Amino Acid Modified Xanthone Derivatives: Novel, Highly Promising

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Amino Acid Modified Xanthone Derivatives: Novel, Highly Promising Membrane-Active Antimicrobials for Multidrug-Resistant GramPositive Bacterial Infections Jun-Jie Koh,†,‡,○ Shuimu Lin,†,§,○ Thet Tun Aung,† Fanghui Lim,† Hanxun Zou,†,§ Yang Bai,†,# Jianguo Li,†,∥ Huifen Lin,† Li Mei Pang,†,● Wee Luan Koh,† Shuhaida Mohamed Salleh,† Rajamani Lakshminarayanan,†,⊥ Lei Zhou,†,⊥ Shengxiang Qiu,× Konstantin Pervushin,# Chandra Verma,†,∥,#,∞ Donald T. H. Tan,‡ Derong Cao,*,§ Shouping Liu,*,†,§,⊥ and Roger W. Beuerman*,†,⊥ †

Singapore Eye Research Institute, 11 Third Hospital Avenue, 168751, Singapore Department of Ophthalmology, Yong Loo Lin School of Medicine, National University of Singapore, 119074, Singapore § School of Chemistry and Chemical Engineering, South China University of Technology, Guangzhou 510641, China ∥ Bioinformatics Institute, 138671, Singapore ⊥ SRP Neuroscience and Behavioral Disorders, Duke-NUS Medical School, 169857, Singapore # School of Biological Sciences, Nanyang Technological University, 637551, Singapore ∞ Department of Biological Sciences, National University of Singapore, 117543, Singapore × South China Botanical Garden, Guangzhou 510650, China ‡

S Supporting Information *

ABSTRACT: Antibiotic resistance is a critical global health care crisis requiring urgent action to develop more effective antibiotics. Utilizing the hydrophobic scaffold of xanthone, we identified three components that mimicked the action of an antimicrobial cationic peptide to produce membrane-targeting antimicrobials. Compounds 5c and 6, which contain a hydrophobic xanthone core, lipophilic chains, and cationic amino acids, displayed very promising antimicrobial activity against multidrug-resistant Gram-positive bacteria, including MRSA and VRE, rapid time−kill, avoidance of antibiotic resistance, and low toxicity. The bacterial membrane selectivity of these molecules was comparable to that of several membrane-targeting antibiotics in clinical trials. 5c and 6 were effective in a mouse model of corneal infection by S. aureus and MRSA. Evidence is presented indicating that 5c and 6 target the negatively charged bacterial membrane via a combination of electrostatic and hydrophobic interactions. These results suggest that 5c and 6 have significant promise for combating lifethreatening infections.

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INTRODUCTION Increasing antibiotic resistance has led to the failure of current antimicrobial therapies, with concomitant mortality and healthcare costs.1 Because many antibiotics are no longer effective against bacterial infections that were once easily treated, there is an urgent medical need for a sustainable supply of new, effective, safe antimicrobials without cross-resistance with currently used antibiotics.2 For instance, methicillinresistant S. aureus (MRSA), a leading cause of nosocomial infection worldwide, has acquired resistance against a wide range of antibiotic classes, including β-lactams, fluoroquinolones, tetracyclines, macrolides, lincosamides, aminoglycosides, and even the newest drugs licensed to treat MRSA infections, linezolid and daptomycin.3 Moreover, MRSA infections are © XXXX American Chemical Society

associated with higher hospital mortality rates than methicillinsusceptible S. aureus (MSSA) infections.1 Cationic antimicrobial peptides (CAMPs) are host defense proteins that represent an evolutionarily ancient component of the innate immune system that has remained effective against multiple classes of microbes.4 CAMPs usually comprise hydrophobic and cationic groups to create an amphipathic structure.4 We previously demonstrated that the non-natural branched CAMP B2088 adopts a compact structure in which the hydrophobic residues are buried in the core, while the cationic residues are distributed on the surface to disrupt the Received: August 22, 2014

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dx.doi.org/10.1021/jm501285x | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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Scheme 1. Synthesis of the Xanthone Analogues without Isoprenyl Groups and Reduced Isoprenyl Groupsa

Reaction conditions for the synthesis of xanthone analogues without isoprenyl groups (1a, 2a and 3a): (i) P2O5−CH3SO3H, 80 °C, 1 h; (ii) K2CO3, 1,4-dibromobutane, acetone, reflux overnight; (iii) diethylamine, DMSO, rt, 4 h. Reaction conditions for the synthesis of xanthone analogues with reduced isoprenyl groups (1b, 2b and 3b): (iv) H2, Pd−C, ethanol, rt, 2 h; (v). 1,4-dibromobutane, K2CO3, acetone, reflux; (vi) diethylamine, DMSO, rt. The MIC value of each xanthone analogue is displayed at the bottom of the structure. a

Figure 1. Components required for the design of a small-molecule membrane-targeting antimicrobial. Each component is highlighted using different colors: red, hydrophobic core; blue, lipophilic chain; black, cationic moieties. All compounds used in this study are shown.

bacterial membrane.5 CAMPs have considerable advantages over conventional antibiotics, such as a rapid bacterial killing, selectivity toward the bacterial membrane, and a low propensity for bacterial resistance.5 However, high manufacturing costs, poor stability, and a loss of activity in the presence of salts pose significant challenges to the therapeutic development of CAMPs.6 Consequently, the development of small-moleculebased membrane-targeting antimicrobials that maintain the essential key characteristics of CAMPs has received considerable attention.7 Small antimicrobial peptidomimetics that mimic the structure and antibacterial action of CAMPs have been developed. Peptidomimetic design introduces amide bond isosteres or peptide backbone modifications via heteroatoms or non-natural side chains to mimic a peptide structure or function.6,8 Classes of antimicrobial peptidomimetics include β-peptides, peptoids, arylamides, AApeptides, and γ-peptide-based oligomers.8 However, nonpeptidic small molecular membrane-targeting agents that are facially amphiphilic can also be derived from other scaffolds.9 Ceragenins are semisynthetic antimicrobial cationic amphiphiles with a hydrophobic steroid scaffold derived from bile acid.10 Modifications of benzophenone11

and aminoglycoside12 scaffolds to obtain membrane-targeting antimicrobial cationic amphiphiles have also been reported. Membrane-targeting antibiotics in clinical trials15 include 7 (LTX-109; synthetic antimicrobial peptidomimetic),13 brilacidin (PMX-30063; acrylamide),14 and 8 (CSA-13; ceragenin).10 The major limitations of these compounds are their membrane selectivity, complex synthesis, and high cost of production.16 The development of effective small-molecule-based membranetargeting antimicrobials with high membrane selectivity from simple scaffolds and synthetic routes would have an important positive impact on antibiotic development. Xanthones are natural polyphenolic compounds that are widely produced by higher plants, fungi, and lichens as secondary metabolites.17 Their interesting structural scaffolds and significant biological activities have prompted many groups to isolate and modify xanthones for the development of new drug candidates, particularly as anticancer drugs.17,18 We hypothesized that the incorporation of appropriate substituents may permit the tuning of the antibacterial activities of xanthone analogs to mimic those of CAMPs. We previously reported that α-mangostin and its synthetic derivative, 9 (AM-0016), which are characterized by a hydrophobic xanthone core and an amphiphilic structure B



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In contrast to α-mangostin and 9, the xanthone analogues without isoprenyl groups (1a, 2a, and 3a) did not exhibit activity against the bacterial strains tested (MIC > 50 μg/mL). Therefore, lipophilic chains are key components for enhancing the interaction of small-molecule xanthone-based membraneactive antimicrobials with the bacterial membrane. Compound 1b displayed 25-fold weaker antibacterial activities (MIC = 50 μg/mL) against clinical isolates S. aureus DM4001 compared to α-mangostin (MIC = 2 μg/mL). Compound 3b displayed MIC values of 3 μg/mL, an approximately 1.5- to 6-fold reduction in antibacterial activity compared to 9 (MIC = 0.5−2 μg/mL). These data suggest that the hydrophobicity of the lipophilic chains (for instance, an isoprenyl group or its reduced form) may be a critical factor in the penetration of amphiphilic xanthone analogues into the cytoplasmic membrane. Synthesis of Xanthone Analogues Conjugated with Basic Amino Acids and Assessment of Their Antimicrobial Activities and Membrane Selectivity. Our results indicated that isoprenyl groups are critical for bacterial killing. Next we examined the role of cationic moieties (component 3). As illustrated in Scheme 2, a new series of α-mangostin

(Scheme 1), can disrupt the inner bacterial membrane.19 However, these compounds exhibited unsatisfactory toxicity. Thus, in the present study, we sought to identify a set of components that mimic the action of an antimicrobial cationic peptide to further decrease toxicity and increase bacterial membrane selectivity while maintaining satisfactory antimicrobial activity in vivo. As part of a minimalist strategy to design a small molecule with bacterial membrane-targeting activity (Figure 1), we first identified the utility of the hydrophobic xanthone core (component 1). In this study, xanthone was used as a template to understand and design effective membrane-targeting smallmolecule antimicrobials with promising membrane selectivity. To elucidate the effects of isoprenyl groups or lipophilic chains (component 2) on antimicrobial activity, six xanthone analogues, 1−3, with varied isoprenyl groups at the C2 and C8 positions on the xanthone core were synthesized. Next, a series of xanthone-based membrane-targeting molecules, 5a−f and 6, were designed to investigate the role of cationic amino acid residues (component 3) in antimicrobial activity. Cationic amino acids were used to improve selectivity because we previously demonstrated that these amino acids improve the membrane selectivity of CAMPs.5,20 Finally, the antimicrobial activity, in vitro time−kill kinetics, multipassage resistance selection, membrane selectivity, and efficacy in a mouse model of corneal infection of these molecules were systemically studied. The results reported herein are the first to provide guidelines for the design and synthesis of xanthone analogues with CAMP properties that target bacterial membranes with high selectivity and therapeutic levels of antimicrobial activity in vitro and in vivo.

Scheme 2. General Synthetic Routes for a Family of Amphiphilic Xanthones Using Cationic Amino Acids (5a−f, 6)a

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RESULTS AND DISCUSSION Synthesis and Antibacterial Activity of Xanthone Analogues with Varied Lipophilic Groups. Isoprenyl groups have been demonstrated to enhance interactions with biological membranes.21 We previously employed molecular dynamics (MD) simulations and a model bacterial membrane to demonstrate that α-mangostin molecules preferentially penetrate the lipid tail region of the bacterial membrane.19b In this study, no penetration of molecule 1a (analogue without isoprenyl groups) was observed, and all molecules remained on the membrane surface (see Supporting Information, Figure S1). MD simulations suggested that two isoprenyl groups or lipophilic chains enhanced the interaction with the bacterial membrane, which further facilitated the penetration of the molecule into the lipid membrane. To explore and confirm the role of the lipophilic chains or isoprenyl groups, a series of compounds with modified isoprenyl groups were synthesized (Scheme 1). The xanthone analogue 1a without isoprenyl groups at the C2 and C8 positions was synthesized via the condensation of 2,4dihydroxybenzoic acid and phloroglucinol in the presence of Eaton’s reagent.22 Then bis-α,ω-dibromoalkane-substituted xanthone 2a was synthesized using a Williamson ether synthesis followed by an amination reaction with diethylamine to form compound 3a. To synthesize tetrahydro-α-mangostin 1b, the double bonds of the isoprenyl groups were reduced by catalytic hydrogenation using a protocol similar to that previously reported by Sudta et al.23 Compounds 2b and 3b were prepared from 1b using similar protocols as for the preparation of 2a and 3a.

a Reagents and conditions: (i) BrCH2CO2Me, KOH, anhydrous EtOH, reflux, 24 h; (ii) LiOH, THF, H2O, rt, 2 h. (iii) For 5a: HLys(Fmoc)-OMe·HCl, DIC, HOBt, anhydrous DMF, rt, overnight; then piperidine, DMF, rt, 20 min. (iv) For 5b−f and 6: corresponding basic amino acid, DIC, HOBt, anhydrous DMF, rt, overnight.

analogues were synthesized by a chemical modification with basic amino acid residues through functionalization of the two phenolic groups at the C3 and C6 positions of α-mangostin. The hydroxyl group at the C1 position is less reactive because of the potential for intramolecular hydrogen bond formation between the C1 hydroxyl group and the C9 carbonyl group.19a Initial alkylation with methyl bromoacetate produced the corresponding esters. The esters were subsequently hydrolyzed with lithium hydroxide to obtain acid 4, which was further coupled with Lys, Arg, or His derivatives using N,N′diisopropylcarbodiimide (DIC) and N-hydroxybenzotriazole (HOBt) in anhydrous DMF at room temperature to produce 5b−f. 5a was obtained through a two-step process in which 4 C



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These results suggest that increasing the hydrophobicity of the arginine-containing analogues induced stronger hemolytic activities. This observation is consistent with previous reports that the introduction of hydrophobic substituents confers hemolytic activity on cationic antimicrobial peptides and peptidomimetics.20,26 Compound 4, a xanthone analogue coupled with two carboxylic acids, was not active against any of the bacterial strains tested. This observation further indicates a crucial role of cationic moieties in increasing the selectivity of membrane-targeting antimicrobials. An additional set of arginines was coupled to 5c to yield 6. Compound 6 (also coded as AM-0218) was obtained through a two-step process in which 5c was first hydrolyzed using lithium hydroxide and then coupled with H-Arg-OMe·2HCl in the presence of DIC and HOBt. Compound 6 exhibited excellent antimicrobial activity (MIC = 0.5−3 μg/mL) against a panel of Gram-positive bacteria and reduced hemolytic activity (HC50 = 277 ± 4 μg/mL), as shown in Tables 1 and 3. By contrast, 10 (MSI-78),27 an analogue of magainin containing a 23-residue helical peptide, exhibited a HC50 value of 120 μg/mL. Notably, the selectivities (HC50/MIC) of 5c (selectivity = 77−116) and 6 (92−554) (Table 2) were significantly improved compared to α-mangostin (selectivity = 5) and 9 (selectivity = 7−40) (Table 3). In addition, the selectivities of 5c and 6 were also comparable or superior to those of other membrane-active drugs in clinical trials, such as 7, 8, and 10 (Table 3). 5c and 6 were further examined as small-molecule membrane-active antibiotics against a panel of 37 Grampositive pathogens, including MSSA, MRSA, and vancomycinresistant enterococci (VRE) (Table 4). The MIC values of these compounds were determined by Quotient Bioresearch Ltd. (U.K.). 5c and 6 were effective against Gram-positive bacteria exhibiting resistance to vancomycin, teicoplanin, and macrolides and against multidrug resistant (MDR) strains. The MICs of 5c and 6 were 2−4 μg/mL against all strains tested, with the exception of Streptococcus, Corynebacterium jeikeium, and Listeria monocytogenes (MIC = 4−8 μg/mL). More importantly, because the MICs of 5c and 6 were not significantly higher for strains with known resistance to established antibacterial agents, cross resistance is not anticipated. This study identified three important structural components for the design of a successful small-molecule with properties similar to a CAMP. First, a rigid hydrophobic core with two or more aromatic rings is needed. A relatively small size and conformationally constrained structure might facilitate the penetration of various physical barriers in the outer membrane of Gram-positive bacteria.28 Second, cationic moieties are required to form an amphiphilic structure and to discriminate the bacterial membrane from the mammalian membrane. We and others have previously demonstrated that amphiphilic structures are important for appropriate physical−chemical interactions between a membrane-targeting molecule and the bacterial membrane.11,29 In this study, our data revealed that a cationic group with a more dispersed positive charge, such as arginine, is preferred for enhanced selectivity. Cationic moieties are also critical to ensure rapid access to the cytoplasmic membrane via electrostatic interactions. Positively charged residues, particularly arginine, also facilitate peptide entry into cells.30 However, the hydrophobic core and cationic moieties are not sufficient for disruption of the membrane bilayer by xanthones. Therefore, a third component, a lipophilic chain in the form of an isoprenyl group or the reduced form of an

was reacted with H-Lys(Fmoc)-OMe·HCl in the presence of DIC and HOBt and then treated with 20% piperidine/DMF to remove the protecting group. The antibacterial activities of 5a−f were tested against a panel of Gram-positive bacteria, including clinical strains of MRSA (Table 1). Coupling high pKa amino acids (5a, lysine, Table 1. In Vitro Antibacterial (μg/mL) and Hemolytic Activities (μg/mL) of 4, 5a−f, and 6 Compared to 10 bacterial straina compd

A

B

C

D

HC50 b (μg/mL)

4 5a 5b 5c 5d 5e 5f 6 α-mangostin 9 10

>50 6 >50 2 6 6 12 0.5 2 2 12.5

>50 12 >50 3 6 1 12 2 2 0.5 12.5

>50 6 >50 3 12 6 12 3 2 2 25

>50 12 >50 2 12 6 12 3 2 3 12.5

>400 39 ± 1 >400 232 ± 8 56 ± 6 128 ± 5 79 ± 6 277 ± 4 9±2 20 ± 3 120c

a Clinical isolates S. aureus DM4001 (A), MRSA DM21455 (B), MRSA DM09809R (C), Bacillus cereus ATCC 11778 (D). bThe HC50 value is the concentration required to lyse 50% of red blood cells after 1 h. c Value from a published report.27

pKa of ε-NH2 of 10.54; 5c (also coded as AM-0052), arginine, pKa of guanidine of 12.48)24 yielded antimicrobials with MICs of 6−12 μg/mL for 5a and 2−3 μg/mL for 5c. A histidinecontaining analogue (5b, pKa of side chain histidine of 6.10) exhibited greatly reduced antibacterial activity (MIC > 50 μg/ mL). In general, arginine-containing analogues, exhibited less hemolytic activity than lysine-containing analogue and 9. The positive charge of the guanidinium group of arginine is more dispersed than that of the single ε-amino group of lysine (5a) and the diethylamino group (9), increasing the selectivity of 5c−e toward the anionic bacterial membrane.25 Because 5c exhibited potent antimicrobial activity, the structure of the arginine-containing form was further altered (5d−f), and the effect of hydrophobicity on the interaction with mammalian membranes was determined by measuring the concentration needed to induce lysis in 50% of red blood cells (HC50). In general, the selectivity of arginine-containing analogues for bacterial membranes increased as the hydrophobicity decreased (Table 2). For example, 5c had a higher HC50 value (232 ± 8 μg/mL) than the more hydrophobic analogues, such as 5d and 5f, which caused hemolysis at lower concentrations (HC50 of 56 ± 6 and 79 ± 6 μg/mL, respectively), as shown in Table 1. Table 2. Hydrophobicity, MIC Range, HC50, and Selectivity Range of Compounds Containing Arginine Analogues compda 5c 5d 5e 5f 6

tR (min)

% ACN required

MIC range (μg/mL)

HC50 (μg/mL)

selectivity range

± ± ± ± ±

66.9 68.5 64.6 73.0 61.2

2−3 6−12 1−6 12 0.5−3

232 56 128 79 277

77−116 5−9 13−128 7 92−554

7.96 8.38 7.40 9.51 6.49

0.01 0.01 0.01 0.01 0.01

a

Only compounds containing arginine analogues were tested for molecular hydrophobicity. D



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Table 3. Selectivity of Xanthone Analogues and Selected Membrane-Active Antimicrobials in Clinical Trials compd α-mangostin 9 5c 6 7 8 10 a

company

MIC

HC50 (μg/mL)

selectivity (HC50/MIC)

ref

9 20 232 277 175 29a 120

5 7−40 77−116 92−554 44 73 2−8

19b 19a

Lytix Biopharma, Norway Ceragenix Pharma, U.S. Dipexium Pharma, U.S.

2 0.5−3 2−3 0.5−3 4 0.4 16−64

51 52 53

The reported value is the minimum hemolytic concentration.

Table 4. MIC Range for 5c, 6, and Vancomycin against a Panel of Gram-Positive Bacteria, As Determined by Quotient Bioresearch Ltd. (U.K.) bacterial strains (number of strains)a Staphylococcus MSSA (7) VISAb (1) MRSA (10) EMRSAb (3) teicoplanin-RIb (1) MDRb S. epidermidis (2) Enterococcus VSEb (3) VREb (5) Streptococcus (4) othersc (2)

vancomycin

5c

6

2−4 4 2−4 2−4 2 2 2

2−4 2 2 2 2 2 2

1−2 1 1 1−8 2 2 2

2−4 2−4 4−8 4−8

2−4 1−2 2−8 2−4

1−2 8→32 0.5−2 0.5−1

Figure 2. Time−kill kinetics of (a) 5c and (b) 6 against MRSA and 6 against (c) vanA VRE and (d) vanB VRE. MIC against MRSA DM21455: 5c = 3 μg/mL, 6 = 2 μg/mL. MIC against VRE: 6 = 2 μg/ mL, vancomycin (VAN) > 32 μg/mL. Test concentration of VAN = 2048 μg/mL. The control is the culture without any antimicrobial treatment.

a

See Table S1 in the Supporting Information for the MIC of each particular strain. bVISA, vancomycin-intermediate S. aureus; EMRSA, epidemic MRSA; RI, resistant intermediate; MDR, multidrug resistant; VSE, vancomycin-sensitive enterococci; VRE, vancomycin-resistant enterococci. cOthers: Corynebacterium jeikeium and Listeria monocytogenes.

isoprenyl group, is needed to provide sufficient driving force for the penetration of the bulky xanthone into the cytoplasmic membrane. Time−Kill Kinetics. Time−kill kinetic studies of 5c and 6 against MRSA and VRE were performed to confirm and explore the membrane-targeting characteristics of the small molecules. Time−kill kinetics revealed that 5c killed bacteria rapidly, achieving a 3 log reduction (99.9% of bacteria killed) in 1 h at 1 × MIC and in 30 min at 2 × MIC against MRSA DM21455 (Figure 2a). 6 also induced rapid bacterial cell death; a 3 log reduction was achieved in 2 h at 4 × MIC (Figure 2b). 6 also induced rapid killing of VRE; a 3 log reduction was achieved in 1 h at 2 × MIC and 4 × MIC (Figure 2c and Figure 2d, respectively). By contrast, 2048 μg/mL vancomycin induced negligible log reductions at 2 and 24 h. Multipassage Resistance Selection Studies. The avoidance of resistance is a hallmark of natural antimicrobial peptides as membrane-active antimicrobials.31 We examined the ability of 5c and 6 to avoid endogenous mutational resistance during prolonged passages at subinhibitory concentrations of S. aureus (Figure 3). Fluoroquinolones are among the most prescribed antibiotics in the United States. Norfloxacin and gatifloxacin are two broad-spectrum fluoroquinolones to which resistance has been reported.32 Therefore, these two antibiotics served as useful comparisons to investigate the potential of 5c and 6 to induce resistance. Resistance is usually defined as a >4-fold increase in the original MIC.33

Figure 3. Multistep passage resistance studies of 5c and 6 against S. aureus. The ability of 5c and 6 to avoid endogenous mutational resistance was investigated in multipassage resistance selection studies. The fourth- and first-generation fluoroquinolones gatifloxacin and norfloxacin were selected as reference antibiotics. MICs against S. aureus ATCC29213 are the following: 5c = 3 μg/mL; 6 = 2 μg/mL; gatifloxacin = 0.1 μg/mL; norfloxacin = 2 μg/mL. Resistance was defined as a >4-fold increase in the original MIC.

Resistance to 5c and 6 did not develop over 17 passages. By contrast, a 64- to 128-fold increase in the MIC values of gatifloxacin and norfloxacin was observed. The results of this multistep passage resistance study clearly demonstrated that 5c and 6 averted the induction of antibiotic resistance, an important characteristic of an antimicrobial peptide. Farrell et E



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al. reported that bacterial resistance to daptomycin, a nonlytic membrane-active agent, emerged at the fifth passage.34 Membrane Selectivity Study of Vesicle Leakage from Calcein-Loaded LUVs. The selectivity of 5c and 6 for bacterial or mammalian model membranes was evaluated. Initially, we analyzed the levels of leakage of calcein encapsulated in large unilamellar vesicles (LUV) with a membrane lipid composition of DOPE/DOPG = 75/25 (mimicking a negatively charged bacterial cytoplasmic membrane) or DOPC (mimicking a neutral mammalian cytoplasmic membrane). As shown in Figure 4, 5c induced strong calcein

Figure 5. Investigation of membrane selectivity of 5c by NMR spectroscopy. 1H spectra of 5c in (A) DOPE/DOPG liposomes, (B) DOPC liposomes, and (C) water. 1H resonances are assigned. The spectra in each column share the common axis labeled at the bottom.

of 5c was likely free in solution rather than embedded in the lipid phase. The preferential binding of 5c to the bacterial membrane mimic rather than to the mammalian cell membrane mimic supports its high selectivity. MD simulations confirmed that compound 5c was more selective against bacterial membranes (see Supporting Information, Figure S2). Antimicrobial Properties in the Presence of Bovine Serum Albumin. We have demonstrated that a xanthonebased membrane-active molecule exhibits improved selectivity and potent antimicrobial properties. However, binding to plasma proteins such as albumin may reduce the effective concentration of most cationic antimicrobial peptides or peptoids.35 Therefore, we next investigated the antibacterial activities of 5c and 6 in the presence of bovine serum albumin (BSA). A 2-fold increase in the MIC was observed, indicating weak binding of 5c to BSA (Table 5). A 4- to 8-fold reduction in the antibacterial activity of 6 was observed in the presence of BSA. By contrast, a 16-fold increase was observed in the MICs of 10 and daptomycin, a lipopeptide antibiotic used in the treatment of systemic and life-threatening infections caused by Gram-positive organisms. A relatively weak binding to plasma proteins is clearly an important consideration for the therapeutic use of 5c and 6. Evaluation of in Vivo Toxicity. In vivo toxicity is a major challenge in the use of membrane-targeting antimicrobials. Applications of 5c and 6 (5 times/day) for 4 days at a concentration of 3 mg/mL (0.3% solution) did not produce any deleterious effects in mouse eyes, as determined by the examination of living eyes with a biomicroscope and by the inspection of histological sections. The rabbit corneal epithelial abrasion model of wound healing was also used to investigate the topical toxicity of 5c and 6 and to assess the potential adverse effects on normal physiological wound healing processes.36 Normal corneal clarity and transparency were observed upon the application of 0.3% solutions of 5c and 6. There were no signs of corneal inflammation and no evidence of an inflammatory response or retardation of wound healing in

Figure 4. Leakage (%) induced by compounds 5c and 6 in calceinloaded liposomes. Liposome compositions of 75:25 DOPE/DOPG and DOPC only were used to mimic the bacterial and mammalian membranes, respectively. For all measurements, each point represents the mean ± SD of three independent experiments.

leakage in DOPE-DOPG vesicles, as indicated by a leakage value of 68 ± 3% at a compound to lipid (C/L) ratio of 1/16. At the same C/L ratio, a leakage value of 32 ± 2% was observed in DOPC liposomes. Striking differences were observed in the ability of 6 to induce leakage in DOPE-DOPG and DOPC liposomes. At C/L ratios of 1/16, 1/32, and 1/64, 99.2 ± 8.1%, 67.9 ± 6.4%, and 24.6 ± 0.5% leakage, respectively, were observed in the DOPE-DOPG vesicles, while the leakage was less than 15% in DOPC vesicles at all C/L ratios tested. These results strongly suggest that the higher cationic charge density of xanthone analogues 5c and 6 is critical for the desired membrane interaction and high membrane selectivity. Membrane Selectivity Study Using NMR Spectroscopy and MD Simulations. To further characterize the compound−membrane interactions, 1D 1H NMR spectra of 5c with liposomes were obtained. Significant differences in the chemical shift perturbations were observed when 5c was mixed with liposomes mimicking bacterial or mammalian cell membranes (Figure 5). Compared to 5c in aqueous solution, substantial line broadening of the Arg resonances and resonances in the aliphatic region of the 1H spectrum of 5c were observed during mixing with the DOPE-DOPG liposome medium. The line widths of a majority of the 5c resonances were comparable to those of the resonances of the aliphatic protons of the liposomes, indicating a full partitioning of 5c into the lipid phase and the maintenance of a significant degree of mobility of 5c in the model bacterial membrane. By contrast, the relatively sharp and unperturbed 1H resonances of 5c in the presence of the DOPC liposomes indicated that a large fraction F



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Table 5. Antimicrobial Testing of Compounds 5c and 6 and Selected Comparison Compounds in the Presence and Absence of 4% BSA MIC (μg/mL) S. aureus ATCC29213

MRSA DM21455 compd

without 4% BSA

with 4% BSA

increment

without 4% BSA

with 4% BSA

increment

5c 6 10 vancomycin daptomycin

1.56 3.125 12.5 0.78 0.39

3.125 12.5 200 0.78 6.25

2-fold 4-fold 16-fold 1-fold 16-fold

3.125 1.56 12.5 0.78 0.39

6.25 12.5 200 1.56 6.25

2-fold 8-fold 16-fold 2-fold 16-fold

the standard rabbit corneal epithelial abrasion model.37 No significant differences in the wound defect area (p > 0.05) compared to the control group (10 mM PBS at pH 7) were observed at all time points (Figure 6). In summary, 5c and 6

infection model (p < 0.001). By comparison, a 0.3% solution of gatifloxacin produced a 2.06 log reduction in viable bacteria (p = 0.037). By contrast, 9 induced a less than 1 log reduction at the tolerated concentration of 0.02% in solution (p = 0.731). Encouragingly, 6 demonstrated a clear therapeutic efficacy. The potency of 6 was also investigated in the mouse model of corneal infection using MRSA as the pathogen. As shown in Figure 7B, a 0.3% solution of 6 exhibited excellent in vivo efficacy (2.9 log reduction, p = 0.004) compared to a 5% solution of vancomycin (2.5 log reduction; p = 0.015). A high concentration of vancomycin is used in corneal infections because of its poor tissue penetration.40 The results of these studies confirm the appropriateness of our molecular design strategy for developing a new class of xanthone-based antibiotics with efficient therapeutic characteristics.

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CONCLUSION We have provided design principles for a new class of antibiotics with a molecular structure consisting of a rigid, hydrophobic core derived from a natural product. Despite interest in developing small-molecule synthetic mimics of antimicrobial peptides, the design principles and molecular components necessary for small-molecule membrane-active molecules with excellent selectivity have not been elucidated.19a,41 Modifying the hydrophobic core with cationic amino acids and lipophilic chains produced a compound with an optimal set of characteristics that was active in vivo. The design considerations provided here may be useful to address the critical need for new antimicrobials to combat the increasing prevalence of resistant pathogens.42 The promising antimicrobial activity, low toxicity in vivo and in vitro, and activity in a mouse model of corneal infection of xanthones 5c and 6 suggest that these molecules have immense potential as new templates in the therapeutics pipeline against some of the most serious forms of Gram-positive bacteria, including MRSA, and to avert drug resistance.

Figure 6. Effects of 5c and 6 on wound healing in rabbit corneas. (A) The topical application of 5c and 6 as 0.3% solutions did not interfere with wound closure. (B) Slit lamp photography after treatment with 5c and 6 as 0.3% solutions revealed no clinical signs of toxicity or corneal opacity.

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had no adverse effects on normal corneas or on cornea wound healing when applied topically to the eye at typical therapeutic concentrations. By contrast, fourth-generation fluoroquinolones, such as ofloxacin, norfloxacin, and gatifloxacin, have been reported to induce damage to corneal cells and interfere with wound healing.38 Evaluation of in Vivo Efficacy Using a Mouse Model of Corneal Infection. Antimicrobials with good in vitro activity may be inactive in vivo.39 We therefore investigated the efficacy of 5c and 6 in a mouse model of corneal infection (Figure 7). The mouse cornea was infected with S. aureus ATCC29213 or MRSA ATCC700699. After 1 day, antimicrobials were applied topically 5 times daily. As 0.3% solutions, compounds 5c and 6 effectively reduced the number of viable bacteria by 2.56 log (99.7%) and 3.03 log (99.9%), respectively, in the cornea

EXPERIMENTAL SECTION

General Chemistry. α-Mangostin (purity of 99.4%, HPLC) was purchased from Chengdu Biopurify Phytochemicals Ltd. (Chengdu, China). All other chemical reagents and solvents were obtained from commercial sources and used without further purification. 1H and 13C NMR spectra were collected on a Bruker Avance 400 MHz instrument using CD3OD, DMSO-d6, or CDCl3 as the solvent and tetramethylsilane as an internal reference. Coupling constants (J) are reported in hertz (Hz), while peak multiplicities are reported as singlet (s), doublet (d), triplet (t), quadruplet (q), broad (br), and multiplet (m), where applicable. APCI mass spectra were recorded on a Bruker amaZonX spectrometer, and ESI mass spectra were measured using an API2000 LC/MS/MS system. Chromatographic separation was achieved by preparative reverse phase HPLC on a Shimadzu LC20AP with a C18 column (Phenomenex, 150 mm × 21.2 mm, 5 μm, G



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Figure 7. Evaluation of the in vivo efficacy of selected antimicrobials. The in vivo efficacy of selected antimicrobials in the inhibition of bacterial growth on mouse corneas. (A) Infection by S. aureus ATCC 29213 (keratitis model). Concentration used: 9, 200 μg/mL, p = 0.731; 5c, 6, and gatifloxacin (GAT), 0.3% solution. MIC: 9, 0.5 μg/mL; 5c and 6, 3 μg/mL. (B) Infection by MRSA ATCC700699. Concentration used: 6, 0.3% solution, MIC = 2 μg/mL; vancomycin (Van), 5% solution. PBS was used as a negative control. (∗∗∗) p < 0.001 compared to control, except gatifloxacin. (∗∗) p = 0.004 compared to control, except vancomycin. The p-values of gatifloxacin and vancomycin compared to the control are 0.037 and 0.015, respectively. 100 Å), a flow rate of 10 mL/min, and UV detection at 254 nm. A mixture of water and methanol (both containing 0.1% formic acid) was used as the eluent using a gradient elution method. The solvent was then removed using a Heidolph rotary evaporator. The purity of all compounds was >98% based on HPLC analysis. Synthesis and Characterizations of Compounds. 1,3,6Trihydroxy-9H-xanthen-9-one (1a). 2,4-Dihydroxybenzoic acid (1.55 g, 10.0 mmol) and phloroglucinol (1.26 g, 10.0 mmol) were dissolved in Eaton’s reagent (25.0 mL). The reaction mixture was stirred at 80.0 °C for 1 h. Upon cooling to room temperature, the reaction mixture was poured into ice and stirred for 2 h. A thin slurry was formed. The solids were collected after filtration and washed with water. The crude product was then purified by silica gel chromatography with petroleum ether/ethyl acetate (1.5:1, v/v) to afford the desired product as a light yellow solid (1.62 g, 66%). Rf = 0.25 (1.5:1, petroleum ether/EtOAc). 1H NMR (400 MHz, DMSOd6) δ 13.06 (br, 1H, OH), 11.00 (br, 2H, 2 × OH), 7.98 (d, J = 7.2 Hz, 1H, Ar-H), 6.91 (d, J = 8.8 Hz, 1H, Ar-H), 6.83 (s, 1H, Ar-H), 6.36 (s, 1H, Ar-H), 6.19 (s, 1H, Ar-H). 13C NMR (101 MHz, DMSO-d6) δ 179.99, 166.05, 165.13, 163.76, 158.36, 158.27, 128.07, 114.89, 113.25, 102.96, 102.56, 98.88, 94.85. HRMS (ESI+): calcd for C13H9O5 [M + H]+ 245.0444, found 245.0437 (error 3.2 ppm). 1,3,6-Trihydroxy-2,8-diisopentyl-7-methoxy-9H-xanthen-9one (1b). α-Mangostin (500 mg, 1.22 mmol) was dissolved in ethanol (6.00 mL). Palladium−carbon catalyst (15.0% w/w) was added, and the reaction mixture was stirred at room temperature for 2 h under hydrogen atmosphere. Upon completion of the reaction, the reaction mixture was filtered and diluted with ethyl acetate. The mixture was concentrated under vacuum and purified through silica gel chromatography with petroleum ether/ethyl acetate (6:1, v/v), affording yellow solids (478 mg, 95%) as the desired product. Rf = 0.35 (6:1, petroleum ether/EtOAc). 1H NMR (400 MHz, CDCl3) δ 13.82 (s, 1H, OH), 6.76 (s, 1H, Ar-H), 6.28 (s, 1H, Ar-H), 3.83 (s, 3H, OCH3), 3.38−3.31 (m, 2H, CH2), 2.70−2.63 (m, 2H, CH2), 1.78−1.72 (m, 1H, CH), 1.60−1.67 (m, 1H, CH), 1.50−1.41 (m, 4H, 2 × CH2), 1.01 (s, 3H, CH3), 0.99 (s, 3H, CH3), 0.97 (s, 3H, CH3), 0.95 (s, 3H, CH3). 13C NMR (101 MHz, CDCl3) δ182.30, 161.34, 161.19, 156.08, 154.92, 154.85, 142.80, 139.64, 112.51, 111.60, 103.82, 101.65, 92.91, 62.42, 40.60, 38.33, 29.19, 28.62, 25.81, 22.95 (2 × CH3), 22.84 (2 × CH3), 20.55. HRMS (ESI+): calcd for C24H31O6 [M + H]+ 415.2115, found 415.2111 (error 0.9 ppm). 3,6-Bis(4-bromobutoxy)-1-hydroxy-9H-xanthen-9-one (2a). To a solution of 1a (300 mg, 1.23 mmol) in acetone (5.00 mL) was added potassium carbonate (849 mg, 6.15 mmol) and dibromobutane (2.17 mL 18.45 mmol), and the reaction mixture was stirred and refluxed overnight. Upon completion of the reaction, the mixture was concentrated under vacuum. The resulting brown viscous oil was diluted with ethyl acetate and washed three times with sodium bicarbonate and saturated NaCl consecutively. The organic

layer was dried over anhydrous sodium sulfate, concentrated under vacuum, and purified by silica gel chromatography with petroleum ether/ethyl acetate (8:1, v/v) to afford the desired product as a light yellow solid (228 mg, 36%). Rf = 0.30 (8:1, petroleum ether/EtOAc). 1 H NMR (400 MHz, CDCl3) δ 12.97 (s, 1H, OH), 8.13 (d, J = 8.9 Hz, 1H, Ar-H), 6.91 (dd, J = 8.8 Hz, 2.4 Hz, 1H, Ar-H), 6.81 (d, J = 2.4 Hz, 1H, Ar-H), 6.37 (d, J = 2.4 Hz, 1H, Ar-H), 6.31 (d, J = 2.0 Hz, 1H, Ar-H), 4.13−4.06 (m, 4H, 2 × CH2), 3.53−3.48 (m, 4H, 2 × CH2), 2.15−1.95 (m, 8H, 4 × CH2). 13C NMR (101 MHz, CDCl3) δ 180.08, 165.50, 164.52, 163.55, 157.96, 157.73, 127.39, 114.37, 113.38, 103.61, 100.67, 97.31, 93.20, 67.65, 67.51, 33.15, 33.11, 29.30, 29.28, 27.64 (2 × CH2). HRMS (APCI+): calcd for C21H23Br2O5 [M + H]+ 512.9907, found 512.9915 (error −1.5 ppm). 3,6-Bis(4-bromobutoxy)-1-hydroxy-2,8-diisopentyl-7-methoxy-9H-xanthen-9-one (2b). 1b (150 mg, 0.36 mmol) was dissolved in 8 mL of acetone. Thereafter, potassium carbonate (250 mg, 1.8 mmol) and 1,4-dibromobutane (0.65 mL, 5.4 mmol) were added. The reaction mixture was refluxed for 24 h. After the reaction was completed, the solvent was removed under reduced pressure. The oil residue was diluted with EtOAc and washed twice with saturated NaCl and once with water. The organic phase was dried over anhydrous Na 2SO 4 and then purified via silica gel column chromatography with petroleum ether/ethyl acetate (20:1, v/v), affording 191 mg of product 2b as a light yellow solid in 77% yield. Rf = 0.38 (20:1, petroleum ether/EtOAc). 1H NMR (400 MHz, CDCl3) δ 13.63 (s, 1H, OH), 6.69 (s, 1H, Ar-H), 6.27 (s, 1H, Ar-H), 4.12 (t, 2H, CH2), 4.07 (t, 2H, CH2), 3.83 (s, 3H, OCH3), 3.55−3.49 (m, 4H, 2 × CH2), 3.37−3.33 (m, 2H, CH2), 2.67−2.63 (m, 2H, CH2), 2.16− 2.00 (m, 8H, 4 × CH2), 1.80−1.73 (m, 1H, CH), 1.66−1.57 (m, 1H, CH), 1.47−1.36 (m, 4H, 2 × CH2), 1.01−0.96 (m, 12H, 4 × CH3). 13 C NMR (101 MHz, CDCl3) δ 182.31, 162.91, 160.39, 157.40, 155.64, 155.28, 144.19, 139.88, 113.18, 112.56, 104.25, 98.81, 89.33, 68.12, 67.50, 61.54, 40.64, 38.53, 33.52, 33.48, 29.76 (2 × CH3), 29.20, 28.71, 28.11, 27.97, 25.47, 23.00 (2 × CH3), 22.88 (2 × CH3), 20.61. HRMS (APCI+): calcd for C32H45Br2O6 [M + H]+ 683.1577, found 683.1588 (error −1.5 ppm). 3,6-Bis[4-(diethylamino)butox)]-1-hydroxy-9H-xanthen-9one (3a). To a solution of 2a (100 mg, 0.195 mmol) in dimethyl sulfoxide (4.00 mL) was added diethylamine (4.00 mL). The mixture was stirred at room temperature for 4 h. Upon completion of the reaction, the reaction mixture was diluted with ethyl acetate, followed by washing thrice with sodium bicarbonate and saturated NaCl consecutively. The organic phase was then dried over anhydrous sodium sulfate and concentrated under vacuum. The resulting crude oil was purified by silica gel chromatography (ethyl acetate/methanol/ triethylamine, 100:2:1, v/v/v) to afford the desired product as a light yellow solid (60.7 mg, 63%). Rf = 0.20 (100:2:1, EtOAc/MeOH/ Et3N). 1H NMR (400 MHz, CDCl3) δ 12.98 (s, 1H, OH), 8.12 (d, J = 8.8 Hz, 1H, Ar-H), 6.91 (dd, J = 8.8, 2.4 Hz, 1H, Ar-H), 6.81 (d, J = H



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washed with NaCl solution (3 × 25 mL). The organic phase was dried over anhydrous sodium sulfate. The solvent was evaporated to generate a crude residue, which was purified by HPLC to give 5a (81.6 mg, 53%) as a yellow solid. HPLC (eluent, methanol/water 65:35 to 95:5 for 29 min, 0.1% formic acid), tR = 4.5 min. 1H NMR (400 MHz, MeOD) δ 6.89 (s, 1H, Ar-H), 6.41 (s, 1H, Ar-H), 5.27−5.20 (m, 2H, 2 × CH), 4.79 (s, 2H, CH2), 4.73 (s, 2H, CH2), 4.59−4.55 (m, 2H, 2 × CH), 4.10 (d, J = 6 Hz, 2H, CH2), 3.86 (s, 3H, OCH3), 3.74 (s, 6H, 2 × CH3), 3.40 (d, J = 7.2 Hz, 2H, CH2), 2.97−2.86 (m, 4H, 2 × CH2), 1.98−1.93 (m, 2H, CH2), 1.87−1.80 (m, 6H, 2 × CH3), 1.76− 1.64 (m, 10H, 2 × CH3, 2 × CH2), 1.52−1.43 (m, 4H, 2 × CH2), 1.33−1.27 (m, 2H, CH2). 13C NMR (101 MHz, MeOD) δ 183.23, 173.44, 173.35, 170.20, 169.83, 162.97, 160.96, 157.81, 156.44, 156.34, 145.60, 138.79, 132.75, 132.37, 124.52, 123.41, 113.76, 113.12, 105.27, 101.29, 91.27, 68.65, 68.50, 61.77, 53.20, 53.10, 53.02 (2 × CH3), 40.43 (2 × CH2), 32.21, 32.13, 28.06 (2 × CH2), 27.07, 26.01, 25.99, 23.72, 23.70, 22.36, 18.38, 18.12. HRMS (ESI+): calcd for C42H59N4O12 [M + H]+ 811.4124, found 811.4153 (error −3.6 ppm). Dimethyl 2,2′-((2,2′-((1-Hydroxy-7-methoxy-2,8-bis(3-methylbut-2-en-1-yl)-9-oxo-9H-xanthene-3,6-diyl)bis(oxy))bis(acetyl))bis(azanediyl))bis(3-(1H-imidazol-5-yl)propanoate) (5b). HOBt (94.9 mg, 0.703 mmol) was added to 4 (147.8 mg, 0.281 mmol) in anhydrous DMF (5 mL). DIC (88.7 mg, 0.703 mmol) and H-His-OMe·2HCl (170.2 mg, 0.703 mmol) were added and stirred at 0 °C for 1 h. After stirring at room temperature overnight, the mixture was diluted with butanol and washed with NaCl solution (3 × 25 mL). The organic phase was dried over anhydrous sodium sulfate. The solvent was evaporated to generate a crude residue, which was purified by HPLC to give 5b (98.8 mg, 42%) as a yellow solid. HPLC (eluent, methanol/water 50:50 to 95:5 for 20 min, 0.1% formic acid), tR = 14.5 min. 1H NMR (400 MHz, MeOD) δ 8.08 (s, 1H, Ar-H), 8.05 (s, 1H, Ar-H), 7.07 (s, 1H, Ar-H), 7.04 (s, 1H, Ar-H), 6.85 (s, 1H, Ar-H), 6.36 (s, 1H, Ar-H), 5.25−5.17 (m, 2H, 2 × CH), 4.91−4.88 (m, 2H, CH), 4.75 (s, 2H, CH2), 4.69 (s, 2H, CH2), 4.11 (d, J = 5.2 Hz, 2H, CH2), 3.80 (s, 3H, OCH3), 3.74 (s, 6H, 2 × OCH3), 3.38 (d, J = 4.8 Hz, 2H, CH2), 3.28−3.21 (m, 2H, CH2), 3.19−3.11 (m, 2H, CH2), 1.85 (s, 3H, CH3), 1.77 (s, 3H, CH3), 1.68 (s, 3H, CH3), 1.65 (s, 3H, CH3). 13 C NMR (101 MHz, MeOD) δ 183.20, 172.19, 172.14, 170.19, 169.76, 162.85, 160.94, 157.66, 156.35, 156.23, 145.55, 138.75, 135.71, 135.68, 132.68, 132.36 (2 × CH), 132.30, 124.54, 123.30, 118.25, 118.12, 113.77, 113.12, 105.27, 101.24, 91.24, 68.63, 68.52, 64.64, 61.74, 53.26 (2 × CH2), 52.98, 28.72, 28.65, 27.06, 25.99, 25.94, 22.35, 18.38, 18.06. HRMS (ESI+): calcd for C42H49N6O12 [M + H]+ 829.3403, found 829.3439 (error −4.4 ppm). (2S,2′S)-Dimethyl 2,2′-((2,2′-((1-Hydroxy-7-methoxy-2,8-bis(3-methylbut-2-en-1-yl)-9-oxo-9H-xanthene-3,6-diyl)bis(oxy))bis(acetyl))bis(azanediyl))bis(5-guanidinopentanoate) (5c). HOBt (64.1 mg, 0.475 mmol) was added to 4 (100 mg, 0.19 mmol) in anhydrous DMF (5 mL). DIC (59.9 mg, 0.475 mmol) and H-Arg-OMe·2HCl (124 mg, 0.475 mmol) were added and stirred at 0 °C for 1 h. After stirring at room temperature overnight, the mixture was diluted with butanol and washed with NaCl solution (3 × 25 mL). The organic phase was dried over anhydrous sodium sulfate. The solvent was evaporated to generate a crude residue, which was purified by HPLC to give 5c (83 mg, 50%) as a yellow solid. HPLC (eluent, methanol/water 20:80 to 95:5 for 15 min, 0.1% formic acid), tR = 12.5 min. 1H NMR (400 MHz, MeOD) δ 6.89 (s, 1H, Ar-H), 6.42 (s, 1H, Ar-H), 5.27−5.20 (m, 2H, 2 × CH), 4.78 (s, 2H, CH2), 4.73 (s, 2H, CH2), 4.61−4.57 (m, 2H, 2 × CH), 4.11 (d, J = 6.4 Hz, 2H, CH2), 3.86 (s, 3H, OCH3), 3.75 (s, 6H, 2 × CH3), 3.40 (d, J = 7.4 Hz, 2H, CH2), 3.23−3.17 (m, 4H, 2 × CH2), 2.02−1.96 (m, 2H, CH2), 1.85− 1.80 (m, 8H, 2 × CH3, CH2), 1.69−1.62 (m, 10H, 2 × CH3, 2 × CH2). 13C NMR (101 MHz, MeOD) δ 183.09, 173.31, 173.20, 170.18, 169.81, 162.79, 160.85, 158.75, 158.73, 157.63, 156.29, 156.18, 145.48, 138.64, 132.74, 132.30, 124.59, 123.41, 113.66, 113.01, 105.19, 101.18, 91.22, 68.54, 68.44, 61.76, 53.20, 53.10, 53.02 (2 × CH3), 41.84, 41.82, 29.93, 29.84, 27.09, 26.21, 26.16, 26.03, 25.99, 22.39, 18.42, 18.16. HRMS (ESI+): calcd for C42H59N8O12 [M + H]+ 867.4247, found 867.4279 (error −3.7 ppm).

2.4 Hz, 1H, Ar-H), 6.37 (d, J = 2.4 Hz, 1H, Ar-H), 6.31 (d, J = 2.0 Hz, 1H, Ar-H), 4.11−4.03 (m, 4H, 2 × CH2), 2.57−2.48 (m, 12H, 6 × CH2), 1.92−1.78 (m, 4H, 2 × CH2), 1.69−1.58 (m, 4H, 2 × CH2), 1.03 (t, J = 7.2 Hz, 12H, 4 × CH3). 13C NMR (101 MHz, CDCl3) δ 180.10, 165.76, 164.78, 163.49, 158.00, 157.74, 127.26, 114.19, 113.45, 103.48, 100.62, 97.32, 93.21, 68.62, 68.48, 52.49 (2 × CH2), 46.85 (4 × CH2), 27.07 (2 × CH2), 23.58, 23.53, 11.66 (4 × CH3). HRMS (ESI+): calcd for C29H43N2O5 [M + H]+ 499.3166, found 499.3146 (error 4.1 ppm). 3,6-Bis(4-(diethylamino)butoxy)-1-hydroxy-2,8-diisopentyl7-methoxy-9H-xanthen-9-one (3b). To a solution of 2b (114 mg, 0.167 mmol) in DMSO (4 mL), diethylamine (4 mL) was added. The mixture was stirred at room temperature for 4 h. After the end of the reaction, the mixture was diluted with 50 mL of ethyl acetate, then washed with aqueous NaHCO3 and saturated NaCl (each three times). The organic phase was dried over anhydrous Na2SO4 and concentrated under vacuum. The residual crude oil was purified via silica gel column chromatography (EtOAc/MeOH/Et3N, 100/2/1, v/ v), affording 99.1 mg of pure product 3b as a yellow oil in 89% yield. Rf = 0.25 (100:2:1, EtOAc/MeOH/Et3N). 1H NMR (400 MHz, CDCl3) δ 13.65 (s, 1H, OH), 6.70 (s, 1H, Ar-H), 6.28 (s, 1H, Ar-H), 4.10 (t, 2H, CH2), 4.04 (t, 2H, CH2), 3.83 (s, 3H, OCH3), 3.37−3.33 (m, 2H, CH2), 2.67−2.63 (m, 2H, CH2), 2.60−2.52 (m, 12H, 6 × CH2), 1.95−1.58 (m, 10H, 2 × CH, 4 × CH2), 1.47−1.37 (m, 4H, 2 × CH2), 1.07−1.03 (t, 12H, 4 × CH3), 0.95−1.01 (t, 12H, 4 × CH3). 13 C NMR (101 MHz, CDCl3) δ 182.33, 163.17, 160.33, 157.59, 155.69, 155.33, 144.20, 139.69, 113.13, 112.38, 104.13, 98.80, 89.36, 69.03, 68.48, 61.45, 52.73, 52.69, 47.10 (2 × CH2), 47.08 (2 × CH2), 40.65, 38.51, 29.18, 28.66, 27.62, 27.46, 25.45, 23.90, 23.70, 22.99 (2 × CH3), 22.88 (2 × CH3), 20.57, 11.82 (4 × CH3). HRMS (ESI+): calcd for C40H65N2O6 [M + H]+ 669.4837, found 669.4852 (error −2.3 ppm). 2,2′-((1-Hydroxy-7-methoxy-2,8-bis(3-methylbut-2-en-1-yl)9-oxo-9H-xanthene-3,6-diyl)bis(oxy))diacetic Acid (4). A mixture of α-mangostin (500 mg, 1.22 mmol), methyl bromoacetate (1120 mg, 7.3 mmol), and KOH (341.6 mg, 6.1 mmol) in ethanol (30 mL) was refluxed for 24 h. After cooling, the mixture was diluted with ethyl acetate and washed with NaCl solution (3 × 50 mL). The organic phase was dried over anhydrous sodium sulfate and evaporated under reduced pressure. The crude residue was dissolved in THF (10 mL), and a 5% LiOH aqueous solution (5 mL) was added. After stirring at room temperature for 2 h, the reaction mixture was acidified with acetic acid, diluted with butanol, and washed with NaCl solution (3 × 50 mL). The organic phase was dried over anhydrous sodium sulfate. Subsequently, the solvent was evaporated to generate a crude residue, which was purified by column chromatography (silica gel, petroleum ether/ethyl acetate/acetic acid, 3:1:0.04) to yield 4 (427.2 mg, 67%) as a yellow solid. Rf = 0.20 (1:1:0.04, petroleum ether/ethyl acetate/acetic acid). 1H NMR (400 MHz, MeOD) δ 6.62 (s, 1H, ArH), 6.18 (s, 1H, Ar-H), 5.30−5.15 (m, 2H, 2 × CH), 4.80−4.67 (m, 4H, CH2), 4.05−3.93 (m, 2H, CH2), 3.83−3.79 (m, 3H, OCH3), 3.30−3.24 (m, 2H, CH2), 1.83 (s, 3H, CH3), 1.79 (s, 3H, CH3), 1.69 (s, 3H, CH3), 1.66 (s, 3H, CH3). 13C NMR (101 MHz, MeOD) δ 183.07, 171.70, 171.41, 163.12, 160.71, 157.84, 156.17, 156.09, 145.41, 138.19, 132.04 (2 × C), 124.78, 123.55, 113.21, 112.92, 104.88, 100.40, 90.79, 66.31, 66.20, 61.39, 27.02, 26.02, 25.97, 22.32, 18.37, 18.02. HRMS (ESI+): calcd for C28H30NaO10 [M + Na]+ 549.1731, found 549.1747 (error −2.8 ppm). Dimethyl 2,2′-((2,2′-((1-Hydroxy-7-methoxy-2,8-bis(3-methylbut-2-en-1-yl)-9-oxo-9H-xanthene-3,6-diyl)bis(oxy))bis(acetyl))bis(azanediyl))bis(6-aminohexanoate) (5a). HOBt (64.1 mg, 0.475 mmol) was added to 4 (100 mg, 0.19 mmol) in anhydrous DMF (5 mL). DIC (59.9 mg, 0.475 mmol) and H-Lys(Fmoc)-OMe· HCl (199 mg, 0.475 mmol) were added and stirred at 0 °C for 1 h. After stirring at room temperature overnight, the mixture was diluted with ethyl acetate and washed with NaCl solution (3 × 25 mL). The organic phase was dried over anhydrous sodium sulfate and evaporated under reduced pressure. The crude residue was dissolved in 20% piperidine/DMF (5 mL), and the reaction mixture was stirred for 20 min at room temperature. The mixture was diluted with CH2Cl2 and I



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(2S,2′S)-Diethyl 2,2′-((2,2′-((1-Hydroxy-7-methoxy-2,8-bis(3methylbut-2-en-1-yl)-9-oxo-9H-xanthene-3,6-diyl)bis(oxy))bis(acetyl))bis(azanediyl))bis(5-guanidinopentanoate) (5d). This compound was prepared using the method for 5c. 4 (100 mg, 0.19 mmol) and H-Arg-OEt·2HCl (130.7 mg, 0.475 mmol) were utilized to yield the coupled product 5d (41.5 mg, 24%) as a yellow solid. HPLC (eluent, methanol/water 20:80 to 95:5 for 18 min, 0.1% formic acid), tR = 16.2 min. 1H NMR (400 MHz, MeOD) δ 6.86 (s, 1H, Ar-H), 6.37 (s, 1H, Ar-H), 5.30−5.19 (m, 2H, 2 × CH), 4.77 (s, 2H, CH2), 4.71 (s, 2H, CH2), 4.60−4.52 (m, 2H, 2 × CH), 4.28−4.14 (m, 4H, 2 × CH2), 4.06 (d, J = 6.4 Hz, 2H, CH2), 3.84 (s, 3H, OCH3), 3.38 (d, J = 6.8 Hz, 2H, CH2), 3.25−3.16 (m, 4H, 2 × CH2), 2.04−1.95 (m, 2H, CH2), 1.86−1.62 (m, 16H, 4 × CH3, 2 × CH2), 1.30−1.22 (m, 8H, 2 × CH3, CH2). 13C NMR (101 MHz, MeOD) δ 183.16, 172.84, 172.72, 170.18, 169.81, 162.86, 160.91, 158.75 (2 × C), 157.70, 156.38, 156.27, 145.54, 138.74, 132.77, 132.35, 124.54, 123.39, 113.72, 113.06, 105.24, 101.24, 91.22, 68.61, 68.46, 62.79, 62.77, 61.80, 53.19, 53.10, 41.83 (2 × CH2), 29.98, 29.89, 27.08, 26.20, 26.16, 26.02, 25.99, 22.43, 18.40, 18.15, 14.51 (2 × CH3). HRMS (ESI+): calcd for C44H63N8O12 [M + H]+ 895.4560, found 895.4585 (error −2.8 ppm). (2S,2′S)-2,2′-((2,2′-((1-Hydroxy-7-methoxy-2,8-bis(3-methylbut-2-en-1-yl)-9-oxo-9H-xanthene-3,6-diyl)bis(oxy))bis(acetyl))bis(azanediyl))bis(5-guanidinopentanamide) (5e). This compound was prepared using the method for 5c. 4 (150 mg, 0.285 mmol) and H-Arg-NH2·2HCl (175.4 mg, 0.713 mmol) were utilized to yield the coupled product 5e (147.5 mg, 62%) as a yellow solid. HPLC (eluent, methanol/water 15:85 to 95:5 for 24 min, 0.1% formic acid), tR = 20.0 min. 1H NMR (400 MHz, MeOD) δ 6.71 (s, 1H, ArH), 6.25 (s, 1H, Ar-H), 5.27−5.15 (m, 2H, 2 × CH), 4.71 (s, 2H, CH2), 4.68 (s, 2H, CH2), 4.61−4.49 (m, 2H, 2 × CH), 3.99 (d, J = 5.5 Hz, 2H, CH2), 3.80 (s, 3H, OCH3), 3.35 (d, J = 11.1 Hz, 2H, CH2), 3.28−3.16 (m, 4H, 2 × CH2), 1.97−1.91 (m, 2H, CH2), 1.87−1.76 (m, 8H, 2 × CH3, CH2), 1.74−1.60 (m, 10H, 2 × CH3, 2 × CH2). 13C NMR (101 MHz, MeOD) δ = 182.94, 175.86, 175.70, 169.97, 169.57, 162.58, 160.77, 158.74, 158.72, 157.41, 156.18, 156.08, 145.39, 138.55, 132.77, 132.24, 124.64, 123.35, 113.55, 112.87, 105.09, 101.06, 91.14, 68.46, 68.40, 61.81, 53.56, 53.38, 41.94 (2 × CH2), 30.96, 30.91, 27.11, 26.16, 26.10, 26.05, 26.02, 22.43, 18.45, 18.24. HRMS (ESI+): calcd for C40H57N10O10 [M + H]+ 837.4254, found 837.4251 (error 0.3 ppm). (2S,2′S)-Di-tert-butyl 2,2′-((2,2′-((1-Hydroxy-7-methoxy-2,8bis(3-methylbut-2-en-1-yl)-9-oxo-9H-xanthene-3,6-diyl)bis(oxy))bis(acetyl))bis(azanediyl))bis(5-guanidinopentanoate) (5f). This compound was prepared using the method for 5c. 4 (100 mg, 0.19 mmol) and H-Arg-OtBu·2HCl (144 mg, 0.475 mmol) were utilized to yield the coupled product 5f (83.8 mg, 46%) as a yellow solid. HPLC (eluent, methanol/water 20:80 to 95:5 for 24 min, 0.1% formic acid), tR = 21.0 min. 1H NMR (400 MHz, MeOD) δ 6.82 (s, 1H, Ar-H), 6.36 (s, 1H, Ar-H), 5.27−5.19 (m, 2H, 2 × CH), 4.75 (s, 2H, CH2), 4.70 (s, 2H, CH2), 4.51−4.39 (m, 2H, 2 × CH), 4.06 (d, J = 5.6 Hz, 2H, CH2), 3.86 (s, 3H, OCH3), 3.39 (d, J = 6.4 Hz, 2H, CH2), 3.27−3.15 (m, 4H, 2 × CH2), 1.97−1.91 (m, 2H, CH2), 1.88− 1.75 (m, 8H, 2 × CH3, CH2), 1.73−1.61 (m, 10H, 2 × CH3,2 × CH2), 1.47 (s, 18H, 6 × CH3). 13C NMR (101 MHz, MeOD) δ 183.17, 172.04, 171.89, 170.01, 169.68, 162.79, 160.95, 158.74, 158.72, 157.69, 156.41, 156.30, 145.56, 138.80, 132.83, 132.36, 124.53, 123.39, 113.76, 113.07, 105.27, 101.23, 91.17, 83.61, 83.55, 68.64, 68.45, 61.84, 53.81, 53.67, 41.84, 41.82, 30.19, 30.06, 28.46 (6 × CH3), 27.08, 26.18, 26.12, 26.03, 26.00, 22.40, 18.41, 18.19. HRMS (ESI+): calcd for C48H71N8O12 [M + H]+ 951.5186, found 951.5221 (error −3.7 ppm). Dimethyl 2,2′-((2,2′-((2,2′-((1-Hydroxy-7-methoxy-2,8-bis(3methylbut-2-en-1-yl)-9-oxo-9H-xanthene-3,6-diyl)bis(oxy))bis(acetyl))bis(azanediyl))bis(5-guanidinopentanoyl))bis(azanediyl))bis(5-guanidinopentanoate) (6). To a solution of 5c (100 mg, 0.115 mmol) in THF (4 mL) was added a 5% LiOH aqueous solution (2 mL). After stirring at room temperature for 2 h, the reaction mixture was acidified with acetic acid, diluted with butanol, and extracted with saturated NaCl (3 × 25 mL). The organic phase was dried over anhydrous sodium sulfate and evaporated under reduced pressure. The crude residue was dissolved in anhydrous DMF (5 mL). HOBt (38.8 mg, 0.288 mmol), DIC (36.3 mg, 0.288 mmol),

and H-Arg-OMe·2HCl (75.1 mg, 0.288 mmol) were added, and the mixture was stirred at 0 °C for 1 h. After stirring at room temperature overnight, the mixture was diluted with butanol and washed with NaCl solution (3 × 25 mL). The organic phase was dried over anhydrous sodium sulfate. The solvent was evaporated to generate a crude residue, which was purified by HPLC to afford 6 (87.3 mg, 64%) as a yellow solid. HPLC (eluent, methanol/water 20:80 to 95:5 for 30 min, 0.1% formic acid), tR = 16.5 min. 1H NMR (400 MHz, MeOD) δ 6.96 (s, 1H, Ar-H), 6.47 (s, 1H, Ar-H), 5.31−5.15 (m, 2H, 2 × CH), 4.81− 4.73 (m, 4H, 2× CH2), 4.57−4.42 (m, 4H, 4 × CH), 4.20−4.06 (m, 2H, CH2), 3.86 (s, 3H, OCH3), 3.78−3.65 (m, 6H, OCH3), 3.49−3.37 (m, 2H, CH2), 3.27−3.07 (m, 8H, 4 × CH2), 2.00−1.87 (m, 4H, 2 × CH2), 1.87−1.78 (m, 6H, 2 × CH3), 1.76−1.60 (m, 14H, 2 × CH3, 4 × CH2), 1.34−1.15 (m, 4H, 2 × CH2). 13C NMR (101 MHz, MeOD) δ 183.33, 173.58 (2 × C), 170.44 (2 × C), 170.09, 169.70, 162.96, 161.05, 158.72 (4 × C), 157.81, 156.58, 156.46, 145.66, 138.90, 132.84, 132.44, 124.48, 123.28, 113.85, 113.16, 105.35, 101.35, 91.24, 68.58, 68.41, 61.85, 57.69, 57.47, 53.95, 53.82, 53.32, 52.92, 41.97(2 × CH2), 41.80 (2 × CH2), 30.61 (2× CH2), 29.46 (2× CH2), 27.07, 26.22 (2× CH3), 26.00 (4× CH2), 22.38, 18.38, 18.14. HRMS (ESI+): calcd for C54H83N16O14 [M + H]+ 1179.6269, found 1179.6315 (error −3.9 ppm). Bacterial Strains and Growth Conditions. Inoculum suspensions were prepared from isolated colonies using the direct colony suspension method as described by the Clinical and Laboratory Institute (CLSI). The colonies were selected from an 18−20 h tryptic soy agar (TSA) plate. Determination of Antimicrobial Activity. Susceptibility testing was performed in Mueller Hinton broth (MHB) using broth macrodilution in accordance with the CLSI guidelines and our previous report.19a To determine the antimicrobial activity of the compounds in the presence of BSA, BSA was added to bacterial suspensions at a final concentration of 4% w/v. Then the MIC was determined as described. Determination of Hemolytic Activity. The hemolysis assay was performed as described previously.19a Briefly, fresh rabbit red blood cells (RBCs) were isolated. All procedures for isolating blood from New Zealand white rabbits were approved by IACUC Singhealth and performed according to the standards of the Association for the Research in Vision and Ophthalmology. Molecular Hydrophobicity Analysis. The molecular hydrophobicity of the synthesized analogues was characterized in terms of retention time and % ACN by HPLC (Waters 2695 separation module) on a Waters Delta-Pak CA 300 Å column. The experiments were conducted under the same conditions for all analogues. The samples were injected at a concentration of 10 μg/mL with an injection volume of 20 μL and a flow rate of 1 mL/min. The gradient profile was 5−55% ACN over 5 min, followed by 55−95% over the next 15 min. Time−Kill Studies. The bacteria used in the time−kill studies were isolated from the TSA plate after 18−20 h. The inoculum was then suspended and adjusted in CA-MHB to obtain a bacterial suspension of 105−106 CFU/mL. Then the inoculum was treated with various concentrations of gatifloxacin, 5c, or 6. The mixtures were incubated at 35 °C. Culture aliquots were removed at specific time points for viable plate counts. The aliquots were serially diluted at 10-fold ratios using D/E neutralization broth, and 20 μL of each dilution was plated on TSA plates using the surface-spread plate method. The plates were incubated at 35 °C for 48−72 h. Cell viability was assessed by counting the resulting colonies on the plates. The bacterial strains used in this study were S. aureus ATCC 29213, MRSA DM21455, vanA VRE, and vanB VRE. Multipassage Resistance Selection Studies. Multipassage resistance selection studies were performed using S. aureus ATCC29213 with 5c and 6. Dilution series of eight concentrations of 5c or 6, gatifloxacin, and norfloxacin were prepared. The resistance of the strains against 5c or 6, gatifloxacin, and norfloxacin was determined based on the progressive increase in the MIC of the bacteria over the passages. Bacterial growth in each dilution was examined after 20−22 h of incubation. The bacteria that grew in the J



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medium at 0.5 × MIC of each antimicrobial agent were repassaged in a fresh dilution series of eight concentrations. Each antimicrobial agent was repassaged for 17 passages in each culture. In this study, resistance was defined as an increase in the initial MIC of more than 4-fold.33 Vesicle Leakage from Calcein-Loaded LUVs. All phospholipids used in this assay were purchased from Avanti Polar Lipids, Inc. (Alabaster) and used without further purification. The phospholipids used in this study were 1,2-di-(9Z-octadecenoyl)-sn-glycero-3phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phospho(1′-rac-glycerol) (sodium salt) (DOPG), and E. coli total lipid extract. Calcein-loaded large unilamellar vesicles (LUVs) were prepared using the film hydration method, as described previously.19a Briefly, the phospholipids (DOPE/DOPG = 75/25) were dissolved in methanol− chloroform (1:2 by volume) and transferred to a test tube. Then the phospholipid solution was dried gently using a constant stream of nitrogen gas and placed under vacuum for at least 2 h. The calcein solution (80 mM calcein, 50 mM HEPES, 100 mM NaCl, and 0.3 mM EDTA; pH 7.4) was added to the dried phospholipid film to obtain a final phospholipid concentration of 30 mM. The solution was then frozen in liquid nitrogen and warmed in a water bath for seven cycles. A miniextruder (Avanti Polar Lipid Inc.) was used to prepare 100 nm homogeneous LUVs. The extrusion was repeated for 15 cycles using a polycarbonate membrane (Whatman) with a pore size of 100 nm. The vesicles were passed through a Sephadex G-50 gel filtration column to remove excess free calcein. The concentration of the calceinencapsulated liposomes was determined using a total phosphorus assay. An aliquot of the calcein-encapsulated LUVs was transferred to a stirred cuvette. The desired concentrations of the xanthone analogues were prepared in DMF and added to the lipid to obtain compound to lipid ratios of 1/16, 1/32, and 1/64. The final concentration of phospholipids was 50 μM. The percentage of DMF in any experiment was less than 0.2%. Control experiments containing 0.2% DMF demonstrated that the LUVs were not lysed by the presence of DMF. Triton X-100 (0.1%) was added to determine the intensity at 100% lysis. The fluorescence emission intensity was monitored using a TECAN Infinite M200Pro at an excitation wavelength of 490 nm and an emission wavelength of 520 nm for 30 min. Percentage leakage (% L) was calculated using the following formula: %L = [(It − Io)/(I∞ − Io)] × 100, where Io and It are the intensities before and after the addition of the xanthone analogues, respectively, and I∞ is the intensity after the addition of 0.1% Triton X-100. Membrane Selectivity Study Using Nuclear Magnetic Resonance Spectroscopy. The liposomes used in the nuclear magnetic resonance study were prepared using a method similar to that used for the calcein leakage experiments. The compositions of the liposomes in this study were 30 mM 75:25 DOPE/DOPG and 30 mM DOPC. NMR experiments were performed using a Bruker Avance II 600 MHz spectrometer equipped with a TXI cryoprobe at 25 °C. 1D 1 H spectra of 1 mM 5c were acquired in Milli-Q water, 30 mM DOPE/DOPG mixed liposomes, and 30 mM DOPC liposomes. In Vivo Topical Toxicity Tests. Wild-type C57BL6 mice (6−8 weeks old; 20−30 g) were purchased from the National University of Singapore. All animals were utilized after 1 week of acclimatization and were kept in air-conditioned rooms with a controlled temperature (23 ± 2 °C), 12 h light−dark cycles, and a humidity of 55−60%. All animal experiments were conducted in compliance with the ARVO statement for the Use of Animals in Ophthalmic and Vision Research and the guide for the Care and Use of Laboratory Animals (National Research Council) and under the supervision of the SingHealth Experimental Medical Centre (SEMC). Ethics approval was obtained from SingHealth IACUC. To investigate corneal toxicity, three normal, healthy wild-type mice were selected randomly and treated with 0.5, 1, or 3 mg/mL 5c or 6 in 10 mM PBS. The respective drug was applied topically 5 times/day for 4 days. Corneal clarity was examined by slit lamp biomicroscopy every day through day 4, after the last application. To study acute systemic toxicity, 5c and 6 were administered by bolus injection via intravenous and intraperitoneal routes. Two mice were used for each route and were monitored carefully over 24 h to observe and record any sign of discomfort, mortality, morbidity, or toxicity signs. Gross necropsy was performed on any animal that died.43

Rabbit Corneal Wound Healing Model. Twelve rabbits were randomly assigned to four groups (three rabbits per group). Two groups were treated with 10 mM PBS at pH 7 (groups A and B) as control groups, and two groups of rabbits were treated with 5c (3 mg/ mL in 10 mM PBS; group C) and 6 (3 mg/mL in 10 mM PBS; group D). The rabbits were tranquilized by intraperitoneal injection of 1 mL of ketamine (100 mg/mL) and 0.5 mL of xylazil (20 mg/mL). The corneas were anesthetized by topical administration of 1% xylocaine. A 5 mm trephine was used to outline the wound margin, and epithelial cells were removed mechanically using a sterile miniblade (BDBeaver) while leaving the basal lamina intact.25,38 Rabbits in groups C and D were treated by topical administration of the respective antimicrobials 3 times per day. The cornea wound was visualized by staining with fluorescein sodium, which is used in ophthalmology clinics to visualize wounds on the ocular surface, with the help of a slitlamp biomicroscope equipped with a cobalt-blue filter.36,44 Measurements of the residual wound area were performed during the reepithelialization process using Image-J, version 1.44o. A Mann− Whitney test was employed to evaluate differences in re-epithelialization between the two groups. The statistical calculations were performed using PASWstatistic18. In Vivo Efficacy Testing of Xanthone Analogues. S. aureus (ATCC 29213) was grown overnight on TSA plates at 35 °C. A few colonies were selected and suspended in United States Pharmacopeia (USP) phosphate buffer to a concentration of 1 × 108 CFU/mL and used for the animal study. All mouse eyes were examined previously under slit-lamp examination. Four healthy mice were chosen for each treatment group. The antimicrobials and concentrations used in this study were gatifloxacin (0.3% solution), 9, 5c, and 6; each xanthone analogue was used at a specific concentration. Vancomycin was used as a 5% solution in the MRSA ATCC700699 treatment. The animals were anesthetized subcutaneously with 0.2 mL of ketamine (100 mg/ mL) and 0.1 mL of xylazine (20 mg/mL) mixed with 0.7 mL of normal saline (0.08 mL/mice). Superficial scratches of the cornea (n = 3, each 1 mm long) were created with a sterile miniblade (BD-Beaver) under the microscope on one eye and did not penetrate beyond the superficial stroma, whereas the other eye remained uninvolved.45 Then 10 μL of the bacterial suspension was applied topically to the surface of the wounded eye. All animals were treated with the respective antimicrobials 5 times/day starting from day 1 postinfection. At days 1, 2, and 3 post-treatment, four animals from each group were sacrificed. The wounded corneas were dissected, and the number of viable bacteria was quantified after plating. For this purpose, individual corneas were homogenized in sterile 0.9% NaCl containing 0.25% BSA,46 serially diluted, and plated in duplicate on TSA plates. The plates were incubated for 48 h at 35 °C. The number of viable bacteria in an individual cornea was determined by counting individual colonies on plates from the various dilutions and multiplying the number of colonies by the appropriate dilution. The results were reported as log10 of number of CFU/cornea. The data were examined statistically by one-way analysis of variance (ANOVA) and a post hoc test (Bonferroni test, PASW Statistics 18). The Kruskal−Wallis nonparametric test was employed, and a post hoc test (pairwise comparison test) was performed to determine if a difference in colony forming units existed among the different groups. Molecular Dynamic (MD) Simulations. MD Simulation Part 1: Investigation of the Role of the Lipophilic Chain. In each simulation, nine molecules of 1a and 3a were placed close to the bacterial membrane and solvated with water and counterions. Next, each simulation was subjected to 500 steps of energy minimization, and 200 ns of MD simulations were then performed. MD Simulation Part 2: Investigation of the Selectivity of 5c. MD simulations were performed to study the interactions of xanthone analogues with model membranes. The GROMACS 53a6 force field was used with an SPC water model in all simulations. To study the selectivity of 5c, the interactions of 5c with model bacterial and human membranes were investigated. The bacterial membrane was modeled using 128 lipids composed of 96 zwitterionic POPE and 32 anionic POPG lipid molecules.47 The human membrane contained 128 lipids, with 96 POPC and 32 cholesterol molecules.47 To examine the K



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concentration-dependent effects of 5c, simulations were performed with varying numbers of 5c in the vicinity of both membranes. Initially, one, four, and eight 5c molecules were placed in random orientations on top of the two model membranes, after which the system was solvated in a simulation box with approximately 6000 water molecules, and counterions were added to neutralize the system. MD simulations of 200 ns were then conducted for each system. The surface potential of the model membranes was calculated using the APBS plugin for PYMOL.48 During the simulations, the covalent bonds involving hydrogen atoms were constrained using the LINCS algorithm, which enabled a time step of 2 fs to be used in all simulations. A cutoff distance of 0.9 nm was used for real-space electrostatic interactions, and the particle-mesh Ewald algorithm was employed to calculate the long-range electrostatic interactions in reciprocal space.49 LJ potentials were cut off at 1.44 nm. The Nose−Hoover coupling method was used to maintain the target temperatures at 310 K,50 and the semi-isotropic Parrinello−Rahman method was used to maintain the pressure at 1 atm in the NPT ensemble.

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ASSOCIATED CONTENT

S Supporting Information *

Molecular dynamics simulations (Figure S1 and S2) and MICs of 5c and 6 against a panel of Gram-positive bacteria (Table S1). This material is available free of charge via the Internet at http://pubs.acs.org.

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Article

AUTHOR INFORMATION

Corresponding Authors

*D.C.: phone, (86) 20-8711-0245; fax, (86) 20-8711-0245; email, [email protected]. *S.L.: phone, (+65) 66012464; fax, (+65) 68723818; e-mail, [email protected]. *R.W.B.: phone, (+65) 63224544; fax, (+65) 63224599; e-mail, [email protected]. Present Address ●

L.M.P.: School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 63755, Singapore.

Author Contributions ○

J.-J.K. and S.L. contributed equally to this work. All authors contributed to the preparation of this manuscript. All authors have given approval to the final version of the manuscript.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank BII and A*STAR Computational Resource Center for providing computational facilities. The authors also thank ACRC at A*STAR and CSC in Finland for providing computational facilities. This work was supported by Grant SHF/FG538P/2013 (Grant R1137/39/2014), SHF/FG603S/ 2013 R1136, NMRC/NIG R1159, Exploit Flagship funding Grants X031, NMRC/TCR/002-SERI/2088R618, NSFC/ 21072064, and NMRC-CBRG 14may012.

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ABBREVIATIONS USED CLSI, Clinical and Laboratory Standards Institute; MRSA, methicillin-resistant S. aureus; MSSA, methicillin-susceptible S. aureus; CAMP, cationic antimicrobial peptide; TSA, tryptic soy agar; MIC, minimum inhibitory concentration; CFU, colony forming units; VRE, vancomycin-resistant enterococci; BSA, bovine serum albumin; NP, natural product; ATCC, American Type Culture Collection L



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