Semisynthetic Flavone-Derived Antimicrobials with ... - ACS Publications

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Semisynthetic Flavone-Derived Antimicrobials with Therapeutic Potential against Methicillin-Resistant Staphylococcus aureus (MRSA) Shuimu Lin,†,‡,∥ Jun-Jie Koh,‡ Thet Tun Aung,‡ Wan Ling Wendy Sin,‡ Fanghui Lim,‡ Lin Wang,†,∥ Rajamani Lakshminarayanan,‡,§ Lei Zhou,‡,§ Donald T. H. Tan,‡,⊥ 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, 169856 Singapore, Singapore § SRP Neuroscience and Behavioral Disorders, Duke−NUS Graduate Medical School, 169857 Singapore, Singapore ∥ National Engineering Research Center for Tissue Restoration and Reconstruction, Guangzhou 510006, China ⊥ Singapore National Eye Center, 11 Third Hospital Avenue, 168751 Singapore, Singapore # School of Chemistry and Chemical Engineering, South China University of Technology, Guangzhou 510641, China ‡

S Supporting Information *

ABSTRACT: A new series of semisynthetic flavone-based small molecules mimicking antimicrobial peptides has been designed from natural icaritin to combat drug-resistant Grampositive bacterial infections. Compound 6 containing two arginine residues exhibited excellent antibacterial activity against Gram-positive bacteria, including MRSA, and very low toxicity to mammalian cells, resulting in a high selectivity of more than 511, comparable to that of several membraneactive antibiotics in clinical trials. Our data show for the first time that icaritin derivatives effectively kill bacteria. Meanwhile, this is the first study deploying a biomimicking strategy to design potent flavone-based membrane targeting antimicrobials. 6 showed rapid bactericidal activity by disrupting the bacterial membrane and can circumvent the development of bacterial resistance. Importantly, 6 was highly efficacious in a mouse model of corneal infection caused by MRSA and Staphylococcus aureus.

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INTRODUCTION

compounds are also mainly limited by membrane selectivity and complex synthesis protocols.15,17 Recently, our group discovered several α-mangostin analogues that mimic the structure of AMPs show promising antibacterial activity (Figure 1).19−21 These compounds can disrupt the bacterial membrane, leading to cell lysis and death.19−21 Recent studies have shown that biomimetic polyamine analogues have potent antibacterial activity.22,23 They can disrupt bacterial membrane integrity rapidly and promote biofilm dispersal.22,23 In spite of several advantages of these compounds, the membrane selectivity between mammalian and bacterial membranes is still not sufficient to be applied clinically. Flavones are present in vegetables and fruits, and they have beneficial effects on human health without major side effects.24 Flavones have been found to exhibit a wide variety of remarkable biological activities such as antioxidant,25 antimicrobial,26 antimalarial,27 antiviral,28 and anticancer activities.29,30 Icaritin, an active prenylflavone isolated from epimedium plants, exhibits various pharmacological and

The increasing development of bacterial resistance to antibiotics is a major obstacle to the successful treatment of infectious diseases caused by bacteria.1,2 The rapid spread of MRSA poses huge threats to public health.1 In view of the serious situation, MRSA was recently classified as one of the 12 listed priority pathogens which pose the greatest threat to human health by World Health Organization (WHO).3 There is an urgent need for novel antimicrobial agents without crossresistance to currently used antibiotics.4,5 Antimicrobial peptides (AMPs) act as part of the innate immune system and have gained considerable attention as a class of promising antibiotics because of their excellent antibacterial activity and low probability of developing drug resistance.6−8 However, AMPs have some disadvantages, including high manufacturing costs, high cytotoxicity levels, and poor in vivo stability.9−11 These disadvantages hamper their clinical application.9−11 To potentially address these problems, synthetic antimicrobial peptidomimetics that maintain the critical biophysical characteristics of AMPs have been developed.12−15 1 (LTX-109)16 and 2 (PMX 30063) 17,18 are successful examples of peptidomimetics undergoing clinical trials. Nevertheless, these © 2017 American Chemical Society

Received: March 10, 2017 Published: June 21, 2017 6152

DOI: 10.1021/acs.jmedchem.7b00380 J. Med. Chem. 2017, 60, 6152−6165

Journal of Medicinal Chemistry

Article

Figure 1. Design concept for flavone-based antimicrobials biomimicking antimicrobial peptides and structures of our previously designed amphiphilic xanthones as antimicrobial agents.

selectivity. We examined the structural requirements for enhancing membrane selectivity and provided evidence of the mode of action for these analogues. We found that compound 6, containing two arginine residues, displayed rapid time-kill, excellent in vitro and in vivo antibacterial activity, and very low toxicity toward mammalian cells.

biological activities, including those useful in the treatment of cancer and osteoporosis.31−33 Icaritin is an excellent natural scaffold for further drug discovery and development and has entered into clinical trials for the treatment of advanced breast cancer (phase 1, ClinicalTrials.gov identifier NCT01278810), advanced solid tumors (phase 1, ClinicalTrials.gov identifier NCT02496949) and advanced hepatocellular carcinoma (phase 2, ClinicalTrials.gov identifier NCT01972672). Because icaritin has a very good safety profile, it would be a good natural chemical entity for further modification to obtain potent compounds with significantly improved antimicrobial activities and low toxicity. Herein, we report the design and synthesis of a novel series of semisynthetic flavone amphiphiles as antimicrobial peptidomimetics starting from natural icaritin. To the best of our knowledge, this is the first report on the antibacterial study of icaritin and its derivatives and is the first study deploying a biomimicking strategy to design potent flavone-based membrane targeting antimicrobials. We hypothesized that the hydrophobic moieties of icaritin containing a flavone core and lipid chains can enhance membrane interactions and facilitate the penetration of flavone peptidomimetics into the lipid membrane. Additionally, the incorporation of cationic charges would ensure the electrostatic attraction of flavone amphiphiles to negatively charged bacterial membranes and enhance the selectivity by distinguishing anionic bacterial membranes from zwitterionic mammalian membranes (Figure 1). Icaritin could be used as one special type of unnatural hydrophobic amino acid residue to design antimicrobials with enhanced membrane selectivity and improved stability by biomimicking AMPs. As shown in Figure 1, our previous studies indicated that cationic moieties modified xanthone derivatives with the spacer lengths of 2 < n < 8 displayed potent antimicrobial activities against Gram-positive bacterial strains and that highly basic side chain groups with high pKa values (e.g., guanidinium of the arginine side chain) were advantageous for potent antimicrobial activity and high membrane selectivity.20,34 In the present study, we modified flavone derivatives based on structure−activity relationship (SAR) studies of xanthones mentioned above in order to obtain promising compounds of amphiphilic flavone derivatives with potent antimicrobial activity, low toxicity, and high membrane

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RESULTS AND DISCUSSION Synthesis. Two cationic moieties were incorporated into the flavone scaffold to produce flavone amphiphiles and induce strong bacterial membrane disruption (Schemes 1 and 2). Initially, the alkylation of the parent compound icaritin with ethyl iodoacetate afforded compound 3. The hydroxyl groups at C3 and C7 of the flavone scaffold were more active than that at C5 due to stable intramolecular hydrogen bond formation between the C4 carbonyl group and the C5 hydroxyl group.35 Ester 3 was hydrolyzed by LiOH to provide the key acid 4, which was further coupled to corresponding basic amino acids using 2-(3H-[1,2,3]triazolo[4,5-b]pyridin-3-yl)-1,1,3,3-tetramethylisouronium hexafluorophosphate (HATU) or N,N′-diisopropylcarbodiimide (DIC) combined with HOBt to obtain compounds 5−8. Deprotection of the N-Fmoc of compound 8 yielded compound 9. Compound 11 was obtained through a two-step reaction in which methyl ester 6 was first hydrolyzed by LiOH to yield compound 10, and then compound 10 was reacted with H-Arg-OMe·2HCl in the presence of HATU. Icaritin was alkylated with ethyl iodoacetate to produce intermediate 12. The treatment of ester 12 with LiOH produced acid 13. Then acid 13 was coupled to H-Arg-OMe· 2HCl using HATU to yield compound 14. Icaritin was alkylated with different α,ω-dibromoalkanes to yield compounds 15−18, followed by amination with diethylamine or dimethylamine to produce compounds 19−23. Compound 24 was obtained through a two-step process in which compound 16 was reacted with 1-iodopentane in the presence of Cs2CO3 and then treated with diethylamine. All final compounds were purified by HPLC to >95% purity and characterized by HRMS and NMR. Antibacterial and Hemolytic Activity. The biological activities of flavone compounds in comparison with vancomycin and 25 (MSI-78)36,37 (a membrane-targeting antimicrobial peptide applied as a topical antibiotic in phase 3 clinical trials) 6153



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Scheme 1. Synthesis of Amino Acids Modified Flavone Compoundsa

a

Reagents and conditions: (a) ethyl iodoacetate, K2CO3, acetone, reflux, 12 h; (b) LiOH, THF, H2O, RT, 1.5 h; (c) corresponding basic amino acid, DIC, HOBt, anhydrous DMF, RT, overnight, His = histidine, Arg = arginine, Lys = lysine; (d) corresponding basic amino acid, HATU, DIPEA, anhydrous DMF, RT, overnight; (e) piperidine, DMF, RT, 20 min.

demonstrating the great importance of cationic moieties with high pKa values in enhancing the antibacterial activity of flavones. For the effect of spacer length to be examined, diethylamine-modified compounds 19, 20, 22, and 23 with different spacer lengths of 2, 4, 6, and 8 were synthesized. Compound 22 (n = 6) also displayed enhanced antibacterial activities against all tested strains with MICs in the range of 3.13−12.5 μg/mL, comparable to that of compound 20 (n = 4; MICs = 6.25−25 μg/mL). Surprisingly, compounds 19 (n = 2) and 23 (n = 8) displayed poor antibacterial activities with MICs of ≥50 μg/mL. In general, the longer the spacer lengths, the higher the lipophilicity. The cLogP values of compounds 19− 23 are 5.76, 6.90, 5.83, 8.30, and 10.50, respectively. Compound 24 containing an additional lipid chain at the C5 position of the flavone core would also enhance its hydrophobicity, with cLogP value of 9.12. These results indicated that

are summarized in Tables 1 and 2. In vitro antibacterial activity was determined against four Gram-positive bacterial strains, including MRSA, Bacillus cereus, and Staphylococcus aureus. The hemolytic activity toward mammalian cells was determined using rabbit erythrocytes. The uncharged parent compound icaritin was weakly active against the Gram-positive bacteria, with a minimum inhibitory concentration (MIC) of 50 μg/mL. Notably, icaritin did not display any hemolytic activity against mammalian cells, even up to 400 μg/mL. Two types of aliphatic amines (diethylamine for compounds 20 and 24 and dimethylamine for compound 21) with high pKa values (pKa = 10.98 and 10.64, respectively)34 were selected to be linked with a ω-halobutyl-substituted flavone to form amphiphilic structures. Compounds 20 and 21 showed improved antibacterial activities with MICs in the range of 6.25−25 μg/mL compared with the parent compound icaritin, 6154



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Scheme 2. Synthesis of Aliphatic Amines Modified Flavone Compoundsa

Reagents and conditions: (a) α,ω-dibromoalkanes (15 equiv), K2CO3 (5 equiv), acetone, reflux, 12 h; (b) diethylamine or dimethylamine, DMSO, RT, 4 h; (c) 1-iodopentane, Cs2CO3, acetone, reflux, 12 h, then diethylamine, DMSO, RT, 4h.

a

Table 1. In Vitro Antibacterial and Hemolytic Activities (μg/mL) of Aliphatic Amines Modified Flavone Compounds

bacterial strains (μg/mL) compd icaritin 19 20 21 22 23 24 vancomycin 15

R R1 R1 R1 R1 R1 R1

= = = = = =

C2H5; R2 = H C2H5; R2 = H CH3; R2 = H C2H5; R2 = H C2H5; R2 = H C2H5; R2 = C5H11

n 2 4 4 6 8 4

S. aureus DM4001R

MRSA DM 9808R

MRSA DM 21455

B. cereus ATCC 11778

cLogPa

50 50 25 25 6.25 >50 25 1.56 12.5

50 >50 12.5 12.5 3.13 >50 12.5 3.13 25

50 50 12.5 12.5 3.13 >50 12.5 1.56 12.5

50 >50 6.25 25 12.5 >50 25 1.56 12.5

NDc 5.76 6.90 5.83 8.30 10.50 9.12

HC50b (μg/mL) >400 >400 >400 379.2 ± 6.2 165.3 ± 6.4 70.9 ± 2.3 236.6 ± 3.4 NDc 120d

a cLogP = calculated LogP, generated using ACD/Percepta software. bHC50 value is the concentration required to lyse 50% of rabbit red blood (RBCs) cells. Values of variation 1600 μg/mL), whereas compound 9 showed increased hemolytic activity (HC50 = 103.5 ± 2.5 μg/mL) relative to icaritin. Compound 7 (cLogP = 6155



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Table 2. In Vitro Antibacterial and Hemolytic Activities (μg/mL) of Amino Acids Modified Flavone Compounds

bacterial strains, μg/mL (μM) compd

R

S. aureus DM4001R

MRSA DM 9808R

MRSA DM 21455

B. cereus ATCC 11778

cLogPa c

HC50,b μg/mL (μM)

5

R1 = His-OMe

>50 (>63.5)

>50 (>63.5)

>50 (>63.5)

>50 (>63.5)

ND

>400 (>508.4)

6

R1 = Arg-OMe

1.56 (1.9)

3.13 (3.8)

1.56 (1.9)

3.13 (3.8)

1.65

>1600 (>1939.6)

7

R1 = Arg-OtBu

3.13 (3.4)

3.13 (3.4)

6.25 (6.8)

3.13 (3.4)

3.84

>400 (>440.0)

9

R1 = Lys-OMe

25 (32.5)

25 (32.5)

25 (32.5)

25 (32.5)

NDc

103.5 ± 2.5 (134.5 ± 3.3)

11

R1 = Arg-Arg-OMe

6.25 (5.5)

6.25 (5.5)

12.5 (11.0)

12.5 (11.0)

−0.50

>400 (>351.7)

14

R2 = Arg-OMe

3.13 (5.2)

3.13 (5.2)

6.25 (10.4)

6.25 (10.4)

2.82

>400 (670.4)

vancomycin

1.56 (1.1)

3.13 (2.2)

1.56 (1.1)

1.56 (1.1)

NDc

15

12.5 (5.0)

25 (10.0)

12.5 (5.0)

12.5 (5.0)

120d (48.4)

a

cLogP = calculated LogP, generated using ACD/Percepta software. bHC50 value is the concentration required to lyse 50% of rabbit red blood (RBCs) cells. Values of variation 50 μg/mL) and had no effect on hemolytic activity. To elucidate the effect of overall charge, compound 11 (charge of +4) and compound 14 (charge of +1) were synthesized in comparison with compound 6, containing two charges and the uncharged parent compound icaritin. Compounds 11 and 14 showed increased antimicrobial activity (MICs = 3.13−12.5 μg/mL) relative to icaritin and retained an extremely low hemolytic activity against mammalian cells (HC50 > 400 μg/mL). However, the antibacterial activity of compound 11 was 2−8 times lower than that of compound 6. Compound 14 displayed MIC values of 3.13−6.25 μg/mL, a 2fold reduction in antibacterial activity compared to that of 6 (MIC = 1.56−3.13 μg/mL). The cLogP values of flavone compounds should be affected by the incorporation of charges. The more the overall charges, the lower the hydrophobicity. The cLogP values of compounds 6, 11, and 14 are 1.65, −0.50, and 2.82, respectively. The results indicated that the charge− hydrophobicity balance in these flavone analogues is an

important factor in determining antibacterial activity. The increased overall charges would improve the antibacterial activity; however, if the overall charge becomes too large or too small, the compound would disrupt the charge−hydrophobicity balance and have a reduced affinity for bacterial membranes, leading to reduced antimicrobial activity.38 Compound 6 was the most active of all the tested compounds. Among all the tested flavone derivatives, only compound 6 and 11 were water-soluble. The greatest improvement in antimicrobial activity was attributed to the strong electrostatic interaction between compound 6 and the negatively charged bacterial membrane. The positive charge of the guanidinium group on arginine is more dispersed than that of the tertiary amine and −NH2 groups,20 greatly enhancing the electrostatic interaction of compound 6 toward the bacterial membrane. Compound 6 was further screened against 12 Gram-positive bacterial strains, including MRSA (Table 3). The MICs were in the range of 1.56−3.13 μg/mL, which are comparable to those of vancomycin (MICs = 0.78−1.56 μg/mL). Additionally, compound 6 showed excellent activity against drug-resistant strains, indicating that there was no drug cross-resistance for compound 6 with other antibiotic classes. The selectivity (HC50/MICs) of compound 6 (>511) was higher than that of several membrane-targeting antibiotics in clinical trials, including 1, 2, and 25, demonstrating the potency of compound 6 as a membrane-targeting antibiotic (Table 4). 6156



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drug resistance. Because drug resistance for membranetargeting antimicrobials commonly requires bacteria to have large-scale changes in their membrane compositions, but it is extremely hard for bacteria to keep alive while remodelling their membranes.34 These results indicate that compound 6 can kill bacteria quickly and avert the development of drug resistance. Mechanism of Action. To investigate the mode of action of flavone compounds, we performed a SYTOX Green assay. SYTOX Green is a high-affinity nucleic acid binding dye that penetrates only bacteria with damaged membranes, enhancing its fluorescence intensity.34,39 Figure 4 displays the effect of the flavone derivatives 5, 6, 9, and icaritin at 12.5 μg/mL against MRSA DM9808R incubated with SYTOX Green. The potent compound 6 showed a strong ability to permeabilize the bacterial inner membranes, resulting in the highest increase in fluorescence intensity. The moderately active compound 9 also induced an obvious enhancement in fluorescence intensity, indicating that compound 9 also caused obvious bacterial membrane permeabilization at 12.5 μg/mL in the buffer. Unlike compounds 6 and 9, which kill MRSA via membrane disruption, the inactive compound 5 and icaritin did not affect the fluorescence intensity due to the lack of interaction between the two compounds and the bacterial membrane. These results are in accordance with their in vitro antibacterial activities. A calcein release assay using large unilamellar vesicles (LUVs) was performed to further confirm the membranetargeting properties of flavones against bacteria. The LUVs were prepared from 75% 1,2-di(9Z-octadecenoyl)-sn-glycero-3phosphoethanolamine (DOPE) and 25% 1,2-dioleoyl-snglycero-3-phospho-(1′-rac-glycerol) (DOPG), mimicking negatively charged inner bacterial membranes.20 As shown in Figure 5, the potent compound 6 caused a significant leakage of the entrapped calcein dye from the vesicles, with leakage values of 46.0 ± 1.6% (C/L ratios = 1/4) and 53.5 ± 1.2% (C/L ratios = 1/8), respectively, suggesting a very strong interaction between compound 6 and the bacterial membranes. Approximately 30% dye leakage was measured for the moderately active compound 9 (MICs = 25 μg/mL), indicating the bacterial membrane was also sensitive to compound 9. Striking differences were observed for the inactive compound 5 and icaritin (MICs ≥ 50 μg/mL), which showed much lower

Table 3. Antimicrobial Activities: MIC (μg/mL) Values of Compound 6 against a Panel of Gram-Positive Bacteria Compared with Vancomycin organisms

compounds

Staphylococcus aureus

6

vancomycin

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

1.56 3.13 3.13 3.13 1.56 1.56 3.13 3.13 3.13 1.56 3.13 3.13

1.56 0.78 1.56 1.56 0.78 0.78 0.78 1.56 0.78 0.78 1.56 1.56

Time-Kill Kinetics. In an additional study, compound 6 rapidly killed MRSA DM21455 and MRSA DM9808R in a concentration-dependent manner and circumvented the development of bacterial resistance. A 3 log (CFU/mL) reduction (99.9% of bacteria killed) was observed in 30 min at 2× and 4× MIC against MRSA DM21455 (Figure 2A). The rate of killing of compound 6 was greater and quicker than that of the previous report for vancomycin against MRSA DM21455.34 Compound 6 also displayed rapid killing of MRSA DM9808R; a 3 log reduction was achieved in 2 h at 2× and 4× MIC (Figure 2B). These results indicate that compound 6 can kill MRSA quickly. Drug Resistance Studies. As shown in Figure 3, serial passaging S. aureus ATCC 29213 in the presence of sublethal concentrations of compound 6 over a period of 17 days failed to yield resistant mutants, suggesting that the target is nonspecific. There was no >4-fold enhancement in the MIC for compound 6 after 17 passages. In contrast, the MICs for gatifloxacin and norfloxacin were increased by 64- and 128-fold after 15 passages. The rapid bactericidal activity of compound 6 was resulted from its membrane-targeting action. This important feature contributes to the avoidance of developing

Table 4. Selectivity of Compound 6 and Several Membrane-Targeting Antimicrobials (1, 2, and 25) in Clinical Trials

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Figure 2. Time-kill kinetics of compound 6 against MRSA DM21455 (A) and MRSA DM9808R (B); detection limit Log CFU/mL = 2.

Figure 5. Percent leakage caused by compounds 5, 6, 9, and icaritin in calcein-loaded LUVs. Liposome composition of DOPE/DOPG (3:1, w/w) was used to mimic bacterial membranes.

Figure 3. Bacterial resistance studies of compound 6, norfloxacin, and gatifloxacin against S. aureus ATCC 29213.

assay. These results revealed that compound 6 had very low cytotoxicity toward mammalian cells up to 100 μM, suggesting that compound 6 was able to selectively kill Gram-positive bacteria, including MRSA. In Vivo Antibacterial Efficacy. The topical administration of antimicrobial agents for treating corneal infections has many advantages over the systemic administration, including avoiding adverse systemic side effects, increasing the drug concentration at the target site of infection, and reducing the probability of developing drug resistance.42,43 Recently, the incidence of bacteria-induced keratitis is increasing.44,45 S. aureus is the predominant pathogen isolated from ocular infections.44,45 In 2009, MRSA-induced keratitis represents up to 39.0% of S. aureus-induced keratitis cases in the United States.46 In New York, the percentage of MRSA strains among all ocular S. aureus isolates was increased from 7.2% in 1997 to 41.6% in 2008.47 In this study, the in vivo efficacy of compound 6 was investigated topically by using an infected mouse keratitis model. 6 displayed good safety profile toward corneal fibroblasts in the LDH and ATP assays. In brief, the mice corneas (four mice per group) were infected with ∼6 × 106 CFUs of MRSA ATCC 700699. After 1 day of infection, each group of mice was treated 5 times daily for 3 days with 0.5% compound 6, 5% vancomycin, or phosphate-buffered saline (PBS; used as a negative control). As shown in Figure 7A, compound 6 and vancomycin produced 2.95 log (99.89%) and 3.20 log (99.94%) reductions in the bacterial burden, respectively. The concentration of vancomycin used in corneal

Figure 4. Cytoplasmic membrane permeabilization of compounds 5, 6, 9, and icaritin at 12.5 μg/mL against MRSA DM9808R.

bacterial membrane-lytic activities (95%, as confirmed by HPLC analysis. Melting points were recorded on a Mel-Temp 1101D melting point apparatus. Temperatures were expressed in degrees celsius (°C). Atmospheric pressure chemical ionization (APCI) mass spectra were recorded on a Bruker AmaZon X spectrometer, and electrospray ionization mass spectra were obtained on an API2000 liquid chromatography−tandem mass spectrometry system. Diethyl 2,2′-{[5-Hydroxy-2-(4-methoxyphenyl)-8-(3-methylbut-2en-1-yl)-4-oxo-4H-chromene-3,7-diyl]bis(oxy)}diacetate (3). Icaritin (736 mg, 2 mmol) was dissolved in acetone (15 mL), and ethyl iodoacetate (497.1 μL, 4.2 mmol) and potassium carbonate (828 mg, 6 mmol) were then added. The reaction mixture was refluxed for 12 h. After cooling to room temperature, the mixture was diluted with ethyl acetate and washed with brine three times. The organic phase was

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CONCLUSIONS A novel series of semisynthetic flavone-based antibiotics as mimics of AMPs were designed and evaluated. Compound 6 showed potent in vitro activity against drug-resistant Grampositive bacterial strains, including MRSA; its antimicrobial activity was comparable to that of vancomycin and better than that of 25. In addition, compound 6 displayed very low hemolytic activity and cytotoxicity levels toward mammalian cells. This compound killed bacteria rapidly with high membrane selectivity by disrupting bacterial membrane integrity and hampered the development of bacterial resistance. Furthermore, compound 6 displayed potent in vivo efficacy in a mouse keratitis model of MRSA and S. aureus infections, thereby demonstrating that compound 6 has potential as a therapeutic antimicrobial agent for overcoming drug resistance. Importantly, the results reported here can provide a promising new molecular platform for the further design of potent 6159



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dried over anhydrous Na2SO4, filtered, and evaporated to dryness under reduced pressure. The crude product was purified by column chromatography (silica gel, hexanes/ethyl acetate, 4:1, v/v) to afford compound 3 as a yellow solid (849.2 mg, 79%); mp 147−148 °C. 1H NMR (400 MHz, CDCl3) δ = 12.58 (s, 1H, OH), 8.19 (d, J = 9.0 Hz, 2H, 2 × Ar-H), 7.01 (d, J = 9.1 Hz, 2H, 2 × Ar-H), 6.24 (s, 1H, Ar-H), 5.24 (t, J = 6.9 Hz, 1H, CH), 4.79 (s, 2H, OCH2), 4.70 (s, 2H, OCH2), 4.27 (q, J = 7.1 Hz, 2H, OCH2), 4.19 (q, J = 7.1 Hz, 2H, OCH2), 3.89 (s, 3H, OCH3), 3.58 (d, J = 6.8 Hz, 2H, CH2), 1.79 (s, 3H, CH3), 1.68 (s, 3H, CH3), 1.31 (t, J = 7.1 Hz, 3H, CH3), 1.24 (t, J = 7.1 Hz, 3H, CH3). 13C NMR (101 MHz, CDCl3) δ = 178.5, 168.9, 167.9, 161.8, 160.8, 160.2, 156.0, 153.6, 136.7, 132.2, 130.7 (2 × CH), 123.0, 122.1, 114.0 (2 × CH), 108.6, 106.1, 95.4, 68.6, 65.6, 61.6, 61.1, 55.4, 25.7, 21.8, 18.0, 14.1 (2 × CH3). HRMS (APCI+): calculated for C29H33O10 [M + H]+ 541.2068, found 541.2064. Dimethyl 2,2′-[(2,2′-{[5-Hydroxy-2-(4-methoxyphenyl)-8-(3methylbut-2-en-1-yl)-4-oxo-4H-chromene-3,7-diyl]bis(oxy)}bis(acetyl))bis(azanediyl)](2S,2′S)-bis[3-(1H-imidazol-5-yl)propanoate] (5). Compound 3 (149 mg, 0.276 mmol) was dissolved in THF (4 mL), and a 5% LiOH aqueous solution (2 mL) was then added. The reaction mixture was stirred at room temperature for 1.5 h. After adding acetic acid, the mixture (pH < 7) was diluted with butanol and washed with brine three times. The organic phase was dried over Na2SO4, filtered, and evaporated to dryness under reduced pressure to yield the desired compound 4, which was used in the next reaction without further purification. Compound 4 was dissolved in anhydrous dimethylformamide (DMF, 5 mL). The mixture was cooled to 0 °C, and HATU (314.6 mg, 0.828 mmol), DIPEA (288.4 μL, 1.656 mmol), and H-His-OMe·2HCl (200.5 mg, 0.828 mmol) were added. The reaction mixture was stirred at 0 °C for 1 h and was then stirred at room temperature overnight. The mixture was then diluted with butanol and extracted with brine three times. The organic phase was dried over anhydrous Na2SO4, filtered, and evaporated to dryness under reduced pressure. Subsequently, the crude product was purified by HPLC to yield compound 5 as a yellow solid (173 mg, 80%); mp 128−129 °C. 1H NMR (400 MHz, DMSO-d6) δ = 8.71 (d, J = 7.7, 1H, Ar-H), 8.65 (d, J = 7.7, 1H, Ar-H), 8.11−8.06 (m, 2H, 2 × Ar-H), 7.97 (d, J = 16.1, 2H, 2 × Ar-H), 7.09 (d, J = 9.1, 2H, 2 × Ar-H), 7.02 (d, J = 7.1, 2H, 2 × Ar-H), 6.42 (s, 1H, Ar-H), 5.22−5.15 (m, 1H, CH), 4.74 (s, 2H, OCH2), 4.69−4.61 (m, 2H, 2 × CH), 4.49 (s, 2H, OCH2), 3.86 (s, 3H, OCH3), 3.62 (s, 6H, 2 × OCH3), 3.56 (d, J = 6.6, 2H, CH2), 3.07−2.95 (m, 4H, 2 × CH2), 1.70 (s, 3H, CH3), 1.62 (s, 3H, CH3). 13C NMR (101 MHz, DMSO-d6) δ = 178.0, 171.2, 171.1, 167.6, 167.0, 162.9, 161.6, 160.8, 159.2, 155.9, 152.9, 136.3, 134.6, 132.3, 132.0, 131.4, 130.3 (2 × CH), 122.1, 122.0, 116.4, 116.1, 114.2 (2 × CH), 107.7, 105.0, 96.1, 70.2, 67.3, 55.5 (2 × CH), 52.0, 51.8, 51.6, 27.9 (2 × CH2), 25.4, 21.3, 17.8. HRMS (ESI+): calculated for C39H43N6O12 [M + H]+ 787.2933, found 787.2937. Methyl {2-[(7-{[(S)-7-Guanidino-4-(methoxyamino)-2,3dioxoheptyl]oxy}-5-hydroxy-2-(4-methoxyphenyl)-8-(3-methylbut2-en-1-yl)-4-oxo-4H-chromen-3-yl)oxy]acetyl}-L-argininate (6). Compound 6 was prepared from compound 3 (90.4 mg, 0.167 mmol), HOBt (67.7 mg, 0.501 mmol), DIC (77.6 μL, 0.501 mmol), and H-Arg-OMe·2HCl (130.8 mg, 0.501 mmol) using the same synthesis procedure as described for compound 5. This product was obtained as a yellow solid (105.6 mg, 77%); mp 138−139 °C. 1H NMR (500 MHz, CD3OD) δ = 8.03 (d, J = 8.3, 2H, 2 × Ar-H), 7.08 (d, J = 8.3, 2H, 2 × Ar-H), 6.38 (s, 1H, Ar-H), 5.25−5.12 (m, 1H, CH), 4.73 (s, 2H, OCH2), 4.60 (dd, J = 13.2, 8.1, 2H, 2 × CH), 4.52− 4.41 (m, 2H, OCH2), 3.90 (s, 3H, OCH3), 3.78 (s, 6H, 2 × OCH3), 3.60−3.49 (m, 2H, CH2), 3.29−3.16 (m, 4H, 2 × CH2), 2.07−1.96 (m, 2H, CH2), 1.93−1.63 (m, 12H, 3 × CH2, 2 × CH3). 13C NMR (126 MHz, CD3OD) δ = 178.5, 172.0, 171.9, 169.8, 168.7, 168.7, 162.4, 161.1, 159.8, 157.4, 156.7, 153.4, 137.0, 132.0, 130.2 (2 × CH), 122.0, 121.8, 114.1 (2 × CH), 108.5, 105.4, 95.8, 70.9, 67.2, 54.7, 51.7, 51.7, 51.6, 51.6, 40.5, 40.4, 28.4, 28.4, 24.9, 24.8, 24.5, 21.3, 17.0. HRMS (ESI+): calculated for C39H53N8O12 [M + H]+ 825.3777, found 825.3780. Di-tert-butyl 2,2′-[(2,2′-{[5-Hydroxy-2-(4-methoxyphenyl)-8-(3methylbut-2-en-1-yl)-4-oxo-4H-chromene-3,7-diyl]bis(oxy)}bis-

(acetyl))bis(azanediyl)](2S,2′S)-bis(5-guanidinopentanoate) (7). Compound 7 was prepared from compound 3 (59.4 mg, 0.11 mmol), HATU (125.5 mg, 0.33 mmol), DIPEA (95.8 μL, 0.55 mmol), and H-Arg-OtBu·2HCl (100 mg, 0.33 mmol) using the same synthesis procedure as described for compound 5. This product was obtained as a yellow gel (82.6 mg, 83%). 1H NMR (400 MHz, CD3OD) δ = 8.02 (d, J = 8.5 Hz, 2H, 2 × Ar-H), 7.07 (d, J = 8.6 Hz, 2H, 2 × Ar-H), 6.38 (s, 1H, Ar-H), 5.19 (t, J = 5.8 Hz, 1H, CH), 4.77−4.63 (m, 2H, 2 × CH), 4.54−4.37 (m, 4H, 2 × OCH2), 3.88 (s, 3H, OCH3), 3.53 (d, J = 8.1 Hz, 2H, CH2), 3.29−3.14 (m, 4H, 2 × CH2), 2.05−1.90 (m, 2H, CH2), 1.85−1.60 (m, 12H, 3 × CH2, 2 × CH3), 1.54−1.38 (m, 18H, 6 × CH3). 13C NMR (101 MHz, CD3OD) δ = 180.0, 172.2, 172.1, 171.3, 170.0, 163.9, 162.6, 161.4, 158.9 (2 × C), 158.3, 154.9, 138.6, 133.5, 131.7 (2 × CH), 123.5, 123.4, 115.7 (2 × CH), 110.0, 107.0, 97.3, 83.7, 83.5, 72.5, 68.7, 56.3, 53.9, 50.0, 42.0, 42.0, 30.3, 30.1, 28.4 (6 × CH3), 26.4, 26.3, 26.1, 22.9, 18.6. HRMS (ESI+): calculated for C45H65N8O12 [M + H]+ 909.4716, found 909.4729. Dimethyl 2,2′-[(2,2′-{[5-Hydroxy-2-(4-methoxyphenyl)-8-(3methylbut-2-en-1-yl)-4-oxo-4H-chromene-3,7-diyl]bis(oxy)}-bis(acetyl))bis(azanediyl)](2S,2′S)-bis[6-({[(9H-fluoren-9-yl)methoxy]carbonyl}-amino)hexanoate] (8). Compound 8 was prepared from compound 3 (113.3 mg, 0.21 mmol), HOBt (85.1 mg, 0.63 mmol), DIC (97.6 μL, 0.63 mmol), and H-Lys(Fmoc)-OMe·HCl (263.9 mg, 0.63 mmol) using the same synthesis procedure as described for compound 5. This product was obtained as a yellow solid (200.2 mg, 79%); mp 114−117 °C. 1H NMR (400 MHz, CDCl3) δ = 12.38 (s, 1H), 7.93 (d, J = 9.0, 2H, 2 × Ar-H), 7.72 (d, J = 7.5, 4H, 4 × Ar-H), 7.59−7.51 (m, 4H, 4 × Ar-H), 7.39−7.32 (m, 4H, 4 × Ar-H), 7.30− 7.27 (m, 3H, 3 × Ar-H), 7.01 (d, J = 9.0, 2H, 2 × Ar-H), 6.91 (d, J = 8.2, 1H, Ar-H), 6.28 (s, 1H, Ar-H), 5.18 (t, J = 6.7, 1H, CH), 4.73− 4.61 (m, 2H, 2 × CH), 4.56−4.50 (m, 2H, 2 × CH), 4.44−4.26 (m, 6H, 3 × OCH2), 4.21−4.09 (m, 2H, OCH2), 3.87 (s, 3H, OCH3), 3.74 (s, 6H, 2 × OCH3), 3.52 (d, J = 6.5, 2H, CH2), 3.24−3.05 (m, 4H, 2 × CH2), 2.05−1.65 (m, 12H, 2 × CH3, 3 × CH2), 1.48−1.09 (m, 6H, 3 × CH2). 13C NMR (101 MHz, CDCl3) δ = 178.7, 172.3, 172.0, 168.7, 166.97, 162.2, 160.3, 160.2, 157.0, 156.4 (2 × C), 153.5, 144.0 (2 × C), 143.9 (2 × C), 143.9 (2 × C), 141.2 (2 × C), 138.0, 132.9, 130.2 (2 × CH), 127.6 (4 × CH), 127.6 (4 × CH), 127.0 (4 × CH), 125.0, 124.9, 122.0, 121.6, 119.9, 119.9, 114.5 (2 × CH), 108.4, 106.0, 96.0, 72.5, 67.6, 66.5, 55.5 (2 × CH), 52.5, 52.3, 51.8, 51.5, 47.2 (2 × CH), 40.7, 40.5, 32.0, 31.8, 29.3, 25.6 (2 × CH2), 22.6, 22.3, 21.8, 18.1. HRMS (APCI+): calculated for C69H73N4O16 [M + H]+ 1213.5016, found 1213.5011. Methyl (2-{[7-(2-{[(R)-6-Amino-1-methoxy-1-oxohexan-2-yl]amino}-2-oxoethoxy)-5-hydroxy-2-(4-methoxyphenyl)-8-(3-methylbut-2-en-1-yl)-4-oxo-4H-chromen-3-yl]oxy}acetyl)-L-lysinate (9). Compound 8 (200.2 mg, 0.165 mmol) was dissolved in 20% piperidine/DMF (5 mL). After stirring at room temperature for 20 min, the mixture was diluted with butanol and extracted with brine three times. The organic phase was dried over Na2SO4, filtered, and evaporated to dryness under reduced pressure. The crude product was purified by HPLC to yield compound 9 as a yellow solid (62.8 mg, 50%); mp 137−138 °C. 1H NMR (400 MHz, CD3OD) δ = 8.06 (d, J = 8.8, 2H, 2 × Ar-H), 7.12 (d, J = 8.9, 2H, 2 × Ar-H), 6.42 (s, 1H, ArH), 5.23 (t, J = 6.7, 1H, CH), 4.76 (s, 2H, OCH2), 4.58 (dd, J = 8.9, 5.1, 2H, 2 × CH), 4.47 (s, 2H, OCH2), 3.92 (s, 3H, OCH3), 3.77 (s, 6H, 2 × OCH3), 3.59 (d, J = 6.5, 2H, CH2), 3.01−2.86 (m, 4H, 2 × CH2), 2.05−1.92 (m, 2H, CH2), 1.87−1.62 (m, 12H, 3 × CH2, 2 × CH3), 1.62−1.38 (m, 4H, 2 × CH2). 13C NMR (101 MHz, CD3OD) δ = 178.6, 172.1, 172.0, 169.8, 168.7, 162.5, 161.2, 159.8, 157.0, 153.5, 137.1, 131.9, 130.2 (2 × CH), 122.0, 121.9, 114.1 (2 × CH), 108.7, 105.5, 95.9, 71.0, 67.2, 54.7, 51.7, 51.7, 51.6, 51.6, 39.1, 39.1, 30.7 (2 × CH2), 26.7, 26.7, 24.5, 22.3 (2 × CH2), 21.3, 16.9. HRMS (ESI+): calculated for C39H53N4O12 [M + H]+ 769.3654, found 769.3658. Dimethyl 2,2′-({(2S,2′S)-2,2′-[(2,2′-{[5-Hydroxy-2-(4-methoxyphenyl)-8-(3-methylbut-2-en-1-yl)-4-oxo-4H-chromene-3,7-diyl]bis(oxy)}-bis(acetyl))bis(azanediyl)]bis(5-guanidinopentanoyl)}bis(azanediyl))-(2S,2′S)bis(5-guanidinopentanoate) (11). Compound 6 (252 mg, 0.305 mmol) was dissolved in THF (4 mL)/methanol (4 mL), and a 5% LiOH aqueous solution (4 mL) was then added. The reaction mixture was stirred at room temperature for 1.5 h. After 6160



Article

2H, CH2), 1.85−1.76 (m, 3H, CH3), 1.69 (s, 3H, CH3). 13C NMR (101 MHz, CDCl3) δ = 179.0, 162.1, 161.3, 160.5, 156.7, 153.8, 137.2, 132.2, 130.9 (2 × CH), 123.0, 122.4, 114.3 (2 × CH), 108.5, 106.1, 95.8, 72.2, 68.6, 55.7, 30.1, 28.9, 26.0, 22.0, 18.3. HRMS (ESI+): calculated for C25H27Br2O6 [M + H]+ 581.0169, found 581.0169. 3,7-Bis(4-bromobutoxy)-5-hydroxy-2-(4-methoxyphenyl)-8-(3methylbut-2-en-1-yl)-4H-chromen-4-one (16). Compound 16 was prepared from icaritin (36.8 mg, 0.1 mmol), 1,4-dibromobutane (179.1 μL, 1.5 mmol), and potassium carbonate (69.1 mg, 0.5 mmol) using the same synthesis procedure as described for compound 3. This product was obtained as a yellow solid (55.6 mg, 87%); mp 111−113 °C. 1H NMR (400 MHz, CDCl3) δ = 12.73 (s, 1H, OH), 8.05 (d, J = 9.1, 2H, 2 × Ar-H), 7.02 (d, J = 9.1, 2H, 2 × Ar-H), 6.36 (s, 1H, ArH), 5.21−5.13 (m, 1H, CH), 4.08 (t, J = 5.9, 2H, OCH2), 4.00 (t, J = 6.1, 2H, OCH2), 3.90 (s, 3H, OCH3), 3.51−3.42 (m, 6H, 3 × CH2), 2.12−1.96 (m, 6H, 3 × CH2), 1.90−1.83 (m, 2H, CH2), 1.77 (s, 3H, CH3), 1.69 (s, 3H, CH3). 13C NMR (101 MHz, CDCl3) δ = 179.2, 161.9, 161.6, 160.4, 156.3, 153.5, 137.5, 131.8, 130.3 (2 × CH), 123.2, 122.4, 114.0 (2 × CH), 107.8, 105.6, 95.4, 71.5, 67.7, 55.5, 33.5, 33.1, 29.4, 29.3, 28.6, 27.8, 25.8, 21.7, 18.1. HRMS (APCI+): calculated for C29H35Br2O6 [M + H]+ 637.0795, found 637.0797. 3,7-Bis[(6-bromohexyl)oxy]-5-hydroxy-2-(4-methoxyphenyl)-8(3-methylbut-2-en-1-yl)-4H-chromen-4-one (17). Compound 17 was prepared from icaritin (73.6 mg, 0.2 mmol), 1,6-dibromohexane (461.5 μL, 3.0 mmol), and potassium carbonate (138.2 mg, 1.0 mmol) using the same synthesis procedure as described for compound 3. This product was obtained as a yellow solid (108.5 mg, 78%); mp 89−90 °C. 1H NMR (400 MHz, CDCl3) δ = 8.08 (d, J = 9.0 Hz, 2H, 2 × ArH), 7.01 (d, J = 9.1 Hz, 2H, 2 × Ar-H), 6.36 (s, 1H, Ar-H), 5.23−5.14 (m, 1H, CH), 4.04 (t, J = 6.3 Hz, 2H, OCH2), 3.99 (t, J = 6.5 Hz, 2H, OCH2), 3.90 (s, 3H, OCH3), 3.48 (d, J = 6.8 Hz, 2H, CH2), 3.42 (t, J = 6.8 Hz, 2H, CH2), 3.37 (t, J = 6.8 Hz, 2H, CH2), 1.95−1.64 (m, 14H, 4 × CH2, 2 × CH3), 1.58−1.48 (m, 4H, 2 × CH2), 1.46−1.38 (m, 4H, 2 × CH2). 13C NMR (101 MHz, CDCl3) δ = 179.4, 162.3, 161.7, 160.5, 156.3, 153.7, 137.8, 131.8, 130.5 (2 × CH), 123.6, 122.7, 114.1 (2 × CH), 107.9, 105.7, 95.6, 72.8, 68.7, 55.6, 34.0, 33.8, 32.9, 32.9, 30.1, 29.2, 28.1 (2 × CH2), 26.0, 25.5, 25.3, 21.9, 18.2. HRMS (ESI+): calculated for C33H43Br2O6 [M + H]+ 693.1421, found 693.1420. 3,7-Bis[(8-bromooctyl)oxy]-5-hydroxy-2-(4-methoxyphenyl)-8-(3methylbut-2-en-1-yl)-4H-chromen-4-one (18). Compound 18 was prepared from icaritin (73.6 mg, 0.2 mmol), 1,8-dibromooctane (552.5 μL, 3.0 mmol), and potassium carbonate (138.2 mg, 1.0 mmol) using the same synthesis procedure as described for compound 3. This product was obtained as a yellow solid (112.3 mg, 75%); mp 81 °C. 1 H NMR (400 MHz, CDCl3) δ = 8.09 (d, J = 9.0 Hz, 2H, 2 × Ar-H), 7.01 (d, J = 9.0 Hz, 2H, 2 × Ar-H), 6.36 (s, 1H, Ar-H), 5.34−5.02 (m, 1H, CH), 4.03 (t, J = 6.4 Hz, 2H, OCH2), 3.98 (t, J = 6.7 Hz, 2H, OCH2), 3.90 (s, 3H, CH3), 3.49 (d, J = 6.8 Hz, 2H, CH2), 3.40 (td, J = 6.8, 5.5 Hz, 4H, 2 × CH2), 1.91−1.66 (m, 14H, 4 × CH2, 2 × CH3), 1.51−1.26 (m, 16H, 8 × CH2). 13C NMR (101 MHz, CDCl3) δ = 179.5, 162.3, 161.7, 160.5, 156.3, 153.7, 137.9, 131.8, 130.5 (2 × CH), 123.7, 122.7, 114.1 (2 × CH), 107.8, 105.6, 95.6, 73.1, 68.9, 55.6, 34.1, 34.1, 33.0 (2 × CH2), 30.2, 29.3 (2 × CH2), 29.3, 28.9, 28.9, 28.3, 28.3, 26.1, 26.0, 26.0, 21.9, 18.2. HRMS (ESI+): calculated for C37H51Br2O6 [M + H]+ 749.2047, found 749.2050. 3,7-Bis[2-(diethylamino)ethoxy]-5-hydroxy-2-(4-methoxyphenyl)-8-(3-methylbut-2-en-1-yl)-4H-chromen-4-one (19). Compound 15 (22.4 mg, 0.038 mmol) was dissolved in DMSO (3 mL), and diethylamine (3 mL) was then added. After stirring at room temperature for 4 h, the mixture was diluted with ethyl acetate and washed with brine three times. The organic phase was dried over Na2SO4, filtered, and evaporated to dryness under reduced pressure. The crude product was purified by HPLC to yield compound 19 as a yellow gel (15.1 mg, 69%). 1H NMR (400 MHz, CD3OD) δ = 8.09 (d, J = 8.7 Hz, 2H, 2 × Ar-H), 7.10 (d, J = 8.7 Hz, 2H, 2 × Ar-H), 6.52 (s, 1H, Ar-H), 5.15 (t, J = 6.1 Hz, 1H, CH), 4.22 (t, J = 5.5 Hz, 2H, OCH2), 4.15 (t, J = 5.5 Hz, 2H, OCH2), 3.90 (s, 3H, OCH3), 3.52 (d, J = 6.1 Hz, 2H, CH2), 3.19 (t, J = 4.9 Hz, 2H, CH2), 3.10−2.93 (m, 6H, 3 × CH2), 2.72 (q, J = 7.2 Hz, 4H, 2 × CH2), 1.77 (s, 3H, CH3),

adding acetic acid, the mixture (pH < 7) was diluted with butanol and washed with brine three times. The organic phase was dried over Na2SO4, filtered, and evaporated to dryness under reduced pressure to yield the desired compound 10, which was used in the next reaction without further purification. Compound 10 was dissolved in anhydrous DMF (5 mL). The mixture was cooled to 0 °C, and HATU (348 mg, 0.915 mmol), DIPEA (318.7 μL, 1.83 mmol), and HArg-OMe·2HCl (239 mg, 0.915 mmol) were added. The reaction mixture was stirred at 0 °C for 1 h and was then stirred at room temperature overnight. The mixture was then diluted with butanol and extracted with brine three times. The organic phase was dried over anhydrous Na2SO4, filtered, and evaporated to dryness under reduced pressure. Subsequently, the crude product was purified by HPLC to yield compound 11 as a yellow solid (159 mg, 46%); mp 184−186 °C. 1 H NMR (400 MHz, CD3OD) δ = 8.05 (d, J = 8.9, 2H, 2 × Ar-H), 7.11 (d, J = 9.0, 2H, 2 × Ar-H), 6.42 (s, 1H, Ar-H), 5.20 (t, J = 6.7, 1H, CH), 4.76 (s, 2H, OCH2), 4.58−4.39 (m, 6H, 4 × CH, OCH2), 3.90 (s, 3H, OCH3), 3.74 (s, 3H, OCH3), 3.73 (s, 3H, OCH3), 3.58 (d, J = 6.3, 2H, CH2), 3.29−3.12 (m, 8H, 4 × CH2), 2.04−1.89 (m, 4H, 2 × CH2), 1.88−1.61 (m, 18H, 6 × CH2, 2 × CH3). 13C NMR (101 MHz, CD3OD) δ = 178.5, 172.5, 172.3, 172.2, 170.0, 169.2, 168.6, 162.5, 161.2, 157.4 (5 × C), 157.0, 153.6, 137.3, 132.0, 130.2 (2 × CH), 122.1, 121.8, 114.1 (2 × CH), 108.6, 105.5, 95.8, 71.2, 67.1, 54.8, 52.5, 52.5, 52.0, 51.9, 51.6, 51.5, 40.6, 40.6, 40.5 (2 × CH2), 29.2, 28.9, 28.2, 28.0, 24.9, 24.9, 24.8, 24.6, 24.5, 21.3, 17.0. HRMS (ESI+): calculated for C51H77N16O14 [M + H]+ 1137.5800, found 1137.5803. Ethyl 2-{[3,5-Dihydroxy-2-(4-methoxyphenyl)-8-(3-methylbut-2en-1-yl)-4-oxo-4H-chromen-7-yl]oxy}acetate (12). Compound 12 was prepared from icaritin (73.6 mg, 0.2 mmol), ethyl iodoacetate (23.6 μL, 0.2 mmol), and potassium carbonate (69.1 mg, 0.5 mmol) using the same synthesis procedure as described for compound 3. This product was obtained as a yellow solid (59.0 mg, 65%); mp 167−170 °C. 1H NMR (400 MHz, CDCl3) δ = 12.47 (s, 1H, OH), 8.16 (d, J = 9.0 Hz, 2H, 2 × Ar-H), 7.01 (d, J = 9.1 Hz, 2H, 2 × Ar-H), 6.56 (s, 1H, OH), 6.30 (s, 1H, Ar-H), 5.26 (t, J = 7.0 Hz, 1H, CH), 4.78 (s, 2H, OCH2), 4.20 (q, J = 7.1 Hz, 2H, OCH2), 3.88 (s, 3H, OCH3), 3.54 (d, J = 6.9 Hz, 2H, CH2), 1.82 (s, 3H, CH3), 1.74 (s, 3H, CH3), 1.24 (t, J = 7.1 Hz, 3H, CH3). 13C NMR (101 MHz, CDCl3) δ = 178.6, 169.3, 162.0, 160.9, 160.1, 155.8, 154.0, 136.8, 135.0, 130.8 (2 × CH), 123.2, 121.6, 114.2 (2 × CH), 106.0, 105.5, 99.6, 68.7, 61.4, 55.6, 26.0, 22.1, 18.2, 14.3. HRMS (ESI+): calculated for C25H27O8 [M + H]+ 455.1700, found 455.1711. Methyl (2-{[3,5-Dihydroxy-2-(4-methoxyphenyl)-8-(3-methylbut2-en-1-yl)-4-oxo-4H-chromen-7-yl]oxy}acetyl)-L-argininate (14). Compound 14 was prepared from compound 12 (55 mg, 0.121 mmol), HATU (69.2 mg, 0.182 mmol), DIPEA (63.2 μL, 0.363 mmol), and H-Arg-OMe·2HCl (47.5 mg, 0.182 mmol) using the same synthesis procedure as described for compound 5. This product was obtained as a yellow gel (60.5 mg, 84%). 1H NMR (400 MHz, CD3OD) δ = 7.97 (d, J = 8.7 Hz, 2H, 2 × Ar-H), 7.03 (d, J = 8.7 Hz, 2H, 2 × Ar-H), 6.24 (s, 1H, Ar-H), 5.16 (t, J = 6.4 Hz, 1H, CH), 4.65−4.55 (m, 1H, CH), 4.48−4.31 (m, 2H, OCH2), 3.87 (s, 3H, OCH3), 3.76 (s, 3H, OCH3), 3.41 (d, J = 6.5 Hz, 2H, CH2), 3.28− 3.16 (m, 2H, CH2), 2.10−1.81 (m, 2H, CH2), 1.79−1.68 (m, 5H, CH2, CH3), 1.65 (s, 3H, CH3). 13C NMR (101 MHz, CD3OD) δ = 179.7, 173.5, 171.5, 163.7 (2 × C), 160.7, 158.9, 157.8, 155.7, 138.5, 132.8, 131.5 (2 × CH), 123.7, 123.6, 115.5 (2 × CH), 108.3, 105.8, 99.7, 72.6, 56.2, 53.2, 53.1, 42.0, 29.9, 26.4, 26.0, 22.6, 18.3. HRMS (ESI+): calculated for C30H37N4O9 [M + H]+ 597.2555, found 597.2560. 3,7-Bis(2-bromoethoxy)-5-hydroxy-2-(4-methoxyphenyl)-8-(3methylbut-2-en-1-yl)-4H-chromen-4-one (15). Compound 15 was prepared from icaritin (73.6 mg, 0.2 mmol), 1,2-dibromoethane (258.2 μL, 3.0 mmol), and potassium carbonate (138.2 mg, 1.0 mmol) using the same synthesis procedure as described for compound 3. This product was obtained as a yellow solid (62.6 mg, 54%); mp 167−169 °C. 1H NMR (400 MHz, CDCl3) δ = 12.63 (s, 1H, OH), 8.2−8.08 (m, 2H, 2 × Ar-H), 7.08−6.98 (m, 2H, 2 × Ar-H), 6.34 (s, 1H, Ar-H), 5.25−5.16 (m, 1H, CH), 3.92−3.88 (m, 3H, OCH3), 3.68 (t, J = 6.0 Hz, 2H, OCH2), 3.59 (t, J = 6.3 Hz, 2H, OCH2), 3.53 (d, J = 6.9 Hz, 6161



Article

131.6 (2 × CH), 124.5, 123.9, 115.2 (2 × CH), 109.1, 106.4, 96.5, 74.0, 70.0, 56.2, 53.4 (2 × CH2), 48.2 (4 × CH2), 31.2, 30.5, 30.4, 30.4, 30.4, 30.3, 28.1, 28.1, 27.2, 27.1, 26.1, 25.7, 25.6, 22.8, 18.5, 9.9 (4 × CH3). HRMS (ESI+): calculated for C45H71N2O6 [M + H]+ 735.5307, found 735.5289. 3,7-Bis[4-(diethylamino)butoxy]-2-(4-methoxyphenyl)-8-(3methylbut-2-en-1-yl)-5-(pentyloxy)-4H-chromen-4-one (24). Compound 16 (40 mg, 0.063 mmol) was dissolved in acetone (10 mL), and 1-iodopentane (41.1 μL, 0.315 mmol) and Cs2CO3 (102.6 mg, 0.315 mmol) were then added. The reaction mixture was refluxed for 12 h. After cooling to room temperature, the mixture was diluted with ethyl acetate and washed with brine three times. The organic phase was dried over anhydrous Na2SO4, filtered, and evaporated to dryness under reduced pressure. The crude residue was dissolved in DMSO (3 mL), and diethylamine (3 mL) was then added. After stirring at room temperature for 4 h, the mixture was diluted with ethyl acetate and washed with brine three times. The organic phase was dried over Na2SO4, filtered, and evaporated to dryness under reduced pressure. The crude product was purified by HPLC to give compound 24 as a yellow gel (26 mg, 60%). 1H NMR (400 MHz, CD3OD) δ = 8.06 (d, J = 9.0, 2H, 2 × Ar-H), 7.10 (d, J = 9.0, 2H, 2 × Ar-H), 6.62 (s, 1H, ArH), 5.17 (t, J = 6.6, 1H, CH), 4.27−4.19 (m, 2H, OCH2), 4.16 (t, J = 6.5, 2H, OCH2), 3.95 (t, J = 5.6, 2H, OCH2), 3.91 (s, 3H, OCH3), 3.56 (d, J = 6.4, 2H, CH2), 3.24−3.12 (m, 6H, 3 × CH2), 3.10−3.01 (m, 6H, 3 × CH2), 1.99−1.82 (m, 10H, 5 × CH2), 1.79 (s, 3H, CH3), 1.69 (s, 3H, CH3), 1.62−1.54 (m, 2H, CH2), 1.51−1.42 (m, 2H, CH2), 1.32 (t, J = 7.3, 6H, 2 × CH3), 1.27 (t, J = 7.2, 6H, 2 × CH3), 1.00 (t, J = 7.3, 3H, CH3). 13C NMR (101 MHz, CD3OD) δ = 176.5, 163.2, 162.3, 160.3, 156.9, 155.7, 140.5, 132.9, 131.2 (2 × CH), 124.2, 123.7, 115.2 (2 × CH), 110.8, 109.7, 94.9, 73.1, 70.6, 69.3, 56.0, 53.1, 52.9, 48.4 (2 × CH2), 48.1 (2 × CH2), 29.9, 29.4, 28.3, 27.9, 25.9, 23.5, 22.8, 22.6, 22.5, 18.4, 14.4, 9.8 (2 × CH3), 9.4 (2 × CH3). HRMS (ESI+): calculated for C42H65N2O6 [M + H]+ 693.4837, found 693.4840. Antibacterial Assay. In vitro antimicrobial activities were determined on the basis of MIC values. The antibacterial activities of all compounds were determined in Mueller−Hinton broth (MHB) by standard broth microdilution methods according to the Clinical and Laboratory Standards Institute guidelines, as previously described.19 Compounds were dissolved in H2O or DMF and then diluted in PBS buffer to prepare 1000 μg/mL stock solutions. Serial 2-fold dilutions of the compounds were prepared from the stock solutions and diluted with cation-adjusted MHB in test tubes (final concentration of DMF = 2.5%). All tested organisms were grown in Tryptic Soy Agar (TSA) plates at 35 °C for 20 h and adjusted to approximately 5× 105 CFU/ mL. These bacterial suspensions were added to test tubes containing serial 2-fold dilutions of the compounds. The test tubes were then incubated at 35 °C for 24 h. The MIC values of these compounds were assessed by showing no visible growth compared with the control. All MICs were determined with biological replicates. Hemolytic Activity. The hemolytic activity of the antimicrobial compounds against RBCs in PBS buffer was used to evaluate their cytotoxic activity against mammalian cells. The hemolysis of RBCs was measured according to a previously reported protocol.20 Fresh New Zealand white rabbit RBCs were washed with PBS buffer three times and centrifuged at 3000 rpm for 10 min. Two-fold serial dilutions of the flavone analogues dissolved in PBS or DMF (final concentration of DMF = 0.5%) were incubated together with the 4% RBCs (final concentration v/v) at 37 °C for 1 h. These mixtures were then centrifuged at 3000 rpm for 3 min. The PBS were used as negative controls, while 2% Triton X-100 was used as a positive control. Supernatant (100 μL) aliquots of each sample were transferred into a 96-well plate, and the absorbance was measured at 576 nm using a TECAN infinite 200 microplate reader. The percent hemolysis was calculated using the following equation: % hemolysis = (Abssample − Absnegative control)/(Abspositive control − Absnegative control) × 100. All measurements were performed with biological replicates. Time-Kill Study. Various concentrations of compound 6 (0.5×, 1×, 2×, and 4× MIC) were used to inoculate MRSA DM21455 and MRSA DM9808R suspensions that had a starting inoculum of 105−

1.67 (s, 3H, CH3), 1.22 (t, J = 7.2 Hz, 6H, 2 × CH3), 1.12 (t, J = 7.2 Hz, 6H, 2 × CH3). 13C NMR (101 MHz, CD3OD) δ = 180.5, 163.9, 163.86, 161.6, 158.7, 155.1, 138.4, 133.0, 131.7 (2 × CH), 124.0, 123.8, 115.5 (2 × CH), 109.6, 106.5, 97.0, 69.8, 68.6, 56.2, 53.8, 52.6, 48.9 (4 × CH2), 26.0, 22.7, 18.5, 11.8 (2 × CH3), 10.6 (2 × CH3). HRMS (ESI+): calculated for C33H47N2O6 [M + H]+ 567.3429, found 567.3435. 3,7-Bis[4-(diethylamino)butoxy]-5-hydroxy-2-(4-methoxyphenyl)-8-(3-methylbut-2-en-1-yl)-4H-chromen-4-one (20). Compound 20 was prepared from compound 16 (30.9 mg, 0.048 mmol) and diethylamine (3 mL) using the same synthesis procedure as described for compound 19. This product was obtained as a yellow gel (23.5 mg, 78%). 1H NMR (400 MHz, CD3OD) δ = 8.09−8.00 (m, 2H, 2 × ArH), 7.14−7.04 (m, 2H, 2 × Ar-H), 6.49−6.41 (m, 1H, Ar-H), 5.19− 5.11 (m, 1H, CH), 4.18−4.08 (m, 2H, OCH2), 4.00 (t, J = 4.9, 2H, OCH2), 3.94−3.89 (m, 3H, OCH3), 3.53−3.43 (m, 2H, CH2), 3.14− 2.94 (m, 12H, 6 × CH2), 1.94−1.76 (m, 11H, 4 × CH2, CH3), 1.68 (s, 3H, CH3), 1.31−1.21 (m, 12H, 4 × CH3). 13C NMR (101 MHz, CD3OD) δ = 179.0, 168.9, 162.1, 160.0, 156.6, 153.4, 137.1, 131.4, 130.1 (2 × CH), 122.7, 122.3, 113.8 (2 × CH), 107.7, 105.0, 95.2, 71.6, 68.0, 54.7 (2 × CH2), 51.5, 46.9 (2 × CH2), 46.7 (2 × CH2), 26.9, 26.4, 24.5, 21.3, 21.2, 21.0, 17.0, 8.6 (2 × CH3), 8.3 (2 × CH3). HRMS (ESI+): calculated for C37H55N2O6 [M + H]+ 623.4055, found 623.4056. 3,7-Bis[4-(dimethylamino)butoxy]-5-hydroxy-2-(4-methoxyphenyl)-8-(3-methylbut-2-en-1-yl)-4H-chromen-4-one (21). Compound 21 was prepared from compound 16 (24.7 mg, 0.039 mmol) and dimethylamine (3 mL) using the same synthesis procedure as described for compound 19. This product was obtained as a lightyellow gel (20.6 mg, 94%). 1H NMR (400 MHz, CD3OD) δ = 8.01 (d, J = 8.8, 2H, 2 × Ar-H), 7.05 (d, J = 8.8, 2H, 2 × Ar-H), 6.38 (s, 1H, Ar-H), 5.11 (s, 1H, CH), 4.12−4.02 (m, 2H, OCH2), 3.99−3.92 (m, 2H, OCH2), 3.88 (s, 3H, OCH3), 3.42 (d, J = 5.8, 2H, CH2), 2.77 (t, J = 6.8, 2H, CH2), 2.69- 2.61 (m, 2H, CH2), 2.55 (s, 6H, 2 × CH3), 2.46 (s, 6H, 2 × CH3), 1.88−1.70 (m, 11H, 4 × CH2, CH3), 1.65 (s, 3H, CH3). 13C NMR (101 MHz, CD3OD) δ = 179.0, 169.0, 162.1, 160.0, 156.6, 153.4, 137.2, 131.3, 130.1 (2 × CH), 122.8, 122.3, 113.8 (2 × CH), 107.7, 104.9, 95.1, 71.8, 68.2, 58.4, 58.1, 54.7, 43.4 (2 × CH3), 43.0 (2 × CH3), 27.0, 26.5, 24.5, 22.8, 22.4, 21.2, 16.9. HRMS (ESI+): calculated for C33H47N2O6 [M + H]+ 567.3429, found 567.3431. 3,7-Bis{[6-(diethylamino)hexyl]oxy}-5-hydroxy-2-(4-methoxyphenyl)-8-(3-methylbut-2-en-1-yl)-4H-chromen-4-one (22). Compound 22 was prepared from compound 17 (44.5 mg, 0.064 mmol) and diethylamine (3 mL) using the same synthesis procedure as described for compound 19. This product was obtained as a yellow gel (34.7 mg, 80%). 1H NMR (400 MHz, CD3OD) δ = 8.03 (d, J = 8.3 Hz, 2H, 2 × Ar-H), 7.06 (d, J = 8.2 Hz, 2H, 2 × Ar-H), 6.38 (s, 1H, Ar-H), 5.19−5.09 (m, 1H, CH), 4.12−4.01 (m, 2H, OCH2), 3.97 (t, J = 6.1 Hz, 2H, OCH2), 3.91 (s, 3H, OCH3), 3.48−3.39 (m, 2H, CH2), 3.29−3.18 (m, 8H, 4 × CH2), 3.17−3.06 (m, 4H, 2 × CH2), 1.92− 1.66 (m, 14H, 4 × CH2, 2 × CH3), 1.64−1.39 (m, 8H, 4 × CH2), 1.34 (td, J = 7.2, 2.5 Hz, 12H, 4 × CH3). 13C NMR (101 MHz, CD3OD) δ = 180.6, 163.7, 163.5, 161.5, 157.8, 154.8, 138.8, 132.7, 131.6 (2 × CH), 124.4, 123.9, 115.2 (2 × CH), 109.1, 106.4, 96.6, 73.7, 69.9, 56.2 (2 × CH2), 53.0, 48.3 (4 × CH2), 30.9, 30.2, 27.6, 27.5, 26.9, 26.7, 26.1, 25.1, 24.9, 22.7, 18.5, 9.3 (4 × CH3). HRMS (ESI+): calculated for C41H63N2O6 [M + H]+ 679.4681, found 679.4693. 3,7-Bis{[8-(diethylamino)octyl]oxy}-5-hydroxy-2-(4-methoxyphenyl)-8-(3-methylbut-2-en-1-yl)-4H-chromen-4-one (23). Compound 23 was prepared from compound 18 (67 mg, 0.089 mmol) and diethylamine (3 mL) using the same synthesis procedure as described for compound 19. This product was obtained as a yellow gel (45.4 mg, 69%). 1H NMR (400 MHz, CD3OD) δ = 8.01 (d, J = 8.1 Hz, 2H, 2 × Ar-H), 7.02 (d, J = 7.9 Hz, 2H, 2 × Ar-H), 6.33 (s, 1H, Ar-H), 5.17−5.05 (m, 1H, CH), 4.05−3.97 (m, 2H, OCH2), 3.92 (t, J = 6.0 Hz, 2H, OCH2), 3.87 (s, 3H, OCH3), 3.46−3.36 (m, 2H, CH2), 3.08−2.96 (m, 8H, 4 × CH2), 2.94−2.86 (m, 4H, 2 × CH2), 1.83− 1.59 (m, 14H, 4 × CH2, 2 × CH3), 1.52−1.30 (m, 16H, 8 × CH2), 1.23 (td, J = 7.2, 3.5 Hz, 12H, 4 × CH3). 13C NMR (101 MHz, CD3OD) δ = 180.6, 170.4, 163.8, 163.5, 161.5, 154.8, 138.8, 132.6, 6162



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106 CFU/mL at 35 °C. Aliquots of 100 μL were removed from the cell cultures at 10 min, 30 min, 1 h, 2 h, and 4 h, were serially diluted 10fold in PBS buffer, were and plated onto Mueller−Hinton Agar medium. After incubation at 35 °C for 24 h, colonies were counted, and CFU per mL of total bacteria was calculated. All measurements were performed with biological replicates. Drug Resistance Study. The initial MIC values of compound 6 and two control antibiotics (gatifloxacin and norfloxacin) against S. aureus ATCC29213 were determined as described above. Bacteria from duplicate test tubes at a concentration of 0.5× MIC were used to prepare the bacterial dilution (approximately 5 × 105 CFU/mL) for the next MIC assay. Then these bacterial suspensions were incubated with compound 6 and the two control antibiotics. After incubation at 35 °C for 24 h, the fold increase in MIC value was determined. The process was repeated for 17 passages. All measurements were carried out with biological replicates. SYTOX Green Assay. The SYTOX Green assay was used to analyze the membrane integrity of MRSA DM9808R during treatment with the flavone compounds. In brief, bacteria (MRSA DM9808R) were grown on TSA plates to reach the exponential phase. The bacteria were then washed twice with PBS buffer (10 mM, pH 7) and resuspended in PBS to a cell density with an optical density at 600 nm (OD 600) of 0.2. The bacterial suspension was incubated with 0.3 μM SYTOX Green dye in the dark. The fluorescence of the SYTOX Green dye was monitored every 1 s for 50 min using a PTI spectrofluorometer at an excitation wavelength of 504 nm and an emission wavelength of 523 nm. Once the fluorescence signals stabilized, test samples dissolved in PBS or DMF (final concentration of DMF = 0.5%) were added to the SYTOX Green-treated suspensions, and the changes in fluorescence intensity were recorded. All tests included biological replicates. Calcein Leakage Assay. The homogeneous calcein-loaded LUVs (100 nm) were prepared from DOPE/DOPG (3:1, w/w) using the film hydration method as previously reported. The concentration of the calcein-loaded liposome solution (80 mM calcein, 100 mM NaCl, 50 mM HEPES, and 0.3 mM EDTA; pH 7.4) was measured using a total phosphorus determination method. The calcein leakage assays were performed in black immunological 96-well plates at room temperature. The test compounds prepared in DMF at different concentrations were then added to the aliquots of liposomes to achieve liposome-to-compound ratios of 8 and 16. Each well contained the same concentration of liposomes (50 μM) and the same amount of DMF (0.5%, v/v). The fluorescence emission intensity of calcein that had leaked from LUVs was determined using a TECAN infinite M200Pro microplate reader (excitation at 490 nm and emission at 520 nm) every 2 min for 30 cycles. Positive controls consisted of 0.1% Triton X-100, while negative controls consisted of 0.5% DMF. The percentage of leakage was determined according to the following equation: % L = [(Itest compound − Inegative control)/(Ipositive control − Inegative control)] × 100, where Itest compound is the fluorescence intensity after the addition of the test compounds, Inegative control is the fluorescence intensity after the addition of 0.5% DMF, and Ipositive control is the fluorescence intensity after the addition of 0.1% Triton X-100. All experiments were carried out with biological replicates. LDH Assay. The cellular toxicity to human corneal fibroblasts of compound 6 was evaluated by measuring the leakage of LDH in the presence of this compound, according to a previously described standard procedure.34,40 The incubation time with test samples was 24 h. The assay was carried out with biological replicates. Luminescent Cell Viability Assay. Cell viability was evaluated using a luminescent cell viability (ATP) assay according to a previously reported standard procedure.34 The assay was carried out in duplicate in 96-well opaque white plates seeded with human corneal fibroblasts at a density of approximately 10000 cells per well. After a 24 h incubation with a test sample, the plate was equilibrated for 0.5 h at 22 °C, and Cell Titer-Glo reagent (Promega Inc., USA) was added to each well. Finally, cell viability was assessed according to the manufacturer’s instructions. Luminescence was determined using a TECAN Infinite M200 Pro microplate reader. The addition of 1% Triton X-100 was used as a positive control, whereas cells treated with

medium was used as a negative control. The assay was carried out with biological replicates. In Vivo Efficacy. Wild-type 6−8-week-old C57BL6 mice weighing 20−30 g were used for this study. The treatment of all animals was in compliance with the ARVO statement for the Use of Animals in Ophthalmic and Vision Research and the Guide for the Care and Use of Laboratory Animals (National Research Council) and was under the supervision of SingHealth. MRSA ATCC 700699 or S. aureus ATCC 29213 was grown overnight in TSA at 35 °C. Bacterial stock at a concentration of 6 × 108 CFU/mL was prepared for mice corneal infection in PBS by suspending a few colonies from the plates. Slit-lamp photography was performed to screen for mice with good corneal clarity in the experiment. Each treatment group contained four mice in this study. The animals were anesthetized with 0.2 mL of ketamine (100 mg/mL) and 0.1 mL of xylazine (20 mg/mL) mixed with 0.7 mL of normal saline (0.08 mL per mouse). Corneal scratches (n = 3, each 1 mm long) were made using a sterile Mini-Blade (BD Beaver), which did not breach beyond the superficial stroma on the right eye, and the left eye remained untouched.49,50 The bacteria suspension (10 μL) was topically applied to the damaged cornea. Treatment started at day 1 post infection. In the S. aureus infection experiment, the respective drugs (0.5% compound 6, 0.5% levofloxacin, or PBS) dissolved in PBS (12 mM, pH 7.2) were administered totally 4 times at 2 h intervals. In the MRSA infection experiment, the respective drugs (0.5% compound 6, 5% vancomycin, or PBS) dissolved in PBS (12 mM, pH 7.2) were administered 5 times daily for 3 days. Finally, the animals were sacrificed and samples were plated for the determination of viable bacteria. At the end of the experiment, the infected corneas were dissected for the quantification of viable bacteria. In brief, the corneas from the respective groups were homogenized in sterile 0.9% NaCl containing 0.25% bovine serum albumin, serially diluted, and plated in duplicate on TSA plates.51,52 The plates were incubated for 48 h at 35 °C. Statistical tests (Mann−Whitney U test) were used for the statistical evaluations, and p values ≤0.05 were considered statistically significant.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.7b00380. H and and 13C NMR spectra of all final compounds (PDF) Molecular formula strings (CSV) 1

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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; Email, [email protected]. *For L.R.: phone, (+86) 20-39380255; E-mail, psliren@scut. edu.cn. ORCID

Rajamani Lakshminarayanan: 0000-0001-8214-5315 Li Ren: 0000-0003-0604-9166 Shouping Liu: 0000-0003-2415-076X Author Contributions

The manuscript was written through contributions from all authors. All authors have approved the final version of the manuscript. Notes

The authors declare no competing financial interest. 6163



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ACKNOWLEDGMENTS This work was supported by National Medical Research Council and Singhealth Foundation (SHF/FG538P/2013, 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 and Wei Hong Chor (SERI) for technical support in in vivo efficacy studies.

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ABBREVIATIONS USED MRSA, methicillin-resistant S. aureus; MSSA, methicillinsusceptible S. aureus; AMPS, antimicrobial peptides; HPLC, high-performance liquid chromatography; MIC, minimum inhibitory concentrations; RBC, red blood cell; ATP, adenosine triphosphate; LDH, lactate dehydrogenase; LUVs, large unilamellar vesicles; DMF, dimethylformamide; NMR, nuclear magnetic resonance; HATU, 2-(3H-[1,2,3]triazolo[4,5-b]pyridin-3-yl)-1,1,3,3-tetramethylisouronium hexafluorophosphate; DIC, N,N′-diisopropylcarbodiimide; HOBt, N-hydroxybenzotriazole; THF, tetrahydrofuran; CFU, colony forming units; TSA, tryptic soy agar; HRMS, high-resolution mass spectrometry; DOPG, 1,2-dioleoyl-sn-glycero-3-phospho-(1′rac-glycerol) (sodium salt); DOPE, 1,2-di(9Z-octadecenoyl)sn-glycero-3-phosphoethanolamine; CLSI, Clinical and Laboratory Standards Institute

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