Recent development of membrane-active molecules as antibacterial agents

European Journal of Medicinal Chemistry 184 (2019) 111743

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European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech

Mini-review

Recent development of membrane-active molecules as antibacterial agents Nan Zhang, Shutao Ma* Department of Medicinal Chemistry, Key Laboratory of Chemical Biology (Ministry of Education), School of Pharmaceutical Sciences, Shandong University, 44 West Culture Road, Jinan, 250012, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 7 April 2019 Received in revised form 9 May 2019 Accepted 26 September 2019 Available online 27 September 2019

The continuous emergence of drug-resistant bacteria has become a severe threat to the public health. Therefore, the discovery of novel antibacterial mechanisms to combat this jeopardized problem is urgently needed. In the past decades, plenty of new antibacterial modes of action have been discovered continuously, based on which many promising scaffolds have been designed and synthesized. In particular, cationic amphiphilic small-molecules open a door to the new mode of action of bactericidal agents by depolarizing and disturbing the bacteria membrane. The cationic amphiphilic are characterized by high efficacy, resistant-proof, wide-spectrum, and high selectivity toward bacteria. In this review, we summarized recent advances in the discovery of membrane-active small-molecules and their structureactivity relationships (SARs), hoping to provide an evidence for future research and development of new antibacterial agents with new mechanism. © 2019 Elsevier Masson SAS. All rights reserved.

Keywords: Membrane-active molecules Cationic amphiphilic compounds Antibacterial agents Bacterial resistance

1. Introduction Infectious diseases caused by bacterial pathogens have become a severe threat over the years, leading to high morbidity and mortality all over the world [1]. Even though new antibiotics have been developing continuously, which have saved tens of thousands of lives of human race, the rate of bacterial infections has never reduced or stagnated. What’s the worse, the emergence of multidrug resistant bacteria has become one of the biggest catastrophes to public health and economy. According to the O’Neill commission, it is estimated that over 10 million lives will be deprived and 100 trillion dollars be cost to fight against the antimicrobial resistance by 2050 [2]. Drug-resistant bacteria are mainly formed by bacterial adaptation to antibiotics, gene mutation of bacteria and the over expression of efflux pump [3]. This problem is aggravated by widespread use even abuse of antibacterial agents, which is prevalent in most countries in the world. In the period from 1983 to 2012, new systemic antibacterial drugs approved by FDA (Food and Drug Administration of America) kept to decrease while bacteria resistance phenomenon was becoming even worse [4]. Therefore, there is a desperate need of new mechanisms to develop novel

* Corresponding author. E-mail address: [email protected] (S. Ma). https://doi.org/10.1016/j.ejmech.2019.111743 0223-5234/© 2019 Elsevier Masson SAS. All rights reserved.

antibacterial agents in response [5,6]. The bacterial membrane, a crucial structure for cell survival, has attracted quite a lot attention to develop novel antibacterial agents in the past two decades [7]. Firstly, since the bacterial membrane is essential at any phase of any bacteria, it could be an effective strategy to disturb its function as a broad-spectrum method to eradicate bacteria, no matter if the bacteria is prolific or dormant. Secondly, the cell membrane contains almost one third of proteins which play a significant role in bacterial metabolism, incomplete cell membrane will thus interrupt necessary physiological function, conducing to the death of organism. Thirdly, damaging the membrane will affect the maintenance of homeostasis and reduce the possibility of resistance to membrane-acting drugs [8]. Furthermore, membrane permeabilizing drugs are potential to serve as chemosensitizers to increase the activity of other antibacterial agents [9]. For the above reasons, bacterial cell membrane has become a fascinating target to develop new antibacterial agents that fight against the problematic bacterial resistance. Hereunder, we describe bacterial membrane as a non-specific anti-infectious target, and highlight recent progress in the development of membrane-active antibacterial compounds with less toxicity and less probability to induce bacterial resistance.

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2. Bacterial cell membrane and the mode of action Between the bacteria itself, the structure and molecular components of their cell membranes differ from each other. Grampositive bacterial membrane is simpler than that of Gramnegative bacteria, consisting of a cytoplasmic plasma membrane (CM) and lipoteichoic acids (LTA) attaching to a thick layer of peptidoglycan (Fig. 1) [10]. Whereas Gram-negative bacterial membrane consists of an inner membrane (IM), a thin layer of peptidoglycan, and an outer membrane (OM) containing lipopolysaccharides (LPS) (Fig. 1) [11]. Even though they have structural diversity, LTA and LPS are both amphiphilic and anionic, which creates a necessary condition for membrane-active drugs with high selectivity. Despite of these, phosphatidylglycerol (PG) and cardiolipin (CL) are also crucial reasons for the negative-charged property of the bacterial membrane. In a remarkable contrast, in mammalian cell membranes, anionic lipids are sequestered to the monolayer facing the interior of the cell, and its surface is composed of largely zwitterionic sphingomyelin and phosphatidylcholine [12,13]. Generally, agents that disturb the physical integrity of the cell membrane are typically lipophilic and positively charged. Their cationic side is mainly attached to the negativecharged surface of bacterial membranes, and meanwhile the lipophilic side inserts into the lipid bilayer, leading to the instability and disruption of the bacterial membrane and eventually death [14]. Nonetheless, the cationic side cannot be attached to the zwitterionic mammalian cell surface, which creates the adequate condition for selectivity. 3. Membrane-active antibacterial compounds Host defense antimicrobial peptides (AMPs) with cationic amphipathic structures are one of the natural membrane-active compounds which exert their effect on bacterial membrane by disturbing its integrity. However, there are many obstacles that prevent AMPs from being widely used in practice so far. For example, AMPs are not stable to metabolic enzymes, resulting in poor bioavailability and low proteolytic stability in vivo, and they can cause potential immunogenicity in some cases. Moreover, moderate efficacy and high production cost of AMPs also limit their uses in clinic [13]. In order to overcome these weaknesses, a variety of other cationic amphipathic mimics have been designed and optimized, contributing significantly to the development of membrane-active drugs. 3.1. Amines It is noticed that many membrane-active compounds have amino groups that form a hydrophilic area in their molecules, which is essential for their antibacterial property. Herein, the amine

compounds with membrane-disturbing activity published recently are introduced, including alkylamines, aromatic group containing amines and quaternary amines.

3.1.1. Alkylamines A series of dialkyl cationic amphiphilic peptide mimics containing two certain-length lipophilic alkyl chains that attach to the amino acid residues in their structure were synthesized and evaluated for their bioactivities against both drug-sensitive and drugresistant bacteria (Gram-positive and Gram-negative). In the series, compound 1 (Fig. 2) was a lysine-based membrane-active molecule with two different alkyl chains, which displayed MIC values of 5 mM against both Staphylococcus aureus and Escherichia coli, and 10.5 mM against Enterococcus faecium and Acinetobacter baumannii, respectively [15]. However, 1 was found to show slight corrosive toxicity during the first two days in mouse model. Later in 2018, Zhang et al. [16] designed and synthesized another series of novel L-lysine derivatives containing two same alkyl chains. Among them, the most active compound 2 was deserved to be mentioned for the MIC values against methicillin-resistant S. aureus (MRSA), carbapenemase-producing Klebsiella pneumoniae (KPC) and New Delhi metallo-b-lactamase-1-producing Enterobacteriaceae (NDM) were 0.5, 2 and 2 mg/mL, respectively. With a HC50 (concentration corresponding to 50% lysis of the human erythrocytes) value of 133 mg/mL, 2 had a good selectivity (S ¼ HC50/MIC) against S. aureus. Structure-activity relationship (SAR) studies indicated that the length of lipophilic chains of the molecules influenced their antibacterial activity significantly, optimally C7H15 to C9H19, while the kinds of amino acids had also a huge impact on their efficacy. Moreover, both 1 and 2 acted rapidly not only on bacterial membrane but also on bacterial biofilm, giving them less possibility to induce bacterial resistance and more potential to combat drugresistant phenomenon. D-Amino acids were reported to be natural triggers for biofilm disassembly [17], motivated by which, Konai et al. designed and synthesized a series of D-amino acids compounds. Among them, compound 3, obtained by the conjunction of fatty acid and D-lysine, was found to be able to eliminate growing planktonic MRSA at a low concentration (MIC ¼ 3.1e6.3 mg/mL), and to have the capacity to influence non-dividing stationary phase bacteria [18]. Additionally, 3 was also proven to eradicate established biofilms and not to develop bacterial resistance. In vivo studies showed that it had anti-MRSA activity with 2.7 and 3.9 Log reduction at 20 and 40 mg/ kg, respectively, in a superficial skin infectious murine model, but no toxicity detected. Based on the same strategy above, a series of lipopeptides were designed and synthesized, among which compound 4 was the most potent one. 4 showed good antibacterial activity (MIC ¼ 6.3e12.5 mg/mL) against dangerous bacteria like A. baumannii, MRSA and K. pneumoniae, etc., with even no hemolytic activity or cytotoxicity [19]. The SAR analysis indicated that

Fig. 1. Components of Gram-positive and Gram-negative bacteria.

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Fig. 2. Chemical structures of compounds 1e4.

compounds with more density of positive charges possessed more potent activity and less toxicity than those had less density of positive charges. 3.1.2. Aromatic group containing amines In the process of designing and synthesizing simple AMP mimics without complex synthesis steps, compound 5 (Fig. 3), a dimeric lysine N-alkyl aromatic amide, was identified as a broad-spectrum and potent molecule which displayed high efficacy against MRSA and MRSE (methicillin-resistant S. epidermidis) with the MIC values of 0.75e1.5 and 1.5e3 mg/mL, respectively [20]. It also exhibited excellent antibacterial potency against VRE (vancomycin-resistant Enterococcus faecalis), E. coli, and Pseudomonas aeruginosa in the ranges of 3e6, 0.75e1.5, and 3e6 mg/mL, respectively. It was

observed that the balance between its hydrophobicity and hydrophilicity was crucial for optimal antibacterial activity. Moreover, ability to eliminate bacteria quickly and inhibit biofilm formation suggested that 5 could be a new therapeutic agent to combat the bacterial resistance. A class of cationic deacetyl linezolid derivatives were developed and evaluated for their bioactivities by Bai et al., in 2018. Among them, compound 6 not only had the best antibacterial activity (MIC ¼ 2e16 mg/mL) taking linezolid (MIC ¼ 2e>64 mg/mL) as a control against Gram-negative bacteria E. coli and Salmonella enterica, and Gram-positive S. aureus and E. faecalis, but also displayed good MIC values ranging from 2 to 4 mg/mL against 10 different clinical isolated MRSA strains [21]. From the SARs, it could be concluded that amphiphilic structure was essential for broad-

Fig. 3. Chemical structures of compounds 5e14.

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spectrum activity, especially against Gram-negative bacteria, which meant that the length of the alkyl chain was the point to be active, whereas different hydrophilic amino acid residues (lysine and arginine) displayed little influence on their antibacterial activity. Importantly, time-kill kinetics showed that 6 acted on bacteria rapidly and did not induce bacterial resistance. Besides, no toxicity was detected for 6 toward mammalian red blood cells (RBCs) and HeLa cells. Hydantoin pharmacophore has been applied to develop antibacterial agents for a long time, but the action of them is complicated and not fully understood [22]. Su et al. designed and synthesized a new series of membrane-active compounds by combining hydantoin derivatives with hydrophobic tails and cationic charged groups (lysine). Among them, the most active compound 7 displayed enhanced antimicrobial activity (MIC ¼ 0.5e1 mg/mL) compared to nitrofurantoin (a marketed hydantoin antibiotic, MIC ¼ 6.25e>50 mg/mL) against MRSA, MRSE, VREF (vancomycin-resistant E. faecalis), P. aeruginosa and K. pneumoniae, but had limited hemolytic activity [22]. Although the mechanism of the hydantoin is complex, this series still acted on bacterial membrane as well as their potential targets such as DNA and ribosomes synergistically. Being able to kill bacterial pathogens rapidly and prevent the development of resistance under tested conditions, 7 also exhibited excellent in vivo efficacy in a rat model bearing MRSA-induced pneumonia, superior to vancomycin as a control. Since reduced-amide-containing molecules were more stable to proteolytic degradation [23], a class of acylated reduced-amide based scaffold derivatives were designed and synthesized, performing good membrane disintegrating activity. Among them, compound 8 displayed the best antibacterial activity against MRSA with an MIC value of 1.56 mg/mL [24]. The SAR analysis (Fig. 3) was obvious that when R1 and R4 were hydrophobic groups, as well as R2 and R3 were cationic groups, this class showed no activity at all. Kept R1 as the cationic group, compounds with R2 as the hydrophobic group still displayed no efficacy. In remarkable contrast, when R2, R3 and R4 were all hydrophobic groups, they showed broad-spectrum antimicrobial activity, which was improved along with the hydrophobicity of R4. As for the cationic groups, it was noticed that the amino group in structure gave slightly better activity than the guanidino group, and the introduction of cationic charges would reduce hemolytic activity, which had been proven before [25]. On the basis of the above findings, compound 9 was designed and synthesized with one more lysine residue on the R1 position compared with 8 [24]. It had the same MIC value (1.56 mg/ mL) as that of 8 against the Gram-positive bacteria tested. As for the Gram-negative strains, 9 was weaker than 8 in the antibacterial activity in the experimental condition. This suggests that the increase of cationic groups may not be preferred for bacterial killing. Further, replacement of the lysine residue with ornithine residue increased hemolytic activity, while arginine residue did not has much influence. Moreover, 8 and 9 were able to kill MRSA in a dosedependent fashion and effectively inhibit the formation of biofilm. In vivo studies demonstrated that 8 suppressed the acute lung inflammation caused by pneumonia effectively. Afterwards, L-lysine based lipidated biphenyls were also found to display good anti-MRSA effect. Among them, compound 10 emerged to be the most selective one showing an MIC value of 3.1 mg/mL against the ATCC strain of S. aureus and MRSA, and an MIC value of 6.2 mg/mL against three strains of clinical isolates of MRSA [26]. Meanwhile, it was capable to eliminate both stationary phase bacteria and the persister cells of S. aureus, showing a dosedependent effect. Combined with its commendable activity in inhibiting intracellular infection and the stimulation of LTAinduced inflammatory response, 10 was hard to induce bacterial

resistance as expected. Finally, animal experiment demonstrated that 10 was still effective in a skin infectious murine model. In order to simplify the structure of membrane-active compounds, aliphatic norspermidine derivatives were designed and synthesized. Compound 11 was the most potent one in this series with the MIC values of 2.8 and 2.4 mg/mL against E. faecium and VRE, respectively, but showed moderate activity against other bacterial strains [27]. The SAR study indicated that antibacterial activity against Gram-positive bacteria tested was reduced along with the shortage of the aliphatic chain. Later, phenylalanine was introduced to the core, leading to a considerable improvement of its efficacy. For instance, compound 12 displayed excellent MIC values of 3.6 and 10 mg/mL against S. aureus and E. coli, respectively. It also showed superior activity against VRE (MIC ¼ 2.3 mg/mL) and MRSA (MIC ¼ 3.3 mg/mL) to 11. Compound 13, which consisted of D-isomer of phenylalanine, displayed comparable antibacterial activity to 12. Although both of them were rapid-bactericide and had high selectivity ratios, 13 showed less toxicity with a higher HC50 and were more stable in plasma than 12. However, the mechanism of action experiment indicated that 13 had alternative modes of action rather than membrane damaging property, even though it showed nearly the same efficacy with 12. Wang et al. [28] designed and synthesized a series of substituted diamines as rapid bactericide, in which compound 14 was a broadspectrum one, exhibiting MIC99 values of 2.0, 8.1 and 2.2 mg/mL against S. aureus, P. aeruginosa and E. coli, respectively. Likewise, it was effective against five MRSA strains isolated from clinic with the MIC values of 1e2 mg/mL against each isolate. From the SARs, it could be concluded that both the central charged nitrogens and the terminal diaryl moieties were essential for its efficacy, while the (bis)aryl substitutions on the terminal alkyl group were more effective than the monoaryl substituents. Besides, the (bis)arylthioureido analogues showed more efficacy than the (bis)arylureido analogues as the MIC99 reflected. 14 was even comparable to tetracycline and norfloxacin in inhibition of P. aeruginosa biofilm formation and dispersal of the formed biofilm, as well as in avoidance of drug tolerance in both dividing log phase and nondividing stationary phase of bacteria. Moreover, either on HEK293T human kidney embryonic cells and A549 human adenocarcinoma cells, or on Caenorhabditis elegans, 14 showed nearly no toxicity and favorable selectivity for bacteria. Specially, it had a synergistic interaction with kanamycin, which broadened its usage as a promising chemosensitizer. Likewise, Ghosh et al. [29] insisted on the principle of “the simpler the better”, designed and synthesized three series of peptide mimics containing an alkyl chain, an aromatic core and a cationic charged moiety (a lysine). Compound 15 with the anthracene core (Fig. 4) was the most potent one in this series, whose MIC values were 2.2 and 2.9 mg/mL against S. aureus and E. coli, respectively. As for the drug-resistant bacteria, 15 also had an admirable activity with the MIC values of 2.3, 3 and 4.3 mg/mL against MRSA, VRE and P. aeruginosa, respectively. Afterwards, substitution of the anthracene with a methyl naphthalene led to compounds 16 and 17, which had a superior efficacy against all the drug-sensitive bacteria tested (MIC values ranged from 1.6 to 4 mg/ mL). And 16 was specially mentioned for its anti-VRE activity of 1.6 mg/mL. In order to confirm the further impact of the decrease of aromaticity, compounds 18 and 19 were designed and synthesized by using a methyl benzene to substitute the anthracene, showing the MIC values of 2e5 mg/mL against the drug-sensitive bacteria tested. Additionally, 16 and 19 had comparable anti-MRSA activity of 2.5 mg/mL. The SARs indicated that two hydrophobic bulk units and two cationic charges were necessary for antibacterial activity [30,31]. On the one hand, if the aromatic ring was constant, short chain analogues were devoid of any activity or had moderate

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Fig. 4. Chemical structures of compounds 15e25.

activity, and the activity was increasing along with the length of the alkyl chain and finally reached a steady. On the other hand, keeping the alkyl chain constant, it could be concluded that the aromaticity was not the dominant factor while the long chain exerted more important effects. The hemolytic activity showed that all these compounds had high HC50 at a very low concentration, which provided a good selectivity toward mammalian cell. Moreover, plasma and enzyme stability demonstrated their potentiality to be novel antibacterial agents. Naphthoquinone and anthraquinone cores were already proved to uncouple mitochondria oxidative phosphorylation, interrupting bacteria via redox chemistry. Thus, a series of cationic quinone analogues were designed and synthesized by combining the cationic with naphthoquinone or anthraquinone to possess two different modes of action. Compound 20 was a cationic anthraquinone analog, showing excellent antibacterial activity against S. aureus (MIC ¼ 0.032e0.064 mg/mL) [32]. However, 20 had only a moderate activity against Gram-negative bacteria like E. coli. Mechanism assay indicated that this class of molecules exerted effects by inhibiting the redox processes in bacteria which could be the reason for bactericidal effects, while at a higher concentration they could disturb the integrity of membrane, giving them higher selectivity and broader-spectrum activity. Modification was carried out by introducing a longer alkyl chain, leading to compound 21, which manifested admirable MIC values of 0.032e0.5 mg/mL against S. aureus and MRSA, and 2e4 mg/mL against E. coli and K. pneumoniae [33]. Later, dimeric cationic anthraquinones were designed and synthesized, many of which showed a higher MIC value than mono-cationic molecules. Nevertheless, they are still narrow-spectrum similar to 20. With the increasing length of alkyl chain, they began to have broad-spectrum activity against a wide range of bacteria just like other membrane-disturbing molecules. Compound 22 was one of them, showing MIC values of 1e16 mg/mL against all strains of bacteria tested [34]. The cytotoxicity assay indicated that these dimeric cationic anthraquinone analogues manifested little to no cytotoxicity against a human cell line. Alpha-mangostin (compound 23), a natural xanthone, was

extracted and purified from the fruit hull of Garcinia mangostana, which showed good antibacterial activity (MIC ¼ 0.78e1.56 mg/mL) against Gram-positive bacteria including two strains of MRSA isolates [35]. 23 was a rapid concentration-dependent bactericide with no resistance induced. Therefore, cationic modifications were carried out at the free C3 and C6 hydroxyl groups with amine groups to improve its efficacy and selectivity. For instance, compound 24 was synthesized with a better efficacy (MIC ¼ 0.095e1.56 mg/mL against S. aureus and MRSA) and was even comparable to vancomycin and daptomycin against some strains [36]. There was no resistance or cross-resistance showed against 24, and no obvious in vivo toxicity was observed in a mouse cornea. It could be concluded that their antimicrobial spectrum was broaden by adding a cationic-charged moiety to the hydrophobic xanthone core, and the introduction of oxygen- and nitrogencontaining nonaromatic morpholine rings or pyrazole and 1,2,4triazole at the C3 and C6 positions of a-mangostin led to no activity at all. In addition, the isoprenyl group played a significant role in the antibacterial activity of xanthones. And it was clearly demonstrated that amphiphilic modifications with amine moieties were beneficial for their efficacy and selectivity. Thus here comes compound 25, an amphiphilic xanthone derivative with low MIC values (1.56e3.125 mg/mL) against Gram-positive bacteria and low hemolytic activity, which was potential to be further structurally optimized against MRSA infection [37]. 3.1.3. Quaternary amines Small-molecular quaternary ammonium compounds have been used in clinic to solve bacterial infections against certain viruses. Inspired by this, cationic amphiphilc quaternary ammonium compounds were designed and optimized, leading to dimeric amphiphiles. Compound 26 belonged to a class of cationic dimeric amphiphiles, which consisted of two hydrophilic cationic quaternary ammonium head groups and two hydrophobic chains, showing lower MICs (13 mM against E. coli and 10 mM against S. aureus) than their mono counterparts (35 mM against E. coli and 43 mM against S. aureus) [38]. The reason is that the antibacterial

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potency mainly counts on the hydrophilic-hydrophobic balance of the amphiphilic compounds, and the dimeric amphiphiles provide a greater number of positive charges as well as better interaction with the lipid bilayer [39]. Moreover, 26 showed low hemolytic activity and high selectivity against both Gram-positive and Gramnegative bacteria even at a concentration above its MIC value, but with no cytotoxicity against human cells. In addition, 26 was stable to thermal and acidic conditions, giving it more possibility to be used in vivo as well. Afterwards, Hoque et al. [40] designed and synthesized other quaternary amines as membrane-disrupt antibacterial molecules bearing two positive charges, two lipophilic moieties and two nonpeptidic amide groups, which exhibited good efficacy against various drug-sensitive and drug-resistant bacteria. Among them, compounds 27 and 28 (Fig. 5) were the most potent ones, showing the MIC values of 3 and 1 mg/mL against MRSA, respectively. And they also displayed good activity against VRE with the MIC values of 6 and 1 mg/mL, respectively. In vivo assay indicated that both compounds had the capability to disrupt biofilm as well as reduce the viable bacteria in biofilm, and they were less or non-toxic to human erythrocytes and human kidney cells. However, 28 had lower HC50 than 27. Based on this, another series of quaternary amines featuring four non-peptidic amide groups were developed, which also exhibited the property of being wide-spectrum rapid bactericides. Compounds 29 and 30 displayed the MIC values of 2 and 1 mg/mL against MRSA, as well as 4 and 2 mg/mL against both NRSA (norfloxacin-resistant S. aureus) and CREC (colistin-resistant E. coli), respectively [41]. Besides, they were capable of killing bacteria at 2  MIC, by inhibiting the formation of biofilm and dispersing the established bacterial biofilm, and showed negligible toxicity against mammalian cells. The SAR study of 27e30 suggested that a parabolic pattern was observed to describe the antibacterial activity changing with the increase of the lipophilic alkyl chain length. Besides, keeping the alkyl chain constant, the efficacy increased with the length of lipophilic spacer chain. Additionally, it was observed that in the four non-peptidic amide group series, the nature of the culture medium had an impact on the antibacterial activity, which meant that the cationic compounds were found to be more active in MEM (minimum essential medium) than in growth media (e.g. LB medium and nutrient medium), indicating a interaction between the cationic small molecules and the anionic additives like peptone in nutrient medium and tryotone in LB [41]. Another series of cationic small molecules with spatial positioning were designed specially to evaluate the role of hydrophobicity in their activity and toxicity, among which compound 31 had the most potency with the MIC values of 1, 2 and 2 mg/mL against S. aureus, E. coli, and MRSA, respectively [42]. This series were

synthesized following the modification strategy that the methylene groups between the positive centers were gradually increased along with the gradual decrease of methylene groups in the pendent alkyl chains. Thus, their SARs could be concluded that the spatial positioning of hydrophobicity did not influence their antibacterial efficacy but the hemolytic activity, while the molecules with confined hydrophobicity showed higher selectivity than the molecules with pendent hydrophobicity. Moreover, 31 showed no toxicity toward human cells even at the concentration of eradicating the established biofilms. Further, it was tested for its antibacteria and anti-biofilm activity and performed well in a murine model of superficial skin infection. Vancomycin, a glycopeptide antibiotic, is known as the “drug of last resort” to treat infectious diseases caused by different drugresistant bacteria. Nevertheless, the continuous appearance of vancomycin-resistant bacteria asked for a desperate need of other analogous. Compound 32 was a cationic vancomycin analogue with an excellent antibacterial activity against both vancomycinsensitive and vancomycin-resistant bacteria, showing the MIC values of 0.31 mM against MSSA (methicillin-sensitive S. aureus) and MRSA, and the MIC values of 0.4, 0.15, 12.5 mM against VISA (Vancomycin-intermediate-resistant S. aureus), VSE (Vancomycin-sensitive E. faecium) and VRE, respectively [43]. The SAR analysis showed that the permanent positive charged groups performed better than the secondary amines which dissociates into cations under physiological conditions. Besides, the physical mixture of cationic lipophilic portion and vancomycin was inactive, which meant that this sort of conjunction was meaningful. Further study confirmed that 32 was a rapid bactericide even in whole blood and showed excellent in vivo activity against MRSA in infected mouse model. Besides, it had no observed toxicity, and no possibility to induce bacterial resistance. A class of cationic chalcone derivatives were designed and synthesized by Chu et al. [44], in which, the representative compounds 33 (MIC ¼ 1 mg/mL against S. aureus and 0.5 mg/mL against MRSA) and 34 (MIC ¼ 0.5 mg/mL against S. aureus and 0.25 mg/mL against MRSA) showed good antibacterial activity as well as the ability to reduce the viable cell counts in biofilms effectively. It could be observed that their antibacterial activity was significantly associated with the length of their alkyl chain, and showed a parabolic pattern which may be caused by the aggregation tendency of long hydrophobic chain [45]. In addition, the substitution of the phenyl group with a furanyl or thienyl group showed good activity, while the pyridyl group had poor activity. Besides, the presence and position of a fluoride atom was found to have a significant influence on the efficacy. Furthermore, 33 was found not to induce the development of resistance in bacteria, and showed negligible toxicity to mammalian cells.

Fig. 5. Chemical structures of compounds 26e34.

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3.2. Guanidines 3-Amino benzoic acid (3-ABA) was considered as a peptidomimetic element, forming a new series of small cationic peptidomimetics by introducing arginine (Arg) and tryptophan (Trp) amino acid residues. Among them, compounds 35 and 36 (Fig. 6) were found to show good antibacterial activity against both drugsensitive and drug-resistant bacteria, 35 yielding the MIC values of 5e5.5 mg/mL against S. aureus, MRSA, and MRSE, whereas 36 exhibiting the MIC values in the range of 5e6.25 mg/mL against the same bacteria [46]. However, they did not perform well against Gram-negative bacteria. The SARs demonstrated that amphipathic structural framework was essential for the antibacterial activity, and the incorporating of 3-ABA improved significantly in the efficacy. Besides, low hemolytic activity and low toxicity toward mammalian cells gave these rapidly bactericidal compounds better selectivity. Moreover, they were stable both to human blood plasma and trypsin. Introducing lipophilic chains and cationic amino acids into the hydrophobic xanthone core, compound 37 was designed and synthesized, which exhibited promising efficacy (MIC ¼ 0.5e3 mg/mL) against a panel of Gram-positive bacteria, including MRSA and VRE [47]. According to the SAR analysis, a hydrophobic core with aromatic rings and cationic groups were basically needed for its activity, and cationic moieties with more dispersed positive charges were preferable for enhanced selectivity. For instance, the argininecontaining analogues exhibited less hemolytic activity than the lysine-containing analogues. Besides, the introduction of high pKa cationic amino acids were better to both the efficacy and the selectivity. Additionally, 37 induced rapid killing of VRE at 2  MIC, without any drug-resistance or toxicity. Furthermore, compound 37 was also effective in a corneal infected mouse model by both MRSA and S. aureus. Based on this scaffold, multiple optimizations were conducted, leading to synthesis of compounds 38 and 39 through an advantageous method with low cost and synthetic simplicity. 38 and 39 exhibited potent antibacterial activity

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(MICs ¼ 0.78e6.25 mg/mL) against MRSA and VRE [48]. Both of them were rapid bactericidal and resistance-proof, and in particular, 38 was still effective in a mouse model of corneal infection. Icaritin, isolated from epimedium plants, was an active prenylflavone with various pharmacological and biological activity. Further modifications were taken to improve its antibacterial activity and reduce its toxicity. Consequently, compound 40 bearing two arginine residues was found to display the MIC values of 1.56e3.13 mg/mL against MRSA and S. aureus [49]. 40 could also kill MRSA in 2 h at 2  MIC and 4  MIC without the development of drug resistance. And it had very low cytotoxicity toward mammalian cells even at a concentration of 100 mM, which meant that it had high selectivity between bacterial cell and mammalian. Furthermore, it showed high efficacy in the MRSA and S. aureus infected mouse model suffering from a corneal infection. 3.3. Amphiphilic aminoglycosides As an old type of antibiotic, aminoglycosides (AGs) have been used for the treatment of infectious diseases in clinic for more than 70 years, but their potential nephrotoxicity and ototoxicity still limit their application primarily [50]. The certain conventional aminoglycosides work as ribosomal RNA targeting antibacterial agents by binding to 16S rRNA to interrupt the production of proteins whose expression are essential to bacterial metabolism, thereby killing the cell [51]. However, the widespread use in clinic has led to the reduction of their efficacy because of the selection of resistant bacteria. In contrast, amphiphilic aminoglycosides have been proved to possess a totally different mechanism of action. Amphiphilic aminoglycoside antimicrobials (AAAs) are the conjunction of hydrophobic groups to a polycationic aminoglycoside, forming a cationic amphipathic scaffold. Accordingly, the physicochemical characteristics and biological properties of the AAAs are similar to other cationic amphiphiles such as AMPs and AMP mimics [10]. Acting on the bacterial membrane to damage its integrity, the AAAs shed light on unique architectures without the

Fig. 6. Chemical structures of compounds 35e40.

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Fig. 7. Chemical structures of compounds 41e44.

drawbacks of AMPs, which provides a different thought for cationic lipids. The conjunction of alkyl groups to AGs brought to compounds 41 and 42 (Fig. 7), both of which exhibited potent antibacterial activity against various drug-sensitive and drug-resistant bacteria strains [52]. For example, 41 with a dialkyl neamine incorporating two same alkyl groups at the 30 and 6 positions, exhibited the MIC values of 1e8 mg/mL against susceptible and resistant S. aureus and P. aeruginosa strains, whereas 42 bearing two different alkyl chains at the 30 and 6 positions, showed the MIC values of 1e4 mg/mL against the above strains. It was confirmed that its lipophilicity played a key role in the antibacterial activity, indicating that compounds with clogP values between 13.5 and 10.5 performed well. Moreover, replacing the dialkyl with a naphthylalkyl led to less efficacy than the former especially against the Gram-negative bacteria. In contrast, grafting of branched alkyl chains with similar lipophilicities increased or preserved its efficacy, but increased the toxicity as well. The truth is that these compounds show a strong decrease in mammalian cell viability, which may be blocks to their pathway to clinic, even though they have high difficulty to induce resistance. Modifications at the 30 and 40 positions of the dialkyl neamines led to compound 43, which showed the MIC values of 2e16 mg/mL against both drug-sensitive and drug-resistant S. aureus and P. aeruginosa [53]. SAR study demonstrated that the presence of three protonated amines at least were necessary for its activity, and the ring I was not the main pharmacophore. Thus, compounds 44a and 44b, which were dinonyl neosamines obtained by simplifying the ring I and retaining the ring II in structure, showed modest MIC values against P. aeruginosa, Acinetobacter lwoffi, E. coli, and K. pneumoniae strains (MIC ¼ 4e8, 2e8, 4e8, and 32 mg/mL, respectively). Actually all other hydrophilic neosamine derivatives exhibited similar efficacy. Additionally, their MIC values increased slowly after the exposure to P. aeruginosa for a long time compared

to that of ciprofloxacin. 4. Conclusion and prospect Infectious diseases caused by bacteria are getting worse and worse in recent years, together with the continuous emergence of multidrug resistant bacteria, leading to an alarm that antibacterial agents with novel modes of action are needed urgently. Membraneactive compounds give us a new pathway to solve these problems by depolarization and disruption of bacteria cell membrane. Over the years, researchers have been keening on reducing their hemolytic activity, increasing their selectivity and improving their serum stability. In this review, we summarized recently synthesized small molecules targeting the bacterial membrane and their SARs, which demonstrated that the cationic portion determined their selectivity, whereas the lipophilic portion determined their antibacterial activity. As described above, most of these compounds have high efficacy against both Gram-positive and Gram-negative bacteria (drug-sensitive and drug-resistant strains), along with a good selectivity toward mammalian cells and the bacteria. Low cost and stability in plasma condition endowed them much more promising to be a new clinical agents. Moreover, rapid bactericidal activity and low possibility to induce drug-resistance provided a hopeful approach to resolve the multidrug resistant phenomenon. More importantly, some of them have the ability to inhibit bacteria biofilm, thereby combating the notorious biofilm formation. Therefore, these compounds can be potentially applied to tackle various infectious diseases in the near future. Declaration of competing interest The authors declare that this study was carried out only with public funding. There is no funding or no agreement with commercial for profit firms.

N. Zhang, S. Ma / European Journal of Medicinal Chemistry 184 (2019) 111743

Acknowledgements This research was supported financially by the National Natural Science Foundation of China (81973179 and 81673284), and Key Research and Development Project of Shandong Province (2017CXGC1401).

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