Symmetrically Substituted Xanthone Amphiphiles Combat Gram

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Symmetrically Substituted Xanthone Amphiphiles Combat Grampositive Bacterial Resistance with Enhanced Membrane Selectivity Shuimu Lin, Jun-Jie Koh, Thet Tun Aung, Fanghui Lim, Jianguo Li, Hanxun Zou, Lin Wang, Rajamani Lakshminarayanan, Chandra S. Verma, Yingjun Wang, Donald T. H. Tan, Derong Cao, Roger W. Beuerman, Li Ren, and Shouping Liu J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01403 • Publication Date (Web): 25 Jan 2017 Downloaded from http://pubs.acs.org on January 26, 2017

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Journal of Medicinal Chemistry

Symmetrically Substituted Xanthone Amphiphiles Combat Gram-positive Bacterial Resistance with Enhanced Membrane Selectivity ⊥

Shuimu Lin, †,‡,|| Jun-Jie Koh, ‡ Thet Tun Aung, ‡ Fanghui Lim, ‡ Jianguo Li, ‡, Hanxun Zou, ‡ Lin ⊥,

Wang, †,|| Rajamani Lakshminarayanan, ‡, § Chandra Verma, ‡,

#,∞

Yingjun Wang, †,|| Donald T. H.

Tan, ‡, ¤ Derong Cao,× Roger W. Beuerman,*, ‡, § Li Ren,*, †,|| 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,

169856, Singapore §

SRP Neuroscience and Behavioral Disorders, Duke-NUS Graduate Medical School, 169857,

Singapore ||

National Engineering Research Center for Tissue Restoration and Reconstruction, Guangzhou

510006, China ⊥

#

Bioinformatics Institute (A*STAR), 30 Biopolis Street, 07-01 Matrix, 138671, Singapore

School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, 637551,

Singapore ∞

Department of Biological Sciences, National University of Singapore, 14 Science Drive 4,

ACS Paragon Plus Environment

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117543, Singapore ¤

Singapore National Eye Center, 11 Third Hospital Avenue, 168751, Singapore

×

School of Chemistry and Chemical Engineering, South China University of Technology,

Guangzhou 510641, China KEYWORDS: Xanthone, Gram-positive bacteria, membrane active, amphiphiles, antimicrobial agents

ABSTRACT

This study is the first report of the design of a new series of symmetric xanthone derivatives that mimic antimicrobial peptides (AMPs) using a total synthesis approach. This novel design is advantageous because of its low cost, synthetic simplicity and versatility, and easy tuning of amphiphilicity by controlling the incorporated cationic and hydrophobic moieties. Two watersoluble optimized compounds, 6 and 18, showed potent activities against Gram-positive bacteria, including MRSA and VRE (MICs = 0.78-6.25 µg/mL), with a rapid bactericidal effect, low toxicity and no emergence of drug resistance. Both compounds demonstrated enhanced membrane selectivity that was higher than those of most membrane-active antimicrobials in clinical trials or previous reports. The compounds appear to kill bacteria by disrupting their membranes. Significantly, 6 was effective in vivo using a mouse model of corneal infection. These results provide compelling evidence that these compounds have therapeutic potential as novel antimicrobials for multidrug-resistant Gram-positive infections.


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INTRODUCTION The rapid increase in the emergence of multidrug resistance and the declining discovery rate of novel antibiotics are growing threats to public health.1-4 Pharmaceutical companies have significantly reduced their investment in the research and development of anti-infective agents because of the short life-span of antimicrobial drugs and reduced profits.5, 6 As a result, current bacterial infection treatments have become inadequate and are often associated with higher healthcare costs and increased morbidity and mortality.7, 8 Thus, the development of new classes of antimicrobial agents is urgently needed. Antimicrobial peptides (AMPs), also termed host-defense peptides, have been extensively studied and are recognized to be the most promising choice for next-generation anti-infective agents because the probability of resistance emerging against AMPs is very low.9,

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AMPs

generally have a net positive charge and a substantial proportion (30-50%) of hydrophobic residues, which gives them highly amphiphilic topologies that are important for membrane selectivity and antibacterial properties.11-13 AMPs can penetrate through anionic bacterial membranes via hydrophobic interactions and disrupt the membrane integrity, leading to cell death via various mechanisms.11-14 However, several drawbacks, including high manufacturing costs, low in vivo stability, and high toxicity, have prevented AMPs from reaching the market.11, 15-17

To overcome these problems, several research groups have focused on the design and development of membrane-active small-molecule-based antimicrobial peptidomimetics to mimic the structure and antibacterial activity of AMPs.11, 18-25 Successful examples of small molecular mimics for AMPs include Destiny Pharma Ltd’s exeporfinium chloride (XF-73; Phase 1),26 Lytix Biopharma’s 27 (LTX-109; Phase 2),27, 28 and Cellceutix Corporation’s 28 (PMX 30063; Phase


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2).29 Some other examples include m-phenylene ethynylenes (mPEs),30 binaphthyl-based dicationic peptoids,31 cyclic peptides,32 aromatic oligomers,33 β-peptides,34-36 and cationic steroid antibiotics (CSAs).37 Although these molecules show good antibacterial activity, most also cause hemolysis.19, 38 The membrane selectivity of these compounds presents a major obstacle in the development of effective antibiotics. 11 Xanthones are secondary metabolites that are widely found in higher plant families, bacteria, lichens and fungi.39-41 Isoprenylated xanthones constitute a new class of naturally occurring and synthetic xanthone derivatives that have been widely recognized as promising bioactive compounds with remarkable pharmacological activities, including antibacterial,42-44 anticancer,45, 46

antiviral,47, 48 anti-inflammatory,49, 50 and antifungal activities.51, 52 Recently, we reported that a natural xanthone of α-mangostin and its semi-synthesized

xanthone-based derivatives including 29 (AM016), 30 (AM052) and 31 (AM218), that mimic AMPs showed excellent antibacterial activity against Gram-positive bacteria, by disrupting bacterial membrane integrity.38, 44, 53, 54 Although we have improved the membrane selectivity, it is still not sufficiently discriminative to be applied clinically. In this current study, we designed and synthesized a novel series of xanthone amphiphiles with symmetric structures as small antimicrobial peptidomimetics with excellent antibacterial activity and high membrane selectivity. To our knowledge, this is the first report using a total synthesis method to design xanthone-based antimicrobial peptidomimetics. This new design is advantageous because of its low cost, synthetic simplicity, and versatility. This strategy should easily facilitate the generation of a library of amphiphilic xanthone-based compounds and provide more modification options than the semi-synthesis of amphiphilic xanthones using natural α-mangostin which have limited modification options (Figure 1). The designed molecules


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were imbued with hydrophobicity through incorporation of an alkyl chain together with a xanthone scaffold, whereas hydrophilicity and positive charges were contributed by basic amino acids (Figure 1). Fine-tuning the structures and amphiphilic properties of xanthone derivatives was conducted to obtain potent antimicrobials and understand the role of the structural parameters in antibacterial and hemolytic activities. Finally, time-kill kinetics, in vitro toxicity, drug resistance, and mode of action for two compounds (6 and 18) were also studied. These results demonstrated that xanthone-based peptidomimetics had potential as novel, efficient antimicrobial agents. RESULTS AND DISCUSSION Chemistry. Several amphiphilic xanthone analogues were synthesized and the general synthetic routes adopted are shown in Schemes 1-2. Compound 1, which has four symmetric phenolic groups, served as the key building block and was prepared through the condensation of phloroglucinol and 2,4,6-trihydroxybenzoic acid in the presence of Eaton’s reagent (Scheme 1).55 The alkylation of compound 1 with a certain proportion of 3,3-dimethylallyl bromide, geranyl bromide or 1bromopropane in acetone resulted in compounds 2-3, 12-13 and 24; the remaining phenolic groups were alkylated with ethyl iodoacetate together with K2CO3 in acetone to yield compounds 4-5 and 14-15. The hydroxyl groups at the C1 and C8 positions of the xanthone scaffold were less reactive than those at the C3 and C6 positions because of the formation of intramolecular hydrogen bonds between the hydrogen atoms of the C1 and C8 hydroxyl groups and the oxygen atom of the C9 carbonyl group.38, 44 Compounds 4-5 and 14-15 were hydrolysed with lithium hydroxide to produce the required acids, which were further coupled to corresponding basic amino acid residues using N,N’-diisopropylcarbodiimide (DIC) and N-hydroxybenzo-triazole


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(HOBt) as coupling agents in the presence of DMF to yield compounds 6-8, 10-11, and 16. A similar procedure produced compound 19. Compound 8 was then treated with 20% piperidine/DMF to afford compound 9. To synthesize compounds 17-18 and 20-22, compounds 6 and 15 were hydrolysed using lithium hydroxide, and then, the carboxylic groups of xanthone analogues were coupled with H-Arg-OMe•2HCl or corresponding amine residues through amide coupling

using

2-(3H-[1,2,3]triazolo[4,5-b]pyridin-3-yl)-1,1,3,3-tetramethylisouronium

hexafluorophosphate (HATU) in DMF. A similar procedure produced compound 23. Compound 24 was alkylated with ethyl iodoacetate together with CH3ONa to produce corresponding ester. The ester was subsequently hydrolyzed with lithium hydroxide to yield acid 25, which was further coupled with H-Arg-OMe•2HCl using HATU to yield compound 26. All the final compounds were purified by reverse-phase high-performance liquid chromatography (RPHPLC) to more than 95% purity and characterized by nuclear magnetic resonance (NMR) and high-resolution mass spectrometry (HRMS). In vitro antibacterial and hemolytic activities. Systematic variation of the cationic and hydrophobic moieties as outlined in the synthesis above were carried out to fine tune the amphiphilicity and other structural parameters of xanthone derivatives to obtain potent antimicrobial xanthone analogues (Table 1). The antibacterial activities of the xanthone analogues were screened against different Gram-positive bacteria, including methicillin-resistant S. aureus (MRSA), S. aureus and Bacillus cereus, by measuring their minimum inhibitory concentrations (MICs). The hemolytic activities of xanthone analogues towards mammalian cells were measured using rabbit erythrocytes and were represented as HC50 values. Among the final 15 analogues synthesized, 11 of them showed less than 50% hemolytic activity in rabbit erythrocytes at a high concentration of 300 µg/mL.


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Compounds 16 and 20-22 displayed moderately hemolytic activities with HC50 values in the range of 33-48.8 µg/mL, which were higher than their MICs against Gram-positive bacteria. The hemolysis results demonstrated that all the tested xanthones could act selectively against bacterial cells. The membrane selectivity (HC50/MIC) needed to effectively kill bacterial cells without showing obvious cytotoxicity towards mammalian cells is a key biological property relating to the therapeutic potentials of membrane-active antibiotics. Effect of the overall charge. To examine the effect of the overall charge on the antimicrobial and hemolytic activities of the designed compounds, compounds 2, 6, 11, 18-19 and 23 were synthesized (Table 1). While retaining the hydrophobic moieties of two isoprenyl groups, the cationic moieties were varied to produce compounds 2, 6, 18 and 23 with overall charges of 0, +2, +4 and +6, respectively. Compound 2, which had no cationic moiety, showed poor antibacterial activity (MICs > 50 µg/mL) and weak hemolytic activity (HC50 > 400 µg/mL). Additional two and four arginine residues were coupled to compound 2 to yield compounds 6 and 18, respectively. Both compounds (6 and 18) displayed excellent activity against tested Gram-positive bacteria, with MICs in the range of 0.78-3.13 µg/mL; these values are comparable to those of vancomycin and 32 (MSI-78) (a membrane-active antimicrobial agent in Phase 3 clinical trials as a topical antibiotic).18, 56 But compound 18 (charge of +4) gave rise to more than a 2.5-fold decrease in hemolytic activity (HC50 > 2000 µg/mL) as compared to compound 6 (charge of +2). Compound 23 (charge of +6) showed moderately antibacterial activities (MICs = 12.5 µg/mL) and weak hemolytic activity (HC50 > 400 µg/mL). Due to the increased overall charges, compound 23 would become too polar, resulting in reduced antibacterial activity. In contrast, when the isoprenyl groups were replaced with the more hydrophobic geranyl groups (compounds 11 and 19), the antimicrobial activity decreased significantly, suggesting that the


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overall charge is not the sole factor affecting the antibacterial and hemolytic activities of xanthone derivatives. Effect of hydrophobicity. Compounds 6, 11, 18-19 and 26 were also used to investigate the effects of hydrophobicity on the biological activity of the xanthone derivatives (Table 1); the hydrophobic moieties were varied, while the cationic moieties were kept constant. Compound 6, in which the hydrophobic groups consisted of two isoprenyl groups, exhibited excellent antimicrobial activities (MICs = 0.78-3.13 µg/mL). Increasing the alkyl chain length of compound 6 yielded a more hydrophobic compound 11, which had two longer hydrophobic lipids of two geranyl groups, and showed greatly diminished MICs of 25-50 and even > 50 µg/mL. Decreasing the alkyl chain length of compound 6 yielded a less hydrophobic compound 26, which had two propyl groups, and also displayed diminished MICs of 12.5-25 µg/mL. Similarly, compound 19 (MICs = 25 µg/mL), which had increased hydrophobic moieties, also displayed significantly reduced antibacterial activity compared to the less hydrophobic compound 18 (MICs = 1.56-3.13 µg/mL) with the same cationic moieties of four arginine residues. The results suggest that the charge-hydrophobicity balance in these xanthone analogues is a key factor in determining activity. All five compounds 6, 11, 18-19 and 26 exhibited very low hemolytic activities against RBCs with HC50 values of more than 300 µg/mL. The % hemolysis of RBCs caused by 400 µg/mL of compound 6 (42±2%) was much higher than that of compound 11 (< 5%) although the latter is more hydrophobic. However, unlike the results from compounds 6 and 11, the reduced hydrophobicity of compound 18 (HC50 > 2000 µg/mL) gave rise to a more than 6.5-fold reduction in hemolytic activity compared to that of compound 19 (HC50 = 306±26 µg/mL). These data indicate that hydrophobic moieties play an important role in the antibacterial and hemolytic activity of these analogues.


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Effect of pKa values of the amine moiety. The conditions in which these compounds are expected to be active are complex and hence motivated us to examine the pKa values of the cationic moieties for which compounds 6-7, 9-10 and 20-22 were prepared. In these compounds, the hydrophobic moieties consisted of two isoprenyl groups at the C3 and C6 positions of the xanthone scaffold, and the hydrophilic moieties consisted of cationic amino acid residues with different pKa values at the C1 and C8 positions of the xanthone scaffold. Compounds with high pKa amino acids (compound 6 containing arginine side chain, pKa of guanidine = 12.48; compound 9 containing lysine side chain, pKa of ϵ-NH2 = 10.54; compounds 20-22 containing N,N-dialkylamine moieties, pKa = 10.64, 10.98 and 11.25, respectively)38,

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resulted in low

MICs of 0.78-3.13 µg/mL for compound 6 and 3.13-12.5 µg/mL for compounds 9 and 20-22. A histidine-coupled xanthone analogue 10 was also evaluated (pKa of the side chain of histidine = 6.10), but it was inactive, even at the highest concentration tested (> 50 µg/mL). Nonpeptidic compounds 20-22 showed moderately hemolytic activities (HC50 = 38.4-48.8 µg/mL). By contrast, compounds 6, 9 and 10, which had basic amino acids, showed minimal toxicity to mammalian cells (HC50 > 400 µg/mL). Compound 6 bearing the highest pKa amino acids among the three compounds demonstrated increased hemolytic activity (42±2%) at 400 µg/mL compared to compounds 9 and 10 (< 5%). Compound 7 containing two arginine residues capped with -NH2 instead of -OMe also displayed potential antibacterial activity (MICs = 6.25-12.5 µg/mL) and weak hemolytic activity (HC50 > 400 µg/mL, 400 µg/mL. In contrast, compound 6 bearing two isoprenyl groups and two arginine residues displayed low MICs, while replacing one arginine with an isoprenyl group (compound 16) did not affect the antimicrobial activity, but increased the hemolytic activity significantly (HC50 = 33±1 µg/mL), suggesting that higher hydrophobicity leads to higher membrane toxicity. These data clearly imply that a potential enhance in the antibacterial activity and membrane selectivity of these compounds can be obtained by balancing their cationicity and hydrophobicity. After systematically tuning the cationicity and hydrophobicity, compounds 6 and 18 were identified as potent compounds because of their high membrane selectivity (120-962 for compound 6 and > 639 for compound 18) and excellent antibacterial activity against Grampositive bacteria. Compounds 6 and 18 were further screened against a panel of Gram-positive pathogens including MRSA and vancomycin resistant enterococci (VRE) strains (Table 2). The MICs of compounds 6 and 18 were in the range of 0.78-6.25 µg/mL against all the tested strains. Additionally, their antimicrobial activities were mostly comparable to that of vancomycin (MICs = 0.78-1.56 µg/mL) and were far superior to vancomycin against the 3 VRE strains tested. More importantly, compounds 6 and 18 were water soluble and displayed excellent activity against drug-resistant strains, suggesting that there was no cross-resistance for compounds 6 and 18. As shown in Table 3, the selectivity of compounds 6 and 18 was higher than those of several


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membrane-active antimicrobial agents in clinical trials including compounds 27-28, 32 and 33 (CSA-13), and our previously reported α-mangostin derivatives 29-31. Thus, these results indicate that compounds 6 and 18 are immensely powerful as membrane-active antibiotics and hold promise as novel therapeutics. Thus far, this study has illustrated a strategy to design and synthesize a series of small molecule mimics of AMPs. Their antibacterial and hemolytic activities resulted from a balance between several structural parameters including amphiphilicity, overall positive charge, the pKa values of the cationic moieties, and hydrophobicity. It is clear that, the xanthone core is a relatively small and conformationally rigid hydrophobic core, which favors penetration into the membrane of Gram-positive bacteria.33, 57 In addition, a highly amphiphilic topology appears essential for membrane-active compounds, in which the hydrophobic and hydrophilic cationic moieties are spatially separated.11,

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The overall positive charges endow the molecule with

favorable electrostatic interactions that drive these peptidomimetics for rapid adsorption on to the negatively charged bacterial membranes, and help it distinguish the anionic bacterial membranes from the zwitterionic mammalian cell membranes, thus imparting selectivity for bacterial membranes to these compounds.58,

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This electrostatic selectivity towards the bacteria is

complemented by the hydrophobic moieties that offer a driving force for their penetration into the bacterial membrane, ultimately resulting in the leakage of cell contents and death of bacterial cells.38 It is clear that the hydrophobic and cationic moieties must be well balanced to increase the antimicrobial activity and minimize toxicity. Enhancing the molecular hydrophobicity beyond a certain extent results in high toxicity of compounds towards mammalian cells, reduced water solubility and poor antibacterial activity. Similarly, increasing the hydrophilicity beyond a certain extent also leads to loss of antibacterial potency.


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Based on our earlier studies, we had found that the hydrophobic interactions of the isoprenyl groups triggered the insertion of α-mangostin into the hydrophobic regions of the bacterial membranes.38, 44 In the process, it lowers the free energy barrier of penetration, and the molecule is rapidly absorbed onto the membranes, accelerating its bactericidal action.38, 44 This motivated us to select isoprenyl groups as the key hydrophobic lipid groups in this study. In this study, compounds 1-2 and 17 were not active against all tested bacterial strains, with MICs ≥ 50 µg/mL. Compounds 1 and 2, which had no net charge and amphiphilic conformations, could not effectively interact with the bacterial membrane. However, compound 17 including three positive charges could not produce a strong interaction with bacterial membranes because of the lack of balance between its cationic and hydrophobic moieties. Compounds 11, 17 and 19 containing amphiphilic configurations exhibited moderate or poor antibacterial activities towards Gram-positive bacteria. It appears that the higher hydrophobicity of compounds 11 or 19 bearing two geranyl groups and the higher hydrophilicity of compound 17 including three arginine residues and one isoprenyl group do not contain the appropriate balance between hydrophobicity and hydrophilicity, leading to that those compounds are not able to successfully penetrate the bacterial membranes. Compound 16, which had three isoprenyl groups and one arginine group, displayed excellent antimicrobial activities with MICs of 0.781.56 µg/mL, and also showed a moderate hemolytic activity with a HC50 value of 33±1 µg/mL, 21-42 fold higher than its MICs. The moderate hemolytic activity of compound 16 might be caused by the overly strong hydrophobicity of the structure. Hence, in agreement with this hypothesis, two structurally optimized xanthone compounds, 6 and 18, displayed excellent antibacterial activity against Gram-positive bacteria and very low hemolytic activity, due to the balance between the factors described above.


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Bactericidal kinetics. An in vitro time-kill assay of compounds 6 and 18 against MRSA at four different concentrations (0.5× MIC, 1× MIC, 2× MIC and 4× MIC) was carried out. The results displayed that compound 6 killed bacteria quickly and produced more than 3-log reduction (99.9% of bacteria killed) in 10 minutes at 4× MIC and in 2 h at 2× MIC in growth medium (Mueller Hinton broth [MHB]) against MRSA DM21455 (Figure 2). Compound 18 also exhibited impressively rapid bactericidal activity, achieving a 3-log reduction in 10 min at 4× MIC and in 30 min at 2× MIC in the same growth medium (Figure 2). In contrast, according to a recent report, vancomycin (non-membrane-active), at 4× MIC, induced only negligible bacterial killing of MRSA DM21455 up to 300 min, clearly demonstrating the advantage of membrane active antimicrobials.44 Multipassage resistance selection studies. The propensity of bacteria to develop drug resistance can be assessed through the serial exposure of bacteria to antibiotics.60 S. aureus was serially passaged with sub-inhibitory concentrations of compounds 6, 18, norfloxacin and gatifloxacin, and new MICs were determined during passages of 17-30 days (Figure 3). During the course of the passage studies, no significant change in the MIC values of compounds 6 and 18 was observed, whereas those of norfloxacin and gatifloxacin increased by 128- and 64-fold after 15 passages. These results demonstrated that compounds 6 and 18 had major advantages over conventional antibiotics and did not result in drug resistance. The rapid bactericidal activity of the two optimized compounds resulting from their membrane-targeting actions towards bacteria might contribute to the lack of drug resistance. Because it is difficult for bacteria to remodel their membranes while they are alive.19, 32, 58 Cytotoxicity towards mammalian cells. The in vitro cytotoxic effects of the two optimized compounds, 6 and 18, were investigated with a lactate dehydrogenase (LDH) assay. The results


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showed that compound 6 induced negligible cytotoxicity up to concentration of 250 µM, which is greater than 70-fold higher than its MIC. At 100 µM, almost no cytotoxicity was detected for compound 18, whereas at 250 µM, exceeding its MIC by over 90-fold, only 27.4 ± 1.5% cytotoxicity was observed (Figure 4). Consistent with the hemolysis assay, the LDH assay demonstrated that compounds 6 and 18 possessed low cytotoxicity towards mammalian cells, and hold therapeutic potential. Mode of action Vesicle leakage from calcein-loaded LUV. In this study, the extent of membrane disruption was evaluated by measuring the leakage of calcein from large unilamellar vesicles (LUVs). The abilities of amphiphilic xanthones to perturb the membranes and result in leakage of a fluorescent dye loaded within the LUVs was tested at a compound-to-lipid (C/L) ratio of 1/16. We prepared calcein-loaded liposomes with different compositions, including DOPC/cholesterol (3:1, w/w) and DOPE/DOPG (3:1, w/w), to mimic the neutral mammalian membrane and negatively charged bacterial cytoplasmic membrane, respectively.38 As shown in Figure 5, compounds 6, 9 and 18 induced strong calcein leakage from vesicles mimicking bacterial membranes, with leakage values at 58.7 ± 5.4%, 48.2 ± 9.8% and 63.3 ± 7.7%, respectively, suggesting potentially high sensitivity of the bacterial membrane to these three compounds. In contrast, the inactive compounds 1 and 10, with MICs > 50 µg/mL (Table 1), resulted in only 11.7 ± 1% and 8.9 ± 0.9% leakage upon exposure to the DOPE-DOPG liposomes, indicating the interactions between these two compounds and the bacterial membranes were very weak. This further confirmed our observations that membrane disruption was the critical event that induced bacterial cell death. All five compounds induced very low (less than 20%) calcein leakage from DOPC/cholesterol vesicles. This lower calcein leakage from mammalian membrane mimics


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indicated that these compounds had very weak interactions with the mammalian membrane, which would also explain why these compounds displayed very promising membrane selectivity and were not toxic to mammalian cell lines. Overall, the lytic activities of these compounds correlated very well with their MICs and HC50 values. SYTOX Green assay. In addition to the calcein leakage assay using artificial membranes, a SYTOX Green assay was performed using living bacterial cells, to further investigate the membrane disruption properties of these amphiphilic xanthones. SYTOX Green is a high-affinity nucleic acid dye which is not able to penetrate living cells with intact membranes, but can easily penetrate cells with damaged plasma membranes. Figure 6 showed the effect of several xanthone derivatives at 2× MIC against clinical isolates S. aureus DM4001R which were incubated with SYTOX Green. The results showed that active compounds 6, 9 and 18 clearly permeabilized the bacterial inner membrane at a concentration of 2× MIC. In contrast, inactive compounds 1 and 10 caused very low membrane permeabilization at a concentration of 100 µg/mL (MICs > 50 µg/mL). Together, these data further demonstrated that compounds 6, 9 and 18 disrupted the membranes of Gram-positive bacteria, resulting in their death. ATP leakage assay. We next carried out an ATP assay to further investigate the membrane targeting properties of the amphiphilic xanthone derivatives, using ATP determination kits. ATP leakage results from the bacterial intracellular region upon disruption of the bacterial membranes. As shown in Table 4, active compounds 6, 9 and 18 caused strong ATP leakage from S. aureus DM4001R at a concentration of 4× MIC. In contrast, only a low level of ATP leakage was measured for inactive compounds 1 and 10 (MICs > 50 µg/mL) at a high concentration of 200 µg/ml. These observations are consistent with the SYTOX green uptake assay, suggesting that the xanthone derivatives target the bacterial membrane. After the xanthones permeabilized


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and disrupted the bacterial inner membrane, ATP leakage from the cells results in cell starvation, which further lead to cells death. Molecular dynamics simulations. To gain detailed atomistic insights into the membrane interactions of the cationic xanthone amphiphilies, we carried out molecular dynamics simulations of compound 6 with model bacterial and mammalian membranes. As shown in Figure 7, compound 6 adsorbs onto bacterial membranes rapidly, as revealed by the distances between compound 6 and the membrane, which is driven by favorable electrostatic interactions between the cationic arginine side chains of compound 6 and the anionic head groups of the lipids. As a result, compound 6 can accumulate to a high surface concentration on the bacterial membrane. The interactions are further strengthened by the formation of multi-dentate hydrogen bonds between the guanidinium groups with the head groups of lipid molecules.61 The isoprenyl group was found to penetrate into the lipid tail region of the bacterial membrane, forming an amphiphilic structure that perturbs the membrane-water interface.54 At high concentrations (e.g., the concentrations above the MIC value), large number of such amphiphilic structures at the membrane-water interface will result in significant membrane perturbations. In contrast, the affinity of compound 6 to the zwitterionic mammalian membrane is low due to weaker electrostatic interactions and lack of hydrogen bonds, leading to a low surface concentration of compound 6 on mammalian membrane. In addition, compound 6 was found to remain at the surface of the mammalian membrane without penetrating into the lipid tail region, as evidenced by its larger distance from the mammalian membrane. In vivo efficacy To determine the potential of these compounds as therapeutics, efficacy of compound 6 in a mouse model of cornea was investigated. In this study, mice were first infected with S. aureus


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ATCC 29213 or MRSA ATCC 700699 in the cornea. After 1 day of infection, antimicrobial agents were applied topically at 2 hour interval for 4 times. PBS was used as a control for the mice without any antimicrobial treatment. It is noteworthy that the marketed 0.5% solution of levofloxacin is commonly applied to treat corneal ulcers caused by bacteria, including S. aureus. As shown in Figure 8a, compound 6 (0.5% solution) and levofloxacin (0.5% solution) significantly decreased the number of viable bacteria in infected cornea by 1.82-log (98.5%) and 2.12-log (99.2%), respectively (p = 0.001). The results demonstrated that compound 6 had excellent in vivo efficacy against S. aureus comparable to the levofloxacin. To evaluate the potential of compound 6 against drug-resistant bacterial infection, a MRSA corneal infection model was further evaluated. As shown in Figure 8b, compound 6 (0.5% solution) and 32 (0.5% solution; MSI-78; Phase 3) reduced the bacterial burden in infected cornea by 92.8% (p = 0.021) and 93.3% (p = 0.001), respectively, clearly demonstrating that compound 6 had potent in vivo antibacterial activity against MRSA comparable to compound 32. These results indicated that compound 6 had therapeutic potential as novel antimicrobial agents for drug-resistant Grampositive bacterial infections. CONCLUSIONS In summary, we successfully used a total synthesis method to design and synthesize a new series of symmetrically substituted amphiphilic xanthones as antimicrobial peptidomimetics, and demonstrated that these compounds are promising novel membrane-targeting antimicrobials. After systematically tuning the cationic and hydrophobic moieties, two water-soluble optimized compounds, 6 and 18, were found to show high membrane selectivity and excellent antibacterial activity against Gram-positive bacterial strains, including MRSA and VRE. Their membrane selectivities were higher than those of several membrane-active antimicrobial agents in clinical


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trials and our previously reported α-mangostin derivatives. The mechanism studies suggested that the mode of action of these compounds arose from an electrostatic discrimination in favour of the negatively charged bacterial membranes over the zwitterionic mammalian membranes, resulting in the ability of these compounds to rapidly adsorb onto, and penetrate into the bacterial membrane. The perturbation of the bacterial membrane results in leakage of critical intracellular components, leading to rapid cell death (cell death of 99.9% MRSA in 10 min). Compound 6 displayed excellent in vivo antibacterial efficacy against Gram-positive bacteria, including MRSA and S. aureus. We speculate that this rapidity of action combined with the inability of bacteria to rapidly remodel their membranes, results in the lack of drug resistance emerging. This comprehensive study outlines a rationally devised minimalist strategy to design potent smallmolecule-based antimicrobial agents that mimic AMPs. Through our study, we have generated two compounds, 6 and 18, which have promising therapeutic potential for efficiently combating infections caused by Gram-positive bacteria. EXPERIMENTAL SECTION General chemistry. All solvents and chemicals were purchased from commercial suppliers, and were used without further purification. Chromatographic separation was achieved by using a preparative RP-HPLC on a Shimadzu LC-20AP instrument with a C18 column (Phenomenex, 150 × 21.2 mm, 5 µ, 100 Å), ultraviolet (UV) detection at 254 nm and a flow rate of 12 mL/min. A mixture of methanol (HPLC grade) and distilled water (both containing 0.1% formic acid) was used as a mobile phase with a gradient elution method. The purity of all final compounds was > 95% based on HPLC analysis with a C18 column (Phenomenex, 150 × 4.6 mm, 3 µm, 110 Å). 1

H and

13

C NMR spectra were recorded on a Bruker Avance 400 MHz instrument using

tetramethylsilane as an internal reference and CDCl3 or CD3OD as the specified deuterated


18

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solvent. Chemical shifts (δ) were reported in parts per million (ppm) and coupling constants (J) were given in hertz (Hz), and peak multiplicities were reported as singlet (s), doublet (d), triplet (t), quadruplet (q), broad (br), and multiplet (m). ESI mass spectra were recorded on an API2000 LC/MS/MS system and APCI mass spectra were measured using a Bruker amaZonX spectrometer. Synthesis, structures and characterizations of compounds. 1,3,6,8-Tetrahydroxy-9H-xanthen-9-one (compound 1). Compound 1 was prepared from 2,4,6-trihydroxybenzoic acid (1.88 g, 10.0 mmol) and phloroglucinol (1.26 g, 10.0 mmol) in Eaton’s reagent (25.0 mL) using a reported method.55 The product was obtained as a yellow solid (0.937 g, 36%). HRMS (ESI+): calcd for C13H8NaO6 [M + Na]+ 283.0213, found 283.0214 (error -0.4 ppm). 1,8-Dihydroxy-3,6-bis((3-methylbut-2-en-1-yl)oxy)-9H-xanthen-9-one (compound 2). To a solution of compound 1 (101.0 mg, 0.388 mmol) and potassium carbonate (160.9 mg, 1.164 mmol) in acetone (10 mL), and 3,3-dimethylallyl bromide (89.6 µL, 0.776 mmol) was added. The mixture was refluxed for 12 hours. After cooling, the mixture was diluted with ethyl acetate and washed with brine three times. The organic phase was dried over anhydrous sodium sulfate, filtered, and evaporated under reduced pressure. Subsequently, the crude residue was purified by column chromatography (silica gel, petroleum ether/ethyl acetate, 7:1, v/v) to yield compound 2 (103.0 mg, 67%) as a yellow solid. 1H NMR (400 MHz, CDCl3) δ = 12.01 (s, 2H, O-H), 6.35 (d, J = 2.0 Hz, 2H, 2×Ar-H), 6.30 (d, J = 1.6 Hz, 2H, 2×Ar-H), 5.50 (t, J = 6.6 Hz, 2H, 2×CH), 4.58 (d, J = 6.8 Hz, 4H, 2×CH2), 1.82 (s, 6H, 2×CH3), 1.77 (s, 6H, 2×CH3).

13

C NMR (101 MHz,

CDCl3) δ = 183.15, 166.06 (2×C), 162.70 (2×C), 157.47 (2x C), 139.31 (2×C), 118.51 (2×CH), 102.10 (2×CH), 97.81 (2×CH), 93.63 (2×C), 65.50 (2×CH2), 25.80 (2×CH3), 18.26 (2×CH3).


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HRMS (ESI+): calcd for C23H24NaO6 [M + Na]+ 419.1465, found 419.1467 (error -0.4 ppm). 3,6-Bis(((E)-3,7-dimethylocta-2,6-dien-1-yl)oxy)-1,8-dihydroxy-9H-xanthen-9-one (compound 3). Compound 3 was prepared from compound 1 (100 mg, 0.384 mmol) and geranyl bromide (137.2 µL, 0.691 mmol) using the same method as described for compound 2. The product was obtained as an orange solid (90.0 mg, 44%). 1H NMR (400 MHz, CDCl3) δ 12.01 (s, 2H, O-H), 6.34 (d, J = 2.0 Hz, 2H, 2×Ar-H), 6.29 (d, J = 2.0 Hz, 2H, 2×Ar-H), 5.49-5.46 (m, 2H, 2×CH), 5.11-5.08 (m, 2H, 2×CH), 4.60 (d, J = 6.4 Hz, 4H, 2×OCH2), 2.15-2.08 (m, 8H, 4×CH2), 1.76 (s, 6H, 2×CH3), 1.68 (s, 6H, 2×CH3), 1.61 (s, 6H, 2×CH3). 13C NMR (101 MHz, CDCl3) δ = 182.13, 165.08 (2×C), 161.68 (2×C), 156.45 (2×C), 141.33 (2×C), 130.95 (2×C), 122.62 (2×CH), 117.32 (2×CH), 101.08 (2×CH), 96.84 (2×CH), 92.62 (2×C), 64.57 (2×CH2), 38.50 (2×CH2), 25.23 (2×CH2), 24.64 (2×CH3), 16.69 (2×CH3), 15.73 (2×CH3). HRMS (ESI+): calcd for C33H41O6 [M + H]+ 533.2898, found 533.2893 (error 0.8 ppm). Diethyl

2,2'-((3,6-Bis((3-methylbut-2-en-1-yl)oxy)-9-oxo-9H-xanthene-1,8-

diyl)bis(oxy))diacetate (compound 4). To a solution of compound compound 2 (911.5 mg, 2.299 mmol) in acetone (10 mL), ethyl iodoacetate (816.3 µL, 6.897 mmol) and potassium carbonate (794.4 mg, 5.748 mmol) were added. The mixture was refluxed for 12 hours. After cooling, the mixture was diluted with ethyl acetate and washed with brine three times. The organic layer was dried over anhydrous sodium sulfate, filtered, and evaporated under reduced pressure. Subsequently, the crude residue was purified by column chromatography (silica gel, petroleum ether/ethyl acetate, 1:1, v/v) to yield compound 4 as a yellow solid (999.5 mg, 76%). 1

H NMR (400 MHz, CDCl3) δ 6.47 (d, J = 2.4 Hz, 2H, 2×Ar-H), 6.29 (d, J = 2.4 Hz, 2H, 2×Ar-

H), 5.50 (t, J = 6.8 Hz, 2H, 2×CH), 4.77 (s, 4H, 2×OCH2), 4.56 (d, J = 6.8 Hz, 4H, 2×OCH2), 4.27 (q, J = 7.1 Hz, 4H, 2×OCH2), 1.82 (s, 6H, 2×CH3), 1.76 (s, 6H, 2×CH3), 1.29 (t, J = 7.2 Hz,


20

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6H, 2×CH3).

13

C NMR (101 MHz, CDCl3) δ = 172.94, 167.67 (2×C), 161.96 (2×C), 158.92

(2×C), 157.43 (2×C), 138.43 (2×C), 117.54 (2×CH), 107.85 (2×CH), 97.90 (2×C), 93.69 (2×C), 66.12 (2×CH2), 64.30 (2×CH2), 60.26 (2×CH2), 24.82 (2×CH3), 17.24 (2×CH3), 13.14 (2×CH3). HRMS (APCI+): calcd for C31H37O10 [M + H]+ 569.2381, found 569.2388 (error -1.2 ppm). Diethyl

2,2'-((3,6-Bis(((E)-3,7-dimethylocta-2,6-dien-1-yl)oxy)-9-oxo-9H-xanthene-1,8-

diyl)bis(oxy))diacetate (compound 5). Compound 5 was prepared from compound 3 (389.5 mg, 0.731 mmol), ethyl iodoacetate (259.6 µL, 2.193 mmol) and potassium carbonate (252.6 mg, 1.828 mmol) using the same method as described for compound 4. The product was obtained as a yellow gel (363.3 mg, 71%). 1H NMR (400 MHz, CDCl3) δ 6.46 (d, J = 2.2 Hz, 2H, 2×Ar-H), 6.28 (d, J = 2.2 Hz, 2H, 2×Ar-H), 5.51-5.43 (m, 2H, 2×CH), 5.13-5.05 (m, 2H, 2×CH), 4.77 (s, 4H, 2×OCH2), 4.57 (d, J = 6.6 Hz, 4H, 2×OCH2), 4.28-4.20 (m, 4H, 2×OCH2), 2.17-2.05 (m, 8H, 4×CH2), 1.75 (s, 6H, 2×CH3), 1.67 (s, 6H, 2×CH3), 1.60 (s, 6H, 2×CH3), 1.27 (t, J = 7.1 Hz, 6H, 2×CH3). 13C NMR (101 MHz, CDCl3) δ 172.98, 167.68 (2×C), 162.00 (2×C), 158.89 (2×C), 157.43 (2×C), 141.42 (2×C), 130.95 (2×C), 122.63 (2×CH), 117.33 (2×CH), 107.82 (2×CH), 97.90 (2×C), 93.69 (2×CH), 66.11 (2×CH2), 64.39 (2×CH2), 60.27 (2×CH2), 38.52 (2×CH2), 25.25 (2×CH2), 24.65 (2×CH3), 16.70 (2×CH3), 15.72 (2×CH3), 13.14 (2×CH3). HRMS (APCI+): calcd for C41H53O10 [M + H]+ 705.3633, found 705.3631 (error 0.3 ppm). Dimethyl

2,2'-((2,2'-((3,6-Bis((3-methylbut-2-en-1-yl)oxy)-9-oxo-9H-xanthene-1,8-

diyl)bis(oxy))bis(acetyl))bis(azanediyl))(2S, 2'S)-bis(5-guanidinopentanoate) (compound 6). To a solution of compound 4 (146 mg, 0.257 mmol) in THF (4 mL) was added a 5% LiOH solution (2 mL). After stirring at room temperature for 1.5 h, the reaction mixture was acidified with acetic acid, diluted with butanol, and extracted with brine 3 times. The organic phase was dried over anhydrous sodium sulfate and evaporated under reduced pressure. The crude residue


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was dissolved in anhydrous DMF (5 mL). HOBt (104.2 mg, 0.771 mmol), DIC (119.4 µL, 0.771 mmol) and H-Arg-OMe•2HCl (201.3 mg, 0.771 mmol) were added, and the reaction mixture was stirred at 0 °C for 1 h. After stirring at room temperature overnight, the mixture was diluted with butanol and washed with brine for 3 times. The organic phase was dried over anhydrous sodium sulfate, filtered, and evaporated under reduced pressure. Subsequently, the crude residue was purified by HPLC to afford compound 6 as a yellow solid (138.1 mg, 63%). 1H NMR (400 MHz, MeOD) δ 6.71-6.66 (m, 2H, 2×Ar-H), 6.54 (d, J = 1.9 Hz, 2H, 2x Ar-H), 5.52-5.46 (m, 2H, 2×CH), 4.80-4.76 (m, 4H, 2×OCH2), 4.68 (d, J = 6.7 Hz, 4H, 2×OCH2), 4.42-4.36 (m, 2H, 2×CH), 3.74 (s, 6H, 2×OCH3), 3.21-3.12 (m, 4H, 2×CH2 ), 2.11-1.94 (m, 4H, 2×CH2), 1.82 (s, 6H, 2×CH3), 1.80 (s, 6H, 2×CH3), 1.71-1.63 (m, 4H, 2×CH2).

13

C NMR (101 MHz, MeOD) δ

173.55 (2×C), 171.20 (2×C), 170.06, 165.74 (2×C), 160.86 (2×C), 159.89 (2×C), 158.66 (2×C), 140.06 (2×C), 120.00 (2×CH), 108.63 (2×CH), 99.90 (2×C), 96.24 (2×CH), 70.12 (2×CH2), 66.87 (2×CH2), 53.68 (2×CH), 52.95 (2×CH3), 41.95 (2×CH2), 29.31 (2×CH2), 26.46 (2×CH2), 25.88 (2×CH3), 18.36 (2×CH3). HRMS (ESI+): calcd for C41H57N8O12 [M + H]+ 853.4090, found 853.4087 (error 0.3 ppm). (2S,2'S)-2,2'-((2,2'-((3,6-Bis((3-methylbut-2-en-1-yl)oxy)-9-oxo-9H-xanthene-1,8diyl)bis(oxy))bis(acetyl))bis(azanediyl))bis(5-guanidinopentanamide)

(compound

7).

Compound 7 was prepared from compound 4 (69.1 mg, 0.122 mmol), HOBt (49.4 mg, 0.366 mmol), DIC (56.7 µL, 0.366 mmol) and H-Arg-NH2•2HCl (90.1 mg, 0.366 mmol) using the same method as described for compound 6. The product was obtained as a light yellow solid (69 mg, 69%). 1H NMR (400 MHz, MeOD) δ 6.62-6.55 (m, 2H, 2x Ar-H), 6.49-6.42 (m, 2H, 2x ArH), 5.53-5.41 (m, 2H, 2×CH), 4.73 (s, 4H, 2×OCH2), 4.64 (d, J = 6.2 Hz, 4H, 2×OCH2), 4.404.32 (m, 2H, 2×CH), 3.24-3.13 (m, 4H, 2×CH2), 2.03-1.76 (m, 16H, 4×CH3, 2×CH2), 1.73-1.64


22

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(m, 4H, 2×CH2).

13

C NMR (101 MHz, MeOD) δ 174.92, 169.57 (2×C), 168.63 (2×C), 164.24

(2×C), 159.30 (2×C), 158.50 (2×C), 157.27 (2×C), 138.67 (2×C), 118.60 (2×CH), 107.33 (2×CH), 98.26 (2×C), 94.72 (2×CH), 68.51 (2×CH2), 65.45 (2×CH2), 53.00 (2×CH), 40.65 (2×CH2), 28.96 (2×CH2), 24.99 (2×CH3), 24.47 (2×CH2), 16.95 (2×CH3). HRMS (ESI+): calcd for C39H55N10O10 [M + H]+ 823.4097, found 823.4092 (error 0.7 ppm). Dimethyl


diyl)bis(oxy))bis(acetyl))bis(azanediyl))bis(6-((((9H-fluoren-9-yl)methoxy) carbonyl)amino)hexanoate) (compound 8). To a solution of compound 4 (63.4 mg, 0.112 mmol) in THF (4 mL) was added a 5% LiOH solution (2 mL). After stirring at room temperature for 1.5 h, the reaction mixture was acidified with acetic acid, diluted with butanol, and extracted with brine 3 times. 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 (45.4 mg, 0.336 mmol), DIC (52.0 µL, 0.336 mmol) and H-Lys(Fmoc)-OMe•HCl (140.8 mg, 0.336 mmol) were added, and the reaction mixture was stirred at 0 °C for 1 h. After stirring at room temperature overnight, the mixture was diluted with ethyl acetate and washed with brine for 3 times. The organic phase was dried over anhydrous sodium sulfate, filtered, and evaporated under reduced pressure. Subsequently, the crude residue was purified by column chromatography (silica gel, petroleum ether/ethyl acetate, 1:1, v/v) to yield compound 8 as a yellow solid (106 mg, 77%). 1H NMR (400 MHz, CDCl3) δ 7.73-7.69 (m, 4H, 4×Ar-H), 7.51-7.46 (m, 4H, 4×ArH), 7.38-7.31 (m, 4H, 4×Ar-H), 7.25-7.20 (m, 4H, 4×Ar-H), 6.43-6.39 (m, 2H, 2×Ar-H), 6.336.29 (m, 2H, 2×Ar-H), 5.46-5.39 (m, 2H, 2×CH), 5.19 (br, 2H, 2×CH), 4.73-4.59 (m, 4H, 2×OCH2), 4.50 (d, J = 6.7 Hz, 4H, 2×OCH2), 4.47-4.40 (m, 2H, 2×CH), 4.25 (d, J = 6.9 Hz, 4H, 2×OCH2), 3.68 (s, 6H, 2×OCH3), 3.15-3.04 (m, 4H, 2×CH2), 1.99-1.88 (m, 4H, 2×CH2), 1.80 (s,


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6H, 2×CH3), 1.74 (s, 6H, 2×CH3), 1.55-1.35 (m, 8H, 4×CH2).

Page 24 of 62

13

C NMR (101 MHz, CDCl3) δ

173.74, 168.58 (2×C), 168.51 (2×C), 167.86 (2×C), 163.08 (2×C), 161.65 (4×C), 160.10 (2×C), 159.90 (2×C), 158.38 (2×C), 158.27 (2×C), 139.36 (2×C), 118.54 (8×CH), 109.60 (2×CH), 108.77 (2×CH), 98.90 (2×C), 98.85 (4×CH), 94.74 (4×CH), 94.67 (2×CH), 67.19 (2×CH2), 67.05 (2×CH2), 65.37 (2×CH2), 65.34 (2×CH), 61.66 (2×CH3), 61.29 (2×CH), 61.25 (2×CH2), 25.77 (4×CH2), 18.22 (2×CH2), 14.12 (4×CH3). HRMS (APCI+): calcd for C71H77N4O16 [M + H]+ 1241.5329, found 1241.5326 (error 0.2 ppm). Dimethyl


diyl)bis(oxy))bis(acetyl))bis(azanediyl))(2S,2'S)-bis(6-aminohexanoate)

(compound

9).

Compound 8 (64.8 mg, 0.052 mmol) 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 ethyl acetate and washed with brine for 3 times. The organic phase was dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure to generate a crude residue, which was purified by HPLC to give compound 9 as a yellow gel (28.4 mg, 69 %). 1H NMR (400 MHz, MeOD) δ 6.69-6.65 (m, 2H, 2×Ar-H), 6.52 (d, J = 1.5 Hz, 2H, 2×Ar-H), 5.52-5.45 (m, 2H, 2×CH), 4.80-4.71 (m, 4H, 2×OCH2), 4.68 (d, J = 6.7 Hz, 4H, 2×OCH2), 4.42-4.36 (m, 2H, 2×CH), 3.73 (s, 6H, 2×OCH3), 2.93-2.77 (m, 4H, 2×CH2), 2.05-1.92 (m, 4H, 2×CH2), 1.85-1.76 (m, 12H, 4×CH3), 1.73-1.60 (m, 4H, 2×CH2), 1.56-1.43 (m, 4H, 2×CH2). 13C NMR (101 MHz, MeOD) δ 177.38, 173.56 (2×C), 171.17 (2×C), 165.86 (2×C), 160.85 (2×C), 159.99 (2×C), 140.24 (2×C), 119.92 (2×CH), 108.67 (2×CH), 99.89 (2×C), 96.22 (2×CH), 70.03 (2×CH2), 66.89 (2×CH2), 53.81 (2×CH), 52.86 (2×CH3), 40.39 (2×CH2), 31.59 (2×CH2), 28.16 (2×CH2), 25.85 (2×CH3), 23.99 (2×CH2), 18.32 (2×CH3). HRMS (ESI+): calcd for C41H56N4NaO12 [M + Na]+ 819.3787, found 819.3785 (error 0.2 ppm).


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Dimethyl


diyl)bis(oxy))bis(acetyl))bis(azanediyl))(2S,2'S)-bis(3-(1H-imidazol-5-yl)propanoate) (compound 10). Compound 10 was prepared from compound 4 (71.5 mg, 0.126 mmol), HOBt (51.1 mg, 0.378 mmol), DIC (58.5 µL, 0.378 mmol) and H-His-OMe•2HCl (91.5 mg, 0.378 mmol) using the same method as described for compound 6. The product was obtained as a light yellow solid (64.6 mg, 63%). 1H NMR (400 MHz, MeOD) δ 7.87 (s, 2H, 2×Ar-H), 7.02 (s, 2H, 2×Ar-H), 6.55-6.49 (m, 2H, 2×Ar-H), 6.39-6.33 (m, 2H, 2×Ar-H), 5.45 (t, J = 6.0 Hz, 2H, 2×CH), 4.73-4.62 (m, 6H, 2×OCH2, 2×CH), 4.59 (d, J = 6.3 Hz, 4H, 2×OCH2), 3.72 (s, 6H, 2×OCH3), 3.26 (d, J = 7.0 Hz, 4H, 2×CH2), 1.80 (s, 6H, 2×CH3), 1.78 (s, 6H, 2×CH3). 13C NMR (101 MHz, MeOD) δ 176.89, 172.76 (2×C), 170.97 (2×C), 165.65 (2×C), 160.52 (2×C), 159.76 (2×C), 140.04 (2×C), 135.99 (2×C), 132.95 (2×CH), 120.00 (2×CH), 118.83 (2×CH), 108.39 (2×CH), 99.35 (2×C), 96.11 (2×CH), 69.53 (2×CH2), 66.83 (2×CH2), 53.87 (2×CH), 53.01 (2×CH3), 42.71, 28.94, 25.87, 23.53, 18.36 (2×CH3). HRMS (ESI+): calcd for C41H46N6NaO12 [M + Na]+ 837.3066, found 837.3068 (error -0.3 ppm). Dimethyl

2,2'-((2,2'-((3,6-Bis(((E)-3,7-dimethylocta-2,6-dien-1-yl)oxy)-9-oxo-9H-

xanthene-1,8-diyl)bis(oxy))bis(acetyl))bis(azanediyl))(2S,2'S)-bis(5-guanidinopentanoate) (compound 11). Compound 11 was prepared from compound 5 (294 mg, 0.417 mmol), HOBt (169 mg, 1.251 mmol), DIC (193.7 µL, 1.251 mmol) and H-Arg-OMe•2HCl (326.7 mg, 1.251 mmol) using the same method as described for compound 6. The product was obtained as a light yellow solid (283.2 mg, 69%). 1H NMR (400 MHz, MeOD) δ 6.68-6.60 (m, 2H, 2×Ar-H), 6.556.47 (m, 2H, 2×Ar-H), 5.47 (t, J = 5.9 Hz, 2H, 2×CH), 5.14-5.06 (m, 2H, 2×CH), 4.80-4.74 (m, 4H, 2×OCH2), 4.69 (d, J = 6.3 Hz, 4H, 2×OCH2), 4.44-4.35 (m, 2H, 2×CH), 3.74 (s, 6H, 2×OCH3), 3.22-3.10 (m, 4H, 2×CH2), 2.19-1.95 (m, 12H, 6×CH2), 1.80 (s, 6H, 2×CH3), 1.72-


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1.58 (m, 16H, 2×CH2, 4×CH3). 13C NMR (101 MHz, MeOD) δ = 177.37, 173.56 (2×C), 171.22 (2×C), 165.79 (2×C), 160.89 (2×C), 159.91 (2×C), 158.63 (2×C), 143.37 (2×C), 132.74 (2×C), 124.83 (2×CH), 119.95 (2×CH), 108.62 (2×CH), 100.02 (2×C), 96.30 (2×CH), 70.17 (2×CH2), 66.88 (2×CH2), 53.68 (2×CH), 52.96 (2×CH3), 41.94 (2×CH2), 40.54 (2×CH2), 29.29 (2×CH2), 27.31 (2×CH2), 26.46 (2×CH2), 25.86 (2×CH3), 17.79 (2×CH3), 16.81 (2×CH3). HRMS (ESI+): calcd for C51H73N8O12 [M +H]+ 989.5342, found 989.5345 (error -0.3 ppm). 1-Hydroxy-3,6,8-tris((3-methylbut-2-en-1-yl)oxy)-9H-xanthen-9-one

(compound

12).

Compound 12 was prepared from compound 1 (100 mg, 0.384 mmol), potassium carbonate (238.8 mg, 1.728 mmol) and 3,3-dimethylallyl bromide (133.1 µL, 1.152 mmol) using the same method as described for compound 2. The product was obtained as a light yellow solid (133.8 mg, 75 %). 1H NMR (400 MHz, CDCl3) δ 13.48 (s, 1H, O-H), 6.41 (d, J = 1.5 Hz, 1H, Ar-H), 6.32 (d, J = 2.1 Hz, 1H, Ar-H), 6.29 (d, J = 2.1 Hz, 1H, Ar-H), 6.27 (d, J = 2.2 Hz, 1H, Ar-H), 5.61- 5.55 (m, 1H, CH), 5.52-5.45 (m, 2H, CH), 4.67 (d, J = 6.3 Hz, 2H, CH2), 4.60-4.53 (m, 4H, 2×CH2), 1.84-1.73 (m, 18H, 6×CH3).

13

C NMR (101 MHz, CDCl3) δ 180.34, 164.92, 164.42,

163.68, 161.04, 159.63, 156.57, 139.45, 139.03, 137.86, 119.17, 118.79, 118.50, 105.70, 103.85, 97.40, 96.87, 93.44, 92.63, 66.54, 65.39, 65.29, 25.83 (2×CH3), 25.80, 18.41, 18.26, 18.23. HRMS (ESI+): calcd for C28H33O6 [M +H]+ 465.2272, found 465.2276 (error -1.0 ppm). 1,3,8-Trihydroxy-6-((3-methylbut-2-en-1-yl)oxy)-9H-xanthen-9-one

(compound

13).

Compound 13 was prepared from compound 1 (467.3 mg, 1.796 mmol), potassium carbonate (372.3 mg, 2.694 mmol) and 3,3-dimethylallyl bromide (207.5 µL, 1.796 mmol) using the same method as described for compound 2. The product was obtained as a yellow solid (222.4 mg, 38%).1H NMR (400 MHz, MeOD) δ 6.28 (d, J = 2.2 Hz, 1H, Ar-H), 6.19 (d, J = 2.1 Hz, 1H, ArH), 6.17 (d, J = 2.2 Hz, 1H, Ar-H), 6.10 (d, J = 2.1 Hz, 1H, Ar-H), 5.49-5.40 (m, 1H, CH), 4.56


26

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(d, J = 6.6 Hz, 2H, CH2), 1.80 (s, 3H, CH3), 1.77 (s, 3H, CH3). 13C NMR (101 MHz, MeOD) δ 184.23, 167.49, 167.28, 164.03, 163.67, 159.09, 158.78, 139.70, 120.18, 102.80, 102.22, 99.42, 98.74, 95.28, 94.38, 66.62, 25.84, 18.27. HRMS (ESI+): calcd for C18H16NaO6 [M + Na]+ 351.0839, found 351.0843 (error -1.0 ppm). Ethyl

2-((3,6,8-Tris((3-methylbut-2-en-1-yl)oxy)-9-oxo-9H-xanthen-1-yl)oxy)acetate

(compound 14). Compound 14 was prepared from compound 12 (103.7 mg, 0.223 mmol), ethyl iodoacetate (52.8 µL, 0.446 mmol) and potassium carbonate (61.6 mg, 0.446 mmol) using the same method as described for compound 4. The product was obtained as a yellow solid (88.2 mg, 72%). 1H NMR (400 MHz, CDCl3) δ 6.44 (d, J = 2.3 Hz, 1H, Ar-H), 6.36 (d, J = 2.3 Hz, 1H, Ar-H), 6.30 (d, J = 2.2 Hz, 1H, Ar-H), 6.24 (d, J = 2.3 Hz, 1H, Ar-H), 5.62-5.56 (m, 1H, CH), 5.52 -5.43 (m, 2H, 2×CH), 4.77 (s, 2H, OCH2), 4.63 (d, J = 6.3 Hz, 2H, OCH2), 4.58-4.51 (m, 4H, 2×OCH2), 4.27-4.19 (m, 2H, OCH2), 1.84-1.70 (m, 18H, 6×CH3), 1.29-1.23 (m, 3H, CH3). 13

C NMR (101 MHz, CDCl3) δ 174.06, 168.74, 163.08, 162.72, 161.07, 159.93, 158.52, 158.40,

139.28, 139.08, 136.77, 119.95, 118.79, 118.64, 108.92, 108.47, 98.59, 96.99, 94.58, 92.89, 67.07, 66.47, 65.25, 65.17, 61.20, 25.79 (2×CH3), 25.71, 18.32, 18.23, 18.21, 14.13. HRMS (APCI+): calcd for C32H39O8 [M +H]+ 551.2639, found 551.2645 (error -1.0 ppm). Triethyl

2,2',2''-((6-((3-Methylbut-2-en-1-yl)oxy)-9-oxo-9H-xanthene-1,3,8-

triyl)tris(oxy))triacetate (compound 15). Compound 15 was prepared from compound 13 (133.1 mg, 0.405 mmol), ethyl iodoacetate (215.8 µL, 1.823 mmol) and potassium carbonate (251.9 mg, 1.823 mmol) using the same method as described for compound 4. The product was obtained as a yellow solid (186.2 mg, 78%). 1H NMR (400 MHz, CDCl3) δ 6.43 (d, J = 2.2 Hz, 1H, Ar-H), 6.38 (d, J = 2.4 Hz, 1H, Ar-H), 6.33 (d, J = 2.3 Hz, 1H, Ar-H), 6.26 (d, J = 2.2 Hz, 1H, Ar-H), 5.49-5.41 (m, 1H, CH), 4.78-4.73 (m, 4H, 2×OCH2), 4.64 (s, 2H, OCH2), 4.53 (d, J =


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6.8 Hz, 2H, OCH2), 4.31-4.19 (m, 6H, 3×OCH2), 1.80 (s, 3H, CH3), 1.75 (s, 3H, CH3), 1.32-1.22 (m, 9H, 3×CH3). 13C NMR (101 MHz, CDCl3) δ 173.74, 168.58, 168.51, 167.86, 163.08, 161.65, 160.10, 159.90, 158.38, 158.27, 139.36, 118.54, 109.60, 108.77, 98.90, 98.85, 94.74, 94.67, 67.19, 67.05, 65.37, 65.34, 61.66, 61.29, 61.25, 25.77, 18.22, 14.12 (3×CH3). HRMS (APCI+): calcd for C30H35O12 [M +H]+ 587.2123, found 587.2130 (error -1.3 ppm). Methyl (2-((3,6,8-Tris((3-methylbut-2-en-1-yl)oxy)-9-oxo-9H-xanthen-1-yl)oxy)acetyl)-Largininate (compound 16). Compound 16 was prepared from compound 14 (99.2 mg, 0.18 mmol), HOBt (36.5 mg, 0.27 mmol), DIC (41.8 µL, 0.27 mmol) and H-Arg-OMe•2HCl (70.5 mg, 0.27 mmol) using the same method as described for compound 6. The product was obtained as a light yellow solid (85.2 mg, 68 %). 1H NMR (400 MHz, MeOD) δ 6.45 (d, J = 7.0 Hz, 2H, 2×Ar-H), 6.40-6.30 (m, 2H, 2×Ar-H), 5.57-5.38 (m, 3H, 3×CH), 4.70-4.50 (m, 9H, 4×OCH2, CH), 3.75 (s, 3H, OCH3), 3.29-3.23 (m, 2H, CH2), 2.15-2.00 (m, 2H, CH2), 1.86-1.71 (m, 20H, CH2, 6×CH3).

13

C NMR (101 MHz, MeOD) δ 176.74, 173.41, 171.11, 165.40, 165.33, 165.30,

162.05, 160.07, 159.58, 158.66, 139.74, 139.69, 138.62, 121.00, 120.26, 120.16, 108.74, 108.43, 98.53, 98.40, 95.67, 94.72, 69.25, 67.66, 66.75, 66.60, 53.17, 52.94, 41.88, 29.33, 26.43, 26.03, 25.87 (2×CH3), 18.46, 18.35 (2×CH3). HRMS (ESI+): calcd for C37H49N4O9 [M + H]+ 693.3494, found 693.3496 (error -0.3 ppm). Trimethyl

2,2',2''-((2,2',2''-((6-((3-Methylbut-2-en-1-yl)oxy)-9-oxo-9H-xanthene-1,3,8-

triyl)tris(oxy))tris(acetyl))tris(azanediyl))(2S,2'S,2''S)-tris(5-guanidinopentanoate) (compound 17). To a solution of compound 15 (144.9 mg, 0.247 mmol) in THF (4 mL) was added a 5% LiOH solution (2 mL). After stirring at room temperature for 1.5 h, the reaction mixture was acidified with acetic acid, diluted with butanol, and extracted with brine 3 times. The organic phase was dried over anhydrous sodium sulfate and evaporated under reduced


28

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pressure. The crude residue was dissolved in anhydrous DMF (5 mL). HATU (422.8 mg, 1.112 mmol), DIPEA (193.7 µL, 1.112 mmol) and H-Arg-OMe•2HCl (290.4 mg, 1.112 mmol) were added, and the reaction was stirred at 0 °C for 1 h. After stirring at room temperature overnight, the mixture was diluted with butanol and washed with brine 3 times. The organic phase was dried over anhydrous sodium sulfate, filtered, and evaporated under reduced pressure. Subsequently, the crude residue was purified by HPLC to afford compound 17 as a yellow gel (96.8 mg, 39%). 1H NMR (500 MHz, MeOD) δ 6.67 (d, J = 1.9 Hz, 1H, Ar-H), 6.63 (d, J = 2.0 Hz, 1H, Ar-H), 6.61 (d, J = 2.0 Hz, 1H, Ar-H), 6.53 (d, J = 2.0 Hz, 1H, Ar-H), 5.53-5.47 (m, 1H, CH), 4.86-4.76 (m, 6H, 3×OCH2), 4.67 (d, J = 6.6 Hz, 2H, OCH2), 4.59- 4.55 (m, 1H, CH), 4.454.39 (m, 2H, 2×CH), 3.79-3.74 (m, 9H, 3×OCH3), 3.27-3.15 (m, 6H, 3×CH2), 2.11-1.94 (m, 5H, CH2, CH3), 1.90-1.79 (m, 7H, 2×CH2, CH3), 1.75- 1.65 (m, 6H, 3×CH2).

13

C NMR (126 MHz,

MeOD) δ 175.79, 172.19, 172.07, 169.79, 169.67, 168.86, 168.39, 164.50, 162.97, 159.55, 159.51, 158.48, 158.31, 157.39, 157.27, 157.23, 138.75, 118.57, 107.90, 107.26, 98.59, 98.20, 95.33, 94.90, 68.74, 68.67, 66.76, 65.52, 52.30, 52.28, 51.88, 51.63, 51.60, 51.58, 40.60, 40.55, 40.48, 37.51, 28.08, 27.94, 25.07 (2×CH2), 24.98, 24.49, 16.98. HRMS (ESI+): calcd for C45H65N12O15 [M + H]+ 1013.4687, found 1013.4686 (error 0.1 ppm). Dimethyl

2,2'-(((2S,2'S)-2,2'-((2,2'-((3,6-Bis((3-methylbut-2-en-1-yl)oxy)-9-oxo-9H-

xanthene-1,8-diyl)bis(oxy))bis(acetyl))bis(azanediyl))bis(5guanidinopentanoyl))bis(azanediyl))(2S,2'S)-bis(5-guanidino-pentanoate) (compound 18). Compound 18 was prepared from compound 6 (23.9 mg, 0.028 mmol), HATU (31.9 mg, 0.084 mmol), DIPEA (14.6 µL, 0.084 mmol) and H-Arg-OMe•2HCl (21.9 mg, 0.084 mmol) using the same method as described for compound 17. The product was obtained as a light yellow gel (12.0 mg, 37%). 1H NMR (400 MHz, MeOD) δ 6.70- 6.63 (m, 2H, 2×Ar-H), 6.55-6.49 (m, 2H,


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2×Ar-H), 5.50 (t, J = 6.2 Hz, 2H, 2×CH), 4.77 (s, 4H, 2×OCH2), 4.69 (d, J = 6.6 Hz, 4H, 2×OCH2), 4.53-4.44 (m, 2H, 2×CH), 4.39- 4.30 (m, 2H, 2×CH), 3.74 (s, 6H, 2×OCH3), 3.283.13 (m, 8H, 4×CH2), 2.08-1.90 (m, 6H, 2×CH3), 1.87-1.65 (m, 22H, 8×CH2, 2×CH3). 13C NMR (126 MHz, MeOD) δ 175.86, 172.61 (2×C), 172.15 (2×C), 169.69 (2×C), 168.95 (2×C), 164.35 (2×C), 159.45 (2×C), 158.58 (2×C), 157.33 (2×C), 138.74 (2×C), 118.58 (2×CH), 107.34 (2×CH), 98.39 (2×C), 94.75 (2×CH), 68.57 (2×CH2), 65.45 (2×CH2), 53.50 (2×CH), 51.97 (2×CH), 51.52 (2×CH3), 40.73 (2×CH2), 40.47 (2×CH2), 28.59 (2×CH2), 28.15 (2×CH2), 24.84 (2×CH2), 24.80 (2×CH3), 24.47 (2×CH2), 16.94 (2×CH3). HRMS (ESI+): calcd for C53H81N16O14 [M + H]+ 1165.6113, found 1165.6118 (error -0.5 ppm). Dimethyl

2,2'-(((2S,2'S)-2,2'-((2,2'-((3,6-Bis(((E)-3,7-dimethylocta-2,6-dien-1-yl)oxy)-9-

oxo-9H-xanthene-1,8-diyl)bis(oxy))bis(acetyl))bis(azanediyl))bis(5guanidinopentanoyl))bis(azanediyl))(2S,2'S)-bis(5-guanidinopentanoate) (compound 19). Compound 19 was prepared from compound 11 (97.9 mg, 0.099 mmol), HOBt (40.1 mg, 0.297 mmol), DIC (46.0 µL, 0.297 mmol) and H-Arg-OMe•2HCl (77.6 mg, 0.297 mmol) using the same method as described for compound 6. The product was obtained as a light yellow solid (92.8 mg, 72%). 1H NMR (400 MHz, MeOD) δ 6.62 (d, J = 2.1 Hz, 2H, 2×Ar-H), 6.50 (d, J = 2.0 Hz, 2H, 2×Ar-H), 5.47 (t, J = 6.1 Hz, 2H, 2×CH), 5.14-5.06 (m, 2H, 2×CH), 4.74 (s, 4H, 2×OCH2), 4.69 (d, J = 6.4 Hz, 4H, 2×OCH2), 4.50-4.43 (m, 2H, 2×CH), 4.36-4.29 (m, 2H, 2×CH), 3.72 (s, 6H, 2×OCH3), 3.27-3.14 (m, 8H, 4×CH2), 2.19-2.08 (m, 8H, 4×CH2), 2.02-1.89 (m, 6H, 3×CH2), 1.84-1.67 (m, 16H, 5×CH2, 2×CH3), 1.64 (s, 6H, 2×CH3), 1.61 (s, 6H, 2×CH3). 13

C NMR (101 MHz, MeOD) δ 177.21, 174.01 (2×C), 173.54 (2×C), 171.07 (2×C), 169.83

(2×C), 165.68 (2×C), 160.81 (2×C), 159.92 (2×C), 158.75 (2×C), 143.33 (2×C), 132.73 (2×C), 124.84 (2×CH), 119.99 (2×CH), 108.71 (2×CH), 99.83 (2×C), 96.17 (2×CH), 69.95 (2×CH2),


30

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66.85 (2×CH2), 54.91 (2×CH), 53.37 (2×CH), 52.92 (2×CH3), 42.11 (2×CH2), 41.85 (2×CH2), 40.55 (2×CH2), 29.98 (2×CH2), 29.54 (2×CH2), 27.32 (2×CH2), 26.23 (2×CH2), 26.19 (2×CH3), 25.86 (2×CH2), 17.79 (2×CH3), 16.81 (2×CH3). HRMS (ESI+): calcd for C63H97N16O14 [M + H]+ 1301.7365, found 1301.7363 (error 0.1 ppm). 2,2'-((3,6-Bis((3-methylbut-2-en-1-yl)oxy)-9-oxo-9H-xanthene-1,8-diyl)bis(oxy))bis(N-(3(dimethylamino)propyl)acetamide) (compound 20). To a solution of compound 4 (92.0 mg, 0.162 mmol) in THF (4 mL) was added a 5% LiOH solution (2 mL). After stirring at room temperature for 1.5 h, the reaction mixture was acidified with acetic acid, diluted with butanol, and extracted with brine 3 times. The organic phase was dried over anhydrous sodium sulfate and evaporated under reduced pressure. The crude residue was dissolved in anhydrous DMF (5 mL). HATU (184.8 mg, 0.486 mmol), DIPEA (141.1 µL, 0.810 mmol) and N,N-dimethyl-1,3propanediamine (61.1 µL, 0.486 mmol) were added. After stirring at room temperature overnight, the mixture was diluted with ethyl acetate and washed with brine 3 times. The organic phase was dried over anhydrous sodium sulfate, filtered, and evaporated under reduced pressure. Subsequently, the crude residue was purified by HPLC to afford compound 20 as a light yellow solid (85.6 mg, 78%). 1H NMR (400 MHz, MeOD) δ 6.54 (s, 2H, 2×Ar-H), 6.40 (s, 2H, 2×ArH), 5.46 (t, J = 6.3 Hz, 2H, 2×CH), 4.67 (s, 4H, 2×OCH2), 4.61 (d, J = 6.5 Hz, 4H, 2×OCH2), 3.46 (t, J = 6.5 Hz, 4H, 2×CH2), 3.14-3.03 (m, 4H, 2×CH2), 2.78 (s, 12H, 4×CH3), 2.09- 1.95 (m, 4H, 2×CH2), 1.82 (s, 6H, 2×CH3), 1.79 (s, 6H, 2×CH3). 13C NMR (101 MHz, MeOD) δ 176.86, 171.19 (2×C), 165.56 (2×C), 160.52 (2×C), 159.86 (2×C), 140.02 (2×C), 119.99 (2×CH), 108.52 (2×CH), 99.33 (2×C), 95.87 (2×CH), 69.70 (2×CH2), 66.82 (2×CH2), 56.71 (2×CH2), 43.65 (4×CH3), 37.31 (2×CH2), 26.23 (2×CH2), 25.88 (2×CH3), 18.35 (2×CH3). HRMS (ESI+): C37H52N4 NaO8 [M + Na]+ 703.3677, found 703.3676 (error 0.2 ppm).


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2,2'-((3,6-Bis((3-methylbut-2-en-1-yl)oxy)-9-oxo-9H-xanthene-1,8-diyl)bis(oxy))bis(N-(3(diethylamino)propyl)acetamide) (compound 21). Compound 21 was prepared from compound 4 (70.0 mg, 0.123 mmol), HATU (140.3 mg, 0.369 mmol), DIPEA (107.1 µL, 0.615 mmol) and N,N-diethyl-1,3-propanediamine (58.2 µL, 0.369 mmol) using the same method as described for compound 21. The product was obtained as a light yellow solid (61.4 mg, 68%). 1H NMR (400 MHz, MeOD) δ 6.61 (s, 2H, 2×Ar-H), 6.46 (s, 2H, 2×Ar-H), 5.47 (t, J = 6.1 Hz, 2H, 2×CH), 4.70 (s, 4H, 2×OCH2), 4.65 (d, J = 6.6 Hz, 4H, 2×OCH2), 3.47 (t, J = 6.5 Hz, 4H, 2×CH2), 3.11-3.00 (m, 12H, 6×CH2), 2.04-1.93 (m, 4H, 2×CH2), 1.82 (s, 6H, 2×CH3), 1.80 (s, 6H, 2×CH3), 1.20 (t, J = 7.3 Hz, 12H, 4×CH3). 13C NMR (101 MHz, MeOD) δ 177.08, 171.16 (2×C), 165.75 (2×C), 160.63 (2×C), 159.95 (2×C), 140.11 (2×C), 119.96 (2×CH), 108.51 (2×CH), 99.47 (2×C), 95.99 (2×CH), 69.84 (2×CH2), 66.85 (2×CH2), 50.82 (2×CH2), 48.18 (4×CH2), 37.60 (2×CH2), 25.87 (2×CH2), 25.68 (2×CH3), 18.33 (2×CH3), 9.52 (4×CH3). HRMS (ESI+): calcd for C41H61N4O8 [M + H]+ 737.4484, found 737.4487 (error -0.4 ppm). 2,2'-((3,6-Bis((3-methylbut-2-en-1-yl)oxy)-9-oxo-9H-xanthene-1,8-diyl)bis(oxy))bis(N-(3(dibutylamino)propyl)acetamide) (compound 22). Compound 22 was prepared from compound 4 (70.0 mg, 0.123 mmol), HATU (140.3 mg, 0.369 mmol), DIPEA (107.1 µL, 0.615 mmol) and N,N-dibutyl-1,3-propanediamine (83.2 µL, 0.369 mmol) using the same method as described for compound 22. The product was obtained as a light yellow gel (83.6 mg, 80%). 1H NMR (400 MHz, MeOD) δ 6.65 (s, 2H, 2×Ar-H), 6.49 (s, 2H, 2×Ar-H), 5.48 (t, J = 6.5 Hz, 2H, 2×CH), 4.72 (s, 4H, 2×OCH2), 4.67 (d, J = 6.6 Hz, 4H, 2×OCH2), 3.48 (t, J = 6.2 Hz, 4H, 2×CH2), 3.20 – 3.11 (m, 4H, 2×CH2), 3.09-2.99 (m, 8H, 4×CH2), 2.08-1.95 (m, 4H, 2×CH2), 1.82 (s, 6H, 2×CH3), 1.80 (s, 6H, 2×CH3), 1.66-1.56 (m, 8H, 4×CH2), 1.38-1.27 (m, 8H, 4×CH2), 0.90 (t, J = 7.3 Hz, 12H, 4×CH3). 13C NMR (101 MHz, MeOD) δ 177.15, 171.18 (2×C), 165.87


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(2×C), 160.72 (2×C), 160.00 (2×C), 140.17 (2×C), 119.92 (2×CH), 108.52 (2×CH), 99.63 (2×C), 96.07 (2×CH), 69.97 (2×CH2), 66.89 (2×CH2), 53.85 (4×CH2), 51.65 (2×CH2), 37.33 (2×CH2), 26.80 (4×CH2), 25.87 (2×CH2), 25.24 (2×CH3), 20.95 (4×CH2), 18.33 (2×CH3), 13.90 (4×CH3). HRMS (ESI+): calcd for C49H77N4O8 [M + H]+ 849.5736, found 849.5737 (error -0.1 ppm). Dimethyl 2,2'-(((2S,2'S)-2,2'-(((2S,2'S)-2,2'-((2,2'-((3,6-bis((3-methylbut-2-en-1-yl)oxy)-9oxo-9H-xanthene-1,8-diyl)bis(oxy))bis(acetyl))bis(azanediyl))bis(5guanidinopentanoyl))bis(azanediyl))bis(5-guanidinopentanoyl))bis(azanediyl))(2S,2'S)bis(5-guanidinopentanoate) (compound 23). Compound 23 was prepared from compound 18 (37.5 mg, 0.032 mmol), HATU (36.5 mg, 0.096 mmol), DIPEA (27.9 µL, 0.160 mmol) and HArg-OMe•2HCl (25.1 mg, 0.096 mmol) using the same method as described for compound 17. The product was obtained as a light yellow gel (28.8 mg, 61%). 1H NMR (400 MHz, MeOD) δ 6.67 (s, 2H, 2×Ar-H), 6.49 (s, 2H, 2×Ar-H), 5.49 (t, J = 6.0 Hz, 2H, 2×CH), 4.77 (s, 4H, 2×OCH2), 4.71-4.63 (m, 4H, 2×OCH2), 4.47-4.30 (m, 6H, 3×CH), 3.72 (s, 6H, 2×OCH3), 3.273.07 (m, 12H, 6×CH2), 2.04-1.85 (m, 8H, 4×CH2), 1.85-1.56 (m, 28H, 8×CH2, 4×CH3).

13

C

NMR (101 MHz, MeOD) δ 177.21, 174.14 (2×C), 173.99 (2×C), 173.62 (2×C), 171.24 (2×C), 165.74 (2×C), 160.83 (2×C), 160.04 (2×C), 158.74 (4×C), 140.16 (2×C), 119.97 (2×CH), 108.77 (2×CH), 99.67 (2×C), 96.06 (2×CH), 69.77 (2×CH2), 66.85 (2×CH2), 54.83 (2×CH), 54.40 (2×CH), 53.44 (2×CH), 52.92 (2×CH3), 42.04 (2×CH2), 41.95 (2×CH2), 41.88 (2×CH2), 30.12 (2×CH2), 29.96 (2×CH2), 29.45 (2×CH2), 26.24 (2×CH2), 26.17 (2×CH3), 25.88 (4×CH2), 18.33 (2×CH3). HRMS (ESI+): calcd for C65H107N24O16 [M + 3H]3+ 493.2760, found 493.2750 (error 2.1 ppm). 1,8-Dihydroxy-3,6-dipropoxy-9H-xanthen-9-one (compound 24). Compound 24 was prepared from compound 1 (89 mg, 0.342 mmol) and 1-bromopropane (61.6 µL, 0.718 mmol)


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using the same method as described for compound 2. The product was obtained as a light yellow solid (59.0 mg, 53%). 1H NMR (400 MHz, CDCl3) δ 12.01 (s, 2H, 2×Ar-OH), 6.35-6.32 (m, 2H, 2×Ar-H), 6.30-6.27 (m, 2H, 2×Ar-H), 3.98 (t, J = 6.6 Hz, 4H, 2×OCH2), 1.91-1.75 (m, 4H, 2×CH2), 1.05 (t, J = 7.4 Hz, 6H, 2×CH3). 13C NMR (101 MHz, CDCl3) δ 182.16, 165.33 (2×C), 161.74 (2×C), 156.53 (2×C), 101.07 (2×CH), 96.64 (2×CH), 92.42 (2×C), 69.19 (2×CH2), 21.31 (2×CH2), 9.37 (2×CH3). HRMS (ESI+): calcd for C19H20NaO6 [M + Na]+ 367.1152, found 367.1160 (error -2.3 ppm). 2,2'-((9-Oxo-3,6-dipropoxy-9H-xanthene-1,8-diyl)bis(oxy))diacetic acid (compound 25). To a solution of compound compound 24 (50 mg, 0.145 mmol) in acetone (5 mL), ethyl iodoacetate (51.6 µL, 0.435 mmol) and CH3ONa (39.2 mg, 0.725 mmol) were added. The mixture was refluxed for 24 hours. After cooling, the mixture was diluted with ethyl acetate and washed with brine three times. The organic layer was dried over anhydrous sodium sulfate, filtered, and evaporated under reduced pressure. The crude residue was dissolved in anhydrous THF (4 mL). 5% LiOH solution (2 mL) was added. After stirring at room temperature for 1.5 h, the reaction mixture was acidified with acetic acid, diluted with butanol, and extracted with brine 3 times. The organic phase was dried over anhydrous sodium sulfate and evaporated under reduced pressure. Subsequently, the crude residue was purified by HPLC to afford compound 25 as a yellow solid (47 mg, 70%). 1H NMR (400 MHz, DMSO-d6) δ 6.56 (s, 2H, 2×Ar-H), 6.36 (s, 2H, 2×Ar-H), 5.02-4.64 (m, 4H, 2×OCH2), 4.16-3.94 (m, 4H, 2×OCH2), 1.83-1.67 (m, 4H, 2×CH2), 0.98 (t, J = 7.4 Hz, 6H, 2×CH3).

13

C NMR (101 MHz, DMSO-d6) δ 169.71, 163.07

(2×C), 159.36 (2×C), 157.74 (4×C), 107.30 (2×CH), 97.46 (2×C), 93.73 (2×CH), 69.77 (2×CH2), 66.05 (2×CH2), 21.76 (2×CH2), 10.27 (2×CH3). HRMS (ESI+): calcd for C23H24NaO10 [M + Na]+ 483.1262, found 483.1266 (error -0.9 ppm).


34

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Dimethyl

2,2'-((2,2'-((9-oxo-3,6-dipropoxy-9H-xanthene-1,8-

diyl)bis(oxy))bis(acetyl))bis(azanediyl))(2S,2'S)-bis(5-guanidinopentanoate) (compound 26). Compound 25 (20 mg, 0.043 mmol) was dissolved in anhydrous DMF (5 mL). HATU (49.0 mg, 0.129 mmol), DIPEA (37.5 µL, 0.215 mmol) and H-Arg-OMe•2HCl (33.7 mg, 0.129 mmol) were added, and the reaction was stirred at 0 °C for 1 h. After stirring at room temperature overnight, the mixture was diluted with butanol and washed with brine 3 times. The organic phase was dried over anhydrous sodium sulfate, filtered, and evaporated under reduced pressure. Subsequently, the crude residue was purified by HPLC to afford compound 26 as a yellow gel (16.8 mg, 48%). 1H NMR (400 MHz, MeOD) δ 6.69 (s, 2H, 2×Ar-H), 6.56 (s, 2H, 2×Ar-H), 4.81-4.72 (m, 4H, 2×OCH2), 4.39 (dd, J = 9.3, 5.2 Hz, 2H, 2×OCH), 4.09 (t, J = 6.4 Hz, 4H, 2×OCH2), 3.74 (s, 6H, 2×OCH3), 3.21-3.12 (m, 4H, 2×CH2), 2.13 -1.92 (m, 4H, 2×CH2), 1.901.79 (m, 4H, 2×CH2), 1.73-1.61 (m, 4H, 2×CH2), 1.08 (t, J = 7.4 Hz, 6H, 2×CH3).

13

C NMR

(101 MHz, MeOD) δ 177.57, 173.56 (2×C), 171.29 (2×C), 166.23 (2×C), 161.04 (2×C), 160.09 (2×C), 158.60 (2×C), 108.65 (2×CH), 99.94 (2×C), 96.04 (2×CH), 71.67 (2×CH2), 70.30 (2×CH2), 53.68 (2×CH), 52.95 (2×CH3), 41.97 (2×CH2), 29.26 (2×CH2), 26.49 (2×CH2), 23.40 (2×CH2), 10.74 (2×CH3). HRMS (ESI+): calcd for C37H53N8O12 [M + H]+ 801.3777, found 801.3776 (error 0.2 ppm). Bacterial strains and growth media. Inoculum suspensions were prepared from isolated colonies by applying the direct colony suspension method of the Clinical and Laboratory Standards Institute (CLSI). Isolated colonies from 18- to 20-h tryptic soy agar (TSA) plates were selected to make inoculum suspensions. In vitro antimicrobial activity. All susceptibility tests were performed in cation-adjusted Mueller-Hinton broth (CA-MHB) using the broth macro-dilution method according to CLSI


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guidelines as previously described.54 Compounds used in this assay were first dissolved in DMF and then diluted in phosphate buffer system (PBS) to prepare a 1000 µg/mL stock solution. Serial 2-fold dilutions of the compounds were conducted using CA-MHB in test tubes. The concentration of the inoculum suspension was also adjusted to approximately 5× 105 CFU/mL using CA-MHB. These inoculum suspensions were added to each test tube containing the 2-fold serial dilutions of the compounds. After 24 hours of incubation at 35 °C, the MIC value was defined as the lowest concentration at which no visible growth occurred compared to the control. All measurements were performed with biological replicates. In vitro hemolytic activity. The hemolytic activity of antimicrobial compounds was measured by the amount of hemoglobin released from rabbit erythrocytes after incubating according to the compounds. The hemolysis assay was performed with a previously reported protocol.44, 54 Fresh RBCs isolated from the whole blood of New Zealand white rabbits were used in this assay. All procedures to isolate blood from New Zealand white rabbits were performed 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. RBCs were isolated via centrifugation at 3000 rpm for 10 min. Subsequently, the isolated RBCs were washed twice with sterile PBS and diluted with sterile PBS to the desired concentration. The synthesized compounds were dissolved in DMF or PBS, and mixed with RBCs (final v/v of RBCs = 4%). Triton X-100 (2%) was added to the suspension as a positive control, and the addition of PBS and DMF were used as a negative control. The RBC suspensions which were mixed with serially diluted compounds were incubated at 37 °C for 60 min. Then, the mixtures were centrifuged at 3000 rpm for 3 min. The supernatant (100 µL) was transferred into a 96-well plate, and measurement of the absorbance (abs) at 576 nm was carried out with a TECAN infinite 200


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microplate reader to determine the amount of hemoglobin released from lysed RBCs. The percentage of hemolysis was calculated according to the following equation: % Hemolysis = (Absmixture - Absnegative control) / (Abspositive control - Absnegative control) × 100. All measurements were carried out with biological replicates. Time-kill kinetics. The time-kill kinetics for MRSA DM21455 with various concentrations of compounds 6 and 18 were investigated. MRSA DM21455 was prepared from isolated colonies on an 18-22 h TSA plate and diluted to 105 ~ 106 CFU/mL in PBS. Compounds 5 and 6 with final concentrations of 0.5× MIC, 1× MIC, 2× MIC and 4× MIC were inoculated with bacterial suspension. The mixtures including the control (which contained bacterial suspension and PBS), were incubated at 35 °C. The aliquots were removed from the mixtures at 10 minutes, 30 minutes, 1 hour, 2 hours 4 hours and 8 hours and were serially diluted 10-fold in PBS. Then, 100 µL of each dilution was plated with Mueller-Hinton Agar (MHA) medium. The plates were incubated for 24-48 hours at 35 °C. The number of viable cells was determined by counting the colonies grown on the plates. All measurements were replicated. Multipassage resistance selection studies. The MIC values of 6, 18, gatifloxacin, and norfloxacin were measured against S. aureus ATCC29213 as described in the previous section. The bacteria grown in the medium with 0.5× MIC of each antimicrobial agent were removed and used to prepare bacterial dilutions for another MIC assay. After 24 h incubation at 35 °C, the changes in MICs were determined. This process was repeated for 17 passages except that the process of compound 18 was extended to 30 passages). All measurements were replicated. LDH assay. The cytotoxicity of the two optimized compounds, 6 and 18, toward human corneal fibroblasts was evaluated by determining the leakage of LDH, as previously reported.44 All measurements were replicated.


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Calcein leakage assay. The phospholipids used in this study were 1,2-dioleoyl-sn-glycero-3phospho-(1’-rac-glycerol) phosphoethanolamine

(sodium

(DOPE),

salt)

and

(DOPG),

1,2-di-(9Z-octadecenoyl)-sn-glycero-3-

1,2-di(9Z-octadecenoyl)-sn-glycero-3-phosphocholine

(DOPC). Briefly, lipids with composition of DOPC/cholesterol = 3:1 were used to mimic a mammalian membrane，and lipids with composition of DOPE/DOPG = 3:1 were used to mimic a bacterial membrane. First, the 100 nm homogeneous calcein-loaded LUVs were prepared using a film hydration method as previously described.38, 54 The concentration of the collected calceinloaded LUVs was determined using a total phosphorus determination assay. Aliquots of the calcein-loaded LUVs were transferred to a 96-well black immunological plate. The compounds dissolved in DMF were added to these aliquots to obtain liposomes to compound ratios of 16. The final concentration of DMF in the testing solution was limited to 0.5 / %, and the final concentration of liposomes was 50 µ . Calcein leakage from the LUVs was monitored by measuring the fluorescence emission intensity using a TECAN infinite M200Pro microplate reader at an excitation wavelength of 490 nm and an emission wavelength of 520 nm for 1 hour. The percentage of leakage was calculated using the following equation: %L = [(It – Io) / (I∞ - Io)] × 100, where It is the intensity upon the addition of compounds, Io is the intensity upon the addition of the negative control (DMF) without the compounds, and I∞ is the intensity after the addition of 0.1% triton X-100. All measurements were replicated. SYTOX Green uptake. To investigate the effect of the compounds on bacterial membranes, SYTOX Green was used. SYTOX Green uptake assay was performed as previously described.44 All measurements were replicated. Extracellular ATP bioluminescence assay. An ATP determination kit A22066 (Molecular Probes, Invitrogen) was used to measure the levels of extracellular ATP released from the


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bacteria after disrupting bacterial membranes with xanthone analogues. In this experiment, luciferase reacted with luciferin in the presence of ATP to produce light, which was detected by a luminometer. The determination was carried out as described in the manufacturer’s instructions, with certain modifications. Briefly, S. aureus DM4001 was grown and incubated to its exponential phase, and washed three times with 20 mM PBS (pH 7). The washed bacteria culture was re-suspended in the same buffer at a concentration of OD600 of 0.2. Upon addition of the xanthone compounds with a concentration of 4× MIC, the suspensions were incubated at 37 °C for 1 hour. After incubation, these suspensions were centrifuged at 4000 rpm for 5 min. Then, 180 of the enzyme mixture was prepared according to the determination kit description and added to a sterile white immune-96-well plate. Next, 20 of the supernatant from the centrifuged bacteria suspension was transferred to the well plate containing the enzyme mixture. The concentration of extracellular ATP was determined using a TECAN Infinite M200 Pro microplate luminometer. All measurements were replicated. Molecular dynamics simulations. Molecular dynamics simulations of compound 6 with model bacterial and mammalian membranes were carried out using GROMACS 4.5 package.62 Similar to our previous investigation of the membrane active antimicrobials, the bacterial membrane was modeled using 32 POPG and 96 POPE lipids, while the mammalian membrane was consisted of 32 cholesterol and 96 POPC lipids.61, 63 Both compound 6 and the lipids were modeled using GROMOS 53a6 force field;64 water molecules were treated using the SPC model. In each simulation, one compound 6 molecule was first placed close to the membrane surface and solvated with SPC water molecules. Counter ions were added to neutralize the system. Each system was first subjected to 500 steps of energy minimization, followed by 20 ps NVT simulation. Then a 250 ns MD simulation in NPT ensemble was carried out. LJ interactions were


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cut at a distance of 1.2 nm. Long-range electrostatic interactions were calculated using particlemesh Ewald algorithm.65 To maintain the temperature and pressure at 300K and 1 bar, NoseHoover method and Parrinello-Rahman method were employed, respectively. In vivo efficacy. All animal studies were carried out with wild type C57BL6 (20-30 gram weigh) mice, 6-8 weeks old. The animal studies were conducted in compliance with the ARVO statement for the Use of Animals in Ophthalmic and Vision Research, the guide for the Care and Use of laboratory animals (National Research Council) and under the supervision of Singhealth. Slit-lamp photography was done for screening of good corneal clarity mice for the experiments. Four healthy mice were selected for each treatment group. The antimicrobial agents used in this animal studies were levofloxacin (0.5% solution), compound 6 (0.5% solution) and compound 32 (0.5% solution). 0.1 ml of xylazine (20 mg/mL) and 0.2 ml of ketamine (100 mg/mL), mixed with 0.7 mL of normal saline (0.08 mL/mice) were used to anesthetize the animals. Corneal scratches (n=3, each 1 mm long) were made by a sterile miniblade (BD-Beaver), which did not breach beyond the superficial stroma on the right eye whereas the left eye remained untouched.66, 67

10 µl of 6×108 CFU/mL concentration of bacterial inoculum (S. aureus ATCC 29213 or MRSA

ATCC 700699) was applied topically on the damaged cornea. Treatment started at day 1 postinfection, respective drug was given at 2 hour interval for 4 times. After the fourth time, animals were sacrificed and plated for viable bacteria. Then, the infected corneas were dissected for quantification of viable bacteria. In brief, the cornea from the respective group was homogenized in sterile 0.9% NaCl containing 0.25% BSA and diluted serially, plated in duplicates on TSA plates.68 The plates were incubated for 48 h at 35 °C. Results are expressed as log10 of number of CFU per cornea. Statistical tests (Man-Whitney U test) had been employed for statistical evaluation and a probability value of p ≤ 0.05 was considered statistically significant.


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ASSOCIATED CONTENT AUTHOR INFORMATION Corresponding Author * Tel: (+65) 65767215, Fax: (+65), 62252568, Email: [email protected] (R.W.B); Tel: (+86) 20-39380255, Email: [email protected] (L.R.); Tel: (+65) 65767285, Fax: (+65) 62252568, Email: [email protected] (S.L.). Author Contributions The manuscript was written through contributions from all authors. All authors have approved the final version of the manuscript. Funding Sources This work was supported by the National Medical Research Council and the Singhealth Foundation (SHF/FG538P/2013, NMRC/CBRG/0080/2015 to S.L., NMRC/TCR/R1018 to R.B. and NMRC/BNIG/2016/2014 to J.L.), and National Natural Science Foundation of China (51273072) to L.R. Notes The authors declare no competing ﬁnancial interest. ACKNOWLEDGMENT We thank Prof Jianwen Jiang (NUS), Jaime Chew (SERI), Jia En Chai and Qiao Liu (NUS) for collaboration and technical support in activity, toxicity, and biophysical experiments. We also


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thank Wei Hong Chor and Wan Ling Sin (SERI) for technical support in vivo efficacy studies. ABBREVIATIONS MRSA, methicillin-resistant S. aureus; VRE, vancomycin resistant enterococci; AMPS, antimicrobial peptides; MIC, minimum inhibitory concentrations; LUVs, large unilamellar vesicles; HPLC, high-performance liquid chromatography; MSSA methicillin-susceptible S. aureus; ATP, adenosine triphosphate; CFU, colony forming units; TSA, tryptic soy agar; DMF, dimethylformamide; NMR, nuclear magnetic resonance; DIC, N,N’-diisopropylcarbodiimide; HOBt, N-hydroxybenzo-triazole; HRMS, high-resolution mass spectrometry; HATU, 2-(3H[1,2,3]triazolo[4,5-b]pyridin-3-yl)-1,1,3,3-tetramethylisouronium hexafluorophosphate; DOPG, 1,2-dioleoyl-sn-glycero-3-phospho-(1’-rac-glycerol)

(sodium

salt),

DOPE,

1,2-di-(9Z-

octadecenoyl)-sn-glycero-3-phosphoethanolamine, DOPC, 1,2-di(9Z-octadecenoyl)-sn-glycero3-phosphocholine. MD, molecular dynamics; RBC, red blood cell; ATCC, American type culture collection; CLSI, Clinical and Laboratory Standards Institute. REFERENCES 1.

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FIGURES Figure 1. The design concept for symmetrically substituted xanthone amphiphiles using a total synthesis approach compared with semi-synthesis of xanthone amphiphiles.

Figure 2. Time-kill kinetics of compounds 6 and 18 against MRSA DM21455, limit of detection: Log CFU/mL = 2.


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Figure 3. The development of resistance in S. aureus ATCC29213 towards compounds 6, 18, norfloxacin and gatifloxacin.

Figure 4. In vitro toxicity profiles of compounds 6 and 18 determined using a LDH cytotoxicity assay. The positive control was cells treated with 1% triton X-100.


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Figure 5. Leakage (%) induced by selected xanthone compounds at 6.25 µM in calcein-loaded liposomes. Liposome compositions including DOPC/cholesterol (3:1, w/w) and DOPE/DOPG (3:1, w/w) were used to mimic mammalian and bacterial membranes, respectively.

Figure 6. Cytoplasmic membrane permeabilization of compounds 6, 9 and 18 at 2× MIC and 1, 10 at 100 µg/mL against clinical isolates S. aureus DM4001R as measured by increased fluorescence intensity in the SYTOX Green assay.


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Figure 7. Molecular dynamics simulations of the interactions between compound 6 with model bacterial and mammalian membranes. (a) Distance between compound 6 with bacterial and mammalian membranes. (b) Snapshots of compound 6 with bacterial membrane. (c) Snapshots of compound 6 with mammalian membrane.

Figure 8. In vivo efficacy of compound 6, 32 (MSI-78) and levofloxacin in cornea infection model. (a) Infection by S. aureus ATCC 29213. Concentration used: 6 and levofloxacin, 0.5% solution. PBS was used as control. (**) p = 0.001 compared to control. (b) Infection by MRSA ATCC 700699. Concentration used: 6 and 32, 0.5% solution. PBS was used as control. (***) p < 0.05 compared to control.


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SCHEMES Scheme 1. Synthesis of xanthone-based cationic amphiphiles (6-11 and 18-23).

a

Reagents and conditions: (i) P2O5-CH3SO3H, 80 °C, 1 h; (ii) Alkyl bromide, K2CO3, acetone,

reflux, 12 h; (iii) Ethyl iodoacetate, K2CO3, acetone, reflux, 12 h; (iv) LiOH, THF, H2O, RT, 1.5 h, then corresponding basic amino acid, DIC, HOBt, anhydrous DMF, RT, overnight. Lys = lysine, Arg = arginine, His = histidine; (v) LiOH, THF, H2O, RT, 1.5 h; then corresponding amine group, HATU, DIPEA, anhydrous DMF, RT, overnight. (vi) Piperidine, DMF, RT, 20 min; (vii) LiOH, THF, H2O, RT, 1.5 h; then H-Arg-OMe•HCl, HATU, DIPEA, anhydrous DMF, RT, overnight.


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Scheme 2. Synthesis of xanthone-based cationic amphiphiles (16-17 and 26).

a

Reagents and conditions: (i) 3,3-dimethylallyl bromide, K2CO3, acetone, reflux, 12 h; (ii)

Ethyl iodoacetate, K2CO3, acetone, reflux, 12 h; (iii) LiOH, THF, H2O, RT, 1.5 h, then H-ArgOMe•HCl, DIC, HOBt, anhydrous DMF, RT, overnight; (iv) LiOH, THF, H2O, RT, 1.5 h; then HArg-OMe•HCl, HATU, DIPEA, anhydrous DMF, RT, overnight; (v) Ethyl iodoacetate, CH3ONa, acetone, reflux, 24 h.


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TABLES. Table 1. In vitro antibacterial (µg/mL) and hemolytic activities (µg/mL) of xanthone compounds compared to vancomycin and 32.

Compound

1 2 6 7 9 10 11 16 17 18 19 20 21 22 23 26

Bacterial Strains [a]

R

R1=R2=R3=R4=H R1=R2=Isoprenyl; R3=R4=H R1=R2=Isoprenyl; R3=R4=CH2CO-Arg-OMe R1=R2=Isoprenyl; R3=R4=CH2CO-Arg- NH2 R1=R2=Isoprenyl; R3=R4=CH2CO-Lys-OMe R1=R2=Isoprenyl; R3=R4=CH2CO-His-OMe R1=R2=Geranyl; R3=R4=CH2CO-Arg-OMe R1=R2=R3=Isoprenyl; R4=CH2CO-Arg-OMe R1=Isoprenyl; R2=R3=R4=CH2CO-Arg-OMe R1=R2=Isoprenyl; R3=R4=CH2CO-Arg-Arg-OMe R1=R2=Geranyl; R3=R4=CH2CO-Arg-Arg-OMe R1=R2= Isoprenyl; R3=R4=CH2CONHC3H6N(CH3)2 R1=R2= Isoprenyl; R3=R4=CH2CONHC3H6N(C2H5)2 R1=R2= Isoprenyl; R3=R4=CH2CONHC3H6N(C4H9)2 R1=R2=Isoprenyl; R3=R4=CH2CO-Arg-Arg-Arg-OMe R1=R2= Propyl; R3=R4=CH2CO-Arg-OMe

Vancomycin 32

HC50 [b]

A

B

C

D

>50 >50 0.78

>50 >50 0.78

>50 >50 1.56

>50 >50 3.13

> 400 > 400 750±18

Hemolysis (%)[c] 50

>50

>50

> 400

50

>50

> 400

400

2000

9±1

25

25

25

25

306±26

66±5

6.25

12.5

6.25

12.5

38.4±3.1

ND [d]

6.25

12.5

6.25

25

48.8±0.2

ND [d]

3.13

3.13

3.13

6.25

43.0±2.3

ND [d]

12.5

12.5

12.5

12.5

> 400

0.4±0.5

12.5

12.5

12.5

25

> 400

11±1

1.56 12.5

3.13 25

1.56 12.5

1.56 12.5

ND 120 [e]

ND ND

[a] Clinical Isolates S. aureus DM 4001R (A), MRSA DM 9808R (B), MRSA DM 21455 (C), and Bacillus cereus ATCC 11778 (D). [b] HC50 value is the lowest concentration that causes 50% hemolysis of red blood cells (RBCs).


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[c] % hemolysis of RBCs caused by xanthone analogues at a concentration of 400 µg/mL. [d] ND = Not determined. [e] Literature values obtained from ref 18.18

Table 2. MICs (µg/mL) of compounds 6 and 18 against a panel of Gram-positive bacteria compared to vancomycin. Gram-positive Strains

bacterial

S. aureus S. aureus S. aureus S. aureus S. aureus S. aureus S. aureus S. aureus S. aureus S. aureus S. aureus S. aureus Enterococcus faecium Enterococcus faecium Enterococcus faecium Enterococcus faecalis Enterococcus hirae

Description MSSA DM4583R MSSA DM4400R MSSA DM4299 MSSA ATCC 29213 MSSA ATCC 6538 MSSA ATCC 29737 MRSA DB6506 MRSA DB68004 MRSA DB57964 MRSA ATCC 43300 MRSA ATCC 700699 MRSA ATCC BAA-38 VRE 1006 Van A-resistant VRE 1007 Van A-resistant VRE 1008 Van A-resistant ATCC 29212 ATCC 9790

Compounds 6 1.56 0.78 3.13 3.13 0.78 0.78 1.56 3.13 1.56 1.56 3.13 3.13 6.25 6.25 6.25 3.13 6.25

18 3.13 3.13 1.56 3.13 1.56 1.56 1.56 1.56 1.56 1.56 1.56 3.13 1.56 1.56 3.13 6.25 6.25

Vancomycin 1.56 0.78 1.56 1.56 0.78 0.78 0.78 1.56 0.78 0.78 1.56 1.56 >50 >50 >50 1.56 0.78


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Table 3. Selectivity of xanthone-based compounds and several membrane-targeting antimicrobial agents in clinical trials. MIC (µg/mL)

HC50 (µg/mL)

Selectivity (HC50/MIC)

ref

α-mangostin

2

9

4.5

50

29

0.5 - 3

20

6.7-40

51

30

2-3

232

77-116

35

31

0.5 - 3

277

92-554

35

6

0.78-6.25

750

120-962

18

1.56-3.13

>2000

>639

16-64

120

2-8

53

27

4

175

44

24

28

1

558

558

26

33

0.4

29 α

73

34

Compound

32

α

Structure

GIGKFLKKAKKFGKAFV KILKK

Reported value is the minimum hemolytic concentration.


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Table 4. ATP Leakage (%) caused by compounds 6, 9 and 18 at 4× MIC and 1 and 10 at 200 µg/mL from clinical isolates S. aureus DM4001R. Compound

1

6

9

10

18

% Leakage

6.9 ± 1.6

59.6±2.2

106.7±9.6

21. 3±2.7

66.4±1.2


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TABLE OF CONTENTS GRAPHIC


62

Symmetrically Substituted Xanthone Amphiphiles Combat Gram

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