Investigation of Naphthyl–Polyamine Conjugates as Antimicrobials and Antibiotic Enhancers

As part of our search for new antimicrobials and antibiotic enhancers, a series of naphthyl- and biphenyl-substituted polyamine conjugates have been synthesized. The structurally-diverse library of compounds incorporated variation in the capping end groups and in the length of the polyamine (PA) core. Longer chain (PA-3-12-3) variants containing both 1-naphthyl and 2-naphthyl capping groups exhibited more pronounced intrinsic antimicrobial properties against methicillin-resistant Staphylococcus aureus (MRSA) (MIC ≤ 0.29 µM) and the fungus Cryptococcus neoformans (MIC ≤ 0.29 µM). Closer mechanistic study of one of these analogues, 20f, identified it as a bactericide. In contrast to previously reported diarylacyl-substituted polyamines, several examples in the current set were able to enhance the antibiotic action of doxycycline and/or erythromycin towards the Gram-negative bacteria Pseudomonas aeruginosa and Escherichia coli. Two analogues (19a and 20c) were of note, exhibiting greater than 32-fold enhancement in activity. This latter result suggests that α,ω-disubstituted polyamines bearing 1-naphthyl- and 2-naphthyl-capping groups are worthy of further investigation and optimization as non-toxic antibiotic enhancers.


Introduction
Host-defense peptides (HDPs), produced by a wide variety of organisms in nature, including microorganisms, plants, invertebrates, and mammals, play essential roles as a first line of protection against viral, fungal, and bacterial infections [1][2][3][4][5][6]. A subset of HDPs are the antimicrobial peptides, small (50 amino acids or less) amphipathic peptides of diverse sequence homology and secondary structures [7]. From a structural perspective, antimicrobial peptides are characterized as containing hydrophobic residues on one side of the molecule and hydrophilic cationic residues on the other. These peptides are thought to act directly on bacterial cell membranes, with the cationic charges aiding electrostatic attraction to the negatively charged cell membrane, followed by hydrophobic residue insertion into the membrane leading to disruption, increased membrane permeability, and, ultimately, cell death [7]. Drug development issues associated with HDPs, including susceptibility to proteolysis, high production costs, and, in some case, low to moderate activity under physiological salt conditions, prevent their direct introduction as clinical agents [8]. In order to overcome these deficiencies, an extensive amount of research has been directed towards the synthesis and biological evaluation of synthetic mimics of antimicrobial peptides, so-called SMAMPs [9][10][11][12][13][14][15]. Numerous different structural classes of SMAMPs have been identified, including shorter peptides (e.g., LTX-109 (1)), sterols (e.g., squalamine (2)), and hydantoins (e.g., 3) (Figure 1) [15][16][17]. Although they cover mimics of antimicrobial peptides, so-called SMAMPs [9][10][11][12][13][14][15]. Numerous different structural classes of SMAMPs have been identified, including shorter peptides (e.g., LTX-109 (1)), sterols (e.g., squalamine (2)), and hydantoins (e.g., 3) (Figure 1) [15][16][17]. Although they cover diverse chemo-types, all three of these examples are thought to act via a general bacterial membrane-targeting mechanism, with insertion leading to membrane disruption [16,18,19]. LTX-109 is one example of a number of different antimicrobial peptides that have entered clinical trials for the treatment of microbial infections [20]. The discovery of squalamine (2), a polyamine-containing aminosterol isolated from the dogfish shark Squalus acanthias, and observation of its broad-spectrum activity towards both Gram-positive and Gram-negative bacteria [21] prompted further investigation of the marine environment in the search for new classes of antimicrobials [22]. The marine sponge natural product ianthelliformisamine C (4) exhibits antimicrobial activity and can also enhance the activity of legacy antibiotics towards drug resistant Gram-negative bacteria [23][24][25]. Ianthelliformisamine C has all the structural attributes of an SMAMP, with the secondary amines of the polyamine fragment being protonated at physiological pH and the terminal cinnamate capping group being lipophilic, defining this as a scaffold worthy of attention [22,26].
We recently reported the synthesis and antimicrobial evaluation of a set of α,ω-diacylaryl substituted polyamine analogues, of which examples 5-7 ( Figure 2) are representative [27]. The discovery of squalamine (2), a polyamine-containing aminosterol isolated from the dogfish shark Squalus acanthias, and observation of its broad-spectrum activity towards both Gram-positive and Gram-negative bacteria [21] prompted further investigation of the marine environment in the search for new classes of antimicrobials [22]. The marine sponge natural product ianthelliformisamine C (4) exhibits antimicrobial activity and can also enhance the activity of legacy antibiotics towards drug resistant Gram-negative bacteria [23][24][25]. Ianthelliformisamine C has all the structural attributes of an SMAMP, with the secondary amines of the polyamine fragment being protonated at physiological pH and the terminal cinnamate capping group being lipophilic, defining this as a scaffold worthy of attention [22,26].
We recently reported the synthesis and antimicrobial evaluation of a set of α,ω-diacylaryl substituted polyamine analogues, of which examples 5-7 ( Figure 2) are representative [27].
Analogues bearing a single aryl group at each end of a polyamine (PA) chain, e.g., 5, were found to be almost uniformly inactive towards a panel of Gram-positive and Gram-negative bacteria and fungal strains, with only the longer polyamine chain variants (PA-3-10-3 and PA-3-12-3) exhibiting weak-to-potent activity towards methicillinresistant Staphylococcus aureus (MRSA). Increasing the lipophilicity by inclusion of one or two additional phenyl rings in the capping acid created sets of analogues, e.g., 6 and 7, that demonstrated good to excellent growth inhibition properties towards Staphylococcus aureus (MIC 3.13 µM for both), MRSA (MIC ≤ 0.28 µM and MIC ≤ 0.24 µM, respectively), and Escherichia coli (MIC 2.2 µM and MIC 7.6 µM, respectively). When counter screen cytotoxicity and hemolytic activities were combined with calculated LogP values, it became apparent that optimal selectivity for antimicrobial activity was observed for diaryl-containing capping groups with whole molecule cLogP in the range 7-8.5. Any analogues with cLogP greater than 9-10 appear to breach a 'second hydrophobicity threshold' [28], leading to greater disruption of mammalian membranes and inherent toxicity. Mechanism of ac- tion studies carried out on the diaryl-analogue 6 identified it as a strong disruptor of bacterial cell membranes and to be bactericidal [27], making it a good starting point for further optimization. Analogues bearing a single aryl group at each end of a polyamine (PA) chain, e.g., 5, were found to be almost uniformly inactive towards a panel of Gram-positive and Gramnegative bacteria and fungal strains, with only the longer polyamine chain variants (PA-3-10-3 and PA-3-12-3) exhibiting weak-to-potent activity towards methicillin-resistant Staphylococcus aureus (MRSA). Increasing the lipophilicity by inclusion of one or two additional phenyl rings in the capping acid created sets of analogues, e.g., 6 and 7, that demonstrated good to excellent growth inhibition properties towards Staphylococcus aureus (MIC 3.13 µM for both), MRSA (MIC ≤ 0.28 µM and MIC ≤ 0.24 µM, respectively), and Escherichia coli (MIC 2.2 µM and MIC 7.6 µM, respectively). When counter screen cytotoxicity and hemolytic activities were combined with calculated LogP values, it became apparent that optimal selectivity for antimicrobial activity was observed for diaryl-containing capping groups with whole molecule cLogP in the range 7-8.5. Any analogues with cLogP greater than 9-10 appear to breach a 'second hydrophobicity threshold' [28], leading to greater disruption of mammalian membranes and inherent toxicity. Mechanism of action studies carried out on the diaryl-analogue 6 identified it as a strong disruptor of bacterial cell membranes and to be bactericidal [27], making it a good starting point for further optimization.
Mindful of not exceeding the second hydrophobicity threshold, we chose to prepare new examples of α,ω-disubstituted polyamines that targeted the cLogP range of approximately 5-9 and incorporated aryl-carboxylic acids 8-12 (Figure 3), which contained variations in shape, size, lipophilicity and position of attachment to the polyamine core.  Mindful of not exceeding the second hydrophobicity threshold, we chose to prepare new examples of α,ω-disubstituted polyamines that targeted the cLogP range of approximately 5-9 and incorporated aryl-carboxylic acids 8-12 (Figure 3), which contained variations in shape, size, lipophilicity and position of attachment to the polyamine core. Analogues bearing a single aryl group at each end of a polyamine (PA) chain, e.g., 5, were found to be almost uniformly inactive towards a panel of Gram-positive and Gramnegative bacteria and fungal strains, with only the longer polyamine chain variants (PA-3-10-3 and PA-3-12-3) exhibiting weak-to-potent activity towards methicillin-resistant Staphylococcus aureus (MRSA). Increasing the lipophilicity by inclusion of one or two additional phenyl rings in the capping acid created sets of analogues, e.g., 6 and 7, that demonstrated good to excellent growth inhibition properties towards Staphylococcus aureus (MIC 3.13 µM for both), MRSA (MIC ≤ 0.28 µM and MIC ≤ 0.24 µM, respectively), and Escherichia coli (MIC 2.2 µM and MIC 7.6 µM, respectively). When counter screen cytotoxicity and hemolytic activities were combined with calculated LogP values, it became apparent that optimal selectivity for antimicrobial activity was observed for diaryl-containing capping groups with whole molecule cLogP in the range 7-8.5. Any analogues with cLogP greater than 9-10 appear to breach a 'second hydrophobicity threshold' [28], leading to greater disruption of mammalian membranes and inherent toxicity. Mechanism of action studies carried out on the diaryl-analogue 6 identified it as a strong disruptor of bacterial cell membranes and to be bactericidal [27], making it a good starting point for further optimization.
Mindful of not exceeding the second hydrophobicity threshold, we chose to prepare new examples of α,ω-disubstituted polyamines that targeted the cLogP range of approximately 5-9 and incorporated aryl-carboxylic acids 8-12 (Figure 3), which contained variations in shape, size, lipophilicity and position of attachment to the polyamine core.  Herein, we report the synthesis of a set of new α,ω-disubstituted polyamines that explore variation in the size and shape of the chain end-groups within a narrow band of lipophilicity as well as variation in polyamine length. All analogues were evaluated for antimicrobial activities against a set of Gram-positive and Gram-negative bacteria and two fungi strains, and for the ability to enhance the antibiotic action of doxycycline and erythromycin towards the Gram-negative bacteria Pseudomonas aeruginosa and E. coli, respectively.

Chemistry
Of the five carboxylic acids required for this study (8)(9)(10)(11)(12), two (8 and 10) were commercially available. Carboxylic acids 9, 11, and 12 were prepared by reaction of naphthalen- crobial activities against a set of Gram-positive and Gram-negative bacteria and two fungi strains, and for the ability to enhance the antibiotic action of doxycycline and erythromycin towards the Gram-negative bacteria Pseudomonas aeruginosa and E. coli, respectively.
crobial activities against a set of Gram-positive and Gram-negative bacteria and two fungi strains, and for the ability to enhance the antibiotic action of doxycycline and erythromycin towards the Gram-negative bacteria Pseudomonas aeruginosa and E. coli, respectively.
The structures of the synthesized α,ω-disubstituted polyamine library are shown in Figure 5. The structures of the synthesized α,ω-disubstituted polyamine library are shown in Figure 5.   The structures of the synthesized α,ω-disubstituted polyamine library are shown in Figure 5.

Results and Discussion
The antimicrobial activity of each compound was initially assessed against various bacterial strains, including S. aureus and MRSA, P. aeruginosa, E. coli, Klebsiella pneumoniae, and Acinetobacter baumannii, as well as fungal strains such as Candida albicans and Cryptococcus neoformans ( Table 1). As a set, analogues containing a 1-naphthyl substituted end group (17a-f, 18a-f) were predominantly inactive towards all microbes, with the exceptions being the longer polyamine chain variants 17e (MRSA MIC 9.   The majority of the compounds, 17-21, were evaluated for cytotoxicity towards human kidney epithelial cell line (HEK293) and for hemolytic activity towards human red blood cells (Table 2). Of the set of compounds tested, only biphenyl 21e was considered to be hemolytic (HC 10 6.3 µM), and the observation of cytotoxicity was limited to the four analogues 17e (IC 50 23.7 µM), 19a (IC 50 26 µM), 19c (IC 50 20.5 µM), and 19e (IC 50 4.75 µM). The latter results, in particular, emphasize that cytotoxicity in the current series is not determined by lipophilicity alone, as other analogues with similar cLogP values (e.g., 17a-d, 18a-e, 20a-e) were deemed non-toxic.
>37.6 >37.6 All values presented as the mean (n = 2); a Concentration (IC 50 , µM) of compound at 50% cytotoxicity on HEK293 human embryonic kidney cells with tamoxifen as the positive control (IC 50 24 µM); b Concentration (HC 10 , µM) of compound at 10% hemolytic activity on human red blood cells with melittin as the positive control (HC 10 0.95 µM); c Not tested.
To conduct a detailed analysis of the antibacterial activity and initial assessment of the mechanism of action, analogue 20f was selected due to its strong antibacterial properties, and because it was devoid of any observed cytotoxic or hemolytic effects. To evaluate the kinetics of antibacterial activity, real-time growth inhibition curves were measured for two Gram-positive bacteria (S. aureus (ATCC 25923), MRSA (CF-Marseille) [34]) and the Gram-negative bacterium E. coli (ATCC 25922). The test compound completely inhibited the growth of the Gram-positive bacteria strains at 3.15 µM (3.13 µg/mL) or higher concentrations, with growth observed at the lowest test concentration of 1.57 µM (1.56 µg/mL) ( Figure 6A,B). In the case of the Gram-negative bacterium E. coli ( Figure 6C), bacterial growth inhibition was observed at test concentrations of 6.29 µM (6.25 µg/mL) or higher. MIC values of 3.15 µM (3.13 µg/mL), 3.15 µM (3.13 µg/mL), and 6.29 µM (6.25 µg/mL) were determined for analogue 20f against S. aureus (ATCC 25923), MRSA (CF-Marseille), and E. coli (ATCC 25922), respectively. These values corresponded to the inhibitory concentrations observed at the 18-hour mark in the real-time growth inhibition curve plots. Additionally, the same values were observed for the minimum bactericidal concentration (MBC) of 20f against all three organisms, indicating its bactericidal activity.
The ability of compounds 17-21 to enhance the antibiotic activity of doxycycline against P. aeruginosa (ATCC 27853) and of erythromycin against E. coli (ATCC 25922) were determined (Table 3). In the case of doxycycline, a low-dose fixed concentration of 2 µg/mL (4.5 µM) was used, being 20-fold lower than the intrinsic MIC (40 µg/mL (90 µM)) against P. aeruginosa. Each of the compounds were then evaluated at a range of concentrations, with the upper limit being dependent upon the intrinsic MIC towards P. aeruginosa (Table 1). Two examples of 1-naphthyl-substituted analogues (17b and 18c) were identified as modest enhancers of the action of doxycycline, with MICs of 12.5 µM, representing 16-fold enhancements over their intrinsic MIC values. A further five examples of 2-naphthyl-substituted variants, 19a, 19c, 19d, 20a, 20c, and 20f, exhibited notable levels of enhancement, with MICs of 16.9, 4.0, 15.7, 28, 6.25, and 12.6 µM, respectively. When compared to their intrinsic growth inhibition activities towards P. aeruginosa (Table 1), these represented 40-fold, 8-fold, 8-fold, 20-fold, >32-fold, and >8-fold enhancements, respectively. The ability of the two structurally related 2-naphthyl-substituted PA-3-4-3 (spermine) analogues 19a and 20a to enhance the action of doxycycline towards P. aeruginosa was investigated more closely, revealing a dose-dependent response when the doxycycline concentration was varied from 2 to 8 µg/mL (Table 4). To conduct a detailed analysis of the antibacterial activity and initial assessment of the mechanism of action, analogue 20f was selected due to its strong antibacterial properties, and because it was devoid of any observed cytotoxic or hemolytic effects. To evaluate the kinetics of antibacterial activity, real-time growth inhibition curves were measured for two Gram-positive bacteria (S. aureus (ATCC 25923), MRSA (CF-Marseille) [34]) and the Gram-negative bacterium E. coli (ATCC 25922). The test compound completely inhibited the growth of the Gram-positive bacteria strains at 3.15 µM (3.13 µg/mL) or higher concentrations, with growth observed at the lowest test concentration of 1.57 µM (1.56 µg/mL) ( Figure 6A,B). In the case of the Gram-negative bacterium E. coli ( Figure 6C), bacterial growth inhibition was observed at test concentrations of 6.29 µM (6.25 µg/mL) or higher. MIC values of 3.15 µM (3.13 µg/mL), 3.15 µM (3.13 µg/mL), and 6.29 µM (6.25 µg/mL) were determined for analogue 20f against S. aureus (ATCC 25923), MRSA (CF-Marseille), and E. coli (ATCC 25922), respectively. These values corresponded to the inhibitory concentrations observed at the 18-hour mark in the real-time growth inhibition curve plots. Additionally, the same values were observed for the minimum bactericidal concentration (MBC) of 20f against all three organisms, indicating its bactericidal activity. The ability of compounds 17-21 to enhance the antibiotic activity of doxycycline against P. aeruginosa (ATCC 27853) and of erythromycin against E. coli (ATCC 25922) were determined (Table 3). In the case of doxycycline, a low-dose fixed concentration of 2 µg/mL (4.5 µM) was used, being 20-fold lower than the intrinsic MIC (40 µg/mL (90 µM)) against P. aeruginosa. Each of the compounds were then evaluated at a range of concentrations, with the upper limit being dependent upon the intrinsic MIC towards P. aeruginosa (Table 1). Two examples of 1-naphthyl-substituted analogues (17b and 18c) were identified as modest enhancers of the action of doxycycline, with MICs of 12.5 µM, representing 16-fold enhancements over their intrinsic MIC values. A further five examples of 2-naphthyl-substituted variants, 19a, 19c, 19d, 20a, 20c, and 20f, exhibited notable levels of enhancement, with MICs of 16.9, 4.0, 15.7, 28, 6.25, and 12.6 µM, respectively. When compared to their intrinsic growth inhibition activities towards P. aeruginosa (Table 1), these represented 40-fold, 8-fold, 8-fold, 20-fold, >32-fold, and >8-fold enhancements, respectively. The ability of the two structurally related 2-naphthyl-substituted PA-3-4-3 (spermine) analogues 19a and 20a to enhance the action of doxycycline towards P. aeruginosa was investigated more closely, revealing a dose-dependent response when the doxycycline concentration was varied from 2 to 8 µg/mL (Table 4).    . In addition, they were 8-10-fold more active in combination than when tested alone (Table 3).
Taken together, these studies have identified a number of predominantly 2-naphthylsubstituted polyamines as strong enhancers of the antibiotic action of doxycycline and/or erythromycin towards the Gram-negative bacteria P. aeruginosa, and E. coli. It is interesting to compare these results with our previous investigation of α,ω-diacylarylpolyamines, e.g., 6, which revealed them to be active antimicrobials but with weak antibiotic enhancement properties (e.g., doxycycline vs. P. aeruginosa, 4-fold increase to an MIC of 12.5 µM) [27]. The current results lead us to conclude that substituted naphthyl-polyamines may be worthy of further optimization as antibiotic enhancers. It is pertinent to note that Yasuda et al. have previously reported that naphthylacetylspermine (22) (Figure 7), a synthetic analogue of joro spider toxin, renders E. coli sensitive to hydrophobic antibiotics, including novobiocin and erythromycin, albeit weakly, at doses of 64-128 µg/mL [35].

Chemistry General Methods
Infrared spectra were run as dry films on an ATR crystal and acquired with a Perkin-Elmer 100 Fourier Transform infrared spectrometer equipped with a Universal ATR Sampling Accessory. Mass spectra were acquired on a Bruker micrOTOF Q II mass spectrometer. Melting points were obtained on an Electrothermal melting point apparatus and are uncorrected. The 1 H, 13 C NMR, and 2D NMR spectra were recorded at 298 °K on a Bruker AVANCE AVIII 400 MHz spectrometer at 400.13 and 100.62 MHz, using standard pulse sequences. Proto-deutero solvent signals were used as internal references (DMSO-d6: δH 2.50, δC 39.52; CD3OD: δH 3.31, δC 49.00). For 1 H NMR, the data are quoted as position (δ), relative integral, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, br = broad), coupling constant (J, Hz), and assignment to the atom. Atom positional assignments were made using 2D-NMR data acquired using standard pulse sequences. The 13 C NMR data are quoted as position (δ) and assignment to the atom. Flash column chromatography was carried out using Davisil silica gel (40-60 µm) or LiChroprep RP-8 (40-63 µm) solid support. Silica gel thin layer chromatography (TLC) was conducted on 0.2 mm thick plates of DC-plastikfolien Kieselgel 60 F254 (Merck). Reversed-phase TLC was carried out on 0.2 mm thick plates of DC-Kieselgel 60 RP-18 F254S (Merck). All solvents used were of analytical grade or better and/or purified according to standard procedures. Chemical reagents used were purchased from standard chemical suppliers and used as purchased. All samples were determined to be >95% purity. Protected polyamines di-tert-

General Procedure A-Diamide Bond Formation
The appropriate Boc-protected polyamine 16a-f (1 equiv.) was added to a solution of carboxylic acid (2.2 equiv.), EDC·HCl (2.6 equiv.), HOBt (2.6 equiv.) and DIPEA (4-6 equiv.), that was stirred in anhydrous CH 2 Cl 2 (1.5 mL) at 0 • C for 30 min under N 2 . The mixture was allowed to come to room temperature and was stirred for a further 20 h under N 2 . The reaction mixture was poured into CH 2 Cl 2 (20 mL) and washed with saturated NaHCO 3 (2 × 30 mL) followed by H 2 O (2 × 30 mL), and it was then dried under reduced pressure and purified by silica gel flash column chromatography (0-20% MeOH/CH 2 Cl 2 ) to create the desired products.

General Procedure B-Diamide Bond Formation
The appropriate Boc-protected polyamine 16a-f (1 equiv.) was added to a solution of carboxylic acid (2.5 equiv.) and EDC·HCl (2.8 equiv.) with DMAP (5 equiv.) stirred in anhydrous CH 2 Cl 2 (1.5 mL) at 0 • C for 10 min under N 2 . The mixture was allowed to come to room temperature and stirred for a further 12 h under N 2 . The reaction mixture was poured into CH 2 Cl 2 (20 mL) and washed with saturated NaHCO 3 (2 × 30 mL) followed by H 2 O (2 × 30 mL), and it was then dried under reduced pressure and purified by silica gel flash column chromatography (0-20% MeOH/CH 2 Cl 2 ) to create the desired products.

General Procedure C-Boc Deprotection
A solution of tert-butyl-carbamate derivative in CH 2 Cl 2 (2 mL) and TFA (0.2 mL) was stirred at room temperature under N 2 for 2 h, and it was followed by solvent removal under reduced pressure. The crude product was purified using C 8 reversed-phase flash column chromatography eluting with 0-100% MeOH/H 2 O (0.05% TFA) to create the corresponding polyamine conjugate as the TFA salt.