Isatin Bis-Indole and Bis-Imidazothiazole Hybrids: Synthesis and Antimicrobial Activity

Isatin and its derivatives are important heterocycles found in nature and present in numerous bioactive compounds which possess various biological activities. Moreover, it is an essential building block in organic synthesis. The discovery of novel compounds active against human pathogenic bacteria and fungi is an urgent need, and the isatin may represent the suitable scaffold in the design of biologically relevant antimicrobials. A small library of 18 isatin hybrids was synthetized and evaluated for their antimicrobial potential on three reference strains: S. aureus, E. coli, both important human pathogens infamous for causing community- and hospital-acquired severe systemic infections; and C. albicans, responsible for devastating invasive infections, mainly in immunocompromised individuals. The study highlighted two lead compounds, 6k and 6m, endowed with inhibitory activity against S. aureus at very low concentrations (39.12 and 24.83 µg/mL, respectively).


Introduction
The exploration of privileged heterocyclic frameworks is one of the most significant areas in drug discovery, and isatin is an essential building block in organic synthesis.
Isatin (indoline-2,3-dione) and its derivatives are important heterocycles found in nature and present in numerous bioactive compounds, which can act as anticancer, antitubercular, anti-HIV, antimalarial, and antimicrobial agents [1]. The most attractive application of isatin in organic synthesis is undoubtedly in the highly reactive C-3 carbonyl group that readily undergoes condensation reaction, but the N-1 and C-5 positions are also domains of chemical variations.
In recent years, various isatin derivatives have been screened for their antimicrobial activities, and some of them demonstrated promising in vitro and in vivo activity [2].
Molecular hybridization involves a combination of pharmacophore moieties/bioactive substances to produce a novel compound with better efficacy than potent substrates [13][14][15].
With this in mind and on the basis of our experience on imidazo [2,1-b]thiazoles and indolinones chemistry, we have designed a new series of isatin bis-indole and isatin bisimidazothiazole hybrids; specifically, in the isatin moiety almost all positions have been modified, while only in some cases the indoles have been decorated with a methoxy group in position 5, and the imidazothiazole was maintained unaltered.
The herein synthetized isatin derivatives were screened in vitro for their antibacterial and antifungal activity. In particular, the potential of the compounds was investigated against Staphylococcus aureus and Escherichia coli as representative Gram-positive and Gramnegative bacteria, and Candida albicans as a yeast model.
S. aureus and E. coli are important human pathogens of concern, infamous for causing community-and hospital-acquired infections with high mortality rates in the case of severe systemic infections. Indeed, they are common causes of bacterial infections, but they can also enter the bloodstream and reach different organs and tissues of the human body, thereby causing metastatic infections [16]. The treatment of such infections, unfortunately, has become even more difficult due to the emergence of multiple drug resistance. As for S. aureus, this is best exemplified by the methicillin-resistant S. aureus (MRSA) strains. Regarding E. coli, resistance to critically important antimicrobials, such as third/fourth/fifth-generation cephalosporins and quinolones, and multidrug resistance (MDR) are recognized globally. Recently, the relevance of fungi as human pathogens has been widely recognized because many species, including C. albicans, are responsible for devastating invasive infections, mainly in immunocompromised individuals, such as patients subjected to organ transplantation and those affected by cancer or AIDS. Antifungal therapy failure as well is gradually increasing worldwide because of the diffuse resistance to routinely used antifungal drugs, namely polyenes, azoles, and echinocandins [17].
Combined, the discovery of novel compounds active against human pathogenic bacteria and fungi is an urgent need, and the isatin may represent the suitable scaffold in the design of biologically relevant antimicrobials.

Chemistry
The synthetic route to the designed compounds 4-6 is reported in Scheme 1.
against Staphylococcus aureus and Escherichia coli as representative Gram-positive Gram-negative bacteria, and Candida albicans as a yeast model.
S. aureus and E. coli are important human pathogens of concern, infamous for cau community-and hospital-acquired infections with high mortality rates in the case o vere systemic infections. Indeed, they are common causes of bacterial infections, but t can also enter the bloodstream and reach different organs and tissues of the human bo thereby causing metastatic infections [16]. The treatment of such infections, unfortuna has become even more difficult due to the emergence of multiple drug resistance. As S. aureus, this is best exemplified by the methicillin-resistant S. aureus (MRSA) strains. garding E. coli, resistance to critically important antimicrobials, such as third/fourth/f generation cephalosporins and quinolones, and multidrug resistance (MDR) are re nized globally. Recently, the relevance of fungi as human pathogens has been widely ognized because many species, including C. albicans, are responsible for devastating in sive infections, mainly in immunocompromised individuals, such as patients subjecte organ transplantation and those affected by cancer or AIDS. Antifungal therapy failur well is gradually increasing worldwide because of the diffuse resistance to routinely u antifungal drugs, namely polyenes, azoles, and echinocandins [17].
Combined, the discovery of novel compounds active against human pathogenic teria and fungi is an urgent need, and the isatin may represent the suitable scaffold in design of biologically relevant antimicrobials.

Chemistry
The synthetic route to the designed compounds 4-6 is reported in Scheme 1. For R-R5 see Table 1. For R-R 5 see Table 1.
The structures of the final compounds were confirmed by means of 1 H-NMR, 13 C-NMR and HRMS spectra.
The analysis of the 1 H-NMR spectra allowed us to make some considerations. The derivatives with the isatin core bearing two indole groups have a plane of symmetry so that the protons of the two indoles are in the same chemical environment. In fact, for each proton of the two indoles, the spectra show only one signal integrating for two protons, thus confirming that the two indoles are equivalent.
Moreover, in most of these derivatives a coupling between the indole NH and the proton in position 2 (ind2) is observed, which gives rise to two doublets having the same J. In some cases, the NH signal is a broad singlet, but the coupling is confirmed in the D 2 O exchange spectra: the indole NH protons exchange and the ind2 doublet becomes a singlet.
Conversely, the compounds 6 with the isatin core bearing two imidazothiazoles have no plane of symmetry; thus, the imidazothiazoles are in different chemical environments. Indeed, each imidazothiazole proton gives its own signal, and in compounds 6g and 6m, the benzylic methylene protons form two coupling doublets instead of a singlet because they are diastereotopic.

Biological Evaluation
The multi-substituted isatin derivatives were assayed in vitro against Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, and Candida albicans ATCC 10231. These strains were selected as representative Gram-positive, Gram-negative, and fungal species responsible for a broad spectrum of human infections. All compounds were tested at 100 µM against the three microbial species and on epithelial cells (Vero, ATCC CCL-81) to measure their ability to affect mammalian cell metabolism.
At first glance at the data reported in Table 2 and Figure S1, some general remarks can be drawn. As for the antibacterial activity, the tested compounds interfered with S. aureus and E. coli growths to varying degrees; indeed, several derivatives reduced S. aureus proliferation, and compounds 4c, 6k and 6m proved to be highly active against this Gram-positive strain. Conversely, none of them displayed inhibitory properties toward E. coli. There are many examples of compounds, including clinically used antimicrobial drugs, which are antibacterial against Gram-positive organisms but which are ineffective against Gramnegative ones [30][31][32][33]. It is plausible that the differences in cell envelope structure would present differential barriers to penetration that would be reflected in divergent physicochemical property patterns for molecules effective against the two Gram species. As for the antifungal activity, only 4h significantly reduced the growth of C. albicans when tested at 100 µM (48.93 µg/mL), thus suggesting a generally high resistance of the yeast to the isatin derivatives. Indeed, literature reports scarce data concerning the antifungal properties of the isatin-based compounds [1], and the effectiveness of some small series of derivatives has been mainly demonstrated against filamentous fungi in the range of concentration 20-100 µg/mL [34].
In the frame of a comprehensive analysis on the antimicrobial potential of the isatin derivatives, all compounds were also evaluated for their cytotoxicity on the Vero cell line. These non-malignant cells are internationally recommended as a standard to study cytotoxicity; they have well-defined culturing characteristics in all experimental settings, thus suitable to define the overall safety of the isatin analogues [ISO 10993-5; Biological Evaluation of Medical Devices-Part 5: Tests for In Vitro Cytotoxicity. International Organization for Standardization: London, UK, 2009]. Generally, in vitro assessment of the toxicological profile of pure compounds as well as naturally inspired products must be part of the pipeline in a drug discovery perspective to discriminate a selective antimicrobial effect from a generic cytotoxicity on cells [35,36]. Specifically, as for isatin derivatives, these investigations are crucial as the anti-cancer activity of the isatin "building block" has been widely demonstrated [1,34]. As a matter of fact, in our experimental conditions, eight compounds significantly interfered with Vero metabolism, confirming the overall cytotoxicity of these compounds.
Comparing the antimicrobial activity of 4c, 6k, and 6m with their cytotoxicity results, a selective inhibitory property against the Gram-positive strain was only confirmed for compounds 6k and 6m. Indeed, derivative 4c strongly reduced S. aureus growth as well as Vero cell metabolism, thus excluding its specific antibacterial activity.
Overall, it seems that when the indolinone nucleus is linked to two indoles, the derivatives affect Vero cells proliferation, as in case of compounds 4c, 4e, 4f, 4h, and 4j-l. On the other hand, the substitution of the two indole systems with the imidazothiazole nucleus, leading to compounds 6, determined a lower cytotoxicity against Vero cells and at the same time, when the indolinone is properly substituted, a good efficacy versus S. aureus (compounds 6k and 6m).
Therefore, derivatives 6k and 6m, inhibiting S. aureus growth at a non-toxic concentration, were subjected to dose-response experiments to determine their IC 50 values (Figure 1). The percentage values of the bacterial inhibition defined complete sigmoidal curves ranging from 0 to 100% response, thus indicating the efficacy of the derivatives to completely inhibit bacterial growth at the highest tested concentration (400 µM). Compound 6k showed an IC 50 of 79.95 µM (39.12 µg/mL) (95% confidence interval 73.15-87.38 µM), and compound 6m showed an IC 50 of 50.74 µM (24.83 µg/mL) (95% confidence interval 41.80-61.59 µM). By way of conclusion, these bis-imidazothiazole isatin hybrids demonstrated excellent antibacterial activity, higher than some isatin-azole hybrids [37] and isatin-coumarin hybrids [38], and close to certain isatin-carbohydrazides previously described [2]. investigations are crucial as the anti-cancer activity of the isatin "building block" has been widely demonstrated [1,34]. As a matter of fact, in our experimental conditions, eight compounds significantly interfered with Vero metabolism, confirming the overall cytotoxicity of these compounds.
Comparing the antimicrobial activity of 4c, 6k, and 6m with their cytotoxicity results, a selective inhibitory property against the Gram-positive strain was only confirmed for compounds 6k and 6m. Indeed, derivative 4c strongly reduced S. aureus growth as well as Vero cell metabolism, thus excluding its specific antibacterial activity.
Overall, it seems that when the indolinone nucleus is linked to two indoles, the derivatives affect Vero cells proliferation, as in case of compounds 4c, 4e, 4f, 4h, and 4j-l. On the other hand, the substitution of the two indole systems with the imidazothiazole nucleus, leading to compounds 6, determined a lower cytotoxicity against Vero cells and at the same time, when the indolinone is properly substituted, a good efficacy versus S. aureus (compounds 6k and 6m).
Therefore, derivatives 6k and 6m, inhibiting S. aureus growth at a non-toxic concentration, were subjected to dose-response experiments to determine their IC50 values (Figure 1). The percentage values of the bacterial inhibition defined complete sigmoidal curves ranging from 0 to 100% response, thus indicating the efficacy of the derivatives to completely inhibit bacterial growth at the highest tested concentration (400 µ M). Compound 6k showed an IC50 of 79.95 µM (39.12 µ g/mL) (95% confidence interval 73.15-87.38 µ M), and compound 6m showed an IC50 of 50.74 µM (24.83 µ g/mL) (95% confidence interval 41.80-61.59 µ M). By way of conclusion, these bis-imidazothiazole isatin hybrids demonstrated excellent antibacterial activity, higher than some isatin-azole hybrids [37] and isatin-coumarin hybrids [38], and close to certain isatin-carbohydrazides previously described [2]. Having demonstrated the antibacterial activity of 6k and 6m against the reference strain of S. aureus, 10 clinical isolates presenting different antibiotic susceptibilities and including both methicillin-sensitive (MSSA) and methicillin-resistant strains (MRSA) were tested (Table S1). Isatin derivatives proved to be effective toward MSSA as well as MRSA strains; it is worth noting that IC50 values obtained for the clinical isolates were close to those of the reference bacterial strain and ranging from 73.01 µM to 85.31 µM and from 45.02 µM to 59.38 µM for 6k and 6m, respectively. Having demonstrated the antibacterial activity of 6k and 6m against the reference strain of S. aureus, 10 clinical isolates presenting different antibiotic susceptibilities and including both methicillin-sensitive (MSSA) and methicillin-resistant strains (MRSA) were tested (Table S1). Isatin derivatives proved to be effective toward MSSA as well as MRSA strains; it is worth noting that IC 50 values obtained for the clinical isolates were close to those of the reference bacterial strain and ranging from 73.01 µM to 85.31 µM and from 45.02 µM to 59.38 µM for 6k and 6m, respectively.
Compounds were not recognized by SwissADME software (Lousanne, Switzerland, 2022) [http://www.swissadme.ch/index.php] (accessed on 25 July 2022) as PAINS (Pan Assay INterference compoundS), i.e., molecules containing substructures showing potent response in assays irrespective of the target. This evidence allows to exclude that the studied derivatives interact nonspecifically and yield false positive biological output. Compounds, 4d-i, 4l-m, 5d, 5m, 6c, 6g, 6k, and 6m Isatin (1.72 mmol) was dissolved in 20 mL of isopropanol taken in a reaction vessel, and iodine (5 mmol) was added to it. The appropriate indole or imidazo[2,1-b]thiazole (3.46 mmol) was added to the mixture, and the reaction was continued with constant stirring for 15 min-2 h (according to a TLC test). After completion of reaction, the mixture was concentrated under reduced pressure in a rotary evaporator. It was then extracted with chloroform, and the extract was washed with saturated sodium thiosulphate solution to decompose the remaining iodine. From the aqueous phase during the night a precipitate was formed which was collected by filtration. The crude products were crystallized from ethanol.  13  Microbial inocula were prepared at 0.5 McFarland in PBS and, subsequently, bacterial suspensions were diluted 1:200 in Mueller-Hinton broth (Sigma-Aldrich, St. Louis, MO, USA), while fungal inoculum was diluted 1:20 in RPMI-1640 medium (Gibco ® , Ther-moFisher Scientific Inc., Waltham, MA, USA), containing glucose 2%, 0.3% levo-glutamine buffered to pH 7.0 with 0.165 M 3-(N-morpholino)propanesulfonic acid (MOPS). A total of 100 µL of these microbial suspensions were introduced in a 96-well microplate and treated with 100 µL of the compound at 100 µM or with two-fold serial dilutions of the compound in the range of 400 µM-3.12 µM. All compounds were previously dissolved in 100% DMSO at a concentration of 20 mM. Experiments included controls used to measure the microbial growth in regular medium (positive control) and to check the background turbidity of the reagents and the sterility of the procedures (negative controls). In addition, microbial growths were assessed in the presence of the DMSO solvent in the range of 0.015-2%. The inoculated plate was incubated at 37 • C for 24 h, and subsequently, the optical density at 630 nm was measured by the Multiskan Ascent microplate reader (Thermo Fisher Scientific Inc., Waltham, MA, USA). The effectiveness of the compounds was expressed as percent inhibition relative to the positive growth controls, and the isatin derivatives were defined as active when the inhibition yielded the 50% at 100 µM. At the tested concentrations, the DMSO did not interfere with cell proliferations. These compounds were further evaluated to define their IC 50 values by interpolation of the dose-response curves generated by plotting the percentages of growth inhibition, relative to the positive control (set to 100% of growth), as a function of the tested concentrations (GraphPad Prism version 5.0 for Windows, San Diego, CA, USA). All the experiments were performed on triplicate in at least two independent assays.

Cytotoxicity Test
African green monkey kidney cells (Vero ATCC CCL-81) obtained from ATCC were cultured in Eagle's minimal essential medium (MEM) (Sigma-Aldrich, St. Louis, MO, USA), supplemented with 10% fetal bovine serum (FBS) (Carlo Erba Reagents, Milan, Italy), 100 U/mL penicillin, and 100 µg/mL streptomycin at 37 • C with 5% CO 2 . For the experiments, the cells were seeded into 96-well plates at 10 4 cells/well and incubated at 37 • C for 24 h. Following washes with PBS, the cell monolayer was incubated with 100 µL of medium containing 100 µM of the derivatives. Both untreated cells and cells incubated with the solvent were included in each experiment as controls.
The cell viability was assessed by a WST8-based assay according to the manufacturer's instructions (CCK-8, Cell Counting Kit-8, Dojindo Molecular Technologies, Rockville, MD, USA). After 48 h of incubation, the culture medium was removed from each well, the monolayer was washed with PBS, and 100 µL of fresh medium containing 10 µL of CCK-8 solution were added. Following 2 h at 37 • C, the absorbance was measured at 450/630 nm; data were calculated as the percentage of the cell viability relative to the untreated controls. At the tested concentrations, the DMSO did not interfere with Vero metabolism. Isatin derivatives demonstrating inhibitory activity on Vero cells metabolism superior to 70% at 100 µM were defined as cytotoxic. All the experiments were performed on triplicate in at least two independent assays.

Conclusions
A small library of 18 isatin-based derivatives was synthetized and evaluated for their antimicrobial potential on three reference strains, including S. aureus, E. coli, and C. albicans. The study delivered two lead compounds, 6k and 6m, endowed with excellent inhibitory activity against S. aureus in comparison with other isatin hybrids reported in literature. The compounds were found to be sufficiently soluble for the herein performed in vitro tests (as reported in the section Materials and Methods); however, they are not very soluble as determined by in silico analysis, using the free online software SwissADME (http: //www.swissadme.ch/index.php (accessed on 25 July 2022). From a future perspective, in the next design we will consider the solubility improvement based on the structure of the most interesting ones. The described isatin derivatives showed good antibacterial activity on the reference strain as well as on 10 clinical isolates obtained from different biological specimens indicating that the compounds have in vitro potential also towards human pathogens circulating in the population. As the isatin derivatives revealed a fascinating array of pharmacological activities, including broad-spectrum antiproliferative properties, the herein synthetized molecules were also evaluated for their cytotoxicity on mammalian cells, and the selective inhibitory effect towards bacterial cell was confirmed for 6k and 6m. The overall data therefore indicate the importance of the isatin nucleus in the field of medicinal chemistry as an antibacterial agent. From a future perspective, derivatives demonstrating cytotoxicity on the non-malignant cell model herein used could be investigated on cancer cell lines to measure their anti-cancer potency.