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Article Cite This: J. Med. Chem. 2017, 60, 10135−10150

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Semisynthesis and Biological Evaluation of Xanthone Amphiphilics as Selective, Highly Potent Antifungal Agents to Combat Fungal Resistance Shuimu Lin,†,‡,|| Wan Ling Wendy Sin,‡ Jun-Jie Koh,‡ Fanghui Lim,‡ Lin Wang,†,|| Derong Cao,⊥ Roger W. Beuerman,*,‡,§ Li Ren,*,†,|| and Shouping Liu*,‡,§ †

School of Materials Science and Engineering, South China University of Technology, Guangzhou 510641, China Singapore Eye Research Institute, The Academia, 20 College Road, Discovery Tower Level 6, Singapore 169856, Singapore § SRP Neuroscience and Behavioral Disorders, Duke-NUS Graduate Medical School, Singapore 169857, Singapore || National Engineering Research Center for Tissue Restoration and Reconstruction, Guangzhou 510006, China ⊥ School of Chemistry and Chemical Engineering, South China University of Technology, Guangzhou 510641, China ‡

S Supporting Information *

ABSTRACT: New efficient antifungal agents are urgently needed to treat drug-resistant fungal infections. Here, we designed and synthesized a series of cationic xanthone amphiphilics as antifungal agents from natural α-mangostin to combat fungal resistance. The attachment of cationic residues on the xanthone scaffold of α-mangostin resulted in interesting antifungal agents with a novel mode of action. Two lead compounds (1 and 2) showed potent antifungal activity against a wide range of fungal pathogens, including drugresistant Candida albicans, Aspergillus, and Fusarium strains and low cytotoxicity and hemolytic activity against mammalian cells. Both compounds can kill fungus rapidly by directly disrupting fungal cell membranes and avoid developing drug resistance. Additionally, compound 1 exhibited potent in vivo antifungal activity in the murine model of fungal keratitis. To our knowledge, membrane-targeting xanthone-based antifungals have not been reported previously. These results demonstrated that compounds 1 and 2 may be promising candidates for treating drugresistant fungal infections.

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organisms.16−18 They play an important role in modulating the host innate immune system,18,19 displaying broad spectrum antimicrobial activity against fungi,16,20−22 bacteria,23−25 and viruses.26,27 Unlike conventional antifungal agents with specific target, AMPs kill fungal cells by directly disrupting fungal cell membranes.28,29 This nonspecific mode of action can significantly reduce the probability of developing antifungal resistance.30,31 However, the development of AMPs are hampered by limited stability owing to enzymatic degradation, cytotoxic adverse effects owing to low membrane selectivity, and high production cost.32,33 Peptidomimetics with cationic amphiphilic structure would be an alternative strategy to solve these problems.32−34 Peptidomimetics mimic the fungicidal action of AMPs by directly disrupting negatively charged fungal cell membranes.35−37 Xanthones are secondary metabolites commonly found in lichens, higher plants, and fungi, and they are known to display a wide variety of biological activities.38,39 α-Mangostin, the major xanthone derivative extracted from the pericarp of

INTRODUCTION Fungal infections pose a serious threat to human health, especially to immunocompromised patients.1,2 Fungal infections kill more than 1.5 million people each year.3,4 Recently, the mortality of fungal infections is on the rise because of the increasing drug resistance to the traditional antifungal agents.5−7 This problem is enlarged by a serious lack of new class of antifungal agents.5,8 Over the past six decades, only four classes of compounds, including azoles, polyenes, flucytosine, and echinocandins, are commonly applied in the treatment of fungal infections.5 Since 2001, tavaborole was the only new molecular entity (NME) as antifungal agent approved by U.S. FDA in 2014.9 Tavaborole is a boron-based topical antifungal for treating fungal nail infections.10−12 The drawbacks include the development of drug resistance, fungistatic activity, chronic side effects, or acute toxicity and limited in vivo efficacy due to poor pharmacokinetics.13−15 To address these issues, there is an urgent need to develop novel antifungal agents with new mode of action and no drug-resistance development.8 Antimicrobial peptides (AMPs) with amphipathic structure have attracted much attention as a promising new class of antimicrobial candidates, naturally occurring in various living © 2017 American Chemical Society

Received: September 11, 2017 Published: November 20, 2017 10135

DOI: 10.1021/acs.jmedchem.7b01348 J. Med. Chem. 2017, 60, 10135−10150

Journal of Medicinal Chemistry

Article

Scheme 1. Synthesis of Amphiphilic α-Mangostin Derivatives 1, 2, and 5−29a

a

Reagents and conditions: (i) epibromohydrin, ethanol, KOH, reflux, overnight; (ii) corresponding amine compounds, methanol, reflux, 12 h; (iii) iodomethane, methanol, RT, overnight; (iv) 1H-pyrazole-1-carboxamidine hydrochloride, DIPEA, RT, 12 h.

through the disruption of the bacterial membrane, suggesting that amphiphilic α-mangostin derivatives may be ideal antimicrobial drug candidates.49,50 In this work, we designed and synthesized a new series of cationic xanthone amphiphilics that share common physicochemical characteristics with AMPs, starting from natural αmangostin. To the best of our knowledge, membrane-targeting xanthone-based antifungals have not been reported previously. Fungal cell membranes are negatively charged because they are rich in phosphomannans and negatively charged phospholipids including phosphatidylinositol, phosphatidylserine, and diphosphatidylglycerol.51−53 In contrast, mammalian cell membranes are overall neutral because they are rich in cholesterol and

mangosteen, exhibits diverse pharmacological properties, including antifungal,40,41 anticancer,42 antibacterial,43,44 antioxidant,45,46 antimalarial,47 and anti-inflammatory activities.48 Kaomongkolgit et al. have reported that the antifungal activities of α-mangostin is very poor, with high minimum inhibitory concentration (MIC) values of 1000 μg/mL.41 In recent years, our group has reported that several series of semisynthetic amphiphilic α-mangostin derivatives as the mimics of AMPs displayed potent in vitro and in vivo bactericidal activity against Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE).49,50 These α-mangostin derivatives can avoid the development of drug resistance and kill bacteria rapidly 10136



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Scheme 2. Synthesis of Amphiphilic α-Mangostin derivatives 31−33 and 35a

a

Reagents and conditions: (i) methyl bromoacetate, ethanol, KOH, reflux, 24 h; (ii) LiOH, THF/H2O, RT, 1.5 h; (iii) corresponding Arg compounds, DIC, HOBt, DMF, RT, overnight, Arg = arginine.

yield by the alkylation of the parent compound α-mangostin with epibromohydrin in ethanol in the presence of KOH. Owing to the formation of intramolecular hydrogen bonds between the oxygen atom of C9 carbonyl group and the hydrogen atom of C1 hydroxyl group, the phenolic hydroxyl groups at the C3 and C6 positions of xanthone scaffold were more reactive than the C1 phenolic hydroxyl group.49 The epoxide 4 was then ring-opened with the proper aliphatic amines in methanol to afford the corresponding compounds 2 and 5−25. The tertiary amines 2, 5, and 6 were then converted into the corresponding quaternary ammonium salts 26−28 by reaction with methyl iodide in methanol. The reaction of compound 22 with 1H-pyrazole-1-carboxamidine in DMF in the presence of N,N-diisopropylethylamine (DIPEA) yielded the desired compound 1. Compound 29 was obtained from compound 23 using similar procedure. Arginine-coupled xanthone derivatives 31−35 were synthesized according to the reported methods.49 All final analogues were purified by HPLC to be >95% pure and were characterized by 1H NMR, 13 C NMR, and HRMS. Biological Evaluation. In vitro antifungal activities of all the synthesized xanthone derivatives were determined in comparison with the marketed natamycin and caspofungin as well as the parent compound α-mangostin against two Candida albicans strains, including the drug-resistant strain of C. albicans DF2672R (Table 1−3). C. albicans is the most prevalent pathogenic fungus in humans, causing life-threatening infections in vulnerable patients.37,54 Several potent xanthone

neutral phospholipids including phosphatidylethanolamine and phosphatidylcholine.53 We hypothesize that the incorporated cationic moieties would be effective in facilitating the interaction between xanthone amphiphiles and the negatively charged fungal cell membranes via electrostatic attraction; the isoprenyl groups and xanthone scaffold of α-mangostin served as the hydrophobic moieties would facilitate the penetration of xanthone amphiphiles into fungal lipid membranes, resulting in the leakage of cell constituents and finally fungal death. We optimized α-mangostin analogues by biomimicking AMPs in order to enhance their antifungal potency and the membrane selectivity between zwitterionic mammalian membranes and negatively charged fungal membranes. Finally, the selected potent compounds 1 and 2 were further evaluated by following the time-kill kinetics, multipassage resistance selection, cytotoxicity toward mammalian cells, and mode of action studies. The results demonstrated that the two cationic xanthone amphiphilics had potential for developing as novel antifungal therapeutic agents.

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RESULTS AND DISCUSSION Chemical Synthesis. A series of amphiphilic xanthone derivatives were achieved by attaching cationic residues, such as aliphatic amines and basic amino acids, to the highly hydrophobic xanthone scaffold of α-mangostin. The general synthetic routes employed for the synthesis of cationic amphiphilic xanthone derivatives are depicted in Schemes 1 and 2. The key intermediate epoxide 4 was prepared in good 10137



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Table 1. In Vitro Antifungal and Hemolytic Activities (μg/mL) of Amphiphilic α-Mangostin Derivatives 1−16

a

cLogP = calculated Log P, calculated by using ACD/Percepta software V11.02. bHC50 = the concentration needed to lyse 50% rabbit red blood cells.

Compound 5, in which the cationic moiety was N,Ndimethylamine, showed good activity against C. albicans (MIC = 6.25 μg/mL) and reduced hemolytic activity (HC50 = 30.0 ± 0.7 μg/mL). When N,N-dimethylamine (5) was replaced with N,N-diethylamine (6) and N,N-butylmethylamine (9), the in vitro potency and selectivity was not significantly affected. The replacement of N,N-dimethylamine moiety (5) with N,Ndialkylamine with a total number of carbon atoms >4 (compounds 2, 7, and 8) or N-benzylmethylamine (10) resulted in a dramatic loss of hemolytic activity (HC50 > 400 μg/mL). Compounds 2 and 7 bearing dibutyl group and dipropyl group substituents displayed potent antifungal activities, with MIC values of 3.13 μg/mL. The moderate steric hindrance of the tertiary amines in compounds 2 and 7 (MIC = 3.13 μg/mL) might play a strongly positive role in antifungal activity as compared with the greater steric hindrance of the tertiary amines in compounds 8 and 10 (MIC = 6.25−

derivatives were selected to be evaluated against four more C. albicans strains, six Fusarium strains, and five Aspergillus strains (Table 4). Natamycin is the only approved antifungal agent for ophthalmic applications by U.S. FDA, constituting the first-line therapy for fungal keratitis.55 The in vitro antifungal efficacy was expressed as minimum inhibitory concentration (MIC, the minimum concentration of the compounds needed to inhibit the growth of the fungus). Xanthone derivatives 2 and 5−10 with varied tertiary amine moieties exhibited potent or moderate antifungal activity against two tested C. albicans strains. The parent compound α-mangostin and the intermediate 4 without charges showed poor antifungal activity, with MIC values of >50 μg/mL. The introduction of cationic groups into C3 and C6 position of αmangostin can significantly increase the potency against fungus and reduce the hemolytic activities. 10138



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Table 2. In Vitro Antifungal and Hemolytic Activities (μg/mL) of Amphiphilic α-Mangostin Derivatives 17−29

a

cLogP = calculated Log P, calculated by using ACD/Percepta software V11.02. bHC50 = the concentration needed to lyse 50% rabbit red blood cells.

12.5 μg/mL) as well as the smaller steric hindrance of the tertiary amines in compounds 5 and 6 (MIC = 6.25 μg/mL). In addition, the in vitro antibacterial activities of compounds 1, 2, 6, and 7 against Gram-positive and Gram-negative bacteria were also screened (Supporting Information, Table S1). Compounds 1 and 6 displayed promising activities against Gram-positive bacteria (MICs = 1.56−3.13 μg/mL) and moderate activities against Gram-negative bacteria (MICs = 25−50 μg/mL). Compounds 2 and 7 containing dibutyl group and dipropyl group substituents resulted in reduced anti-Grampositive bacterial activities (MICs > 50 μg/mL for 2 and MICs = 6.25−12.5 μg/mL for 7) and poor anti-Gram-negative bacterial activities (MICs > 50 μg/mL). By contrast,

compounds 2 and 7 displayed potent antifungal activities. These results indicated that the increased hydrophobicity resulted in reduced antibacterial activities and enhanced antifungal activities. Compounds 11−16 were prepared to explore the effects of secondary amine moieties on the antifungal activities. Compounds 11−13 were designed and synthesized to study the effect of linear alkyl chains attached to the terminal amino groups on antifungal activity. When the length of the linear alkyl chains was increased from 3 to 8 carbons, the decrease in MIC values was observed. The MIC values of compounds 11 (n-propyl), 12 (n-hexyl), and 13 (n-octyl) were 6.25, 12.5 and >50 μg/mL, respectively. In addition, the longer the length of 10139



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Table 3. In Vitro Antifungal and Hemolytic Activities (μg/mL) of Amphiphilic α-Mangostin Derivatives 31−33 and 35

MIC values (μg/mL) compd α-mangostin 31 32 33 35 natamycin caspofungin

a

R

cLogP

C. albicans ATCC 10231

C. albicans DF2672R

Arg-NH2 Arg-OMe Arg-OtBu Arg-Arg-OMe

5.07 1.02 2.78 4.76 0.63

>50 6.25 6.25 12.5 6.25 6.25 0.10

>50 6.25 6.25 6.25 12.5 6.25 0.20

HC50b (μg/mL) 9 128 232 79 277 113.0

± ± ± ± ± ±

2 5 8 6 4 0.8

selectivity (HC50/MIC) 400 μg/mL, respectively. Compounds 14 and 15 bearing 4fluorobenzyl group and cyclooctylmethyl group substituents displayed promising antifungal activities (MIC = 6.25 μg/mL) and very low hemolytic activities (HC50 > 180 μg/mL). Compounds 11−16 were also used to investigate the effect of cLogP values. Compound 17 with the lowest cLogP values (1.77) displayed promising antifungal activities (MIC = 6.25 μg/mL) and moderate hemolytic activities (HC50 = 24.3 ± 4.3 μg/mL). In contrast, compound 13 with the highest cLogP values (10.69) leads to poor antifungal and hemolytic activities. These results revealed that excessive cLogP values might lead to reduced antifungal and hemolytic activities. To find the optimal cationic substitution and explore the effect of pKa values of cationic moiety, a series of cyclic amines coupled xanthone derivatives 17−21 were also synthesized and evaluated. Three types of cyclic amines (pyrrolidine, piperidine, and piperazine for compounds 17−19, respectively) with high pKa values (pKa = 11.27, 11.22, and 9.55, respectively)50 were coupled with xanthone derivatives to form amphiphilic structures. Table 2 shows that compounds 17−19 displayed promising antifungal activity with MIC values of 6.25−12.5 μg/ mL. Compounds 17−19 also displayed moderately hemolytic activities, with HC50 values of 26.3 ± 0.4, 31.9 ± 1.5, and 32.8 ± 0.03 μg/mL, respectively. When lower pKa values of cyclic amines (morpholine for compound 20 and imidazole for compound 21) were introduced to xanthone derivatives, no significant effects on the antifungal activities was observed. The results are quite different from their antibacterial activity against Gram-positive bacteria. The lower pKa values of cyclic amine moiety would lead to greatly reduced antibacterial activity (data not shown). In addition, compounds 20 and 21 lost their hemolytic activities, with HC50 values of >400 μg/mL, leading to higher selectivity than that of compounds 17−19. To explore the effect of quaternary ammonium substituents, tertiary amines 2, 5, and 6 were coupled with methyl iodide to generate quaternary ammonium salts 26−28. As shown in Table 2, compounds 26 and 27 retained moderate antifungal and hemolytic activities compared with compounds 5 and 6. As is known to all, quaternary ammonium compounds often lead to increased toxicity. Compound 28 displayed more than 1210140



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Table 4. MIC Values (μg/mL) of 1 and 2 against Various Fungal Strains MIC values (μg/mL) organisms

1

2

7

9

caspofungin

natamycin

C. albicans DF1976R C. albicans ATCC90028 C. albicans ATCC24433 C. albicans ATCC11651 F. solani 52628 F. solani 62877 F. solani MYA3636 F. solani 46492 FO MYA 3461 FO 64530 A. brasiliensis TCC16404 A. flavus TCC 204304 A. flavus TCC MYA3631 A. fumigate TCC MYA3626 A. fumigate TCC 90906

0.78 0.78 0.78 0.78 3.13 3.13 3.13 3.13 1.56 3.13 3.13 6.25 6.25 6.25 6.25

3.13 3.13 3.13 3.13 6.25 6.25 3.13 6.25 3.13 6.25 3.13 12.5 12.5 6.25 12.5

6.25 6.25 6.25 6.25

3.13 3.13 6.25 6.25

0.20 0.10 0.20 0.20 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50

6.25 6.25 6.25 6.25 6.25 25 6.25 6.25 3.13 6.25 6.25 >50 >50 6.25 6.25

Table 5. Combinational Effect between Xanthone Compounds (1 and 2) and Various Classes of Antifungal Agents against C. albicans DF2672R Strain FIC indexa antifungals

MICantifungal alone (μg/mL)

itraconazole miconazole terbinafine posaconazole natamycin amphotericin B

6.25 0.39 3.13 12.5 6.25 0.20

compd 1 1.28 0.88 0.44 1.06 1.08 1.28

± ± ± ± ± ±

0.22 0.25 0.06 0.00 0.05 0.22

compd 2

combinational effectb

± ± ± ± ± ±

additive additive synergistic additive additive additive

0.63 0.81 0.29 0.91 0.91 0.64

0.13 0.25 0.10 0.16 0.16 0.11

a Fractional inhibitory concentration (FIC) index = MIC xanthone compound combi ned with antifungal /MIC xanthone compound al one + MICantifungal combined with xanthone compound/MICantifungal alone, where MICxanthone compound 1 alone is 0.78 μg/mL and MICxanthone compound 2 alone is 3.13 μg/mL. b The combinational effect was considered as follows: synergistic, FIC ≤ 0.5; additive, 0.5 < FIC ≤ 4; antagonistic, FIC > 4.

shown in Table 5. Xanthone compounds (1 and 2) and terbinafine in combination appeared to have strong synergistic interactions (FIC < 0.5), and additive effects were observed for five other tested antifungal agents in combination with xanthone compounds (1 and 2). These results suggested that a combination of xanthone compounds (1 and 2) and terbinafine can be effective in treating fungal infections. Time-Kill Kinetics. To evaluate the fungicidal activity of xanthone derivatives, concentration-dependent time-kill assays of compounds 1, 2, and natamycin were performed against C. albicans DF2672R over a period of 24 h (Figure 1). Compound 1 resulted in 91% killing in 1 h at 1× MIC, whereas 3 log reduction (99.9%) of viable fungi cells within 1 h was observed when the concentration was increased to 4× MIC (Figure 1a). In addition, complete fungal cell death was observed after 24 h for compound 1 at 1× MIC. A concentration-dependent fungicidal activity was observed for compound 2. Compound 2 also displayed rapid fungicidal activity, achieving 3 log reduction (99.9%) in fungi colony count within 4 h at 8× MIC and 6 h at 4× MIC (Figure 1b). In contrast, C. albicans DF2672R treated with natamycin at high concentration of 8× MIC for 24 h showed 64 that are higher than that of natamycin. Compounds 7 and 9 displayed a little higher MIC values against the four tested C. albicans strains than those of compounds 1 and 2. Compounds 1 and 2 were potently active against the tested Fusarium and Aspergillus strains (MICs = 1.56−6.25 and MICs = 3.13−12.5 μg/mL, respectively). By contrast, natamycin and caspofungin displayed moderate or poor activities against the tested Fusarium and Aspergillus strains (MICs ≥ 6.25 and MICs > 50 μg/mL, respectively). The high selectivities and the potent antifungal activities of compounds 1 and 2 indicated that both compounds had the potential to be antifungal agents. Synergy between Xanthone Compounds and Antifungal Agents. The fractional inhibitory concentration (FIC) index was used to evaluate the combinational effect between xanthone compounds (1 and 2) and various classes of antifungal agents. The FIC values determined by using checkerboard assay against C. albicans DF2672R strain are 10141



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Figure 1. Time-kill kinetics of 1 (a), 2 (b), and natamycin (c) against C. albicans DF2672R over a period of 24 h.

Cytotoxicity. A critical factor in the development of antifungal agents is their abilities to selectively target fungal cells without showing toxicity to mammalian cells. The cytotoxicity of antifungal compounds 1 and 2 toward mammalian cells was assessed using human corneal fibroblasts via a lactate dehydrogenase (LDH) assay. Briefly, cells were exposed to compounds 1 and 2 at various concentrations and viable cells were determined. As shown in Figure 3, compound 2 was nontoxic (cell viability >99%) up to a concentration of 100 μM (78.1 μg/mL) against this cell line. Compound 1 had an IC50 (concentration killing 50% of the cells) value >100 μM (64.1 μg/mL) against this cell line. This value was 41−82-fold higher than its antifungal MIC values. At 100 μM, 44.4 ± 2.3% cell viability was observed for compound 1. Consistent with the

Drug Resistance Study. Serial passage experiments were performed to assess the potential to develop drug resistance by repeatedly exposing C. albicans DF2672R to compounds 1 and 2. C. albicans cells taken from the sublethal concentration of tested compound 1 or 2 were served as the inoculum for the MIC measurement of next passage. As shown in Figure 2, MIC

Figure 2. Multipassage resistance selection studies of compounds 1 and 2 against C. albicans DF2672R.

of compound 1 remained constant over the whole 27 passages, and only a 2-fold increase in the MIC was observed for compound 2 over the whole 30 passages. The results demonstrated that both compounds had a very low tendency to develop drug resistance. The reason for avoiding the development of drug resistance might be that compounds 1 and 2 appeared to have multiple nonspecific targets and can disrupt antifungal membranes rapidly.

Figure 3. Cytotoxicity toward mammalian cells of 1 and 2 measured by using the LDH cytotoxicity assays. 10142



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hemolysis assay, compound 1 demonstrated low cytotoxicity profile toward mammalian cells, and compound 2 displayed negligible toxicity under the tested conditions. Antifungal Mechanism. Cationic xanthone compounds might damage the integrity of fungal plasma membranes via electrostatic interaction between the negatively charged fungal membrane and the positively charged xanthone compounds. To identify the mode of antifungal action of cationic xanthone compounds, Sytox Green nucleic acid stain was used to determine the effect of these cationic xanthones on the integrity of the living fungal cell membrane. Sytox Green, a DNAbinding dye, can easily penetrate compromised membranes of dead cells but cannot cross intact membranes of living cells.58,59 The fluorescence of Sytox Green is monitored after the treatment of 4× MIC compounds 1 and 2 to C. albicans DF2672R cell suspensions. As shown in Figure 4, the control

Figure 5. Percentage of calcein leakage induced by 1 and 2 from calcein-loaded LUVs. The investigated liposome with the composition of DOPC/DOPE/PI = 2:1:1 containing 15 wt % ergosterol was used to mimic fungal membranes.

membrane-lytic activities compared with compound 1, in accordance with its MICs values against fungal strains that are 2−4-fold higher than those of compound 1. These results are consistent with SYTOX Green assay using living fungal cells, demonstrating that compounds 1 and 2 kill fungus by disrupting their membranes. In Vivo Efficacy. The therapeutic efficacy of compound 1 compared with natamycin was evaluated via topical administration in the murine model of fungal keratitis. Fungal keratitis is a leading cause of ocular morbidity.60 Antimicrobial agents that were topically administered for the treatment of corneal infections have several advantages over their systemic application, including enhancing the concentration of drugs in the infected area and averting systemic adverse effects.61,62 The mice corneas were infected with ∼1 × 106 CFUs of C. albicans DF2672R. One day after infection, the respective drugs (5% natamycin, 0.2% compound 1, or PBS) were applied topically to the mice every 20 min during the first hour and every 1 h during the next 6 h. As shown in Figure 6, compound 1 displayed potent in vivo antifungal activity, producing 92.8% reductions in the fungal burden compared with the control group. The concentration of compound 1 (0.2%) used in this study is 25-fold lower than that of natamycin (5%). However,

Figure 4. Membrane-permeabilizing properties of compounds 1 and 2 at 4× MIC against C. albicans DF2672R.

(0.5% DMF) did not affect the fluorescence intensity. The most active compound 1 displayed a strong ability to permeabilize the fungal plasma membranes, leading to a significant increase in fluorescence intensity. A modest enhancement in fluorescence intensity was obtained after treatment of compound 2. However, the fluorescence intensity was gradually enhanced with time, within the tested period of 2 h. This finding is in accordance with its slower fungicidal activity compared with compound 1. These results demonstrated that compounds 1 and 2 were able to directly disrupt fungal cell membranes. To further confirm, the mode of action for antifungal xanthones 1 and 2 is membrane targeting, their abilities to induce membrane perturbation in artificial membranes were investigated by measuring the percentage of calcein leakage from calcein-loaded LUVs (Figure 5). The calcein-loaded liposome with composition of DOPC/DOPE/PI = 2:1:1 containing 15 wt % ergosterol was used to mimic a negatively charged fungal membrane. At compound: lipid (C:L) ratio of 1:4 and 1:8, compounds 1 induced a significant calcein leakage from LUVs with the leakage values of 75.6 ± 1.2 and 43.8 ± 3.1% respectively, indicating that the interaction between compound 1 and the fungal membranes is very strong. Under identical conditions, a strong calcein leakage was also observed for compound 2, with leakage values of 55.4 ± 0.7% (C:L ratios = 1:4) and 25.8 ± 1.6% (C:L ratios = 1:8). respectively, suggesting that the fungal membrane was also sensitive to compound 2. Compound 2 showed decreased fungal

Figure 6. In vivo efficacy of compound 1 and natamycin in the murine model of fungal keratitis against C. albicans DF2672R. Concentration used: PBS was used as a negative control; compound 1, 0.2% solution; natamycin, 5% solution; * p < 0.05, ** p < 0.01 vs control. 10143



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(400 MHz, CD3OD) δ 6.83 (s, 1H), 6.39 (s, 1H), 5.28−5.09 (m, 2H), 4.27−4.16 (m, 2H), 4.15−3.97 (m, 6H), 3.81 (s, 3H), 3.59−3.36 (m, 4H), 3.35−3.31 (m, 2H), 1.84 (s, 3H), 1.79 (s, 3H), 1.68 (s, 3H), 1.67 (s, 3H). 13C NMR (100 MHz, CD3OD) δ 183.12, 163.73, 160.64, 159.82, 159.77, 158.49, 156.55, 156.38, 145.36, 138.19, 132.13, 132.12, 124.75, 123.84, 113.12, 112.67, 104.82, 100.53, 90.83, 71.49, 71.00, 69.59, 69.46, 61.65, 45.87, 45.77, 27.03, 26.04, 25.94, 22.32, 18.39, 18.16. HRMS (ESI+): calculated for C32H45N6O8 [M + H]+ 641.3293, found 641.3288. 3,6-Bis(3-(dibutylamino)-2-hydroxypropoxy)-1-hydroxy-7-methoxy-2,8-bis(3-methylbut-2-en-1-yl)-9H-xanthen-9-one (2). To a solution of compound 4 (140 mg, 0.268 mmol, 1.0 equiv) in methanol (5 mL), dibutylamine (346.4 mg, 2.68 mmol, 10.0 equiv) was added. The reaction mixture was refluxed and stirred for 12 h. After reaction completion, the reaction mixture was diluted with 1-butanol and washed with brine. The organic phase was dried over anhydrous Na2SO4 and evaporated under vacuum. The crude product was purified by RP-HPLC, using water and methanol as eluents, both containing 0.1% v/v formic acid. The fractions containing the desired product were concentrated and lyophilized. Compound 2 was obtained as a yellow gel with the yield of 87% (182.3 mg). 1H NMR (400 MHz, CD3OD) δ 6.74 (s, 1H), 6.32 (s, 1H), 5.30−5.14 (m, 2H), 4.47−4.33 (m, 2H), 4.17−3.98 (m, 6H), 3.81 (s, 3H), 3.30− 3.02 (m, 14H), 1.88−1.78 (m, 6H), 1.77−1.64 (m, 14H), 1.49−1.38 (m, 8H), 1.00 (dt, J = 7.3, 4.5 Hz, 12H). 13C NMR (100 MHz, CD3OD) δ 183.06, 163.46, 160.60, 158.38, 156.43, 156.26, 145.34, 138.15, 132.24, 132.10, 124.81, 123.81, 113.11, 112.53, 104.81, 100.55, 90.84, 72.02, 71.54, 66.08 (2 × CH), 61.65, 57.03, 56.82, 55.36 (2 × CH2), 55.27 (2 × CH2), 27.45 (2 × CH2), 27.37 (2 × CH2), 27.09, 26.12, 26.05, 22.42, 21.14 (4 × CH2), 18.48, 18.34, 14.09 (2 × CH3), 14.06 (2 × CH3). HRMS (ESI+): calculated for C46H73N2O8 [M + H]+ 781.5361, found 781.5367. 3,6-Bis(3-(dimethylamino)-2-hydroxypropoxy)-1-hydroxy-7-methoxy-2,8-bis(3-methylbut-2-en-1-yl)-9H-xanthen-9-one (5). The title compound was synthesized using the same procedure for compound 2 from compound 4 and dimethylamine. Compound 5 was obtained as a yellow gel with the yield of 73%. 1H NMR (400 MHz, CD3OD) δ 6.73 (s, 1H), 6.31 (s, 1H), 5.29−5.13 (m, 2H), 4.44−4.27 (m, 2H), 4.16−3.95 (m, 6H), 3.81 (s, 3H), 3.28 (d, J = 5.6 Hz, 2H), 3.12−3.00 (m, 4H), 2.75 (s, 6H), 2.73 (s, 6H), 1.84 (s, 3H), 1.80 (s, 3H), 1.70 (s, 3H), 1.67 (s, 3H). 13C NMR (100 MHz, CD3OD) δ 183.19, 163.76, 160.72, 158.57, 156.57, 156.39, 145.45, 138.22, 132.21, 132.20, 124.98, 124.11, 113.21, 112.76, 104.90, 100.64, 90.96, 72.41, 71.97, 66.72, 66.66, 62.31, 62.05, 61.76, 45.16 (2 × CH3), 45.11 (2 × CH3), 27.20, 26.22, 26.10, 22.48, 18.57, 18.42. HRMS (ESI +): calculated for C34H49N2O8 [M + H]+ 613.3483, found 613.3499. 3,6-Bis(3-(dihexylamino)-2-hydroxypropoxy)-1-hydroxy-7-methoxy-2,8-bis(3-methylbut-2-en-1-yl)-9H-xanthen-9-one (8). The title compound was synthesized using the same procedure for compound 2 from compound 4 and dihexylamine. Compound 8 was obtained as a yellow gel with the yield of 67%. 1H NMR (400 MHz, CD3OD) δ 6.63 (s, 1H), 6.22 (s, 1H), 5.32−5.14 (m, 2H), 4.29−4.18 (m, 2H), 4.05 (d, J = 7.9 Hz, 6H), 3.80 (s, 3H), 3.27 (d, J = 6.2 Hz, 2H), 3.08−2.91 (m, 4H), 2.90−2.73 (m, 8H), 1.84 (s, 3H), 1.80 (s, 3H), 1.73−1.54 (m, 14H), 1.38−1.26 (m, 24H), 0.94−0.84 (m, 12H). 13C NMR (100 MHz, CD3OD) δ 183.11, 163.72, 160.69, 158.59, 156.45, 156.30, 145.42, 138.09, 132.10, 132.03, 125.10, 124.10, 113.09, 112.59, 104.83, 100.51, 90.85, 72.30, 71.69, 67.35 (2 × CH), 61.73, 57.74, 57.57, 56.07 (2 × CH2), 55.94 (2 × CH2), 32.91 (2 × CH2), 32.90 (2 × CH2), 28.08 (4 × CH2), 27.26, 26.77 (4 × CH2), 26.37, 26.32, 23.81 (4 × CH2), 22.62, 18.71, 18.57, 14.57 (4 × CH3). HRMS (ESI+): calculated for C54H89N2O8 [M + H]+ 893.6613, found 893.6621. 3,6-Bis(3-(butyl(methyl)amino)-2-hydroxypropoxy)-1-hydroxy-7methoxy-2,8-bis(3-methylbut-2-en-1-yl)-9H-xanthen-9-one (9). The title compound was synthesized using the same procedure for compound 2 from compound 4 and N-butylmethylamine. Compound 9 was obtained as a yellow gel with the yield of 75%. 1H NMR (400 MHz, CD3OD) δ 6.69 (s, 1H), 6.27 (s, 1H), 5.22−5.08 (m, 2H), 4.52−4.35 (m, 2H), 4.16−3.95 (m, 6H), 3.75 (s, 3H), 3.29−3.28 (m,

the therapeutic efficacy of compound 1 (0.2%) is comparable to that of natamycin (5%). Topical natamycin (5%) is commonly selected as initial therapy for corneal fungal infections.60 The results demonstrated that compound 1 was highly efficacious in the murine model of fungal keratitis.

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CONCLUSIONS In conclusion, a new series of cationic amphiphilic xanthone derivatives were designed and synthesized by biomimicking AMPs, starting from natural α-mangostin. Two lead compounds (1 and 2) were found to display high membrane selectivity and excellent antifungal activity against a wide range of fungal pathogens, including drug-resistant C. albicans, Aspergillus, and Fusarium strains (MICs = 0.78−12.5 μg/mL). The antifungal activity of compounds 1 and 2 were comparable to or better than that of natamycin and caspofungin. Notably, the value of minimal fungicidal concentrations (MFC) for compound 1 against C. albicans DF2672R is the same as its MIC value. Compound 2 was found to show negligible cytotoxicity and hemolytic activity against mammalian cells, while low cytotoxicity and hemolytic activity against mammalian cells were observed for compound 1. Both compounds can kill fungus rapidly by directly disrupting fungal cell membranes and avoid the development of drug resistance. In addition, compound 1 was confirmed to retain potent in vivo antifungal activity in the murine model of fungal keratitis. These results potentially lead to the discovery of a novel class of membrane targeting xanthone-based antifungal agents which can combat drug-resistant fungal pathogens.

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EXPERIMENTAL SECTION

General Chemistry. α-Mangostin was purchased from Chengdu Biopurify Phytochemicals Ltd. (Chengdu, China). All the other reagents and solvents were purchased from commercial sources and used without further purification. Nuclear magnetic resonance (NMR) spectroscopy was recorded on a 400 Bruker Avance instrument using deuterated methanol (CD3OD) or deuterated chloroform (CDCl3) as the solvents and tetramethylsilane as an internal standard. The chemical shifts (δ) are expressed in parts per million (ppm), and the coupling constants (J) are reported in hertz (Hz). Peak multiplicities are denoted as singlet (s), doublet (d), triplet (t), doublet of triplet (dt), and multiplet (m). All final products were purified by a preparative Shimadzu HPLC using reverse phase using a C18 column (Phenomenex, 150 mm × 21.2 mm, 5 μm, 100 Å). The mobile phase was HPLC-grade methanol/distilled water (25−95%) containing 0.1% formic acid. The purity of the final products was above 95%, as determined by HPLC analysis. High-resolution mass spectra (HRMS) were recorded on an API2000 liquid chromatography−tandem mass spectrometry system. Melting points were determined on a Mel-Temp 1101D melting point apparatus without correction. Synthesis of Xanthone Compounds. The synthesis of compounds 4, 6, 7, 16, 23, 25, 29, and 31−35 was reported previously.63 1,1′-(((1-Hydroxy-7-methoxy-2,8-bis(3-methylbut-2-en-1-yl)-9oxo-9H-xanthene-3,6-diyl)bis(oxy))bis(2-hydroxypropane-3,1-diyl))diguanidine (1). To a solution of 22 (61.3 mg, 0.110 mmol) in anhydrous DMF (5 mL), 1H-pyrazole-1-carboxamidine hydrochloride (48.4 mg, 0.330 mmol) and N,N-diisopropylethylamine (0.054 mL, 0.330 mmol) were added. The reaction mixture was stirred at room temperature for 12 h. After reaction completion, diethyl ether was added to the reaction mixture. The resulting suspension was centrifuged and washed with diethyl ether. The crude product was purified by RP-HPLC, using water and methanol as eluents, both containing 0.1% v/v formic acid. The fractions containing the desired product were concentrated and lyophilized. Compound 1 was obtained as a yellow gel with the yield of 71% (50.0 mg). 1H NMR 10144



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HRMS (ESI+): calculated for C46H73N2O8 [M + H]+ 781.5361, found 781.5375. 3,6-Bis(3-((4-fluorobenzyl)amino)-2-hydroxypropoxy)-1-hydroxy7-methoxy-2,8-bis(3-methylbut-2-en-1-yl)-9H-xanthen-9-one (14). The title compound was synthesized using the same procedure for compound 2 from compound 4 and 4-fluorobenzylamine. Compound 14 was obtained as a yellow gel with the yield of 71%. 1H NMR (400 MHz, CD3OD) δ 7.56−7.42 (m, 4H), 7.19−7.07 (m, 4H), 6.72 (s, 1H), 6.31 (s, 1H), 5.22 (t, J = 6.5 Hz, 1H), 5.14 (t, J = 6.8 Hz, 1H), 4.35−4.23 (m, 2H), 4.18−3.95 (m, 10H), 3.71 (s, 3H), 3.23 (d, J = 6.6 Hz, 2H), 3.19−3.10 (m, 2H), 3.07−2.96 (m, 2H), 1.83 (s, 3H), 1.75 (s, 3H), 1.68 (s, 3H), 1.64 (s, 3H). 13C NMR (100 MHz, CD3OD) δ 183.12, 165.55, 163.61, 163.10, 160.63, 158.42, 156.48, 156.31, 145.33, 138.14, 132.75, 132.69, 132.66, 132.61, 132.13, 132.12, 131.77, 131.70, 124.77, 123.69, 116.86, 116.82, 116.64, 116.60, 113.09, 112.63, 104.80, 100.44, 90.78, 72.12, 71.68, 67.65, 67.62, 61.54, 52.33 (2 × CH2), 51.24, 51.20, 27.02, 26.05, 25.98, 22.32, 18.41, 18.17. HRMS (ESI+): calculated for C44H51F2N2O8 [M + H]+ 773.3608, found 773.3617. 3,6-Bis(3-(cyclooctylamino)-2-hydroxypropoxy)-1-hydroxy-7-methoxy-2,8-bis(3-methylbut-2-en-1-yl)-9H-xanthen-9-one (15). The title compound was synthesized using the same procedure for compound 2 from compound 4 and cyclooctylamine. Compound 15 was obtained as a yellow gel with the yield of 78%. 1H NMR (400 MHz, CD3OD) δ 6.91 (s, 1H), 6.47 (s, 1H), 5.28−5.15 (m, 2H), 4.36−4.25 (m, 2H), 4.23−4.03 (m, 6H), 3.82 (s, 3H), 3.35 (d, J = 6.9 Hz, 2H), 3.30−3.22 (m, 4H), 3.19−3.08 (m, 2H), 2.04−1.92 (m, 4H), 1.89−1.44 (m, 36H). 13C NMR (100 MHz, CD3OD) δ 183.26, 163.68, 160.77, 158.52, 156.66, 156.51, 145.48, 138.37, 132.28, 132.23, 124.66, 123.75, 113.27, 112.74, 104.94, 100.63, 90.85, 72.14, 71.58, 67.30, 67.24, 61.66, 60.02 (2 × CH), 60.00 (2 × CH2), 31.58, 31.52, 31.00, 30.98, 27.42 (2 × CH2), 27.39 (2 × CH2), 27.02, 26.92, 26.89, 26.02, 25.98, 25.24, 25.21, 25.18, 25.15, 22.30, 18.36, 18.18. HRMS (ESI+): calculated for C46H69N2O8 [M + H]+ 777.5048, found 777.5070. 1-Hydroxy-3,6-bis(2-hydroxy-3-(pyrrolidin-1-yl)propoxy)-7-methoxy-2,8-bis(3-methylbut-2-en-1-yl)-9H-xanthen-9-one (17). The title compound was synthesized using the same procedure for compound 2 from compound 4 and pyrrolidine. Compound 17 was obtained as a yellow gel with the yield of 92%. 1H NMR (400 MHz, CD3OD) δ 6.78 (s, 1H), 6.35 (s, 1H), 5.29−5.13 (m, 2H), 4.47−4.29 (m, 2H), 4.21−3.98 (m, 6H), 3.82 (s, 3H), 3.43−3.30 (m, 14H), 2.17−2.00 (m, 8H), 1.85 (s, 3H), 1.80 (s, 3H), 1.74−1.62 (m, 6H). 13 C NMR (100 MHz, CD3OD) δ 183.25, 163.73, 160.78, 158.56, 156.62, 156.45, 145.51, 138.31, 132.28 (2 × C), 124.92, 124.06, 113.30, 112.80, 104.97, 100.73, 91.01, 72.28, 71.84, 67.11, 67.06, 61.81, 59.27, 59.06, 55.88 (2 × CH2), 55.84 (2 × CH2), 27.19, 26.21, 26.11, 24.16 (4 × CH2), 22.46, 18.56, 18.41. HRMS (ESI+): calculated for C38H53N2O8 [M + H]+ 665.3796, found 665.3812. 1-Hydroxy-3,6-bis(2-hydroxy-3-(piperidin-1-yl)propoxy)-7-methoxy-2,8-bis(3-methylbut-2-en-1-yl)-9H-xanthen-9-one (18). The title compound was synthesized using the same procedure for compound 2 from compound 4 and piperidine. Compound 18 was obtained as a yellow gel with the yield of 82%. 1H NMR (400 MHz, CD3OD) δ 6.72 (s, 1H), 6.30 (s, 1H), 5.29−5.12 (m, 2H), 4.56−4.42 (m, 2H), 4.06 (d, J = 26.3 Hz, 6H), 3.81 (s, 3H), 3.38−3.35 (m, 2H), 3.29−3.18 (m, 12H), 1.97−1.82 (m, 11H), 1.80 (s, 3H), 1.74−1.63 (m, 10H). 13C NMR (100 MHz, CD3OD) δ 183.16, 163.60, 160.71, 158.45, 156.51, 156.35, 145.44, 138.22, 132.29, 132.22, 124.97, 124.06, 113.23, 112.72, 104.92, 100.69, 90.99, 72.32, 71.92, 65.61 (2 × CH), 61.80, 61.09, 60.93, 55.29 (2 × CH2), 50.00 (2 × CH2), 27.21, 26.24, 26.13, 24.43 (2 × CH2), 24.35 (2 × CH2), 23.17 (2 × CH2), 22.49, 18.60, 18.45. HRMS (ESI+): calculated for C40H57N2O8 [M + H]+ 693.4109, found 693.4127. 1-Hydroxy-3,6-bis(2-hydroxy-3-(piperazin-1-yl)propoxy)-7-methoxy-2,8-bis(3-methylbut-2-en-1-yl)-9H-xanthen-9-one (19). The title compound was synthesized using the same procedure for compound 2 from compound 4 and piperazine. Compound 19 was obtained as a yellow gel with the yield of 75%. 1H NMR (400 MHz, CD3OD) δ 6.66 (s, 1H), 6.23 (s, 1H), 5.32−5.10 (m, 2H), 4.26−4.14 (m, 2H), 4.06 (d, J = 28.3 Hz, 6H), 3.80 (s, 3H), 3.54 (d, J = 32.1 Hz,

4H), 3.26−3.15 (m, 6H), 2.92 (s, 3H), 2.90 (s, 3H), 1.82−1.68 (m, 10H), 1.67−1.58 (m, 6H), 1.46−1.34 (m, 4H), 0.96 (dt, J = 7.3, 4.2 Hz, 6H). 13C NMR (100 MHz, CD3OD) δ 183.07, 163.43, 160.61, 158.31, 156.43, 156.26, 145.32, 138.18, 132.22, 132.13, 124.75, 123.82, 113.17, 112.59, 104.84, 100.60, 90.88, 71.96, 71.53, 65.34 (2 × CH), 61.68, 59.31, 59.18, 57.89, 57.86, 41.76, 41.68, 27.10, 27.03, 26.06, 25.98, 22.34, 20.91 (3 × CH2), 18.43, 18.29, 13.95, 13.94. HRMS (ESI +): calculated for C40H61N2O8 [M + H]+ 697.4422, found 697.4432. 3,6-Bis(3-(benzyl(methyl)amino)-2-hydroxypropoxy)-1-hydroxy7-methoxy-2,8-bis(3-methylbut-2-en-1-yl)-9H-xanthen-9-one (10). The title compound was synthesized using the same procedure for compound 2 from compound 4 and N-benzylmethylamine. Compound 10 was obtained as a yellow gel with the yield of 63%. 1H NMR (400 MHz, CDCl3) δ 13.47 (s, 1H), 7.40−7.28 (m, 10H), 6.67 (s, 1H), 6.26 (s, 1H), 5.27−5.11 (m, 4H), 4.34−4.19 (m, 2H), 4.15−3.96 (m, 6H), 3.83 (d, J = 12.8 Hz, 2H), 3.76−3.64 (m, 5H), 3.30 (d, J = 6.7 Hz, 2H), 2.89−2.65 (m, 4H), 2.43 (s, 6H), 1.84 (s, 3H), 1.78 (s, 3H), 1.69 (s, 3H), 1.67 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 181.96, 162.21, 159.86, 157.00, 155.13, 155.02, 144.00, 137.25, 136.32, 136.06, 131.68, 131.41, 129.54 (2 × CH), 129.49 (2 × CH), 128.65 (2 × CH), 128.63 (2 × CH), 128.00, 127.94, 123.28, 122.71, 112.24, 111.47, 104.07, 99.14, 89.47, 70.98, 70.56, 65.75 (2 × CH), 62.31 (2 × CH2), 60.89, 59.69, 59.33, 42.27, 42.21, 26.19, 25.93, 25.81, 21.48, 18.21, 17.96. HRMS (ESI+): calculated for C46H57N2O8 [M + H]+ 765.4109, found 765.4115. 1-Hydroxy-3,6-bis(2-hydroxy-3-(propylamino)propoxy)-7-methoxy-2,8-bis(3-methylbut-2-en-1-yl)-9H-xanthen-9-one (11). The title compound was synthesized using the same procedure for compound 2 from compound 4 and propylamine. Compound 11 was obtained as a yellow gel with the yield of 77%. 1H NMR (400 MHz, MeOD) δ 6.75 (s, 1H), 6.34 (s, 1H), 5.30−5.13 (m, 2H), 4.40− 4.25 (m, 2H), 4.19−3.98 (m, 6H), 3.81 (s, 3H), 3.30−3.16 (m, 4H), 3.16−3.04 (m, 2H), 2.95 (dd, J = 17.6, 9.6 Hz, 4H), 1.84 (s, 3H), 1.80 (s, 3H), 1.78−1.71 (m, 4H), 1.70 (s, 3H), 1.67 (s, 3H), 1.03 (dt, J = 7.4, 3.3 Hz, 6H). 13C NMR (100 MHz, CD3OD) δ 183.30, 163.79, 160.83, 158.61, 156.66, 156.50, 145.55, 138.34, 132.35, 132.31, 125.00, 124.06, 113.31, 112.83, 105.00, 100.70, 91.01, 72.33, 71.83, 67.43, 67.37, 61.84, 52.00, 51.90, 51.39 (2 × CH2), 27.24, 26.27, 26.16, 22.54, 21.50, 21.45, 18.61, 18.43, 11.66 (2 × CH3). HRMS (ESI+): calculated for C36H53N2O8 [M + H]+ 641.3796, found 641.3783. 3,6-Bis(3-(hexylamino)-2-hydroxypropoxy)-1-hydroxy-7-methoxy-2,8-bis(3-methylbut-2-en-1-yl)-9H-xanthen-9-one (12). The title compound was synthesized using the same procedure for compound 2 from compound 4 and hexylamine. Compound 12 was obtained as a yellow gel with the yield of 62%. 1H NMR (400 MHz, CD3OD) δ 6.78 (s, 1H), 6.37 (s, 1H), 5.29−5.12 (m, 2H), 4.46−4.29 (m, 2H), 4.22−3.98 (m, 6H), 3.81 (s, 3H), 3.39−3.32 (m, 2H), 3.30− 3.14 (m, 4H), 3.07 (dd, J = 16.8, 8.9 Hz, 4H), 1.91−1.60 (m, 16H), 1.47−1.30 (m, 12H), 0.97−0.88 (m, 6H). 13C NMR (100 MHz, CD3OD) δ 183.27, 163.67, 160.81, 158.51, 156.63, 156.47, 145.52, 138.36, 132.33, 132.29, 124.89, 123.97, 113.33, 112.80, 105.01, 100.71, 91.00, 72.10, 71.61, 66.71, 66.67, 61.82, 51.44 (2 × CH2), 51.41 (2 × CH2), 32.60, 32.59, 27.51 (2 × CH2), 27.33, 27.24, 27.18, 26.20, 26.12, 23.62 (2 × CH2), 22.48, 18.56, 18.39, 14.42 (2 × CH3). HRMS (ESI +): calculated for C42H65N2O8 [M + H]+ 725.4735, found 725.4747. 1-Hydroxy-3,6-bis(2-hydroxy-3-(octylamino)propoxy)-7-methoxy-2,8-bis(3-methylbut-2-en-1-yl)-9H-xanthen-9-one (13). The title compound was synthesized using the same procedure for compound 2 from compound 4 and octylamine. Compound 13 was obtained as a yellow gel with the yield of 38%. 1H NMR (400 MHz, CD3OD) δ 6.83 (s, 1H), 6.41 (s, 1H), 5.33−5.10 (m, 2H), 4.41−4.28 (m, 2H), 4.21−4.00 (m, 6H), 3.81 (s, 3H), 3.29−2.94 (m, 10H), 1.84 (s, 3H), 1.80 (s, 3H), 1.74−1.65 (m, 8H), 1.47−1.22 (m, 22H), 0.96− 0.82 (m, 6H). 13C NMR (100 MHz, CD3OD) δ 183.32, 163.75, 160.86, 158.58, 156.70, 156.54, 145.57, 138.40, 132.33, 132.30, 124.88, 123.97, 113.36, 112.84, 105.04, 100.73, 91.00, 72.20, 71.67, 67.03, 66.97, 61.82, 51.64 (2 × CH2), 51.62 (2 × CH2), 33.05 (2 × CH2), 30.42, 30.41, 30.36 (2 × CH2), 27.91 (2 × CH2), 27.73, 27.65, 27.19, 26.20, 26.14, 23.82 (2 × CH2), 22.47, 18.56, 18.38, 14.54 (2 × CH3). 10145



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2H), 3.29−3.19 (m, 8H), 2.94−2.55 (m, 12H), 1.84 (s, 3H), 1.79 (s, 3H), 1.70 (s, 3H), 1.67 (s, 3H). 13C NMR (100 MHz, CD3OD) δ 183.12, 163.99, 163.20, 160.60, 158.82, 156.33, 145.47, 137.96, 132.06 (2 × C), 125.10, 124.22, 112.94, 112.62, 104.71, 100.50, 90.87, 72.67, 72.06, 68.50, 68.38, 61.90, 61.77, 61.66, 55.55, 54.33, 51.88, 51.82, 46.95, 44.94 (2 × CH2), 41.14, 27.22, 26.28, 26.17, 22.52, 18.63, 18.45. HRMS (ESI+): calculated for C38H55N4O8 [M + H]+ 695.4014, found 695.4010. 1-Hydroxy-3,6-bis(2-hydroxy-3-morpholinopropoxy)-7-methoxy2,8-bis(3-methylbut-2-en-1-yl)-9H-xanthen-9-one (20). The title compound was synthesized using the same procedure for compound 2 from compound 4 and morpholine. Compound 20 was obtained as a yellow gel with the yield of 80%. 1H NMR (400 MHz, CD3OD) δ 6.55 (s, 1H), 6.14 (s, 1H), 5.31−5.11 (m, 2H), 4.22 (m, 2H), 4.12−3.87 (m, 6H), 3.85−3.66 (m, 11H), 3.27−3.15 (m, 2H), 2.84−2.51 (m, 12H), 1.83 (s, 3H), 1.78 (s, 3H), 1.70 (s, 3H), 1.66 (s, 3H). 13C NMR (100 MHz, CD3OD) δ 182.89, 163.74, 160.41, 158.50, 156.24, 156.06, 145.21, 137.74, 131.86, 131.84, 125.04, 124.16, 112.78, 112.45, 104.52, 100.28, 90.68, 72.67, 72.13, 67.60 (2 × CH2), 67.58 (2 × CH2), 67.52 (2 × CH), 62.72, 62.48, 61.50, 55.34 (2 × CH2), 55.28 (2 × CH2), 27.10, 26.19, 26.06, 22.40, 18.53, 18.37. HRMS (ESI+): calculated for C38H53N2O10 [M + H]+ 697.3695, found 697.3710. 1-Hydroxy-3,6-bis(2-hydroxy-3-(1H-imidazol-1-yl)propoxy)-7-methoxy-2,8-bis(3-methylbut-2-en-1-yl)-9H-xanthen-9-one (21). The title compound was synthesized using the same procedure for compound 2 from compound 4 and imidazole. Compound 21 was obtained as a yellow gel with the yield of 57%. 1H NMR (400 MHz, CD3OD) δ 8.03 (d, J = 39.9 Hz, 2H), 7.57−6.99 (m, 4H), 6.72 (s, 1H), 6.27 (s, 1H), 5.31−5.15 (m, 2H), 4.48−4.20 (m, 6H), 4.12−3.79 (m, 9H), 3.36−3.35 (m, 2H), 1.84 (s, 3H), 1.79 (s, 3H), 1.69 (s, 3H), 1.66 (s, 3H). 13C NMR (100 MHz, CD3OD) δ 183.07, 163.53, 160.69, 158.40, 156.43, 156.29, 145.41, 138.22, 132.35 (2 × CH), 132.26 (2 × C), 124.99 (2 × CH), 124.32 (4 × CH), 113.21, 112.61, 104.87, 100.59, 90.88, 71.40, 70.56, 69.95, 69.82, 61.80, 51.77, 51.25, 27.25, 26.26, 26.08, 22.52, 18.61, 18.44. HRMS (ESI+): calculated for C36H43N4O8 [M + H]+ 659.3075, found 659.3076. 3,6-Bis(3-amino-2-hydroxypropoxy)-1-hydroxy-7-methoxy-2,8bis(3-methylbut-2-en-1-yl)-9H-xanthen-9-one (22). The title compound was synthesized using the same procedure for compound 2 from compound 4 and ammonia solution. Compound 22 was obtained as a yellow gel with the yield of 78%. 1H NMR (400 MHz, CD3OD) δ 6.76 (s, 1H), 6.38 (s, 1H), 5.21 (dt, J = 24.9, 6.6 Hz, 2H), 4.33−4.24 (m, 2H), 4.17−4.02 (m, 6H), 3.80 (s, 3H), 3.31−3.23 (m, 4H), 3.16− 3.06 (m, 2H), 1.85 (s, 3H), 1.80 (s, 3H), 1.70 (s, 3H), 1.67 (s, 3H). 13 C NMR (100 MHz, CD3OD) δ 183.27, 163.70, 160.78, 158.48, 156.63, 156.46, 145.47, 138.33, 132.37, 132.31, 124.90, 123.92, 113.31, 112.80, 104.99, 100.65, 90.95, 71.92, 71.41, 67.53, 67.46, 61.78, 43.68, 43.62, 27.17, 26.20, 26.07, 22.46, 18.54, 18.32. HRMS (ESI+): calculated for C30H41N2O8 [M + H]+ 557.2857, found 557.2873. 3,6-Bis(3-((2-((2-aminoethyl)amino)ethyl)amino)-2-hydroxypropoxy)-1-hydroxy-7-methoxy-2,8-bis(3-methylbut-2-en-1-yl)-9Hxanthen-9-one (24). The title compound was synthesized using the same procedure for compound 2 from compound 4 and diethylenetriamine. Compound 24 was obtained as a yellow gel with the yield of 75%. 1H NMR (400 MHz, CD3OD) δ 6.89 (s, 1H), 6.43 (s, 1H), 5.30−5.12 (m, 2H), 4.49−3.92 (m, 10H), 3.90−3.44 (m, 11H), 3.37− 3.32 (m, 2H), 3.28−2.73 (m, 10H), 1.84 (s, 3H), 1.80 (s, 3H), 1.74− 1.57 (m, 6H). 13C NMR (100 MHz, CD3OD) δ 183.24, 170.43, 166.88, 164.90, 160.79, 156.64, 145.50, 138.32, 132.34, 132.29, 124.94, 124.01, 113.31, 112.72, 104.93, 100.67, 90.95, 71.27, 68.86, 68.67, 67.47, 61.82, 53.87, 52.18, 50.91, 47.90, 46.46, 42.51, 39.27, 38.92, 38.25, 36.12, 27.20, 26.21, 26.11, 22.50, 18.57, 18.41. HRMS (ESI+): calculated for C38H61N6O8 [M + H]+ 729.4545, found 729.4535. 3,3′-((1-Hydroxy-7-methoxy-2,8-bis(3-methylbut-2-en-1-yl)-9oxo-9H-xanthene-3,6-diyl)bis(oxy))bis(2-hydroxy-N,N,N-trimethylpropan-1-aminium) iodide (26). To a solution of 5 (43 mg, 0.070 mmol) in methanol (5 mL), iodomethane (0.087 mL, 1.4 mmol) was added. The reaction mixture was stirred at room temperature overnight. After reaction completion, diethyl ether was added to the reaction mixture. The resulting suspension was centrifuged and

washed with diethyl ether. The crude product was purified by RPHPLC, using water and methanol as eluents, both containing 0.1% v/v formic acid. The fractions containing the desired product were concentrated and lyophilized. Compound 26 was obtained as a yellow solid with the yield of 72% (45.4 mg); mp 58−60 °C. 1H NMR (400 MHz, CD3OD) δ 6.91 (s, 1H), 6.46 (s, 1H), 5.28−5.15 (m, 2H), 4.76−4.59 (m, 2H), 4.25−4.03 (m, 6H), 3.83 (s, 3H), 3.72−3.59 (m, 4H), 3.35−3.26 (m, 20H), 1.84 (s, 3H), 1.81 (s, 3H), 1.68 (s, 6H). 13 C NMR (100 MHz, CD3OD) δ 183.22, 163.47, 160.78, 158.33, 156.62, 156.46, 145.43, 138.42, 132.40, 132.24, 124.63, 123.84, 113.38, 112.78, 105.03, 100.82, 91.01, 72.29, 71.86, 70.04, 69.81, 65.86, 65.78, 61.78, 55.11 (6 × CH3), 27.03, 26.01, 25.93, 22.29, 18.37, 18.24. HRMS (ESI+): calculated for C36H54I2N2O8 [(M − 2I)/2]+ 321.1935, found 321.1931. 3,3′-((1-Hydroxy-7-methoxy-2,8-bis(3-methylbut-2-en-1-yl)-9oxo-9H-xanthene-3,6-diyl)bis(oxy))bis(N,N-diethyl-2-hydroxy-Nmethylpropan-1-aminium) iodide (27). The title compound was synthesized using the same procedure for compound 26 from compound 6. Compound 27 was obtained as a yellow solid with the yield of 77%; mp 52−53 °C. 1H NMR (400 MHz, CD3OD) δ 6.93 (s, 1H), 6.48 (s, 1H), 5.26−5.14 (m, 2H), 4.73−4.58 (m, 2H), 4.27−4.00 (m, 6H), 3.82 (s, 3H), 3.68−3.48 (m, 12H), 3.34 (d, J = 4.9 Hz, 2H), 3.19 (s, 3H), 3.17 (s, 3H), 1.84 (s, 3H), 1.80 (s, 3H), 1.68 (s, 6H), 1.48−1.35 (m, 12H). 13C NMR (100 MHz, CD3OD) δ 183.21, 163.42, 160.77, 158.29, 156.63, 156.47, 145.42, 138.46, 132.61, 132.25, 124.65, 123.82, 113.38, 112.69, 105.03, 100.85, 91.05, 72.17, 71.80, 65.32, 65.25, 63.97, 63.83, 61.82, 59.28, 59.21, 59.08, 59.02, 49.11 (2 × CH3), 27.05, 26.03, 25.95, 22.33, 18.41, 18.27, 8.35 (4 × CH3). HRMS (ESI+): calculated for C40H62I2N2O8 [(M − 2I)/2]+ 349.2248, found 349.2252. N,N′-(((1-Hydroxy-7-methoxy-2,8-bis(3-methylbut-2-en-1-yl)-9oxo-9H-xanthene-3,6-diyl)bis(oxy))bis(2-hydroxypropane-3,1-diyl))bis(N-butyl-N-methylbutan-1-aminium) iodide (28). The title compound was synthesized using the same procedure for compound 26 from compound 2. Compound 28 was obtained as a yellow gel with the yield of 80%. 1H NMR (400 MHz, CD3OD) δ 7.03 (s, 1H), 6.57 (s, 1H), 5.30−5.15 (m, 2H), 4.70−4.55 (m, 2H), 4.31−4.05 (m, 6H), 3.83 (s, 3H), 3.70−3.36 (m, 14H), 3.26−3.13 (m, 6H), 1.94−1.64 (m, 20H), 1.51−1.38 (m, 8H), 1.09−0.96 (m, 12H). 13C NMR (100 MHz, CD3OD) δ 183.35, 163.44, 160.94, 158.36, 156.79, 156.64, 145.55, 138.68, 132.66, 132.36, 124.52, 123.64, 113.53, 112.78, 105.16, 100.87, 90.98, 72.05, 71.62, 65.32, 65.29, 64.71, 64.67, 64.35, 64.27, 64.19, 64.13, 61.79, 50.15, 50.07, 27.03, 26.02, 26.00, 25.31 (4 × CH2), 22.34, 20.75 (4 × CH2), 18.34, 18.19, 13.92 (2 × CH3), 13.90 (2 × CH3). HRMS (ESI+): calculated for C48H78I2N2O8 [(M − 2I)/2]+ 405.2874, found 405.2876. Antifungal and Antibacterial Susceptibility Testing. The antifungal and antibacterial activities of all compounds were determined in Sabouraud dextrose broth (SDB), Mueller−Hinton broth (MHB), or RPMI 1640 broth by the microbroth dilution method according to the Clinical and Laboratory Standards Institute (CLSI) guidelines with minor modifications.29 Candida cells were inoculated on Sabouraud dextrose agar (SDA) plates at 35 °C for 24 h and adjusted to approximately 5× 103 CFU/mL. Fusarium and Aspergillus cells were inoculated on Potato dextrose agar (PDA) plates at 30 °C for 4 days and adjusted to approximately 5× 103 CFU/mL. Two-fold serial dilutions of the tested compounds prepared from stock solution were put in a 96-well plate using Sabouraud dextrose broth (for anti-Candida activity test) or RPMI-1640 broth (for anti-Fusarium and anti-Aspergillus activity test) to obtain concentrations from 100 to 0.78 μg/mL. An equal volume of cell working suspension was added to each well to mixture with the test compounds. The plates including Candida cells were incubated at 35 °C for 24 h, and those including Fusarium and Aspergillus cells were incubated at 30 °C for 48 h. The minimum inhibitory concentration (MIC) was defined as the lowest concentration of tested compounds required to inhibit fungal growth by visible observations and by measuring the OD600 (Candida cells) and OD530 (Fusarium and Aspergillus cells). All MIC determinations were performed with biological replicates. 10146



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Hemolytic Assays. Hemolysis assays were carried out as described previously,61,64 using fresh New Zealand white rabbit RBCs. The procedures to isolate the blood from New Zealand white rabbits were carried out in accordance with the standards of the Association for the Research in Vision and Ophthalmology and approved by Institutional Animal Care and Use Committee (IACUC) of Singhealth. The RBCs were washed three times with PBS buffer and diluted with PBS to give a cell suspension. Two-fold serial dilutions of the xanthone analogues or antifungal agents dissolved in PBS/DMF (final concentration of DMF = 0.5%) were mixed with the 4% RBCs suspension (final concentration v/v). These mixtures were incubated at 37 °C for 1 h and centrifuged at 3000 rpm for 3 min. Aliquots (100 μL) of the supernatant were transferred to a 96-well plate. The absorbance of the supernatant at 576 nm was measured on a TECAN infinite 200 microplate reader to monitor the release of hemoglobin. RBCs with PBS/DMF (final concentration of DMF = 0.5%) served as the negative control (0% lysis), and RBCs treated with 2% Triton X-100 were used as positive control (100% lysis). All measurements were conducted with biological replicates. Checkerboard Assay. The fractional inhibitory concentration (FIC) index of the combination of xanthone compounds (1 and 2) and various classes of antifungal agents was determined using checkerboard assay according to a previously reported standard procedure.65,66 The FIC was calculated according to the equation: FIC = MICxanthone compound combined with antifungal/MICxanthone compound alone + MICantifungal combined with xanthone compound/MICantifungal alone. The combinational effect was considered as follows: synergistic, FIC ≤ 0.5; additive, 0.5 < FIC ≤ 4; antagonistic, FIC > 4.65 All measurements were performed with biological replicates. Time-Kill Assay. The time-kill kinetics of compounds 1 and 2 were determined against C. albicans DF2672R. A suspension of C. albicans DF2672R cells (105−106 CFU/mL) were incubated with various concentrations of compounds 1, 2, and natamycin (1×, 2×, 4×, and 8× MIC) at 35 °C. The samples (100 μL) were removed from the cell cultures at 0.5, 1, 2, 4, 8, and 24 h and then serially diluted in PBS buffer before plating onto SDA plate. After incubation of the plates for 24 h at 35 °C, colony forming units (CFU) were counted. All measurements were carried out with biological replicates. Drug Resistance Study. Drug resistance study was performed by treating C. albicans repeatedly with antifungal compounds 1 and 2. The multipassage resistance studies were performed according to the previous description with some modifications. The MIC values of compounds 1 and 2 against C. albicans DF2672R were determined with the same broth microdilution method described above. C. albicans cells taken from duplicate test tubes at a concentration of 0.5× MIC were adjusted to approximately 5 × 103 CFU/mL as the inoculum for the MIC measurement of next passage. After incubation with antifungal compounds 1 and 2 for 24 h at 35 °C, the new MIC values were measured. The process was repeated for 27−30 passages. All measurements were performed with biological replicates. Lactate Dehydrogenase (LDH) Assay. The cytotoxicity of compounds 1 and 2 was determined using the LDH assay against human corneal fibroblasts. The LDH assay was performed according to a previously reported standard procedure.64 The density of the used human corneal fibroblasts was 10000 cells per well. Cells treated with 1% Triton X-100 were taken as the positive control, and cells treated with DMSO (0.5%) served as the negative control. This assay was performed with biological replicates. SYTOX Green Assay. The SYTOX Green assay was used to measure the changes in membrane integrity of C. albicans DF2672R after treatment of the xanthone compounds. The SYTOX Green assay was performed as described previously, with some modifications.58,61 In brief, overnight cultures of C. albicans DF2672R were washed three times with PBS buffer and resuspended in PBS (OD600 = 0.2). The C. albicans DF2672R suspension was incubated with 0.3 μM SYTOX Green dye in the dark before the addition of xanthone compounds. Once the fluorescence signals of SYTOX Green-treated suspensions had stabilized, xanthone compounds dissolved in PBS/DMF (final concentration of DMF = 0.5%) were added. The increase in fluorescence was monitored continuously for 1−2 h using a PTI

spectrofluorometer (excitation and emission wavelengths were 504 and 523 nm, respectively). All tests were performed with biological replicates. Calcein Leakage Assay. The investigated liposome with composition of DOPC/DOPE/PI = 2:1:1 containing 15 wt % ergosterol was used to mimic fungal membranes. The LUVs were prepared according to a film hydration method as described previously.29 First, the lipids were dissolved in chloroform/methanol (2:1, v/v) and were dried using nitrogen gas. After freeze-drying overnight, the dried lipids were vortexed with a dye solution (80 mM calcein, 50 mM HEPES, 100 mM NaCl, and 0.3 mM EDTA; pH 7.4) to obtain calcein-loaded LUVs. The lipid mixture was freeze−thawed 10 times, using liquid nitrogen and water bath. The homogeneous calcein-loaded LUVs of about 100 nm were prepared by multiple extrusions (10 cycles) through polycarbonate filters (100 nm pore size; Whatman). Untrapped calcein were removed by a Sephadex G-50 column, with HEPES buffer as eluent. The concentration of calceinloaded LUVs was determined according to a total phosphorus determination method. The xanthone analogues dissolved in DMF/ HEPES buffer with desired concentrations (final concentration of DMF = 0.5%, v/v) were added to calcein-loaded LUVs solution (final phospholipid concentration = 50 μM) to obtain compound to liposomes ratios of 1:4, 1:8, and 1:16. The leakage of calcein from LUVs was determined by monitoring the fluorescence emission intensity at 520 nm using a TECAN infinite M200Pro microplate reader with an excitation wavelength of 490 nm. The percentage of calcein leakage was calculated according to the equation: % calcein leakage = [(Ft − Fmin)/(Fmax − Fmin)] × 100, where Ft presents the measured intensity after adding xanthone compounds, Fmin presents the measured intensity without treatment of xanthone compounds, and Fmax is the measured intensity of the treatment of 1% Triton X100. All measurements were performed in replicates. In Vivo Efficacy. Seven to eight-week-old C57BL6 mice (19−23 g) were used. The animals were maintained and treated in compliance with the Guide for the Care and Use of Laboratory Animals (National Research Council) and the ARVO statement for the Use of Animals in Ophthalmic and Vision Research. The experimental protocol was approved by SingHealth Institutional Biosafety Committee (SingHealth IBC). C. albicans DF2672R was inoculated on Sabouraud dextrose agar plates at 35 °C for 24 h and adjusted to approximately 5 × 107 CFU/mL in PBS (12 mM, pH 7.2) for mice corneal infection. Twelve mice were immunosuppressed with cyclophosphamide (100 mg/kg) via intraperitoneal injection 4 days, 1 day prior to infection, and on day 1 post infection. The mice were anesthetized using xylazine (8 mg/kg) and ketamine (80 mg/kg) via intraperitoneal injection. The cornea of the left eye was scratched with three 1 mm incisions using sterile BD Beaver mini-blades.64 The fungal suspension (20 μL) was applied topically to the damaged cornea surface. The mice were randomly divided into three groups (four mice per group). One day after infection, the respective drugs (5% natamycin, 0.2% compound 1, or PBS) were applied topically to the mice every 20 min during the first hour and every 1 h during the next 6 h. Finally, all mice were sacrificed. The infected corneas were collected and analyzed for the fungal load by serial dilution plating method using Sabouraud dextrose agar. Comparisons were carried out using the Mann−Whitney U-test. The statistical significance was defined as p values ≤0.05.

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S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.7b01348. HPLC traces, 1H and 13C NMR spectra of synthesized xanthone derivatives, and MIC values (μg/mL) of amphiphilic α-mangostin derivatives against various Gram-positive and Gram-negative bacterial strains (PDF) Molecular formula strings (CSV) 10147



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AUTHOR INFORMATION

Corresponding Authors

*For S.L.: phone, (+65) 65767285; fax, (+65) 62252568; Email, [email protected]. *For R.W.B.: phone, (+65) 65767215; fax, (+65), 62252568; E-mail, [email protected]. *For L.R.: phone, (+86) 20-39380255; E-mail, psliren@scut. edu.cn. ORCID

Li Ren: 0000-0003-0604-9166 Shouping Liu: 0000-0003-2415-076X Author Contributions

The manuscript was written through contributions of all authors. 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 This work was supported by National Medical Research Council and Singhealth Foundation (NMRC/CBRG/0080/ 2015, NMRC/TCR/R1018) and National Natural Science Foundation of China (51273072). We thank Jaime Chew (SERI) for technical support in toxicity experiments.

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ABBREVIATIONS USED AMPs, antimicrobial peptides; NME, new molecular entity; FDA, Food and Drug Administration; MIC, minimum inhibitory concentrations; RBC, red blood cell; HPLC, highperformance liquid chromatography; DIPEA, N,N-diisopropylethylamine; LDH, lactate dehydrogenase; DMF, dimethylformamide; LUVs, large unilamellar vesicles; CLSI, Clinical and Laboratory Standards Institute; CFU, colony forming units; SDB, Sabouraud dextrose broth; SDA, Sabouraud dextrose agar; PDA, potato dextrose agar; DOPE, 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine; PI, L-α-phosphatidylinositol (sodium salt); DOPE, 1,2-di(9Z-octadecenoyl)-snglycero-3-phosphoethanol-amine; HRMS, high-resolution mass spectrometry; NMR, nuclear magnetic resonance

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