Exploration of Bis-Cinnamido-Polyamines as Intrinsic Antimicrobial Agents and Antibiotic Enhancers

The marine natural product ianthelliformisamine C is a bis-cinnamido substituted spermine derivative that exhibits intrinsic antimicrobial properties and can enhance the action of doxycycline towards the Gram-negative bacterium Pseudomonas aeruginosa. As part of a study to explore the structure–activity requirements of these activities, we have synthesized a set of analogues that vary in the presence/absence of methoxyl group and bromine atoms and in the polyamine chain length. Intrinsic antimicrobial activity towards Staphylococcus aureus, methicillin-resistant S. aureus (MRSA) and the fungus Cryptococcus neoformans was observed for only the longest polyamine chain examples of non-brominated analogues while all examples bearing either one or two bromine atoms were active. Weak to no activity was typically observed towards Gram-negative bacteria, with exceptions being the longest polyamine chain examples 13f, 14f and 16f against Escherichia coli (MIC 1.56, 7.2 and 5.3 µM, respectively). Many of these longer polyamine-chain analogues also exhibited cytotoxic and/or red blood cell hemolytic properties, diminishing their potential as antimicrobial lead compounds. Two of the non-toxic, non-halogenated analogues, 13b and 13d, exhibited a strong ability to enhance the action of doxycycline against P. aeruginosa, with >64-fold and >32-fold enhancement, respectively. These results suggest that any future efforts to optimize the antibiotic-enhancing properties of cinnamido-polyamines should explore a wider range of aromatic ring substituents that do not include bromine or methoxyl groups.


Introduction
With the increasing occurrence of antibiotic-resistant microbial infections, there is an urgent need to discover new classes of antibiotics with new mechanisms of action [1,2]. The marine environment is noted as being a productive source of small molecules, with over 1400 new molecules reported annually [3] and with a sizable percentage of these fitting chemometric descriptors as being drug-like [4]. There are several notable examples of marine natural products that exhibit antimicrobial properties [5] including squalamine (1), ent-eusynstyelamide B (2) and synoxazolidinone A (3) (Figure 1). The polyamine-containing aminosterol squalamine (1), isolated from the dogfish shark Squalus acanthias, exhibits broad-spectrum activity towards both Gram-positive and Gram-negative bacteria [6] and can enhance the action of different classes of antibiotics towards a range of Gram-negative bacteria. Its mechanism of action is attributed to bacterial membrane disruption [7,8] and more recently, to inhibit the glycosyltransferase activity of Escherichia coli penicillin-binding negative bacteria. Its mechanism of action is attributed to bacterial membrane disruption [7,8] and more recently, to inhibit the glycosyltransferase activity of Escherichia coli penicillin-binding protein PBP1b [9]. Knowledge of the structure and attractive biological activities associated with squalamine has led to wide-spread interest in steroidal-polyamine conjugates as a new class of antimicrobial agents [10][11][12]. The discovery that enteusynstyelamide B (2), isolated from the Arctic bryozoan Tegella cf. spitzbergensis [13], exhibits moderate activity towards Gram-positive bacteria and weaker activity towards Gram-negative bacteria and fungi prompted structure-activity studies that identified examples of easily prepared barbiturate analogues exhibiting potent in vitro and in vivo antibacterial properties [14]. The sub-arctic ascidian-derived natural product synoxazolidinone A (3) was originally reported to exhibit modest antimicrobial activity towards Staphylococcus aureus [15,16] and that simplification of the structure-identified analogues that could disperse S. aureus biofilms and acted synergistically with doxycycline [17]. Investigation of an Australian collection of the marine sponge Suberea ianthelliformis led to the characterization of several new polyamine (PA) containing alkaloids including the α,ω-disubstituted spermine (PA-3-4-3) analogue ianthelliformisamine C (4) [18]. The natural product demonstrated growth inhibition towards S. aureus (MIC 17.5 µM) as well as towards the Gram-negative bacterium Pseudomonas aeruginosa with a MIC of 8.75 µM. Two subsequent studies have explored ianthelliformisamine C-related analogues bearing cinnamide head groups with increased bromination and truncated or aryl-containing amine linkers, though no analogues were found to be more active than the natural product [19,20]. A third study [21] reported that ianthelliformisamine C and related analogues were able to enhance the action of legacy antibiotics doxycycline and cefepime toward P. aeruginosa PAO1. Intrigued by the antibiotic enhancing properties of 4, we have prepared a set of new analogues, exploring variation in cinnamate head group substitution as well as variation in polyamine chain length. All analogues were evaluated for antimicrobial activities against a panel of Gram-positive and Gram-negative bacteria and fungi, and for the ability to enhance the antibiotic activity of doxycycline and erythromycin against the Gram-negative bacteria P. aeruginosa and E. coli, respectively. Investigation of an Australian collection of the marine sponge Suberea ianthelliformis led to the characterization of several new polyamine (PA) containing alkaloids including the α,ω-disubstituted spermine (PA-3-4-3) analogue ianthelliformisamine C (4) [18]. The natural product demonstrated growth inhibition towards S. aureus (MIC 17.5 µM) as well as towards the Gram-negative bacterium Pseudomonas aeruginosa with a MIC of 8.75 µM. Two subsequent studies have explored ianthelliformisamine C-related analogues bearing cinnamide head groups with increased bromination and truncated or aryl-containing amine linkers, though no analogues were found to be more active than the natural product [19,20]. A third study [21] reported that ianthelliformisamine C and related analogues were able to enhance the action of legacy antibiotics doxycycline and cefepime toward P. aeruginosa PAO1. Intrigued by the antibiotic enhancing properties of 4, we have prepared a set of new analogues, exploring variation in cinnamate head group substitution as well as variation in polyamine chain length. All analogues were evaluated for antimicrobial activities against a panel of Gram-positive and Gram-negative bacteria and fungi, and for the ability to enhance the antibiotic activity of doxycycline and erythromycin against the Gram-negative bacteria P. aeruginosa and E. coli, respectively.

Determination of the MICs of Antibiotics in the Presence of Synergizing Compounds
Antibiotic enhancer concentrations were determined using previously reported protocols [28].

Cytotoxicity Assays
Cytotoxicity assays were conducted using the protocols previously reported [28].

Hemolytic Assay
Hemolysis assays were conducted using the protocols previously reported [28].
Hemolysis assays were conducted using the protocols previously reported [28].
Biomolecules 2023, 13, x FOR PEER REVIEW Reaction of cinnamic acids 5-8 with Boc-protected polyamines 12apling reagents EDC·HCl or EDC·HCl/HOBt in anhydrous CH2Cl2 with the deprotected with TFA to afford the target compounds as their di-TFA salts   (Table 1). Overall, the set of compounds tended to exhibit antimicrobial activity the Gram-positive bacteria S. aureus and MRSA and the fungus C. neoformans, and cases C. albicans, but they were only poorly active towards the Gram-negative ba coli and essentially inactive towards P. aeruginosa, A. baumannii and K. pneumoni pronounced activities were observed for bromine-containing analogues 15a-f a which are seemingly independent of polyamine chain length; while for those non ated analogues (13a-f and 14a-f), the trend was that the longer the polyamine c more likely was the observation of activity, e.g., 13f and 14d-f.
The intrinsic antimicrobial activity of the series was evaluated against a range of Grampositive (S. aureus and MRSA) and Gram-negative (E. coli, P. aeruginosa, K. pneumoniae and A. baumannii) bacteria and two fungal strains (C. albicans and C. neoformans) (Table 1). Overall, the set of compounds tended to exhibit antimicrobial activity towards the Gram-positive bacteria S. aureus and MRSA and the fungus C. neoformans, and in some cases C. albicans, but they were only poorly active towards the Gram-negative bacteria E. coli and essentially inactive towards P. aeruginosa, A. baumannii and K. pneumoniae. More pronounced activities were observed for bromine-containing analogues 15a-f and 16a-f which are seemingly independent of polyamine chain length; while for those non-brominated analogues (13a-f and 14a-f), the trend was that the longer the polyamine chain, the more likely was the observation of activity, e.g., 13f and 14d-f.
Cytotoxicity towards HEK293 (human kidney epithelial cell line, IC 50 ) and hemolytic activity against human red blood cells (HC 10 ) of the compound set was also determined ( Table 1). The results trended in a similar manner to intrinsic antimicrobial activities shown in Table 1, with the longer polyamine chain analogues and those containing bromine exhibiting cytotoxicity and/or hemolysis. Interest in the promising antimicrobial activities of the cinnamido-PA-3-12-3 analogue 13f and 4-methoxycinnamido analogues 14e and 14f was tempered somewhat by the observation of cytotoxicity (HEK293 IC 50 19.9 µM for 13f, IC 50 2.3 µM for 14e and IC 50 24.3 µM for 14f) for the three analogues. The midchain-length polyamine analogue 14d is one of the few antimicrobial analogues that does not exhibit cytotoxicity/hemolysis, illustrating in this series of compounds the fine line between a compound being antimicrobial and yet not exhibiting any mammalian cell toxicity.  We then evaluated the set of analogues for the ability to enhance the antibiotic activity of doxycycline against P. aeruginosa ATCC 27853 and of erythromycin against E. coli ATCC 25922 (Table 2). In the case of doxycycline, a fixed concentration of the antibiotic of 4.5 µM, which is twenty-fold lower than the intrinsic MIC 90 µM against this organism, was used. Each of the test compounds was then evaluated at a range of concentrations varying from 3.125 to 50-100 µM, with the upper concentration dependent upon the compounds intrinsic MIC towards P. aeruginosa (Table 1). The natural product ianthelliformisamine C 16a exhibited strong 32-fold enhancement at a concentration of 5.86 µM, in close agreement with that previously reported [21]. The methylene homologues 16b and 16c also exhibited strong activity, at concentrations of about 11 µM. It was interesting to note that while these dibrominated analogues were active as doxycycline enhancers, no activity was observed for the mono-brominated analogues 15a-f, while activity was observed for the non-brominated analogues 13b, 13d, 13f and 14f, all of which exhibited activity with comparable potency to ianthelliformisamine C 16a and with good levels of enhancement (>64-fold, >32-fold, 8-fold and 32-fold, respectively). While the observation of cytotoxicity/hemolytic properties of 16a-c, 14f and 13f reduces our interest in these analogues, analogues 13b and 13d were identified as non-toxic and strong enhancers of the action of doxycycline towards P. aeruginosa. Evaluation of the compounds 13-16 to enhance the action of erythromycin, at a fixed dose of 8 µg/mL (10.69 µM), against E. coli ATCC 25922 revealed the set to be overall weakly active with the best enhancement observed for 13e (MIC 64.5 µM, 10-fold enhancement), an analogue that was also cytotoxic and hemolytic.
We recently reported the results of a study exploring the antimicrobial and antibiotic potentiating activities of a series of α,ω-diacyl substituted polyamines [28]. Two of the compounds included in that study were 17 and 18 (Figure 4), which are tetrahydro analogues of 13b and 13d, respectively. Unlike 13b and 13d, compounds 17 and 18 were devoid of antimicrobial activity and the ability to enhance the action of doxycycline towards P. aeruginosa, highlighting the requirement of the cinnamate olefin for biological activity.
27853. Fold change shown in parentheses is the ratio between the intrinsic MIC of the test compound and the combination MIC; b Concentration (µM) required to restore erythromycin activity at 10.9 µM against E. coli ATCC 25922. Fold change shown in parentheses is the ratio between the intrinsic MIC of the test compound and the combination MIC; c Not tested.
We recently reported the results of a study exploring the antimicrobial and antibiotic potentiating activities of a series of α,ω-diacyl substituted polyamines [28]. Two of the compounds included in that study were 17 and 18 (Figure 4), which are tetrahydro analogues of 13b and 13d, respectively. Unlike 13b and 13d, compounds 17 and 18 were devoid of antimicrobial activity and the ability to enhance the action of doxycycline towards P. aeruginosa, highlighting the requirement of the cinnamate olefin for biological activity. The antibiotic-enhancing activities of 13b, the strongest non-toxic doxycycline enhancer, were further evaluated with an expanded set of antibiotics (minocycline, chloramphenicol, vancomycin, nalidixic acid and erythromycin) against three drug-resistant strains of Gram-negative bacteria (P. aeruginosa, K. pneumoniae and A. baumannii) ( Table 3). No significant potentiating activity was observed with 13b for chloramphenicol, vancomycin, nalidixic acid and erythromycin against the three bacterial strains. However, 13b was able to strongly potentiate the activity of minocycline against P. aeruginosa, K. pneumoniae and A. baumannii (>41-fold, >82-fold and >82-fold, respectively) while also enhancing the activity of doxycycline against P. aeruginosa (>40-fold). The enhancing activity of doxycycline against A. baumannii and K. pneumoniae was not investigated due to its sensitivity to these strains.  The antibiotic-enhancing activities of 13b, the strongest non-toxic doxycycline enhancer, were further evaluated with an expanded set of antibiotics (minocycline, chloramphenicol, vancomycin, nalidixic acid and erythromycin) against three drug-resistant strains of Gram-negative bacteria (P. aeruginosa, K. pneumoniae and A. baumannii) ( Table 3). No significant potentiating activity was observed with 13b for chloramphenicol, vancomycin, nalidixic acid and erythromycin against the three bacterial strains. However, 13b was able to strongly potentiate the activity of minocycline against P. aeruginosa, K. pneumoniae and A. baumannii (>41-fold, >82-fold and >82-fold, respectively) while also enhancing the activity of doxycycline against P. aeruginosa (>40-fold). The enhancing activity of doxycycline against A. baumannii and K. pneumoniae was not investigated due to its sensitivity to these strains.

Conclusions
Polyamines that are α,ω-disubstituted can exhibit antimicrobial properties and enhance the activity of legacy antibiotics towards drug-resistant Gram-negative bacteria. The present study explored structural requirements for both properties of the marine natural product ianthelliformisamine C (4). The majority of the analogues that contained longer chain polyamines, PA-3-8-3, PA-3-10-3 and PA-3-12-3, and any analogues containing one or two bromine atoms per capping end group were found to exhibit antimicrobial activities towards Gram-positive bacteria and the fungus C. neoformans. Unfortunately, these intrinsic antimicrobial activities were usually associated with cytotoxicity and/or red blood cell hemolytic properties. Of more promise was the observation of 32-fold or better levels of enhancement of the action of doxycycline towards the Gram-negative bacterial pathogen P. aeruginosa for simple cinnamido-polyamine analogues 13b and 13d, with the former also enhancing the activity of minocycline against K. pneumoniae and A. baumannii. Comparison with previously reported compounds determined the essentiality of the olefin group for activity, thus any future studies to optimize this class of antibiotic enhancers must retain the core cinnamide structure but avoid incorporation of halogen and methoxyl groups at the meta and para-aryl positions.