Targeted Antibiotic Delivery: Selective Siderophore Conjugation with

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Targeted Antibiotic Delivery: Selective Siderophore Conjugation with Daptomycin Confers Potent Activity against Multidrug Resistant Acinetobacter baumannii Both in Vitro and in Vivo Manuka Ghosh,† Patricia A. Miller,†,‡ Ute Möllmann,† William D. Claypool,§ Valerie A. Schroeder,∥ William R. Wolter,∥ Mark Suckow,∥ Honglin Yu,⊥ Shuang Li,⊥ Weiqiang Huang,⊥ Jaroslav Zajicek,‡ and Marvin J. Miller*,†,‡ †

Hsiri Therapeutics, Innovation Park, 1400 East Angela Boulevard, South Bend, Indiana 46617, United States Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, United States § Hsiri Therapeutics, LLC, Rosetree Corporate Center, 1400 N. Providence Road, Building 1, Suite 115S, Media, Pennsylvania 19063, United States ∥ Frieman Life Sciences Center, University of Notre Dame, Notre Dame, Indiana 46556, United States ⊥ PracticaChem, 5 Lanyuan Road, Room D-603, Huayuan Industrial Park, Tianjin, 300384, China ‡

S Supporting Information *

ABSTRACT: In order to address the dire need for new antibiotics to treat specific strains of drug resistant Gram-negative bacterial infections, a mixed ligand analog of the natural Acinetobacter baumannii selective siderophore, fimsbactin, was coupled to daptomycin, a Gram-positive only antibiotic. The resulting conjugate 11 has potent activity against multidrug resistant strains of A. baumannii both in vitro and in vivo. The study also indicates that conjugation of siderophores to “drugs” that are much larger than the siderophore (iron transport agent) itself facilitates active uptake that circumvents the normal permeability problems in Gram-negative bacteria. The results demonstrate the ability to extend activity of a normally Gram-positive only antibiotic to create a potent and targeted Gram-negative antibiotic using a bacterial iron transport based sideromycin Trojan horse strategy.

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INTRODUCTION Bacterial resistance to antibiotics is now a major medical problem. The situation has been exacerbated by an antibiotic discovery and innovation gap that began in the 1960s.1 Only a few antibiotics have been introduced since 2000 when the oxazolidinones2 were marketed as a new class of antibiotics. Other subsequently approved antibiotics including daptomycin, a lipopeptide,3 and the pleuromutilins4 are still based on previously discovered scaffolds, and no totally new effective Gram-negative antibiotics have been developed in decades.5−7 Projected consequences of antimicrobial resistance are staggering, and by 2050 the problem will cause an estimated 50 million deaths and cost the global economy $100 trillion!8 Thus, the need for development of new antibiotics, delivery systems, and bacterial diagnostics is urgent. © 2017 American Chemical Society

While it is perhaps inevitable that rapidly reproducing bacteria will develop resistance to everything that is therapeutically acceptable, design of new antibiotics should anticipate and circumvent as many of the known resistance mechanisms as possible, including Gram-negative bacterial permeability barriers.9 In so doing, it might also be possible to repurpose antibiotics that are still useful for treatment of Gram-positive infections to also make them effective against Gram-negative bacteria. Careful targeting antibiotics to specific strains of bacteria will also minimize development of broad resistance. Herein, we describe the design, synthesis, and studies of a novel daptomycin conjugate that circumvents daptomycin’s lack of Received: January 23, 2017 Published: March 13, 2017 4577

DOI: 10.1021/acs.jmedchem.7b00102 J. Med. Chem. 2017, 60, 4577−4583

Journal of Medicinal Chemistry

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Figure 1. Generalized sideromycin structure and representative bacterial sideromycins of natural origin.

Figure 2. Fimsbactin (4), the natural siderophore utilized by A. baumannii, synthetic mixed ligand mimic (5), and synthetic antibiotic conjugate (6, Acinetobacter baumannii selective sideromycin).

A number of reports describe efforts to mimic natural sideromycins by design, syntheses, and studies of siderophoreantibiotic conjugates, most of which incorporate well-known antibiotics with the intent of expanding their activity by facilitating active transport of the antibiotic warhead into the targeted bacteria while also circumventing efflux pumps associated with the antibiotic alone. Those that utilize βlactams as the drug component do not require a release mechanism and have shown some efficacy but can suffer from inactivation by β-lactamases. Other attempts to develop conjugates with drugs such as ciprofloxacin that have cytoplasmic targets have, in general, not been effective, since appropriate release mechanisms were not incorporated.17 Daptomycin (9), a negatively charged lipopeptide antibiotic, is a valuable treatment only against Gram-positive bacteria.18,19 While full details of the mechanisms responsible for the Grampositive activity of daptomycin are not yet fully understood,19 it has been found to bind to the bacterial cell membranes and disrupt their function, causing rapid depolarization of the membrane, subsequent ion efflux, and eventual disruption of DNA, RNA, and protein syntheses that is lethal to the susceptible bacteria. Its large size (molecular formula of C72H101N17O26, MW = 1619.7) and polyanionic character indicate that it cannot penetrate Gram-negative bacterial outer membranes. Additional studies concluded that the daptomycin target is less prevalent in Gram-negative bacteria such as E. coli and P. aeruginosa so that even if daptomycin could permeate outer membranes of Gram-negative bacteria, it might not be effective.20 Prior structure−activity relationship (SAR) studies indicated that acylation of the peripheral amine of the ornithine residue in daptomycin was well tolerated and Gram-positive antibacterial activity was retained.21 We hypothesized that coupling of a Gram-negative targeted siderophore to daptomycin could circumvent problems associated with bacterial permeability by actively transporting daptomycin into bacteria. Assessment of the activity of the resulting conjugate would then ultimately determine target availability. Because infections caused by Gram-negative Acinetobacter baumannii are especially problematic and antibiotics that

activity against Gram-negative bacteria by combination with a specifically recognized siderophore analog to actively transport daptomycin into strains of Acinetobacter baumannii. The result is a potent, targeted agent 11 that is effective in vitro and in vivo. Competition for iron between a host and pathogenic bacteria is one of the most important factors in determining the course of a bacterial infection.10 Due to the extreme insolubility of ionic forms of iron, bacteria and fungi have evolved highly specific iron sequestration processes that involve energydependent active transport of relatively low molecular-weight iron chelators called siderophores.11 In Gram-negative bacteria, iron-siderophore complexes are preferentially recognized and bound by specific outer membrane receptors (OMR)/transporters. Binding of the siderophore-iron complexes initiates an energy-dependent active transport process that translocates the iron complex to the periplasm. This is often followed by active transport through the inner membrane.12 Hundreds of structurally distinct microbial siderophores have been identified, and more are discovered and reported frequently.13 The structural diversity is not a biosynthetic waste or redundant but careful evolution based on combination of an ideal match of molecular recognition between the siderophore and the outer membrane receptor/transporter protein to give a selective growth advantage for the producing organism. However, many bacteria do express outer membrane proteins that recognize and then utilize siderophores that are biosynthesized by other bacteria, thus exploiting the biosynthetic efforts of their competitors. As a counter to this iron thievery process, some bacteria synthesize natural siderophore-antibiotic conjugates called sideromycins. A generalized structure, 1, of sideromycins is shown in Figure 1, along with representative natural sideromycins (albomycins, 2,14 and salmycins, 315). The “warhead or drug” of the albomycins is a thionucleoside that is a potent tRNA synthetase inhibitor,16 whereas the salmycins incorporate a novel aminoglycoside.15 In both cases, the warhead must be released from the siderophore transport agent after assimilation in order for it to be recognized by and inhibit its cytoplasmic targets. 4578



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Figure 3. Synthesis of sideromycin 11, a mixed ligand siderophore-daptomycin conjugate.

Table 1. Minimum Inhibitory Concentrations (MICs, in μM) of 11 against Different Bacterial Species vs Daptomycin (Dapto) and Ciprofloxacin (Cipro) compound

A. baumannii ATCC 17961

A. baumannii ARC 3486

P. aeruginosa PAO1

Burkholderia multivorans AU0100

S. aureus SG511

S. aureus ATCC 11632

E. coli DCO

11 Dapto Cipro

0.4 >100 0.2

0.4 >100 >25

>100 >100 0.5

>100 >100 >25

6 0.05 0.5

12.5 0.4 0.5

>100 >100 0.5

Table 2. MICs (in μM) of 11 for Strains of A. baumannii, Including MDR Strains compound

ATCC 17961

ATCC BAA 1710

ATCC BAA 1793

ATCC BAA 1797

ATCC BAA 1800

ARC 3484

ARC 3486

ARC 5079

ARC 5081

ATCC 19606

11 10 amikacin Dapto Cipro

0.4 >50 1.7 >100 0.2

0.8 >50 >50 >100 >25

0.8 >50 >50 >100 >25

0.8 >50 27 >100 >25

0.8 >50 >50 >100 >25

0.4 >50 >27 >100 >25

0.4 >50 >50 >100 >25

0.8 >50 >50 >100 >25

0.4 >50 >50 >100 >25

0.8 >50 3.4 NT 0.8

Acinetobacter baumannii, as planned,24 but is less effective against β-lactamase producing strains unless coadministered with a β-lactamase inhibitor.

specifically target this often multidrug resistant strain of bacteria are desperately needed,22,23 our initial goal was to test our hypothesis by targeting A. baumanni. We previously designed and synthesized a mixed ligand (bis-catechol, monohydroxamate) siderophore mimic, 5, of the natural siderophore, fimsbactin, 4, used by pathogenic strains of Acinetobacter baumannii as an essential iron sequestering virulence factor during infections. Direct coupling of the siderophore mimetic, 5, to a carbacephalosporin gave the corresponding siderophoreantibiotic conjugate sideromycin 6 (Figure 2) that had remarkably selective and potent activity against strains of

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RESULTS AND DISCUSSION With the goal of determining whether daptomycin, which is not a β-lactam and thus not susceptible to β-lactamases, tethered to a siderophore designed to target A. baummanni could be effective, we synthesized the corresponding mixed ligand conjugate 11. As shown in Figure 3, zinc-mediated removal of the troc protecting group from the previously reported25 4579



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and salmycins emphasize that bacterial recognition of sideromycins by outer-membrane receptors/transporters is critical for efficacy. Mutants missing the appropriate recognition and transport proteins are not susceptible to the conjugates; however, in direct competition experiments with wild-type bacteria and its sideromycin resistant mutants, the recovery rate of the mutant was significantly lower than for the wild-type indicating that the mutant had reduced fitness in the mouse model. Thus, sideromycin treatment reduces the bacterial load allowing the immune system to remove residual albomycinresistant bacteria.29 Braun also demonstrated the in vitro and in vivo efficacy of albomycin in mice separately infected with S. pneumoniae or Yersinia enterocolitica.30 In fact, albomycin was used extensively in the Soviet Union during and prior to the 1950s to successfully treat human bacterial infections in adults and children without toxicity31 but was eventually discontinued apparently because of the then difficulty in obtaining adequate amounts of the natural product and the reported total synthesis of albomycins32 and salmycins33 were not practical for large scale production. Thus, biosynthetic sideromycins serve an important role in bacterial warfare in nature while also providing precedent for design of potential synthetic sideromycins. By the choice of daptomycin as the warhead of our synthetic sideromycin (11) to test the feasibility of targeting it against Gram-negative bacteria, the remaining choices were related to the siderophore component and linker. Since, as already indicated, modifications at the terminal amino group of the constituent ornithine residue of daptomycin are well tolerated, in contrast to the salmycins and albomycins, we did not anticipate that it would be necessary for the drug component, daptomycin, to be released from the siderophore after uptake by targeted bacteria. Furthermore, the target of daptomycin is within the cell wall and not in the cytoplasm as in the case of the natural sideromycins that require a drug release process. Thus, in contrast to the natural albomycins and salmycins that require intracellular release of the drug component once sequestered, we were able to incorporate a simple amide linkage between daptomycin and an appropriate siderophore.

pentabenzyl protected version of the mixed ligand siderophore 7 in the presence of glutaric anhydride gave 8, a protected synthetic siderophore with a glutarate linker. Direct reaction of a preformed mixed anhydride of 8 with an aqueous solution of daptomycin (9) gave pentabenzyl protected conjugate 10. Subsequent hydrogenolytic removal of the benzyl groups gave the desired sideromycin 11. The purity and structure of the final conjugate were confirmed by LC/MS and detailed multidimensional NMR studies that assigned each proton, carbon, and nitrogen resonance (see Supporting Information). In vitro assays of 11 by MIC determination (Tables 1 and 2) showed selective activity against several multidrug resistant strains of A. baumannii at submicromolar MIC levels. To demonstrate the importance of iron chelation for activity, intermediate 10, in which the iron binding moieties are protected, was tested and found to be completely inactive. Exposure of 11 to a source of ferric iron [Fe(acac)3] produced the corresponding iron complex, confirming that 11 binds iron stoichiometrically. Daptomycin alone had no activity (MIC > 100 μM). Synthetic sideromycin 11 had activity against Grampositive Staphylococcus aureus at 6−12.5 μM but was not as active as daptomycin alone. The retention of moderate activity against this Gram-positive strain again reflects toleration of the antibiotic target toward modification of the constituent ornithine of daptomycin itself. As with the previously reported mixed-ligand loracarbef conjugate 6, daptomycin conjugate 11 was not active against strains of Pseudomonas aeruginosa, Burkholderia, or E. coli, reflecting synthetic sideromycin-induced selectivity, as designed and expected. In vivo tolerability of 11 was determined by intravenous administration at 250 mg/kg in ICR mice with no observed adverse effect. A preliminary in vivo efficacy study with 11 was performed using an A. baumannii ATCC 17961 sepsis model. Thus, 6- to 8-week-old female ICR mice were infected intraperitoneal (ip) with A. baumannii ATCC 17961 and treatments were administered intravenously (iv) at 30 min and 24.5 h after infection. The treatments included ciprofloxacin (Cipro) at 50 mg/kg, 11 at 25 mg/kg, 10 mg/kg, and 5 mg/kg, daptomycin at 50 mg/kg, and vehicle (saline). All mice (6/6) in the vehicle study and all of the daptomycin treated animals (3/ 3) were dead after day 1. Survival rates for mice treated with 11 were as follows: 11 at 25 mg/kg 80% (4/5), 11 at 10 mg/kg 80% (4/5), 11 at 5 mg/kg 20% (1/5), indicating expected dose dependency. These results indicate that conjugation of siderophores to “drugs” such as daptomycin that are much larger than the siderophore (iron chelator) itself facilitates active uptake that circumvents the normal permeability problems in Gramnegative bacteria. The successful outcome relied on careful consideration of the components of natural sideromycins. All three parts (iron binder, linker, and warhead) are important for the ultimate activity of natural sideromycins. The choice of the siderophore component was important to demonstrate selectivity for a targeted strain of bacteria. Synthetic sideromycins should not just incorporate strong iron binders but consider iron binding stoichiometry (note the natural sideromycins in Figures 1−3 contain three bidentate ligands to fully coordinate iron) and structural aspects of the iron coordination sphere that are often critical for recognition by the transport proteins.26,27 Lack of attention to the details of molecular recognition and assay conditions in design and studies of synthetic sideromycins by other laboratories has minimized their effectiveness.28 Braun’s studies with natural albomycins

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CONCLUSION Synthetic sideromycin, 11, based on a mixed ligand daptomycin conjugate, is specifically recognized by and is highly active in vitro and in vivo against different strains of Gram-negative A. baumannii, including multidrug resistant (MDR) strains. This demonstrates the ability to extend potent activity of a normally Gram-positive only antibiotic to generate a potent Gramnegative antibiotic using the sideromycin Trojan horse strategy even when the warhead or drug is larger than the transporting siderophore component. This exciting result merits elaboration to determine the scope and limitations of development of other targeted sideromycins with daptomycin and other antibiotic warheads.

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EXPERIMENTAL METHODS

General Methods. Reactions were conducted under an atmosphere of dry argon unless otherwise stated. All solvents and reagents were obtained from commercial sources and used without further purification unless otherwise stated. Isobutyl chloroformate (ClCO2iBu) was used from Acros Seal anhydrous bottles. Technical grade tetrahydrofuran (THF) was freshly distilled over sodium before use. Sorbent Technologies silica gel 60 (32−63 μm) was used for all silica gel column chromatography purifications. Reverse phase

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(600 MHz, CDCl3) δ 1.24−1.58 (m, 10 H), 1.89 (t, J = 6 Hz, 1H), 2.34 (t, J = 6 Hz, 2H), 2.45−2.59 (m, 6 H), 3.08−3.24 (m, 9 H), 3.62 (br s, 1 H), 4.75 (br s, 2H), 5.07−5.13 (m, 10 H), 7.09−7.13 (m, 4 H), 7.25−7.39 (m, 11 H), 7.41−7.44 (m, 2 H), 7.62−7.69 (m, 2H), 7.78−8.03 (m, 2H); 13C NMR (150 MHz, CDCl3) δ 19.73 (br), 23.70, 24.85, 25.96, 26.37 (br), 26.61, 26.68, 27.43, 28.60, 28.67, 31.01 (br), 31.31 (br), 33.17 (br), 37.01, 37.07, 38.94, 39.16, 39.49, 43.65, 44.57, 45.71, 45.87, 47.67, 53.49, 71.18, 71.22, 71.24, 71.26, 76.18, 76.29, 76.39, 76.45, 76.52, 116.81, 116.87, 116.99, 117.11, 122.98, 123.01, 123.18, 124.37, 124.44, 124.47, 127.01, 127.09, 127.30, 127.49, 127.64, 127.65, 127.67, 127.71, 128.23, 128.27, 128.58, 128.59, 128.66, 128.67, 128.69, 128.72, 128.73, 128.75, 128.76, 128.77, 128.79, 128.93, 129.24, 134.44, 136.37, 136.41, 136.49, 136.54. LCMS (m/z): [M + H]+ calcd for C70H79N5O12, 1182.5798; found, 1182.5790. Sodium 2,2′-((3S,6S,9R,15S,18R,21S,24S,30S,31R)-30-((S)-2-((R)-4Amino-2-((S)-2-decanamido-3-(1H-indol-3-yl)propanamido)-4-oxobutanamido)-3-carboxylatopropanamido)-3-(2-(2-aminophenyl)2-oxoethyl)-6-((S)-1-carboxylatopropan-2-yl)-24-(7-(3-(2,3dihydroxybenzamido)propyl)-1-(2,3-dihydroxyphenyl)-18-hydroxy1,8,11,19,23-pentaoxo-2,7,12,18,24-pentaazaheptacosan-27-yl)-9(hydroxymethyl)-18,31-dimethyl-2,5,8,11,14,17,20,23,26,29-decaoxo-1-oxa-4,7,10,13,16,19,22,25,28-nonaazacyclohentriacontane15,21-diyl)diacetate (11). Formation of a Mixed Anhydride of 8. Benzyl-protected glutaryl siderophore 8 (780 mg, 0.660 mmol) was dissolved in freshly distilled, anhydrous THF (10 mL) in an HClwashed round-bottom flask, and the solution was cooled to 0 °C (ice bath temperature). N-Methylmorpholine (81 μL, 0.737 mmol, 1.12 equiv) was added to the flask under an argon atmosphere, followed by ClCO2i-Bu (96 μL, 0.735 mmol, 1.11 equiv). The reaction mixture was stirred at 0 °C under argon. After 1 h, the mixed anhydride formation was deemed complete when TLC (65% CH3CN in 10 mM NH4OAc; FeCl3 stain) showed complete consumption of starting siderophore 8. Coupling of the Mixed Anhydride of 8 with Daptomycin. Daptomycin (1.0 g, 0.617 mmol) and NaHCO3 (285 mg, 3.39 mmol, 5.5 equiv) were dissolved in LCMS grade H2O (20 mL) in a separate HCl-washed round-bottom flask under an argon atmosphere, and the resulting solution of daptomycin Na salt was cooled to 0 °C. Freshly prepared isobutyl chloroformate ester described above was filtered into the reaction mixture slowly over 45 min, washing with additional THF (15 mL). The reaction mixture was stirred at 0 °C for 2 h, then at room temperature for 2 h under an argon atmosphere to give the benzyl-protected conjugate, 10. Reaction progress was monitored by reverse phase C18 TLC (65% CH3CN in 10 mM NH4OAc; FeCl3 stain) that showed no remaining starting siderophore 8 or the corresponding isobutyl chloroformate ester after 2 h at room temperature. The reaction mixture was analyzed by LCMS. LCMS (m/z): [M + 2H]2+ calcd for C142H178N22O37, 1393.1450; found, 1393.1399; Rt 15.5 min. Hydrogenolytic Deprotection of the Tetrabenzyl Protected Daptomycin Containing Sideromycin 10. In order to minimize exposure to air and coloration, the reaction mixture containing the protected conjugate was diluted with water (25 mL) in a separate HClwashed round-bottom flask, resulting in a mixed solvent system of THF/water (1:3, 60 mL). The mixture was flushed with argon and then treated with 10% Pd−C (117 mg). The resulting suspension was flushed three times with hydrogen gas using intermediate vacuum evacuation and subjected to hydrogenolysis at room temperature under 1 atm of hydrogen for 8 h. Reaction progress was monitored by reverse phase C18 TLC (65% CH3CN in 10 mM NH4OAc; FeCl3 stain) that showed no remaining starting protected conjugate. The reaction mixture was purged with argon, filtered, washed with water (LCMS grade), and lyophilized to provide the fully deprotected mixed ligand daptomycin conjugate 11 as a white solid (1.5 g, yield >99%, purity 88 area %, UV). Conjugate 11 was further purified in three batches (500 mg each) by three consecutive reverse phase chromatography (RediSep Rf Gold reversed-phase C18 high performance columns, 30 g) using a gradient 10−50% of CH3CN/NH4OAc (10 mM) as the eluent. The desired fractions collected at 20−30% of CH3CN/NH4OAc (10 mM) from three batches were combined and lyophilized to give the polyammonium salt form of 11 as a yellowish fluffy solid (801 mg, yield 53%, purity >99 area %, UV). LCMS neutral

chromatographic purifications were performed on Teledyne Instruments’ 30 g RediSep Rf Gold C18Aq reversed-phase columns (column volume of 26.4 mL, average particle size of 20−40 μm, average pore size of 100 Å) at a flow rate of 35 mL/min. Thin layer chromatography (TLC) was performed with Al-backed Merck 60-F254 or Al-backed Merck RP-C18 F256 silica gel plates using a 254 nm lamp and aqueous FeCl3 for visualization. HPLC−MS mass measurements were used to determine purity as well as structural consistency. All characterized compounds were determined to be ≥95% pure. The HPLC−MS studies were performed with a Bruker MicrOTOF-Q II quadrupole time-of-flight mass spectrometer operating in positive ion mode with acquisition mass range 50−3000 u. Electrospray ionization source parameters were the following: capillary voltage = 2200 V, end slate offset = −500 V, nebulizer gas pressure = 5 bar, dry gas flow rate = 10 L/min, and dry gas temperature = 220 °C. Liquid separation was performed on a Dionex UltiMate 3000 RSLC with mobile phases consisting of water with 0.1% formic acid (A) and acetonitrile with 0.1% formic acid (B). The mobile phase gradient at 0.4 mL/min was 10% of B for 2 min followed by linear ramp of 100% of B at 18 min and a return to initial conditions from 18.1 to 20 min. The mobile phase for the iron complex consisted of water (A) and acetonitrile (B). UV−vis spectra were recorded on a Dionex UltiMate 3000 RS diode array detector over the wavelength range of 190−400 nm. The LC column was a Thermo Scientiffic Acclaim RSLC 120 C18 with 2.2 μm particle size, 120 Å pore size, and 2.1 mm × 100 mm dimensions heated at 40 °C. 1D 1H and 13C{1H} spectra of the compound 8 were recorded on a Varian DirectDrive 600 spectrometer operating at a proton resonance frequency of 599.98 MHz. For compound 11, 1D 1H and 13C{1H}, 2D homo DQF-COSY, TOCSY, NOESY, and heteronuclear 1H−13C HSQC, HSQC-TOCSY, HMBC, 1H−15N HSQC spectra were obtained using a four-channel Bruker AVANCE II 800 spectrometer operating at a 1H resonance frequency of 800.18 MHz and equipped with a 5 mm TCI cryoprobe. All experiments were performed at 25 °C with standard pulse sequences in the Bruker TopSpin 3.2 pl6 software. Suppression of the water signal was accomplished by Watergate or Echo-Antiecho techniques. 1D 1H, 2D homonuclear, and 1H dimension in 2D heteronuclear spectra were referenced to the residual H2O signal, δH = 4.70 ppm. The 1D 13C{1H} and the 13C and 15N dimensions in the 2D heteronuclear spectra were referenced indirectly. The sample for NMR measurements was prepared by dissolving the purified compound 11 to final concentration of 1 mM in solution containing 90% 1H2O and 10% 2H2O, pH = 5.0. The structure of compound 11 was verified by analysis and interpretation of 1D 1H and 13C{1H} and 2D homo DQF-COSY, TOCSY, NOESY and heteronuclear 1H−13C HSQC, HSQC-TOCSY, HMBC, 1H−15N HSQC spectra. Residue specific assignments of the 1 H resonance signals were performed by standard methods using DQF-COSY, TOCSY, and 1H−15N HSQC, followed by sequential assignments through NOE connectivities. 13C resonances corresponding to carbons with directly attached protons were assigned using 1 H−13C HSQC and HSQC-TOCSY. The 1H−13C HMBC spectrum was then used to assign resonances of quaternary carbons and to validate the connectivities established by the other spectra. 15N resonance signals of the amide nitrogens were unambiguously assigned by analysis of the 1H−15N HSQC spectrum. The complete 1H, 13C, and 15N resonance assignments for compound 11 are given in Table S1. 18-(Benzyloxy)-7-(3-(2,3-bis(benzyloxy)benzamido)propyl)-1(2,3-bis(benzyloxy)phenyl)-1,8,11,19-tetraoxo-2,7,12,18-tetraazatricosan-23-oic Acid (8). To a solution of compound 725 (1.0 g, 0.80 mmol) in THF/AcOH (1:1, 20 mL) were added glutaric anhydride (459 mg, 4.02 mmol) and freshly activated zinc dust (504 mg, 8.01 mmol). The reaction mixture was vigorously stirred at room temperature for 10 h, filtered and the solvent removed under reduced pressure. The residue was diluted with EtOAc, and the organic layer was washed with H2O, brine, dried over magnesium sulfate, filtered, and concentrated. The crude compound was purified by silica gel chromatography, eluting with CHCl3/i-PrOH/AcOH (90:10:1) as the eluent to afford compound 8 (852 mg, 90%) as a sticky oil. 1H NMR 4581



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Chart 1

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form (m/z): [M + 2H]2+ calcd for C107H148N22O37, 1168.0276; found, 1168.0278; Rt 11.1 min. Neutralization and Na Salt Formation. The isolated salt form of conjugate 11 (801 mg, 0.343 mmol) was dissolved in CH3CN/H2O (1:1, 10 mL) and acidified with 3 N HCl to pH ≈ 2 to obtain some solid white precipitation of the neutral conjugate 11. The combined mixture, after lyophilization, was passed through a short reverse phase column to remove any inorganic salt. Lyophilization of the fractions containing the product provided the neutral conjugate (700 mg, 87%). Then 700 mg (0.300 mmol) of isolated conjugate was dissolved in CH3CN/H2O (1:1, 20 mL) and the solution was treated with NaHCO3 (100 mg, 1.19 mmol, 5 equiv) in water (2 mL, LCMS grade), and the resulting solution was lyophilized to provide the desired Na salt of 11 in quantitative yield. LCMS as neutral form (m/ z): [M + 2H] 2+ calcd for C 107 H 144 N 22 Na 4 O 37 , calcd for C107H148N22O37, 1168.0276; found, 1168.0307; tR = 11.2 min. Syntheses of the Na Salt of the Mixed Ligand−Daptomycin Conjugate−Iron(III) Complex. For further characterization, conjugate 11 (5.0 mg, 0.002 mmol) in MeOH (1 mL) was treated with a solution of Fe(acac)3 (1.1 mg, 0.003 mmol) in MeOH (1 mL). The dark purple solution was stirred at room temperature for 18 h. The MeOH was removed under reduced pressure, and the desired siderophore-iron(III) complex was isolated as a purple solid (5 mg, 0.015 mmol). LCMS (m/z): [M + 2H]2+ calcd for C107H145N22O37Fe, 1194.4698; found, 1194.4834; tR = 7.2 min. These results indicate that conjugate 11 binds iron stoichiometrically as expected. Structure Confirmation of 11 and Tautomer 11a. The structure of 11 was established by high resolution 1D/2D NMR studies. On the basis of the NMR data (see Supporting Information), two isomeric structural forms, 11 and 11a, of the Na salt are present in solution as shown in Chart 1. Sodium 2,2′-((3S,6S,9R,15S,18R,21S,24S,30S,31R)-30-((S)-2-((R)4-amino-2-((S)-2-decanamido-3-(1H-indol-3-yl)propanamido)-4-oxobutanamido)-3-carboxylatopropanamido)-3-(2-(2-aminophenyl)-2-oxoethyl)-6-((S)-1-carboxylatopropan-2-yl)-24-(7-(3-(2,3-dihydroxybenzamido)propyl)-1-(2,3-dihydroxyphenyl)-18-hydroxy-1,8,11,19,23pentaoxo-2,7,12,18,24-pentaazaheptacosan-27-yl)-9-(hydroxymethyl)18,31-dimethyl-2,5,8,11,14,17,20,23,26,29-decaoxo-1-oxa4,7,10,13,16,19,22,25,28-nonaazacyclohentriacontane-15,21-diyl)diacetate is 11. Sodium 2,2′-((3S,6S,9R,15S,18R,21S,24S,30S,31R)-30-((S)-2-((R)4-amino-2-((S)-2-decanamido-3-(1H-indol-3-yl)propanamido)-4-oxobutanamido)-3-carboxylatopropanamido)-3-(2-(2-aminophenyl)-2-oxoethyl)-6-((S)-1-carboxylatopropan-2-yl)-24-(1-(2,3-dihydroxyphenyl)-7-(3-(((Z)-(2,3-dihydroxyphenyl)(hydroxy)methylene)amino)propyl)-18-hydroxy-1,8,11,19,23-pentaoxo-2,7,12,18,24-pentaazahe ptacosan-27-yl)-9-(hydr oxyme thyl)-18,31-dimethyl2,5,8,11,14,17,20,23,26,29-decaoxo-1-oxa-4,7,10,13,16,19,22,25,28nonaazacyclohentriacontane-15,21-diyl)diacetate is 11a.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.7b00102. Molecular formula strings (CSV) Chemical compound characterization (NMR, LC/MS), assay protocols, and source data (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: 574-631-7571. ORCID

Marvin J. Miller: 0000-0002-3704-8214 Author Contributions

M.G. performed the synthesis of the final conjugate and penultimate precursors. M.G. also provided essential details for the assay protocols. W.H., H.Y., and S.L. performed the syntheses of the siderophore components; P.A.M. performed the microbiological assays; U.M. and W.D.C. contributed to the development and implementation of the research plan; V.A.S., W.R.W., and M.S. performed in vivo assays; J.Z. performed and analyzed multidimensional NMR studies; M.J.M. conceived, designed, and directed the research plan and wrote the manuscript. Notes

The authors declare the following competing financial interest(s): Manuka Ghosh is a full time employee of Hsiri Therapeutics. Patricia A. Miller is a part time employee of Hsiri Therapeutics. Ute Mö llmann is a consultant for Hsiri Therapeutics. William D. Claypool and Marvin J. Miller are founders of and have a financial interest in Hsiri Therapeutics. Weiqiang Huang has a financial interest in Hsiri Therapeutics.

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ACKNOWLEDGMENTS We thank B. Boggess, V. Krchnak, and N. Sevov for LC/MS assistance. This work was supported by the Department of Defense (Grant W81XWH-12-2-0015).

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ABBREVIATIONS USED MDR, multidrug resistant; MIC, minimum inhibitory concentration; OMR, outer membrane receptor; Dapto, daptomycin; Cipro, ciprofloxacin 4582



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(24) Wencewicz, T. A.; Miller, M. J. Biscatecholate−monohydroxamate mixed ligand siderophore−carbacephalosporin conjugates are selective sideromycin antibiotics that target Acinetobacter baumannii. J. Med. Chem. 2013, 56, 4044−4052. (25) Ghosh, M.; Miller, M. J. Synthesis and in vitro antibacterial activity of spermidine-based mixed catechol- and hydroxamatecontaining siderophore-vancomycin conjugates. Bioorg. Med. Chem. 1996, 4, 43−48. (26) Ferguson, A. D.; Hofmann, E.; Coulton, J. W.; Diederichs, K.; Welte, W. Siderophore-mediated iron transport: crystal structure of FhuA with bound lipopolysaccharide. Science 1998, 282, 2215−2220. (27) Zheng, T.; Nolan, E. M. Enterobactin-mediated delivery of βlactam antibiotics enhances antibacterial activity against pathogenic Escherichia coli. J. Am. Chem. Soc. 2014, 136, 9677−9691. (28) Tomaras, A.; Crandon, J. L.; McPherson, C. J.; Banevicius, M. A.; Finegan, S. M.; Irvine, R. L.; Brown, M. F.; O’Donnell, J. P.; Nicolau, D. P. Adaptation-based resistance to siderophore-conjugated antibacterial agents by Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 2013, 57, 4197−4207. (29) Braun, V.; Pramanik, A.; Gwinner, T.; Köberle, M.; Bohn, E. Sideromycins: tools and antibiotics. BioMetals 2009, 22, 3−13. (30) Pramanik, A.; Stroeher, U. H.; Krejci, J.; Standish, A. J.; Bohn, E.; Paton, J. C.; Autenrieth, I. B.; Braun, V. Albomycin is an effective antibiotic, as exemplified with Yersinia enterocolitica and Streptococcus pneumonia. Int. J. Med. Microbiol. 2007, 297, 459−469. (31) Gause, G. F. Recent studies on albomycin, a new antibiotic. Br. J. Med. 1955, 2, 1177−1179. (32) Benz, G.; Schroder, T.; Kurz, J.; Wünsche, C.; Karl, W.; Steffens, G.; Pfitzner, J.; Schmidt, D. Konstitution der deferriform der albomycine δ1, δ2, und ε. Angew. Chem., Int. Ed. Engl. 1982, 21, 527−528. (33) Dong, L.; Roosenberg, J. M., II; Miller, M. J. Total synthesis of desferrisalmycin B. J. Am. Chem. Soc. 2002, 124, 15001−15005.

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