Discovery and Preliminary Structure-Activity Investigation of 3-Substituted-1H-imidazol-5-yl-1H-indoles with In Vitro Activity towards Methicillin-Resistant Staphylococcus aureus

Antibiotics have been the cornerstone of modern medicine saving lives by virtue of being able to cure infectious diseases and to prevent infections in those who are immune compromised. Their intense use has led to a surging increase in the incidence of antibiotic-resistant bacteria resulting in a desperate need for antibiotics with new mechanisms of action. As part of our search for new antimicrobials we have screened an in-house library of compounds and identified two 3-substituted-1H-imidazol-5-yl-1H-indoles as weak growth inhibitors (MIC 16 µg/mL) against methicillin-resistant Staphylococcus aureus (MRSA). An extensive library of analogues was prepared using the Van Leusen three-component reaction, biological evaluation of which led to the identification of two analogues (26 and 32) with favorable anti-MRSA activity (MIC ≤ 0.25 µg/mL) which also lacked cytotoxic or hemolytic properties. The screening campaign also identified two derivatives, a phenethyl-indole-imidazole 57 and a 5-phenyl-1H-imidazole 111 that were non-toxic selective antifungals towards Cryptococcus neoformans. These results have identified 3-substituted-1H-imidazol-5-yl-1H-indoles and 5-phenyl-1H-imidazoles as new structural scaffolds for further investigation as anti-MRSA and anti-C. neoformans agents, respectively.


Introduction
New Zealand has a growing problem with Staphylococcus aureus infection, experiencing higher levels of incidence than other developed countries, with incidences of S. aureusrelated hospitalizations highest in the under-five and over 75 year age groups [1][2][3][4][5][6][7]. Over the ten-year period of 2001-2011, the incidence of methicillin-susceptible S. aureus infections increased to 361 per 100,000, with MRSA infection (covering non-multidrug resistant MRSA and multidrug resistant MRSA) accounting for an additional 12% of cases. In the case of children under the age of 15, annual average hospitalization rates for S. aureus skin and soft tissue infection (SSTI) increased to 522 per 100,000 population (2011), with subpopulation analysis identifying Māori and Pacific children with rates of 1488 and 1215 per 100,000 population, respectively [1][2][3][4][5][6][7].
Front-line treatments for SSTI in New Zealand are the topical antimicrobials fusidic acid and mupirocin. During the 1993-2012 timeframe, community prescribing rates for fusidic acid increased dramatically (likely due to the drug becoming a Governmentsubsidized treatment option) and this was matched with a corresponding increase in fusidic acid-resistant MRSA [8,9]. Unfortunately, the widespread, and largely unregulated, use of this valuable topical antimicrobial resource will ultimately lead to the need for more treatment options.
We have recently screened an in-house library of natural products and synthetic compounds against a panel of bacterial and fungal pathogens [10,11] which led to the discovery of a compound class (Figure 1) that exhibited activity exclusively against methicillinresistant Staphylococcus aureus (MRSA). Of the compounds screened, 3-substituted-1Himidazol-5-yl-1H-indole ("indole-imidazole") compounds 1 and 2 were found to weakly inhibit the growth of MRSA with MIC of 16 µg/mL (Table 1) while a closely related 5-fluoro analogue 3 (Supplementary Figures S1-S3) was devoid of activity. treatment options.
We have recently screened an in-house librar pounds against a panel of bacterial and fungal pa ery of a compound class (Figure 1) that exhibited resistant Staphylococcus aureus (MRSA). Of the com idazol-5-yl-1H-indole ("indole-imidazole") comp hibit the growth of MRSA with MIC of 16 µ g/mL analogue 3 (Supplementary Figures S1-S3) was d Similarly functionalized 1H-imidazol-5-yl-1H active towards crop-related fungi [12] and as 5-H iants with increased substitution such as the ma and nortopsentins [16,17] have been shown to ex activities while analogues of the natural produc properties [18]. Additionally, 1-benzenesulfonylreceptor agonists [19]. With this relatively limited 5-yl-1H-indoles, we undertook a study to further this class of molecule by preparing a more diver influence of the imidazole N-substituent and indo tivity. Herein we report on the synthesis and biol logues of hit compounds 1 and 2. Similarly functionalized 1H-imidazol-5-yl-1H-indoles have been recently reported as active towards crop-related fungi [12] and as 5-HT 7 serotonin receptor agonists [13]. Variants with increased substitution such as the marine natural products topsentins [14,15] and nortopsentins [16,17] have been shown to exhibit cytotoxic, antiviral and antifungal activities while analogues of the natural product meridianin exhibit antibiotic adjuvant properties [18]. Additionally, 1-benzenesulfonyl-1H-indoles have been reported as 5-HT 6 receptor agonists [19]. With this relatively limited literature on 3-substituted 1H-imidazol-5-yl-1H-indoles, we undertook a study to further explore the antimicrobial properties of this class of molecule by preparing a more diverse array of analogues that explored the influence of the imidazole N-substituent and indole halogen substitution on biological activity. Herein we report on the synthesis and biological evaluation of a diverse set of analogues of hit compounds 1 and 2.

Results and Discussion
A set of 39 new analogues were prepared (4-42) (Figure 2 and Supplementary Materials Figures S4-S42) utilizing the Van Leusen three-component reaction [20]. This two-step, one-pot reaction starts with in situ generation of an imine by condensation of an indole-3carbaldehyde with an amine, followed by reaction with p-toluenesulfonylmethyl isocyanide (TosMIC) and K 2 CO 3 at 60 • C for 24 h (Schemes 1 and 2).   alkyl chain length between the aromatic group of the amine and the imidazole linker while p-iodobenzylamine can be used to directly compare with p-methoxybenzylamine the difference in bioactivity in the presence of a bulky halogen.
Analogues 43-116 were evaluated for biological activity in the same manner as the earlier compound sets. Surprisingly, from the 74 analogues tested, none were considered active towards MRSA (MIC 32 µg/mL or greater). This result suggests that the structural requirements for activity of the indole-imidazole scaffold towards MRSA is very precise/narrow, with halogenation on 5-position of the indole and a methoxy phenethyl sidechain, making it difficult to identify ways forward in further optimizing the structure. Of note from this set of compounds was the observation of potent antifungal activity against C. neoformans (MIC values of ≤0.25 µg/mL) for both 6-methoxy-phenethyl-indole-imidazole 57 and 5-phenyl-1H-imidazole 111 (Table 1): the lack of cytotoxicity and hemolytic activity for these two compounds identifies them as selective hits and worthy of further investigation.
Currently, the mechanism of antimicrobial action of this compound class is unknown. Thus, we sought to investigate the mechanism of action of this compound class against S. aureus using compound 6, which exhibited strong activity against S. aureus. In a preliminary investigation, compound 6 exhibited only very weak membrane depolarization of S. aureus, suggesting the cellular target in bacteria is not membrane-related. Further investigation to improve the antimicrobial and antifungal activity and identify the mechanism of action of these compounds is underway.
Antibiotics 2022, 11, 1450 6 of 46 ethyl (73-88) and n-pentyl (89-96) side chains. In addition, analogues 97-102 were p pared that retained a common 5-chloroindole fragment, identified as being associat with activity in set 1, but with a wider variation in R3 fragment (Figure 3), while analogu 103-116 explored replacement of the indole fragment with a phenyl or p-methoxyphen group (Figure 4). A total of 74 compounds (43-116) were prepared in this library (Figu S43-S116).   Analogues 43-116 were evaluated for biological activity in the same manner as the earlier compound sets. Surprisingly, from the 74 analogues tested, none were considered active towards MRSA (MIC 32 µ g/mL or greater). This result suggests that the structural requirements for activity of the indole-imidazole scaffold towards MRSA is very precise/narrow, with halogenation on 5-position of the indole and a methoxy phenethyl sidechain, making it difficult to identify ways forward in further optimizing the structure. Of note from this set of compounds was the observation of potent antifungal activity against C. neoformans (MIC values of ≤0.25 µg/mL) for both 6-methoxy-phenethyl-indoleimidazole 57 and 5-phenyl-1H-imidazole 111 (Table 1): the lack of cytotoxicity and hemolytic activity for these two compounds identifies them as selective hits and worthy of further investigation.
Currently, the mechanism of antimicrobial action of this compound class is unknown. Thus, we sought to investigate the mechanism of action of this compound class against S. aureus using compound 6, which exhibited strong activity against S. aureus. In a preliminary investigation, compound 6 exhibited only very weak membrane depolarization of S. aureus, suggesting the cellular target in bacteria is not membrane-related. Further investigation to improve the antimicrobial and antifungal activity and identify the mechanism of action of these compounds is underway.

General Experimental Procedures
High-resolution mass spectra were recorded using a MicrOTOF-QII mass spectrometer (Bruker Daltonics, Bremen, Germany). Melting points were determined on a Reichert-Hofler block and are uncorrected. Infra-red spectra were recorded on a Perkin Elmer Spectrum 100 Fourier Transform infrared spectrometer equipped with a universal ATR accessory. NMR spectra were recorded using a Bruker Avance 400 MHz or Avance III-HD 500 spectrometer operating at 400 or 500 MHz for 1 H nuclei and 100 or 125 MHz for 13 C nuclei. Proto-deutero solvents signals were use as internal references (DMSO-d6: δH 2.50, δC 39.52; CDCl3: δH 7.26, δC 77.16). For 1 H NMR, the data are quoted as position (δ), relative integral, multiplicity (s = singlet, d = doublet, t = triplet, m = multiplet, dd = doublet of doublets, ddd = doublet of doublet of doublets, tt = triplet of triplets, br = broad), coupling constant (J, Hz), and assignment to the atom. The 13 C NMR data are quoted as position (δ), and assignment to the atom. Standard Bruker pulse sequences were utilized. Pressurized (flash) column chromatography was carried out with Kieselgel 60 0.063-0.200 mesh (Merck, Darmstadt, Germany). Analytical thin layer chromatography (TLC) was carried

General Experimental Procedures
High-resolution mass spectra were recorded using a MicrOTOF-QII mass spectrometer (Bruker Daltonics, Bremen, Germany). Melting points were determined on a Reichert-Hofler block and are uncorrected. Infra-red spectra were recorded on a Perkin Elmer Spectrum 100 Fourier Transform infrared spectrometer equipped with a universal ATR accessory. NMR spectra were recorded using a Bruker Avance 400 MHz or Avance III-HD 500 spectrometer operating at 400 or 500 MHz for 1 H nuclei and 100 or 125 MHz for 13 C nuclei. Proto-deutero solvents signals were use as internal references (DMSO-d 6 : δ H 2.50, δ C 39.52; CDCl 3 : δ H 7.26, δ C 77.16). For 1 H NMR, the data are quoted as position (δ), relative integral, multiplicity (s = singlet, d = doublet, t = triplet, m = multiplet, dd = doublet of doublets, ddd = doublet of doublet of doublets, tt = triplet of triplets, br = broad), coupling constant (J, Hz), and assignment to the atom. The 13 C NMR data are quoted as position (δ), and assignment to the atom. Standard Bruker pulse sequences were utilized. Pressurized (flash) column chromatography was carried out with Kieselgel 60 0.063-0.200 mesh (Merck, Darmstadt, Germany). Analytical thin layer chromatography (TLC) was carried out on 0.2 mm thick plates of Kieselgel F 254 or DC-Kieselgel 60 RP-18 F 254 S (Merck). All samples were determined to >95% purity.

General Procedure
A solution of aldehyde (1 eq.) and amine (1 eq.) in DMF (1 mL) was stirred for 3 h under N 2 atmosphere. To the stirred solution was added solid K 2 CO 3 (1 eq.) and p-toluenesulfonylmethyl isocyanide (1 eq.) before heating to 60 • C for 18 h. The reaction mixture was cooled to room temperature and quenched with H 2 O (30 mL). The aqueous layer was extracted with EtOAc (30 mL), and the organic layer was washed with H 2 O (3 × 30 mL) then brine (3 × 30 mL), dried over anhydrous MgSO 4 and concentrated under reduced pressure.

Hemolytic Assay
Human whole blood was washed three times with 3 volumes of 0.9% NaCl and then resuspended in same to a concentration of 0.5 × 10 8 cells/mL, as determined by manual cell count in a Neubauer hemocytometer. The washed cells were then added to the 384-well compound-containing plates for a final volume of 50 µL. After a 10 min shake on a plate shaker the plates were then incubated for 1 h at 37 • C. After incubation, the plates were centrifuged at 1000 g for 10 min to pellet cells and debris, 25 µL of the supernatant was then transferred to a polystyrene 384-well assay plate. Hemolysis was determined by measuring the supernatant absorbance at 405 mm (OD 405 ). The absorbance was measured using a Tecan M1000 Pro monochromator plate reader. HC 10 and HC 50 (concentration at 10% and 50% hemolysis, respectively) were calculated by curve fitting the inhibition values vs. log(concentration) using a sigmoidal dose-response function with variable fitting values for top, bottom and slope [22].

Membrane Depolarization Assay
S. aureus (ATCC25923) was grown in MH II broth for 24 h at 37 • C. After reaching an OD 600 nm of 0.5, cells were centrifuged (3600× g for 20 min at 20 • C) and washed twice with buffered sucrose solution (250 mM), magnesium sulfate solution (25 mM) and Hepes (5 mM) (pH = 7.2). The fluorescent dye 3,3 -diethylthiacarbocyanine iodide DiSC 3 (5) was added to a final concentration of 5 µM and was incubated with the suspensions for 5 min at 37 • C to allow the dye incorporation into the polarized membranes. 10 µL of compound was then added to 90 µL of the fluorescent suspensions at different concentrations ranging from 200 µM to 25 µM. Fluorescence measurements were recorded for 20 minutes (excitation wavelength 622 nm, emission wavelength 690 nm).
The difference in the relative fluorescence values (RFU) from the control containing only buffer and the control containing bacteria treated only with cetyltrimethylammonium bromide (CTAB 1%) is taken as the maximum level of depolarization. Assays were performed in three independent experiments. Blank determined by using no compound. Maximum RFU determined by using CTAB 0.1% Equation used: (RFU of the compound − RFU of the blank) × 100 RFU of the maximum

Conclusions
In a bid to combat the surging increase of antibiotic-resistant bacteria a screening programme of a library of natural products, semi-synthetics and synthetic compounds was initiated targeting the a subset of the ESKAPE pathogens. Following our discovery of weak anti-MRSA activity associated with two 3-substituted-1H-imidazol-5-yl-1H-indoles we have synthesized a further 99 analogues as well as 14 examples of structurally related 5-phenyl-1H-imidazoles. Surprisingly few examples from the extended set of analogues exhibited antimicrobial properties, with only two examples (26 and 32) identified as being promising non-toxic selective anti-MRSA compounds. Of note was the identification of two analogues, 57 (a phenethyl-indole-imidazole) and 111 (a 5-phenyl-1H-imidazole) as being non-toxic selective antifungals towards C. neoformans. Further elaboration of these anti-MRSA and antifungal compounds will be the subject of future research.