Antimicrobial action of arylsulfonamides bearing (aza)norbornane and related motifs: evaluation of new promising anti-MRSA agents

Arylsulfonamides bearing (aza)norbornane and related motifs were evaluated for: (1) antimicrobial activity toward five key ESKAPE pathogenic bacteria, one Gram‐positive bacteria methicillin‐resistant Staphylococcus aureus (MRSA, ATCC 43300), four Gram‐negative bacteria, Escherichia coli (ATCC 25922), Klebsiella pneumonia (ATCC 700603), Acinetobacter baumannii (ATCC 19606), and Pseudomonas aeruginosa (ATCC 27853), and (2) antifungal activity towards two pathogenic fungal strains - Candida albicans (ATCC 90028) and Cryptococcus neoformans var. Grubii (H99; ATCC 208821). Four compounds with 4-nitrobenzenesulfonamide motif (VP-4556, VP-4604, VP-4605, and VP-4509) demonstrated high toxicity towards methicillin‐resistant Staphylococcus aureus (ATCC 43300) with the MIC value of 14.7–49.3 µM. These compounds also possessed high antibacterial activity towards gram-negative bacteria P. aeruginosa (ATCC9027) with the MIC value of 460–555 µM. According to the results of toxicity studies of the compounds to HEK-293, HaCaT, Balb/c 3T3, red blood cells and normal mitogen-activated lymphocytes, three compounds - VP-4556, VP-4604 and VP-4509 - bearing azanorbornene, octahydro-3,5-methanocyclopenta[b]pyrrole and bio-isosteric piperidine motifs were selected for further studies as biocompatible agents with promising antimicrobial activity.


Introduction
In recent years, infectious diseases have become a major challenge to the global health system as killing millions of people worldwide. In addition, they affect socio-economic stability, contributing not only to the growth of the disease's cases, but also leading to psychological disorders, reduction of the economy and living standard [1][2][3]. A wide variety of pathogens and the constant emergence of new multidrugresistant pathogenic strains complicate the treatment and prevention of infectious diseases. At least 30 new infections have insidiously emerged and spread to threaten the health of billions of people around the world, especially in lowincome countries. Unfortunately, for many of them, there are no successful pharmaceuticals or vaccines [4]. To address the above problems, there is an urgent need to develop new antimicrobial drugs.
A significant progress in the use of сage compounds in the drug discovery was caused by the development of leadoriented synthesis concept [27,28]. Thus, cage compounds were proposed as bio-isosteres for arenes, to reduce the number of the aromatic rings in lead-like molecules [29]. Moreover, these compounds have been identified as valuable conformationally constrained analogues of various biologically active molecules, like piperidine or pyrrolidine alkaloids and proline [30].
In summary, cage-like molecules are attractive for the drugs discovery in terms of modern medical chemistry and new concepts, and, therefore, we set out to design and synthesize such derivatives that would be of interest as the antimicrobial agents.
Chemical absorption, distribution, metabolism, excretion, and toxicity (ADMET), play key roles in drug discovery and development. A high-quality drug candidate should not only have sufficient efficacy against the therapeutic target, but also show appropriate ADMET properties at a therapeutic dose. We performed in silico evaluation all the tested compounds using SwissADME tool [54]. Considering druglikeness, we can conclude that all compounds match parameters for Lipinski, Ghose, Veber, Egan and Muegge rules. Lipophilicity in terms of Log P octanol/water for our set of compounds is in the range 0.64-2.60. All compounds are soluble or moderately soluble in water, have high gastrointestinal absorption and no alerts for PAINS filter. For more detailed information, see ESI.
Scheme 1 Synthetic routes to the target compounds Thus, we proposed methods for obtaining compounds that are convenient, variable and allow rapid optimization of the structure, which is especially valuable in drug discovery.

Antimicrobial screening
The primary screening of arylsulfonamides bearing (aza) norbornane and related motifs against 5 key ESKAPE pathogens and 2 fungi was performed by the Community for the Antimicrobial Drug Discovery (CO-ADD), funded by the Wellcome Trust (UK) and The University of Queensland Australia [55,56]. All synthesized compounds were evaluated at 32 µg/mL dose (approx. 100 µM) for (1) (Table S1).
It is important to underline the relationships between the structure and activity of the tested compounds. In all cases, the compounds with the 4-methyl/bromo-benzenesulfonamide moiety showed low activity. However, several compounds maintenance and electron acceptor nitro group showed excellent activity towards the bacterium Staphylococcus aureus (ATCC 43300). Noteworthy, only nitro derivatives with a para-position of the nitro group possessed high activity.
Bacterial pathogens have rapidly developed resistance to antimicrobial agents. The antimicrobial resistance systems involve the involvement of bacterial molecular and cellular-based machinery [57][58][59][60]. These resulted in an urgent need to develop novel effective antibiotics. To confirm the antibacterial potential, we have examined the toxicity of these drugs towards methicillin-resistant Staphylococcus aureus. Thus, a number of compounds (VP-4556, VP-4604, VP-4605, and VP-4509) were discovered that totally inhibited the growth of methicillin-resistant Staphylococcus aureus with growth inhibition GI > 95% (see Table S1 in ESI). All five highly active compounds contained a 4-nitrobenzenesulfonamide motif, indicating its beneficial role for inducing antimicrobial effect. Among them, there are two cage compounds VP-4556 and VP-4509 and two containing morpholine VP-4604 and piperidine VP-4604 substituents, respectively. This strain of bacteria spread mostly in hospitals and has resistance to multiple antibiotics. Therefore, obtaining substances with antibacterial activity to this strain will reduce the number of nosocomial infections, which provides a lot of death and side effects [61]. Thus, compounds with 4-nitrobenzenesulfonamide motif possessed high growth inhibition activity towards drug-resistant S. aureus and could be promising antimicrobial agents.
The minimal inhibitory concentration (MIC; µM) measurements were performed for compounds with significant microbial growth inhibition (VP-4556, VP-4604, VP-4605 and VP-4509) towards Staphylococcus aureus (ATCC 43300), using ceftriaxone, as a reference drug. At the same time, for the active compounds, primary cytotoxicity towards human embryonic kidney cell line and hemolysis of human red blood cells human cells was determined. Haemolysis assay is the most commonly used for initial toxicity investigation of different agents. Human erythrocytes are the most frequently used for the preliminary in vitro study of the haemolytic activity of antimicrobial and other drugs [62]. Compounds VP-4556, VP-4604, VP-4605, and VP-4509 have the best antibacterial activity, comparable to ceftriaxone. Cytotoxic concentration (CC 50 ) of the compound VP-4605 is approximately 5 times higher (71.4 ± 4.4 µM) for HEK-293 cells than that of MIC (14.7 ± 1.3 µM) towards methicillin-resistant S. aureus. Another tested compounds VP-4556, VP-4604, and VP-4509 were also tolerated and non-toxic for human cells, as the cytotoxic and hemolytic dose was higher than the therapeutic dose ( Table 1) Taking into account the primary screening results, next we checked the antibacterial effect of most active compounds towards not-resistant S. aureus ATCC25923 and another strain of the Gram-negative bacteria P. aeruginosa (ATCC9027) using the MTT assay. This method is based on the changes of a colour reaction due to the metabolism of the MTT reagent in bacteria. The substances under study were solved in DMSO, thus, we used DMSO as a substance for comparison in equal doses like the doses of the studied compound.
The most active compounds were tested towards Staphylococcus aureus ATCC25923. Figure 2 presents the effect of compounds VP-4556, VP-4604, VP-4605, and VP-4509 towards S. aureus. Compound VP-4556 was most active towards S. aureus (Fig. 2) in a low concentration.
In conclusion, the results obtained by screening methicillin-resistant Staphylococcus aureus (MRSA) strain and other microorganisms differ from the results obtained at using the MTT method. These results allow us to argue that 4-nitrobenzenesulfonamide derivatives have a specific effect on bacteria, rather than a general toxic effect. We can suggest that the synthesized 4-nitrobenzenesulfonamides cause the destruction of bacterial walls by interacting with structural elements of the bacterial wall, as well as specifically affect them or some internal mechanisms of bacteria functioning. In future, we are going to identify which of the biomolecules could interact with the studied 4-nitrobenzenesulfonamide.

Cytotoxicity
The antimicrobial agents needs to demonstrate low or no toxicity towards mammalian cells [65,66]. Studied derivatives demonstrated low toxicity towards mitogen-activated lymphocytes isolated from healthy adult human peripheral blood. The CC 50 value of studied compounds VP-4509, VP-4556, VP-4604, and VP-4605 was approximately 2500 μM for mitogen-activated human blood lymphocytes (Fig. 5).
Thus, studied compounds had low toxicity towards pseudo-normal cells of HaCaT line (human keratinocytes) and Balb/c 3T3 line (murine fibroblasts), and mitogenactivated lymphocytes isolated from healthy adult human peripheral blood.
In order to establish a therapeutic potential of derivatives VP-4509, VP-4556, VP-4604, VP-4605, we have calculated the values of the selectivity index (SI) of the tested derivatives towards methicillin-resistant S. aureus compared to normal mammalian cells of HEK-293, HaCaT and Balb/c 3T3 lines, and lymphocytes isolated from healthy adult human peripheral blood. The higher values of SI indicate greater specificity, and the compounds displaying the SI above 10.0 are considered to be selective [67]. The median SI above 10.0 was found for studied compounds VP-4509, VP-4556, VP-4604, VP-4605 towards methicillin-resistant S. aureus (Table 2). Thus, studied derivatives demonstrated high selectivity towards methicillin-resistant S. aureus.

Conclusion
Summarizing the results of the antibacterial activity and toxicity, we found that the 4-nitrobenzenesulfonamides