4.74 − 4.55 (m, 2H). 13C NMR (151 MHz, CDCl3) δ 164.73, 150.26, 141.32, 132.17, 131.49, 131.41, 130.68, 130.58, 129.80, 127.77, 125.94, 121.18, 112.28, 59.85. ESI-HRMS: calcd for C15H9ClF3NO2S [M + H]+, 360.0073; found, 360.0079.
5-chloro-2-((3-methoxybenzyl) sulfinyl) benzo[d]oxazole (6k) yellow solid, yield 75%. 1H NMR (600 MHz, Chloroform-d) δ 7.80 (d, J = 2.1 Hz, 1H), 7.54 (d, J = 8.7 Hz, 1H), 7.44 (dd, J = 8.7, 2.1 Hz, 1H), 7.19 (dd, J = 8.3, 7.5 Hz, 1H), 6.85 (ddd, J = 8.4, 2.6, 0.9 Hz, 1H), 6.79 (dt, J = 7.5, 1.2 Hz, 1H), 6.74 (t, J = 2.1 Hz, 1H), 4.60 − 4.51 (m, 2H), 3.68 (s, 3H). 13C NMR (151 MHz, CDCl3) δ 165.28, 159.92, 150.20, 141.39, 131.30, 130.08, 129.30, 127.57, 122.38, 121.13, 115.40, 115.05, 112.25, 60.91, 55.15. ESI-HRMS: calcd for C15H12ClNO3S [M + H]+, 322.0305; found, 322.0312.
5-chloro-2-((4-methoxybenzyl) sulfinyl) benzo[d]oxazole (6 l) white solid, yield 75%. 1H NMR (600 MHz, Chloroform-d) δ 7.79 (d, J = 2.1 Hz, 1H), 7.53 (d, J = 8.8 Hz, 1H), 7.44 (dd, J = 8.8, 2.1 Hz, 1H), 7.15 − 7.10 (m, 2H), 6.83 − 6.79 (m, 2H), 4.59 − 4.48 (m, 2H), 3.77 (s, 3H). 13C NMR (151 MHz, CDCl3) δ 165.41, 160.29, 150.20, 141.44, 131.47, 131.23, 127.47, 121.11, 119.68, 114.51, 112.23, 60.30, 55.28. ESI-HRMS: calcd for C15H12ClNO3S [M + Na]+, 344.0124; found, 344.0102.
2-(benzylsulfinyl)-6-chlorobenzo[d]oxazole (7a) white solid, yield 78%. 1H NMR (600 MHz, Chloroform-d) δ 7.73 (d, J = 8.6 Hz, 1H), 7.61 (d, J = 1.9 Hz, 1H), 7.42 (dd, J = 8.6, 1.9 Hz, 1H), 7.31 − 7.28 (m, 2H), 7.24 − 7.17 (m, 3H), 4.64 − 4.54 (m, 2H). 13C NMR (151 MHz, CDCl3) δ 164.36, 151.71, 139.07, 130.18, 129.14, 129.05, 127.94, 126.58, 124.08, 121.79, 112.04, 60.74. ESI-HRMS: calcd for C14H10ClNO2S [M + H]+, 292.0199; found, 292.0200.
6-chloro-2-((4-chlorobenzyl) sulfinyl) benzo[d]oxazole (7b) white solid, yield 85%. 1H NMR (600 MHz, Chloroform-d) δ 7.75 (d, J = 8.6 Hz, 1H), 7.65 (d, J = 1.8 Hz, 1H), 7.46 (dd, J = 8.6, 1.9 Hz, 1H), 7.29 (d, J = 8.6 Hz, 2H), 7.17 (dd, J = 13.7, 8.4 Hz, 2H), 4.62 − 4.53 (m, 2H). 13C NMR (151 MHz, CDCl3) δ 164.11, 151.74, 139.06, 135.45, 133.19, 131.51, 129.27, 126.69, 126.50, 121.81, 112.08, 59.76. ESI-HRMS: calcd for C14H9Cl2NO2S [M + H]+, 325.9809; found, 325.9812.
6-chloro-2-((4-methylbenzyl) sulfinyl) benzo[d]oxazole (7c) white solid, yield 82%. 1H NMR (600 MHz, Chloroform-d) δ 7.72 (d, J = 8.5 Hz, 1H), 7.61 (d, J = 1.8 Hz, 1H), 7.42 (dd, J = 8.6, 1.9 Hz, 1H), 7.09 (s, 4H), 4.61 − 4.37 (m, 2H), 2.30 (s, 3H). 13C NMR (151 MHz, CDCl3) δ 164.55, 151.69, 139.15, 132.97, 130.07, 129.76, 126.52, 124.75, 123.87, 121.77, 112.03, 60.51, 21.20. ESI-HRMS: calcd for C15H12ClNO2S [M + H]+, 306.0277; found, 306.0360.
6-chloro-2-((4-nitrobenzyl) sulfinyl) benzo[d]oxazole (7d) yellow solid, yield 86%. 1H NMR (600 MHz, Chloroform-d) δ 8.19 − 8.13 (m, 2H), 7.74 (d, J = 8.6 Hz, 1H), 7.63 (d, J = 1.9 Hz, 1H), 7.44 (dd, J = 13.5, 9.6 Hz, 3H), 4.71 − 4.62 (m, 2H). 13C NMR (151 MHz, CDCl3) δ 163.58, 151.78, 148.33, 138.97, 135.34, 133.43, 131.29, 126.88, 124.02, 121.85, 112.12, 59.32. ESI-HRMS: calcd for C14H9ClN2O4S [M + H]+, 337.0050; found, 337.0054.
6-chloro-2-((4-(trifluoromethyl) benzyl) sulfinyl) benzo[d]oxazole (7e) white solid, yield 77%. 1H NMR (600 MHz, Chloroform-d) δ 7.73 (d, J = 8.6 Hz, 1H), 7.61 (d, J = 1.9 Hz, 1H), 7.56 (d, J = 8.0 Hz, 2H), 7.44 (dd, J = 8.5, 1.9 Hz, 1H), 7.37 (d, J = 8.0 Hz, 2H), 4.70 − 4.58 (m, 2H). 13C NMR (151 MHz, CDCl3) δ 163.87, 151.77, 139.01, 133.31, 132.14, 130.68, 126.78, 125.95, 124.09, 121.83, 112.08, 110.94, 110.53, 59.77. ESI-HRMS: calcd for C15H9ClF3NO2S [M + H]+, 360.0073; found, 360.0071.
6-chloro-2-((4-fluorobenzyl) sulfinyl) benzo[d]oxazole (7f) yellow solid, yield 80%. 1H NMR (600 MHz, Chloroform-d) δ 7.73 (d, J = 8.6 Hz, 1H), 7.62 (t, J = 1.4 Hz, 1H), 7.43 (dt, J = 8.6, 1.4 Hz, 1H), 7.22 − 7.20 (m, 2H), 6.98 (d, J = 8.4 Hz, 2H), 4.62 − 4.43 (m, 2H). 13C NMR (151 MHz, CDCl3) δ 164.16, 162.42, 151.73, 139.05, 133.15, 132.05, 132.00, 126.66, 124.11, 121.79, 116.22, 116.07, 112.06, 59.69. ESI-HRMS: calcd for C14H9ClFNO2S [M + H]+, 310.0105; found, 310.0108.
2-(benzylsulfinyl)-5-methylbenzo[d]oxazole (8a) white solid, yield 79%. 1H NMR (600 MHz, Chloroform-d) δ 7.58 (s, 1H), 7.46 (d, J = 8.4 Hz, 1H), 7.32 − 7.26 (m, 3H), 7.26 − 7.21 (m, 2H), 6.86 (d, J = 10.2 Hz, 1H), 4.62 − 4.54 (m, 2H), 2.49 (s, 3H). 13C NMR (151 MHz, CDCl3) δ 156.28, 141.94, 134.09, 130.25, 129.44, 129.00, 128.39, 123.04, 120.96, 110.80, 110.63, 109.61, 60.71, 21.43. ESI-HRMS: calcd for C15H13NO2S [M + H]+, 272.0745; found, 272.0740.
2-((4-chlorobenzyl) sulfinyl)-5-methylbenzo[d]oxazole (8b) white solid, yield 87%. 1H NMR (600 MHz, Chloroform-d) δ 7.60 − 7.56 (m, 1H), 7.46 (d, J = 8.4 Hz, 1H), 7.29 − 7.22 (m, 3H), 7.15 (d, J = 8.4 Hz, 2H), 4.60 − 4.50 (m, 2H), 2.49 (s, 3H). 13C NMR (151 MHz, CDCl3) δ 163.26, 149.94, 140.52, 135.78, 135.21, 131.54, 129.16, 128.43, 126.80, 120.94, 110.79, 59.67, 21.48. ESI-HRMS: calcd for C15H12ClNO2S [M + H]+, 306.0356; found, 306.0344.
5-methyl-2-((4-methylbenzyl) sulfinyl) benzo[d]oxazole (8c) white solid, yield 84%. 1H NMR (600 MHz, Chloroform-d) δ 7.47 (d, J = 8.4 Hz, 1H), 7.29 − 7.24 (m, 1H), 7.10 (t, J = 8.9 Hz, 4H), 6.87 (d, J = 7.9 Hz, 1H), 4.60 − 4.52 (m, 2H), 2.49 (s, 3H), 2.29 (s, 3H). 13C NMR (151 MHz, CDCl3) δ 163.69, 149.96, 140.60, 138.99, 135.67, 10.15, 129.72, 128.34, 125.05, 120.96, 110.80, 60.45, 21.50, 21.19. ESI-HRMS: calcd for C16H15NO2S [M + H]+, 286.0902; found, 286.0896.
5-methyl-2-((4-nitrobenzyl) sulfinyl) benzo[d]oxazole (8d) yellow solid, yield 81%. 1H NMR (600 MHz, Chloroform-d) δ 8.19 − 8.09 (m, 2H), 7.58 (s, 1H), 7.46 (d, J = 8.5 Hz, 1H), 7.41 (d, J = 8.7 Hz, 2H), 7.29 (dd, J = 8.5, 1.7 Hz, 1H), 4.93 − 4.30 (m, 2H), 2.51 (s, 3H). 13C NMR (151 MHz, CDCl3) δ 162.69, 150.00, 148.23, 140.45, 136.04, 135.62, 131.28, 128.66, 120.99, 110.82, 59.31, 21.50. ESI-HRMS: calcd for C15H12N2O4S [M + H]+, 317.0596; found, 317.0588.
5-methyl-2-((4-(trifluoromethyl) benzyl) sulfinyl) benzo[d]oxazole (8e) white solid, yield 73%. 1H NMR (600 MHz, Chloroform-d) δ 7.60 − 7.58 (m, 1H), 7.54 (s, 1H), 7.46 (d, J = 8.5 Hz, 1H), 7.36 (d, J = 8.0 Hz, 2H), 7.28 (dd, J = 8.4, 1.7 Hz, 1H), 4.67 − 4.55 (m, 2H), 2.50 (s, 3H). 13C NMR (151 MHz, CDCl3) δ 163.08, 150.01, 140.54, 135.90, 132.46, 131.25, 131.03, 130.68, 128.54, 125.88, 120.97, 110.82, 59.81, 21.50. ESI-HRMS: calcd for C16H12F3NO2S [M + H]+, 340.0619; found, 340.0602.
2-((4-fluorobenzyl) sulfinyl)-5-methylbenzo[d]oxazole (8f) white solid, yield 79%. 1H NMR (600 MHz, Chloroform-d) δ 7.58 (dt, J = 1.7, 0.8 Hz, 1H), 7.47 (d, J = 8.4 Hz, 1H), 7.29 − 7.27 (m, 1H), 7.23 − 7.17 (m, 2H), 6.97 (t, J = 8.6 Hz, 2H), 4.62 − 4.46 (m, 2H), 2.49 (s, 3H). 13C NMR (151 MHz, CDCl3) δ 164.00, 163.37, 162.35, 149.95, 140.56, 135.77, 132.07, 132.02, 128.40, 120.93, 116.11, 115.97, 110.79, 59.65, 21.49. ESI-HRMS: calcd for C15H12FNO2S [M + H]+, 290.0651, found, 290.0642.
2-(benzylsulfinyl)-5-methoxybenzo[d]oxazole (9a) white solid, yield 81%. 1H NMR (600 MHz, Chloroform-d) δ 7.49 − 7.46 (m, 1H), 7.32 − 7.27 (m, 3H), 7.24 − 7.22 (m, 3H), 7.05 (dd, J = 9.0, 2.6 Hz, 1H), 4.67 − 4.48 (m, 2H), 3.86 (s, 3H). 13C NMR (151 MHz, CDCl3) δ 164.16, 157.97, 146.34, 141.29, 130.22, 128.99, 128.96, 128.34, 116.33, 111.65, 103.32, 60.75, 55.97. ESI-HRMS: calcd for C15H13NO3S [M + H]+, 288.0694; found, 288.0673.
2-((4-chlorobenzyl) sulfinyl)-5-methoxybenzo[d]oxazole (9b) white solid, yield 88%. 1H NMR (600 MHz, Chloroform-d) δ 7.48 (dd, J = 9.0, 1.3 Hz, 1H), 7.30 − 7.22 (m, 3H), 7.17 − 7.14 (m, 2H), 7.07 (dd, J = 8.9, 2.5 Hz, 1H), 4.62 − 4.45 (m, 2H), 3.88 (d, J = 1.4 Hz, 3H). 13C NMR (151 MHz, CDCl3) δ 163.76, 158.06, 146.38, 141.25, 135.29, 131.54, 129.21, 126.76, 116.49, 111.69, 103.32, 59.75, 56.00. ESI-HRMS: calcd for C15H12ClNO3S [M + H]+, 322.0305; found, 322.0295.
5-methoxy-2-((4-methylbenzyl) sulfinyl) benzo[d]oxazole (9c) white solid, yield 85%. 1H NMR (600 MHz, Chloroform-d) δ 7.48 (d, J = 9.0 Hz, 1H), 7.24 (d, J = 2.5 Hz, 1H), 7.12 − 7.04 (m, 5H), 4.65 − 4.42 (m, 2H), 3.87 (s, 3H), 2.30 (s, 3H). 13C NMR (151 MHz, CDCl3) δ 164.32, 157.97, 146.36, 141.34, 138.97, 130.12, 129.72, 125.11, 116.30, 111.66, 103.34, 60.53, 55.98, 21.20. ESI-HRMS: calcd for C16H15NO3S [M + H]+, 302.0851; found, 302.0833.
5-methoxy-2-((4-nitrobenzyl) sulfinyl) benzo[d]oxazole (9d) yellow solid, yield 81%. 1H NMR (600 MHz, Chloroform-d) δ 8.18 − 8.11 (m, 2H), 7.47 (d, J = 9.0 Hz, 1H), 7.44 − 7.39 (m, 2H), 7.24 (d, J = 2.5 Hz, 1H), 7.07 (dd, J = 9.1, 2.4 Hz, 1H), 4.70 − 4.60 (m, 2H), 3.88 (s, 3H). 13C NMR (151 MHz, CDCl3) δ 163.13, 158.16, 148.23, 146.40, 141.16, 135.57, 131.29, 123.95, 116.70, 111.72, 103.31, 59.32, 56.01. ESI-HRMS: calcd for C15H12N2O5S [M + H]+, 333.0545; found, 333.0545.
5-methoxy-2-((4-(trifluoromethyl) benzyl) sulfinyl) benzo[d]oxazole (9e) white solid, yield 83%. 1H NMR (600 MHz, Chloroform-d) δ 7.60 − 7.54 (m, 2H), 7.49 (d, J = 9.0 Hz, 1H), 7.38 (d, J = 8.0 Hz, 2H), 7.26 (d, J = 2.5 Hz, 1H), 7.11 − 7.08 (m, 1H), 4.69 − 4.57 (m, 2H), 3.89 (s, 3H). 13C NMR (151 MHz, CDCl3) δ 163.56, 158.12, 146.42, 141.24, 132.44, 131.27, 131.05, 130.68, 125.87, 116.58, 111.70, 103.33, 59.84, 56.00. ESI-HRMS: calcd for C16H12F3NO3S [M + H]+, 356.0568; found, 356.0551.
2-((4-fluorobenzyl) sulfinyl)-5-methoxybenzo[d]oxazole (9f) white solid, yield 79%. 1H NMR (600 MHz, Chloroform-d) δ 7.48 (d, J = 9.0 Hz, 1H), 7.24 (d, J = 2.6 Hz, 1H), 7.23 − 7.18 (m, 2H), 7.06 (dd, J = 9.0, 2.6 Hz, 1H), 6.98 (t, J = 8.5 Hz, 2H), 4.61 − 4.49 (m, 2H), 3.87 (s, 3H). 13C NMR (151 MHz, CDCl3) δ 164.02, 163.87, 162.37, 158.04, 146.37, 141.27, 132.07, 124.12, 116.42, 116.13, 115.99, 111.67, 103.31, 59.69, 55.99. ESI-HRMS: calcd for C15H12FNO3S [M + H]+, 306.0600; found, 306.0588.
Biological investigations
Biofilm formation assessment
Biofilms were quantitatively detected by the crystal violet method.Citation35 The overnight culture of P. aeruginosa PAO1 was diluted 1000 times with fresh Luria broth medium. 199 μL of the diluted bacterial solution was added to a 96-well plate, and 1 μL of the 10 mM compound stock solution prepared was added to make the final concentration of the compound 50 μM. The same volume of DMSO was added as a negative control. Then the 96-well plates were incubated at 37 °C for 24 h. Then the OD value at 600 nm was measured by a microplate reader. After removing the upper layer of bacteria, rinse gently 2–3 times with PBS buffer. After dehydration and fixation at 37 °C, 200 μL of 0.1% crystal violet solution was added. After standing for 15 min for staining, the excess crystal violet solution was removed by suction and the remaining crystal violet was gently rinsed. After drying at 37 °C, the pigment was dissolved in 200 μL of 95% ethanol. Finally, the absorbance was measured at 595 nm by the microplate reader. The formula for calculating the biofilm inhibition rate is: OD595control-OD595/OD595control × 100%.
Growth curve analysis
P. aeruginosa PAO1 was cultured overnight, diluted to OD600 = 0.01, and then transferred into 96-well plates with active compounds (to give a final concentration of 50 μM, 20 μM, 10 μM, 5 μM). Bacterial cultures were incubated for at 37 °C, and optical density was measured at 600 nm every hour for 16 h.
CLSM images
P. aeruginosa PAO1was cultured overnight and diluted 100 times, the medium and compounds were added to the plate to make the final concentration 50 μM, 25 μM. The mixture was incubated at 37 °C for 24 h. Supernatants were poured out and washed with water for three times to remove the floating bacteria, then fixed with 4% paraformaldehyde for 15 min, after that stained with 0.01% acridine orange for 15 min (in the dark), and the excess dye was washed away with PBS. The established model was observed by laser confocal microscope (LEICA, TCS SP8). The excitation filter wavelength was detected at 488 nm and the blocking filter wavelength was detected at 515 nm. The signal was received by FITC channel with an objective lens ×63.
GFP reporter strain assay
Compounds were prepared as stock solutions at concentration of 10 mM in 100% dimethyl sulfoxide. The PAO1-1asB-gfp strain was grown in LB medium at 37 °C, 200 rpm for 12–16 h, and the culture was diluted in ABTGC medium to a final optical density of 600 nm (OD600) of 0.02 (2.5 × 10 CFU/mL). Next, the compound stock solution and the diluted bacterial suspension were added to a 96-well microtiter plate to a final concentration of 20 μM. The same amount of a 0.02% DMSO solvent control group was set. The 96-well microtiter plates were incubated in a Molecular Devices SpectraMax microplate reader at 37 °C for at least 12 h, with OD600 and GFP fluorescence signals (excitation 485 nm, emission 535 nm) measured every 20 min. Inhibition assays for all test compounds and controls were performed in triplicate. The test methods of PAO1-rhl-gfp and PAO1-pqs-gfp strains are as above.Citation23,Citation36
Elastase assay
P. aeruginosa PAO1 was grown overnight and diluted to a density of 600 nm (OD600) of 0.01 in LB medium. Compounds were added to various final concentrations of 50 μM, 25 μM, 12.5 μM. Then incubate at 37 °C, shaking at 200 rpm for 24 h. Cultures were centrifuged at 10 000 rpm for 10 min. Aspirate the supernatant and filter sterilise with a disposable filter. The supernatant fraction 100 µL was incubated with 900 µL 2 mg/mL Elastin-Congo Red (ECR) prepared in 0.1mMTris-HCl, the reaction was shaken at 37 °C for 18 h. The reaction was then placed on ice, 100 μL of 0.12 M EDTA was added to terminate the reaction, and centrifuged at 4 °C, 12 000 r/min for 10 min to remove insoluble ECR; the absorbance of the supernatant was measured at 495 nm by a microplate reader.Citation37–38
Pyocyanin quantification assay
The determination of pyocyanin is based on the absorbance of pyocyanin at 520 nm in acidic solution.Citation39 P. aeruginosa PAO1 overnight culture was diluted with LB medium to OD600 = 0.01. Compounds were added to make a final concentration of 50 μM, 25 μM, 12.5 μM. Incubate at 37 °C, with shaking at 200 rpm for 24 h. The culture was then centrifuged at 10 000 rpm for 10 min, and 7.5 mL of the supernatant was transferred to a new centrifuge tube. Add 3 mL of chloroform for extraction (the chloroform layer turns blue); transfer the chloroform layer to a new centrifuge tube, add 1.5 mL of 0.2 M HCl for extraction (they turn pink after the hydrochloric acid mixed reaction). The HCl layer was pipetted into a microtiter plate and the absorbance was measured at 520 nm by Microplate reader.
Rhamnolipid quantification assay
Rhamnolipid production was directly quantified using the orcinol assay according to original protocol by Koch et al.Citation40 P. aeruginosa PAO1 was diluted 100 times from overnight culture into fresh Luria broth medium (OD600=0.01). Compounds were then added to a final concentration of 50 μM, 25 μM, 12.5 μM. The cultures were grown for 24 h at 37 °C, shaking condition (200 rpm). Supernatants were collected by centrifuging at 10 000 rpm for 10 min and extracted with diethyl ether (twice). Organic fractions were concentrated to yield white solids. It was then resuspended in deionised water and added with 0.19% (w/v) orcinol in 50% H2SO4. The resulting mixture was incubated at 80 °C for 30 min to give orange solution. After cooling to room temperature, the absorbance was measured at 421 nm by a microplate reader.
Molecular docking
Molecules (OdDHL, 6b) were drawn with ChemBioDraw Ultra 13.0 software and minimised with MOE (Molecular Operate Environment, 2020) software (Innovation Centre of Pesticide Research, Department of Applied Chemistry, College of Science, China Agricultural University, Beijing, China). The receptor protein LasR in PDB format downloaded from RCSB Protein Data Bank (http://www.pdb.org). LasR X-ray crystal structure with 1.80 A° resolution (PDB ID: 2UV0) was used for docking study.Citation41 And it was prepared by the process of deleting water, adding hydrogen, adding Gasteiger charges and so on by Autodock Tools 15.6 software. The ligand binding site was defined using the bound ligands in the crystal structures. The OdDHL-binding pocket was selected as docking sites and docking environment was set in the solvent. Docking was operated by MOE after setting method (placement: triangle matcher, refinement: rigid receptor), score (placement: London dG, refinement: GBVI/WSA dG) and poses (placement: 30, refinement: 5). The best docking poses were selected to analyse the interaction between LasR and target compounds.
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References
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