Article pubs.acs.org/jmc
Design, Synthesis, and Evaluation of Fimbrolide−Nitric Oxide Donor Hybrids as Antimicrobial Agents Samuel K. Kutty,*,† Nicolas Barraud,‡ Amy Pham,‡ George Iskander,† Scott A. Rice,‡ David StC. Black,† and Naresh Kumar*,† †
School of Chemistry, and ‡School of Biotechnology and Biomolecular Sciences and Centre for Marine Bio-Innovation, The University of New South Wales, Sydney, NSW 2052, Australia S Supporting Information *
ABSTRACT: Fimbrolides from marine algae have shown promising activity against quorum sensing (QS), a chief regulatory and communication system in bacteria controlling biofilm formation and virulence factor. Nitric oxide (NO) at sublethal concentration has also been reported to induce dispersal of bacterial biofilms and increase their susceptibility toward standard biocides and antibiotics. Therefore, the combination of QS inhibitors and NO donors has the potential to control the development of biofilm and promote their dispersion via a nonbactericidal mechanism. Inspired by these ideas, novel fimbrolide−NO donor hybrid compounds were designed and synthesized. Fimbrolide−NO hybrids 6b, 6f, and 14a were found to be particularly effective as antimicrobials compared to the nonhybrid natural fimbrolides as revealed by bioluminescent P. aeruginosa QS reporter assays and biofilm inhibition assays. Significantly, these fimbrolide−NO hybrids represent the first dual-action antimicrobial agent based on the baterial QS inhibition and NO signaling.
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INTRODUCTION Bacteria have evolved a range of adaptive strategies that facilitate their survival in challenging environments, including their ability to coordinate behavior at a population level as well as the formation of protective structure known as biofilms. Within a host environment, such survival mechanisms aid bacteria in colonizing host tissues and promote their resistance to immune responses as well as antibiotic treatments. The longterm colonization by bacteria can lead to chronic inflammatory and infectious conditions, such as in the lungs of cystic fibrosis patients, the wounds of diabetic and burn patients, or on implanted biomedical devices.1 Besides posing major challenges in clinical therapy, biofilms in the environment can act as reservoirs of pathogens leading to serious downstream health risks. For example, biofilm formation can contaminate potable water distribution systems, or cause biofouling and corrosion in sewer pipes and heat exchange systems.2 The expression of virulence factors and biofilm formation are controlled by an array of factors, of which quorum sensing (QS) has been shown to play a key role.3 QS is a coordinated cell-to-cell communication system mediated by a rich lexicon of diffusible molecules called autoinducers, which allows cells to regulate the expression of phenotypes related to virulence and biofilm formation in response to changes in the surrounding cell density.4 The most widely studied QS system is the autoinducer-1 (AI-1) system, which is mediated by N-acylated homoserine lactones (AHL) 1 and is present in many Gramnegative human pathogens such as Pseudomonas aeruginosa.5 © 2013 American Chemical Society
Thus, inhibition of QS is a potential strategy to control virulence factor expression and biofilm development. A wide range of synthetic and natural product-based QS inhibitors have been explored in the last two decades. Fimbrolides 2 are halogenated furanones isolated from the marine red algae Delisea pulchra and are known for their potent QS inhibitory activities.6,7 Fimbrolides and their synthetic derivative, for example 3, have been shown to act by competing with the native QS autoinducers 1 for the cognate receptor and/or to destabilize the receptors.8,9 Consequently, fimbrolides are able to inhibit QS-mediated phenotypes such as virulence factor expression and biofilm formation without killing the bacteria and thereby avoids exerting strong selective pressure on bacteria that leads to resistance.10,11
Another promising strategy in biofilm control is based on the use of nitric oxide (NO) signals to induce the natural dispersal of mature biofilms. NO is an ubiquitous biological signal that plays a key role as endogenous mediator in the human body as Received: June 25, 2013 Published: November 5, 2013 9517
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For the preparation of fimbrolides with ester linkages, the precursor hydroxy fimbrolide 7a was prepared from bromofimbrolide 5b by stirring at room temperature for 72 h in DMSO with a few drops of water (Scheme 2). For the fimbrolide thioester analogues, the thiol derivative 8 was prepared from the bromofimbrolide 5b as described in the literature.23 Mononitrooxy-substituted fimbrolide derivatives 14a−i attached via an ester or thioester linker were synthesized according to the pathways shown in Scheme 3. Bromosubstituted alkanoic acids 9a−f were transformed into the corresponding acyl chlorides 10a,b by thionyl chloride (SOCl2). The acyl chlorides were conjugated to fimbrolide alcohol 7a or thiol 8 to give ester or thioester intermediates 11a−c in high yields. Molecules 11a−c were reacted with AgNO3 in refluxing acetonitrile to afford the corresponding mononitrooxy-substituted fimbrolides 14a,c,h in 67−74% yield. The second strategy for the preparation of the target products 14 was carried out from the nitrooxy-substituted alkanoic acids 12a−d. These acids were transformed into the corresponding acyl chlorides 13a−d by the action of SOCl2. Coupling of 13a− d with fimbrolides 7a and 8 in presence of pyridine gave the products 14b, 14d−g, and 14i. For the synthesis of O2-substituted fimbrolide diazeniumdiolates, C-1′ bromo-substituted fimbrolide derivative 5b was initially reacted with diazeniumdiolate 15a under a variety of conditions. However, the reactions were found to be inefficient and yielded undesired products. The second approach for the preparation of O2-substituted fimbrolide diazeniumdiolate derivatives involved the reaction between bromoacetylsubstituted fimbrolide derivative 11a and the sodium diazeniumdiolate derivatives 15a−c in DMSO, which afforded the desired products 16a−c in moderate yields (Scheme 4). The final strategy involved linking the fimbrolide and the diazeniumdiolate via an amide linker. To achieve this, the fimbrolide acid derivatives 18a−d were prepared by the reaction of hydroxy fimbrolides 7a−c with diacid chloride derivatives 17a,b. The fimbrolide acid derivatives 18a−d and diazeniumdiolate 19 were subsequently coupled using EDC to yield the fimbrolide diazeniumdiolate derivatives 20a−d (Scheme 5). QS Inhibition Assay. The QS inhibitory effects of the fimbrolide−NO hybrids were evaluated using the QS reporter screening system developed by Hentzer et al.11 The reporter strain, P. aeruginosa PlasB::gf p, produces its own AHL signals, and an increase in green fluorescent protein (GFP-ASV) production, as a consequence of QS induction, can be observed during the normal growth of the strain. An addition of a QS inhibitor to this bioreporter will result in a lowered expression of GFP-ASV to an extent that correlates with the efficacy of the inhibitor. In the assay, PlasB::gf p bacteria were incubated with various concentrations of compounds, and half hourly measurements of gf p expression (relative fluorescence units, RFU) and cell growth (OD600) were obtained. Representative data are shown for derivative 6b in Figure 2. The percentage QS inhibition of the compounds was calculated as the percentage difference in RFU value between the sample and the control at the time point when the fluorescence reached its maximum value in the control. The most active QS inhibitors in the nitrooxy series were 14a and 6b, which showed 73% and 71% QS inhibition at 88 μM, respectively (Table 1). By comparison, the fimbrolide diazeniumdiolate hybrid derivatives 16a−c showed 42−50% QS inhibition activity at 88 μM (Table 1). The QS inhibitory
well as a signaling molecule in various bacterial systems.12,13 In biofilms, sublethal, nanomolar concentrations of NO were found to prevent initial biofilm formation and induce the transition from a sessile to a free-swimming, planktonic mode of growth in several bacterial species, including pathogens such as P. aeruginosa and Vibrio cholera as well as in mixed-species biofilms established from water distribution and treatment systems.14,15 Further, exogenous NO has been shown to increase the sensitivity of various biofilms to antimicrobial treatments, transforming resistant biofilm populations into sensitive planktonic cells.14 The molecular mechanisms of NO-mediated biofilm dispersal at low NO concentrations were found to involve the secondary messenger c-di-GMP.16 Gene expression studies revealed that exposing biofilms of P. aeruginosa to low, nontoxic levels of NO resulted in the up-regulation of genes involved in motility and energy metabolism, and the genes involved in attachment and virulence were repressed.16 Studies have also shown evidence that NO and QS systems are inter-related and that NO plays significant role in controlling the QS system in different species such as P. aeruginosa and Vibrio harveyi at the molecular level.17,18 Thus, NO could be used as an agent against biofilm formation and virulence expression. As fimbrolide analogues and NO act to control the biofilm lifecycle and virulence without inhibiting the bacterial growth, hybrid molecules based upon fimbrolide derivatives and NO donors could potentially represent a potent new class of antivirulent or antibiofilm agents.19−21 We describe herein the design, synthesis, and biological efficacy of hybrid compounds based on fimbrolide analogues with nitrooxy or diazeniumdiolate NO donor substituents.
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RESULTS Synthetic Scheme. The fimbrolide analogues 4a−f were synthesized from the sulfuric acid-catalyzed cyclization of levulinic acid derivatives.22 The nitrooxy-substituted fimbrolide derivatives were synthesized according to the pathway illustrated in Scheme 1. Brominated fimbrolide derivatives
Scheme 1. Synthesis of Fimbrolide Nitrooxy Derivativesa
Reagents and conditions: (i) NBS, benzoyl peroxide, ℏν, CCl4, 94− 98%; (ii) AgNO3, CH3CN, 60 °C, 70−81%. a
were prepared through photochemical allylic bromination with N-bromosuccinimide (NBS) as the brominating agent. The corresponding nitrooxy-substituted fimbrolides 6a−f were prepared in high yields from the brominated fimbrolide 5a−f by treatment with AgNO3 in refluxing acetonitrile. Figure 1 shows the crystal structure of 6c and the halogen bonding interactions in the crystal lattice, consistent with our previous report.23 9518
dx.doi.org/10.1021/jm400951f | J. Med. Chem. 2013, 56, 9517−9529
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Figure 1. ORTEP structure and the halogen bond (C−Br···O) interaction in dimeric unit of 6c.
Scheme 2. Synthesis of Fimbrolide Hydroxyl and Thiol Derivativesa
Scheme 4. Synthesis of Fimbrolide Diazeniumdiolate Derivatives via O2 Attachmenta
a Reagents and conditions: (i) DMSO, H2O (a few drops), 72 h, 56− 62%; (ii) KSAc, acetone, rt, then 0.3 M methanolic HCl, 50 °C, 46%.
a
Reagents and conditions: (i) DMSO, NaHCO3, 0−20 °C, 40−58%.
under in vitro experimental conditions and the high detection limit of nitrite (≥1 μM) by the Griess reagent,25,26 relatively high concentrations of 250 μM and 500 μM were required to be within the detection limit. The results showed that fimbrolide nitrooxy hybrids 6f and 14a had the highest nitrite (NO) release of 17.9 and 29.0 μM, respectively, at 500 μM of added compound (Figure 3). Compounds 6e, 14f, 14h, and 14i also exhibited better total nitrite release compared to the reference compound isosorbide dinitrate (ISDN), which
activity of the tested compounds was concentration-dependent, and the compounds had little or no effect on growth at 88 μM. Nitric Oxide Measurement via the Griess Reagent. Quantification of NO release was performed for the fimbrolide nitrate derivatives via treatment of compounds at 37 °C for 1 h with xanthine oxidase, xanthine, and cysteine, which convert the nitrate into NO.24 NO is subsequently converted into nitrite in aqueous media and is quantitated using the Griess reagent. Due to the low percentage conversion of nitrate to nitrite (1−5%)
Scheme 3. Synthesis of Fimbrolide Nitrooxy Derivatives via Linkersa
a
Reagents and conditions: (i) SOCl2, cat. DMF, CH2Cl2; (ii) X = O then 7a or X = S then 8, pyridine, CH2Cl2, 81−95%; (iii) AgNO3, CH3CN, 60 °C, 67−74%. 9519
dx.doi.org/10.1021/jm400951f | J. Med. Chem. 2013, 56, 9517−9529
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Scheme 5. Synthesis of Fimbrolide Diazeniumdiolate Derivatives via an Amide Linkera
a
Reagents and conditions: (i) pyridine, CH2Cl2, 0 °C−rt, then H2O, 75−86%; (ii) EDC, CH2Cl2, rt, 57−80%.
Figure 2. Biological screening assay for QS inhibition by compound 6b. (A) Relative fluorescence units (RFU) and (B) optical density (OD) as a function of time. Concentrations tested: ⧫ 88.8 μM, ■ 29.6 μM, ▲ 9.9 μM, × 3.3 μM, * 1.0 μM, ● control. The monitor strain P. aeruginosa PAO1 harboring the PlasB::gf p (ASV) fusion plasmid was employed.
evident from the negative effect on GFP expression. Isosorbide dinitrate (ISDN) and sodium nitroprusside (SNP) were used as reference NO donors. Nitrate-based NO donor ISDN had no effect on GFP expression, whereas spontaneous NO donor SNP showed 3.5-fold increase in fluorescence. Biofilm Growth and Dispersion. One of the key phenotypes of bacteria is the formation of a biofilm, which protects the bacteria from stresses such as predation and antibiotics. QS inhibitors and NO have been shown to influence maturation and dispersion of biofilms.11,14 In particular, NO controls biofilm properties at picomolar to nanomolar concentrations,15 which implies a low concentration of compound (
