Leveraging Marine Natural Products as a Platform to Tackle Bacterial Resistance and Persistence
CONSPECTUS: Antimicrobial resistance to existing antibiotics represents one of the greatest threats to human health and is growing at an alarming rate. To further complicate treatment of bacterial infections, many chronic infections are the result of bacterial biofilms that are tolerant to treatment with antibiotics because of the presence of metabolically dormant persister cell populations. Together these threats are creating an increasing burden on the healthcare system, and a \xe2\x80\x9cpreantibiotic\xe2\x80\x9d age is on the horizon if significant action is not taken by the scientific and medical communities. While the golden era of antibiotic discovery (1940s\xe2\x80\x931960s) produced most of the antibiotic classes in clinical use today, followed by several decades of limited development, there has been a resurgence in antibiotic drug discovery in recent years fueled by the academic and biotech sectors. Historically, great success has been achieved by developing next-generation variants of existing classes of antibiotics, but there remains a dire need for the identification of novel scaffolds and/or antimicrobial targets to drive future efforts to overcome resistance and tolerance. In this regard, there has been no more valuable source for the identification of antibiotics than natural products, with 69\xe2\x80\x9377% of approved antibiotics either being such compounds or being derived from them. Our group has developed a program centered on the chemical synthesis and chemical microbiology of marine natural products with unusual structures and promising levels of activity against multidrug-resistant (MDR) bacterial pathogens. As we are motivated by preparing and studying the biological effects of these molecules, we are not initially pursuing a biological question but instead are allowing the observed phenotypes and activities to guide the ultimate project direction. In this Account, our recent efforts on the synoxazolidinone, lipoxazolidinone, and batzelladine natural products will be discussed and placed in the context of the field\xe2\x80\x99s greatest challenges and opportunities. Specifically, the synoxazolidinone family of 4-oxazolidinone-containing natural products has led to the development of several chemical methods to prepare antimicrobial scaffolds and has revealed compounds with potent activity as adjuvants to treat bacterial biofilms. Bearing the same 4-oxazolidinone core, the lipoxazolidinones have proven to be potent single-agent antibiotics. Finally, our synthetic efforts toward the batzelladines revealed analogues with activity against a number of MDR pathogens, highlighted by non-natural stereochemical isomers with superior activity and simplified synthetic access. Taken together, these studies provide several distinct platforms for the development of novel therapeutics that can add to our arsenal of scaffolds for preclinical development and can provide insight into the biochemical processes and pathways that can be targeted by small molecules in the fight against antimicrobial-resistant and -tolerant infections. We hope that this work will serve as inspiration for increased efforts by the scientific community to leverage synthetic chemistry and chemical microbiology toward novel antibiotics that can combat the growing crisis of MDR and tolerant bacterial infections.
INTRODUCTION<!>SYNOXAZOLIDINONE NATURAL PRODUCTS<!>2,3-PYRROLIDINEDIONE HETEROCYCLES<!>LIPOXAZOLIDINONE NATURAL PRODUCTS<!>BATZELLADINE NATURAL PRODUCTS<!>CONCLUSION
<p>Regardless of what trend is analyzed, public health indicators aim in the same direction: an antibiotic resistance era is already in place, and it is likely to get even worse unless urgent and aggressive actions are taken.5–7 In the past few decades, the rate of new FDA-approved antibiotic scaffolds has decreased dramatically. For the period 2000–2019, only 28 new antibiotics were approved, compared with 52 new antibiotics that were approved from 1980 to 1999.8–10 Specifically, in the last 5 year period (2015–2019), more than 25% of the new FDA-approved antibiotics were novel combinations of already known antibacterial agents,9,10 highlighting the limited development of new antibiotic classes. An underestimation of the impact of antibiotic resistance clinically, minimal profit projections on new antibiotic pipeline investments, and a considerable reduction in the number of companies with active antimicrobial research divisions are some of the socio-economical causes for this reduction in new antimicrobial development.11 To make this situation even worse, bacterial resistance has been growing at an alarming rate every year.5 Several factors have favored bacterial resistance in the last half-century, including inherent adaptability and horizontal gene transfer as well as the ability of bacteria to mutate.12 However, additional factors have also promoted resistance in bacteria, including antibiotic misuse and overuse,13 inappropriate dosage,14 antibiotic spills into the environment,15 and antibiotic growth promoters used in the livestock industry.16 More alarming is the steep increase in the usage of new and last-resort antibiotics in all countries, including the glycylcyclines, oxazolidinones, carbapenems, and polymixins.17 These factors cumulatively have resulted in a significant reduction of the antibiotic-resistance-free time gap, as during the last decades antibiotics introduced into the market have had clinically observed resistance emerge in 5 years or less (Figure 1A).6</p><p>While antibiotic resistance is the most prevalent bacterial defense mechanism, there are other less frequently discussed adaptation advantages that bacteria employ to overcome antibiotic effects: tolerance and persistence. Unlike antibiotic-resistant bacteria, which rely on their capacity to undergo genomic alterations to overcome antibiotic effects, tolerant bacterial populations can endure antibiotic exposure by slowing down their cellular activity,18 thereby decreasing the bactericidal effect of antibiotics.19 Antibiotic-persistent bacteria, which are often present within tolerant populations, exhibit no metabolic activity during antibiotic exposure, which allows them to bypass the killing by antibiotics without needing to undergo genotypic mutations to survive.18 Once the antibiotic dissipates, the persistent cells reactivate their metabolic machinery.20 Antibiotic tolerance and persistence have significant clinical implications18 since they are niches for antibiotic resistance development and are frequently found in bacterial biofilms.19 Biofilms constitute a major evolutionary bacterial advantage that has been associated with persistent bacteria, and these surface communities are known to be responsible for up to 80% of chronic infections.21</p><p>Multiple studies have highlighted the importance of developing new antibiotic classes to address the spread of antibiotic-resistant bacterial strains.22–24 Developing a new antibiotic costs several hundred million dollars;25 however, the perception that old antibiotics still work "well enough",26 the increasing risk of early market withdrawal,27 low profitability,11,28 and the lack of funds for antibiotic research26 have all contributed to the decline in antibiotic development in recent decades. Historically, natural products have served as a privileged starting point for the development of new drugs. This is even more profound in the area of antibiotic development, where 69–77% of all FDA-approved antibiotics are either natural products or analogues of natural products (Figure 1B).29,30 While natural products have evolved to exist in a biological world, they are not optimized to serve as human therapeutics, and there are limitless opportunities to access analogues with improved activity and properties through synthetic chemistry and synthetic biology.31,32 While there has been a constant goal of identifying new targets, often with limited success and an uncertain future, there is an enormous opportunity to innovate in synthetic chemistry to design and modify natural product scaffolds in ways that overcome resistance and expand pathogen scope.33–35 Natural products have shown that these efforts are limited only by the creativity and focus of the synthetic and chemical biology community and are primed to deliver much-needed relief to the growing crisis of antimicrobial resistance. In this regard, marine natural products have emerged in the last decades as an incredibly rich source of diversity and complexity covering chemical space that easily is more diverse than the chemical space covered by approved drugs (Figure 1C).36</p><p>In this Account, we provide an overview of our group's efforts to leverage innovative chemical syntheses of marine natural products to enable the development of these scaffolds as leads for next-generation antibiotics (Figure 2). Through an integrated program of novel methods development, natural product synthesis, and chemical microbiology, we have advanced several projects toward leads for next-generation antibiotics. Herein we present our efforts toward the natural products lipoxazolidinone A (1), synoxazolidinone A (2) and B (3), and batzelladine D (4) and their application to overcoming antimicrobial resistance and bacterial biofilms.37,38</p><!><p>Synoxazolidinone A (2) and B (3) are bromotyrosine-derived guanidine alkaloids that were isolated by Tadesse and co-workers from the ascidian Synoicum pulmonaria in waters off the coast of Norway in 2010 (Figure 3A).39–41 These molecules are the first documented 4-oxazolidinone-containing natural products and were reported to possess modest antimicrobial and antifouling properties.42 We became interested in leveraging these molecules as inspiration to develop a synthetic approach to these 4-oxazolidine cores and subsequently evaluate the potential of this natural product scaffold for antibiotic development. To this end, we developed two independent routes to prepare 4-oxazolidinones that rely on iminium formation/cyclization to generate the heterocycle from simple building blocks (Figure 3).1,2 At the outset of these efforts, it was unclear whether the desired 4-oxazolidinone would predominate or instead the regioisomeric pyrrolidinedione would be formed (Figure 3C). While some reaction conditions did provide pyrrolidinedione scaffolds, we successfully optimized both an imine acylation approach41 and an acid dehydration approach43 to selectively prepare 4-oxazolidinones bearing benzylidene motifs (Figure 3A,B). Utilizing the imine acylation method, we were able to develop a rapid synthesis of both 2 and 3 from the corresponding aldehydes 14b and 14a and phenylpyruvic acid chloride (15), and we confirmed both the natural product structures and the reported antimicrobial activities (Figure 3A).</p><p>With the natural products in hand, we prepared a number of analogues to explore the structure—activity relationship (SAR) of these molecules, but unfortunately, we were unable to improve their antimicrobial properties. It was at this point that we evaluated our initial 4-oxazolidinone library for their ability to affect a number of other bacterial phenotypes, and gratifyingly, we found that the core scaffold was able to both inhibit and disperse Staphylococcus aureus biofilms (Figure 3D). Given our interest in this area and the relatively few scaffolds that have been developed to date,44 we expanded our 4-oxazolidinone library to evaluate the SAR of the antibiofilm heterocycles (31 and 35–41; Figure 3D,E). These efforts ultimately revealed that electron-deficient phenylpyruvic acid groups and α,α-dichlorinated imines resulted in compounds with improved biofilm inhibition and dispersal properties (see 35 and 38 in Figure 3D,E), although the inhibition properties in some cases are due to the antimicrobial activity of these analogues.45 The lead analogues generated in this effort were of particular interest since they could potentially serve to disperse biofilms and act as adjuvants to front-line antibiotics for the treatment of biofilm-implicated infections.</p><p>Having natural product analogues with activity as inhibitors and dispersal agents against Gram-positive bacteria, we became interested in further exploring the SAR of this scaffold and evaluating its effectiveness as an adjuvant in combination with existing antibiotics against bacterial biofilms. To this end, we prepared a second-generation library of compounds further expanding the structural class, mainly focused on alterations of the western aldehyde-derived domain (42–56; Figure 4A).45 These latest-generation compounds were similar in potency to our first-generation library (Figure 4C). During these efforts we also serendipitously prepared the formaldehyde hemi-aminals of the oxazolidine core (57–59; Figure 4B) and found these compounds to have improved activity, although their lifetimes in cell culture remain unknown.3 At this stage, we evaluated our lead compounds for their ability to eradicate preformed robust biofilms in combination with existing antibiotics. To our delight, compounds such as 59 were extremely effective at clearing biofilms and were synergistic with antibiotics such as doxycycline, reducing the antibiotics' minimal biofilm eradication concentration (MBEC) to just 16 μg/mL with only 8 μg/mL oxazolidinone 59 (Figure 4D). This impressive reduction in MBEC (the MBEC of doxycycline alone is 128–256 μg/mL, and that of 59 is 256 μg/mL) is thought to be the result of 59 serving to promote the release of the bacteria from the biofilm matrix, after which the now planktonic bacteria are killed by both 59 and the antibiotic in solution. Current studies are focused on identifying the mechanism by which the synoxazolidinone analogues trigger biofilm release and further improving the activity and other properties of this class for further development as antibiotic adjuvants. As our goals are biofilm eradication and not simply dispersal with no bacterial killing, the fact that our compounds themselves display antimicrobial activity is an asset. This differentiates this class of compounds from other anti-biofilm strategies that are aimed at affecting the biofilm phenotype in completely non-antimicrobial mechanisms (although it appears that some of our compounds do indeed possess these properties, i.e., 36 and 41 in Figure 2). Mechanistic studies to better understand how these small molecules disrupt biofilms and/or act as antimicrobial agents are ongoing.</p><!><p>2,3-Pyrrolidinedione heterocycles are common in medicinal chemistry and have a long history as both building blocks and members of small-molecule screening collections. During our efforts to develop a method to access 4-oxazolidinones bearing a benzylidene motif (e.g., 62), we encountered conditions that provided predominantly 2,3-pyrrolidinedione heterocycles (e.g., 63) in modest yields (Figure 5A). We subsequently optimized the preparation of these heterocycles via a three-component reaction under extremely mild and scalable conditions to yield a variety of monocyclic and bicyclic heterocycles (Figure 5B).46–48 This multicomponent reaction (MCR) is extremely functional-group-tolerant and has been successfully conducted on a 10 g scale, followed by subsequent conversion to a number of value-added products such as β-lactams and β-amino acids. Furthermore, this MCR has also provided direct access to natural-product-like cores such as bicyclic γ-lactams as well as an efficient route to quaternary substituted hydroindole scaffolds present within crinane alkaloids and enabled the total synthesis of the marine natural product phenopyrrozin and analogues.46–48 As part of these efforts, we also evaluated the antimicrobial activities of the 2,3-pyrrolidinedione heterocycles and were pleased to find that a number of them had modest activity against a panel of Gram-positive bacteria (Figure 5C).49 We are currently expanding the SAR of this class and further evaluating the properties of these molecules, but these results highlight the new chemistry that can emerge from ongoing natural product synthesis projects and the unexpected biology that can subsequently result.</p><!><p>The lipoxazolidinone family of natural products were isolated off the coast of Guam and represent one of the only other classes of 4-oxazoldinone-bearing natural products (Figure 6A).50 Unlike the synoxazolidinones, which bear a benzylidene motif on their eastern side, the lipoxazolidinones contain an unusual unsaturated conjugated carbonyl group that presents different synthetic challenges. Lipoxazolidinone A (1) has been reported to possess potent antimicrobial activity (0.5 μg/mL against MRSA) via an unknown mechanism of action. We envisioned accessing the lipoxazolidinone core through the addition of an α-hydroxy amide to a ketene intermediate followed by cyclization to form the 4-oxazolidinone heterocycle (Figure 6A). Upon generation of the required protected α-hydroxy amide (79) from the corresponding amino acid (78) in three steps, we were able to successfully intercept the in situ generated ketenes to produce our targeted heterocycles in good yields after one-pot deprotection/cyclization with TFA (Figure 6A). This method has proven to be extremely tolerant to functional groups, and we have used it to prepare a large panel of analogues to explore the SAR of this natural product class.2,51 While the natural product 1 itself is extremely nonpolar, we have been able to demonstrate that the core oxazolidinone is the required pharmacophore for activity and that the activity can be retained and in some cases enhanced through modifications of the side chains (Figure 6B–E). Initial efforts in probing the SAR were to confirm that the S enantiomer of 1 was in fact the more potent enantiomer (Figure 6B). Once that was confirmed, we then sought to probe the importance of the eastern side chain trialkyl-substituted olefin and long carbon chain. In doing so, we determined that alkyl chains mimicking a similar carbon length as in the natural product retained activity but allowed simplification of the uniquely unsaturated carbonyl (Figure 6C). We then focused on testing the oxazolidinone alone, removing both long-chain alkyl groups, which resulted in a significant loss of antimicrobial activity. Furthermore, we tested the necessity of the hydrogen-bond-donor properties of the oxazolidinone by substitution with a methyl and a thione bioisostere, both of which significantly diminished the activity. These initial results suggested that the hydrogen bonding of the oxazolidinone amide potentially promotes a desired conformer with the eastern enone side chain (Figure 6D). With these results, we then moved forward with the simplified nonane chain on the eastern side of the molecule and focused on testing the steric tolerance of the western portion of the oxazolidinone. To our delight, we found that branched substituents such as isopropyl (89) and isobutyl (91) provided enhanced activity (Figure 6E). With the improved isopropyl side chain, we then took a step back to probe the eastern side chain with various aryl substituents. Across a number of compounds we retained Gram-positive activity (102, 115) compared with the lead compound (88) or achieved formidable Gram-positive activity while also improving the activity toward Gram-negative bacteria (94, 95, 112, and 113) (Figure 6F,G).</p><p>In addition to the synthetic efforts, we have demonstrated that the lipoxazolidinones inhibit both cell-wall and protein synthesis in a dose-dependent manner and are slow to develop resistance.40 Current efforts in this area are focused on identifying the biological target, exploring the efficacy of this class of compounds, and further improving the pathogen scope to include Gram-negative pathogens (Figure 6G). In pursuit of this final goal, we have demonstrated that efflux is the cause of Gram-positive-only activity, as Δ-TolC Escherichia coli are equally subject to the action of our lead compounds.51</p><!><p>Polycyclic guanidinium alkaloids have attracted the attention of synthetic chemists for decades, with beautiful synthetic approaches from many leaders in the field.52–71 We became interested in this class of natural products because of the limited knowledge of how their diverse structure impacts their biological activity, their often poorly explored biological properties, and a lack of a synthetic approach that would allow for diverse SAR studies across the different families of natural products. We envisioned a synthetic route that would leverage the key bicyclic β-lactam intermediate 121 not only to control the stereochemistry but also to serve as a preactivated coupling partner to join the side chains of the target natural products such as 120 and 122 (Figure 7). This approach, through stereodivergent pathways, would pave the way for all of the members of these natural products to be accessed across both the trans- and cis-pyrrolidine cores and would additionally enable pinpoint modifications of both functional group and stereochemistry around the complex natural product scaffolds.</p><p>To bring our envisioned synthesis to realization required developing a stereoselective and stereodivergent access to the targeted bicyclic β-lactam. We explored a number of approaches to form the bicyclic scaffold either directly or from a monocyclic β-lactam building block but were met with challenges due to the increased strain of the fused four- or five-ring system and the appended side chains at their necessary oxidation states. Fortunately, we found that an aza-Michael approach could be utilized to successfully prepare the target heterocycle.4 To this end, our synthesis began with commercially available β-lactam 123, which was substituted with butenyl Grignard reagent 124, N-protected with TBS, and subsequently acylated to provide 127 in good yield over three steps (Scheme 1A). The reduction of the methyl ketone was required at this stage, and we have developed conditions to access both diastereomers (128 and 129) in good yields, ultimately enabling access to both the natural product 4 and its epimer 141 via subsequent displacement (Scheme 1A). Deprotection of 129 with TBAF followed by cross-metathesis72 with a combination of chlorodicyclohexylborane (10 mol %) and HG-II (7 mol %) delivered enone 131. With 131 in hand, a diastereoselective aza-Michael reaction proceeded smoothly with substoichiometric amounts of base to yield the desired [4.5]-fused bicyclic β-lactam 132. The ketone reduction was necessary prior to the lactam-opening reaction, as otherwise a loss of stereochemical integrity was observed. After screening an array of different combinations of additives and reducing agents, we were gratified to develop a stereoselective method to access both alcohol diastereomers of 133 and 135 in a scalable fashion. With access to all four alcohol diastereomers, several stereoisomers of the natural product core, including (±)-batzelladine D, were prepared through our end-game strategy. Opening the bicyclic β-lactam 135 with alcohol side chain 136 under BF3·OEt2 activation provided dihydroxypyrrolidine 137. Guanylation of 137 using a thiourea derivative was followed in the same pot by mesylation of both alcohols, which were rapidly displaced by the guanidine to install the tricyclic core with final formic acid deprotection of the four Boc groups. This one-pot process allowed us to generate a separable 1.0:1.2 mixture of (±)-batzelladine D (4) and 138 (which resulted from β-mesylate elimination under the reaction conditions) in 41% overall yield in a stereoselective fashion.</p><p>Having a concise racemic synthesis in hand, we then moved our attention to explore an enantioselective version. After multiple failed approaches, we were able to develop two routes that allowed us to generate gram-scale quantities of our key intermediates enantioselectively that intersected our previously developed racemic approach for both enantiomers (Scheme 1B,C). To access the non-natural enantiomer, commercially available β-lactam 139 was chosen because of its accessibility on a large scale due to its use as a building block for commercial antibiotic synthesis. 139 could be readily converted to the requisite enantiomeric bicyclic β-lactam on a gram scale in five steps, which was advanced to the unnatural stereoisomer (+)-4. On the other hand, generation of the natural enantiomer relied on an Ellman auxiliary approach that readily provided (−)-β-lactam 125 in good yield on a gram scale, which was then converted to (−)-4 smoothly (Scheme 1C).</p><p>Via the routes that we have developed, a library of batzelladine analogues were prepared, and their activities were evaluated against a series of ESKAPE pathogens,73–77 including Gram-positive and Gram-negative bacteria (Figure 8). This represents the first antimicrobial evaluation of batzelladine D and its stereoisomers. We were delighted to see that (±)-batzelladine D exhibited moderate levels of activity against MSSA and MRSA, with an MIC of 8 μg/mL. Upon systematic evaluation of the stereoisomers against the ESKAPE pathogens, several compounds showed promising activities, most notably non-natural (+)-13-epi-batzelladine D (134) and the elimination analogue 142, which were found to be our best compounds so far (Figure 8). It was revealed that the stereochemistry altered the antimicrobial activity, with non-natural stereoisomers having superior antimicrobial activity. Currently our efforts are focused on an expanded analogue library to explore the SAR of this family and further study the scope and mechanism of these molecules as potential antimicrobial and antiviral agents.</p><!><p>We have developed stereoselective and practical access to several classes of natural products and through these efforts have developed novel synthetic methods, confirmed natural product structures, enabled thorough SAR studies on the natural product scaffolds, and initiated mechanism-of-action studies to understand the biological pathways that they target. Several projects are now in further stages of translation though partnerships with industry and collaborators. Taken together, these projects serve to highlight the role of chemical synthesis to help drive innovation in antibacterial discovery and the huge opportunity that is available in presently unexplored chemical space. We are optimistic that natural products will continue to serve as the backbone of antibiotic development and will be complemented by many new technologies that are emerging or re-emerging. We are thrilled to be part of that journey in the years to come and hope that our contributions can help propel others forward in the common fight against multidrug-resistant and -tolerant bacterial infections.</p>