α,ω-Diacyl-Substituted Analogues of Natural and Unnatural Polyamines: Identification of Potent Bactericides That Selectively Target Bacterial Membranes

In this study, α-ω-disubstituted polyamines exhibit a range of potentially useful biological activities, including antimicrobial and antibiotic potentiation properties. We have prepared an expanded set of diarylbis(thioureido)polyamines that vary in central polyamine core length, identifying analogues with potent methicillin-resistant Staphylococcus aureus (MRSA), Escherichia coli, Acinetobacter baumannii and Candida albicans growth inhibition properties, in addition to the ability to enhance action of doxycycline towards Gram-negative bacterium Pseudomonas aeruginosa. The observation of associated cytotoxicity/hemolytic properties prompted synthesis of an alternative series of diacylpolyamines that explored aromatic head groups of varying lipophilicity. Examples bearing terminal groups each containing two phenyl rings (15a–f, 16a–f) were found to have optimal intrinsic antimicrobial properties, with MRSA being the most susceptible organism. A lack of observed cytotoxicity or hemolytic properties for all but the longest polyamine chain variants identified these as non-toxic Gram-positive antimicrobials worthy of further study. Analogues bearing either one or three aromatic-ring-containing head groups were either generally devoid of antimicrobial properties (one ring) or cytotoxic/hemolytic (three rings), defining a rather narrow range of head group lipophilicity that affords selectivity for Gram-positive bacterial membranes versus mammalian. Analogue 15d is bactericidal and targets the Gram-positive bacterial membrane.


Introduction
Biogenic polyamines (PA) can disrupt bacterial membranes, inhibit formation of biofilms and act as adjuvants to increase activity of antibiotics towards resistant bacteria [1,2]. While simple polyamines such as spermidine and spermine  have weak mM potency, more elaborately functionalized polyamine analogues, including natural products such as squalamine (1) [3][4][5][6] and ianthelliformisamine C (2) [7][8][9] (Figure 1), are significantly more active, exhibiting enhanced ability to disrupt and/or depolarize bacterial membranes and the enhance action of different classes of antibiotics towards Gram-negative bacteria. Examples of polyamine terminal substituents that imbue intrinsic antimicrobial activities and/or antibiotic adjuvant properties include cinnamic acids [8,9], thioureas [10], indole derivatives [11][12][13] and lipids, including alkanes/alkenes [14][15][16][17], sterols [18][19][20], diterpenes [21] and triterpenes [18,22]. Presence of secondary alkylamines in the polyamine chain, protonated at physiological pH and an essential requirement for activity [10,11], combined with lipophilic end-groups, represents an amphipathic motif satisfying the minimal pharmacophore of synthetic mimics of antimicrobial peptides [23][24][25][26]. While [18][19][20], diterpenes [21] and triterpenes [18,22]. Presence of secondary alkylamines in the polyamine chain, protonated at physiological pH and an essential requirement for activity [10,11], combined with lipophilic end-groups, represents an amphipathic motif satisfying the minimal pharmacophore of synthetic mimics of antimicrobial peptides [23][24][25][26]. While satisfying this pharmacophore model, it is still not clear as to what chemometric attributes the terminal substituent(s) require as far as intrinsic antimicrobial, antibiotic adjuvant or cytotoxic/hemolytic properties. Of note was the recent report of urea-and thiourea-functionalized polyamines, including 3 and 4 ( Figure 2), which exhibited broad-spectrum antimicrobial properties towards both Gram-positive and Gram-negative bacteria, with a mechanism attributed to depolarization of the cytoplasmic membrane and permeabilization of the bacterial outer membrane [10]. Structure-relationship analysis identified essentiality of the mid-chain secondary amines for activity, that diaryl aromatic head groups were more active than mono-aryl, thioureas were equipotent to the corresponding ureas and, from a limited dataset of polyamine midchain lengths, there was little variation in activity between PA-3-4-3 and PA-3-5-3 variants. Closer investigation of the biological properties of 4 identified potent activity towards methicillin-resistant Staphylococcus aureus (MRSA), only weak hemolytic and cytotoxic properties, the ability to depolarize bacterial membrane potential and increase bacterial membrane permeability and to act synergistically with kanamycin towards S. aureus. In continuation of our ongoing interest in discovery of polyamine derivatives that exhibit antibacterial and antibiotic adjuvant properties [11][12][13]21,27], we have prepared a set of five additional analogues of thiourea 3 to explore the effect of polyamine chain length on activity and a further set of twenty-four analogues that explore variation in the thiourea linking group and changes to end-group lipophilicity. All analogues were evaluated for antimicrobial activities against a set of Gram-positive and Gram-negative Of note was the recent report of urea-and thiourea-functionalized polyamines, including 3 and 4 ( Figure 2), which exhibited broad-spectrum antimicrobial properties towards both Gram-positive and Gram-negative bacteria, with a mechanism attributed to depolarization of the cytoplasmic membrane and permeabilization of the bacterial outer membrane [10]. Structure-relationship analysis identified essentiality of the mid-chain secondary amines for activity, that diaryl aromatic head groups were more active than mono-aryl, thioureas were equipotent to the corresponding ureas and, from a limited dataset of polyamine midchain lengths, there was little variation in activity between PA-3-4-3 and PA-3-5-3 variants. Closer investigation of the biological properties of 4 identified potent activity towards methicillin-resistant Staphylococcus aureus (MRSA), only weak hemolytic and cytotoxic properties, the ability to depolarize bacterial membrane potential and increase bacterial membrane permeability and to act synergistically with kanamycin towards S. aureus. [18][19][20], diterpenes [21] and triterpenes [18,22]. Presence of secondary alkylamines in the polyamine chain, protonated at physiological pH and an essential requirement for activity [10,11], combined with lipophilic end-groups, represents an amphipathic motif satisfying the minimal pharmacophore of synthetic mimics of antimicrobial peptides [23][24][25][26]. While satisfying this pharmacophore model, it is still not clear as to what chemometric attributes the terminal substituent(s) require as far as intrinsic antimicrobial, antibiotic adjuvant or cytotoxic/hemolytic properties. Of note was the recent report of urea-and thiourea-functionalized polyamines, including 3 and 4 ( Figure 2), which exhibited broad-spectrum antimicrobial properties towards both Gram-positive and Gram-negative bacteria, with a mechanism attributed to depolarization of the cytoplasmic membrane and permeabilization of the bacterial outer membrane [10]. Structure-relationship analysis identified essentiality of the mid-chain secondary amines for activity, that diaryl aromatic head groups were more active than mono-aryl, thioureas were equipotent to the corresponding ureas and, from a limited dataset of polyamine midchain lengths, there was little variation in activity between PA-3-4-3 and PA-3-5-3 variants. Closer investigation of the biological properties of 4 identified potent activity towards methicillin-resistant Staphylococcus aureus (MRSA), only weak hemolytic and cytotoxic properties, the ability to depolarize bacterial membrane potential and increase bacterial membrane permeability and to act synergistically with kanamycin towards S. aureus. In continuation of our ongoing interest in discovery of polyamine derivatives that exhibit antibacterial and antibiotic adjuvant properties [11][12][13]21,27], we have prepared a set of five additional analogues of thiourea 3 to explore the effect of polyamine chain length on activity and a further set of twenty-four analogues that explore variation in the thiourea linking group and changes to end-group lipophilicity. All analogues were evaluated for antimicrobial activities against a set of Gram-positive and Gram-negative In continuation of our ongoing interest in discovery of polyamine derivatives that exhibit antibacterial and antibiotic adjuvant properties [11][12][13]21,27], we have prepared a set of five additional analogues of thiourea 3 to explore the effect of polyamine chain length on activity and a further set of twenty-four analogues that explore variation in the thiourea linking group and changes to end-group lipophilicity. All analogues were evaluated for antimicrobial activities against a set of Gram-positive and Gram-negative bacteria and for the ability to enhance the antibiotic activity of doxycycline towards Gramnegative bacteria Pseudomonas aeruginosa.
bacteria and for the ability to enhance the antibiotic activity of doxycycline towards Gramnegative bacteria Pseudomonas aeruginosa.
The structures of the synthesized diacylpolyamine library are shown in Figure 5.

Results and Discussion
The intrinsic antimicrobial activity of the series was evaluated against a range of Gram-positive (S. aureus and MRSA) and Gram-negative (Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae and Acinetobacter baumannii) bacteria and two fungal strains (Candida albicans and Cryptococcus neoformans) (Table 1). Cytotoxicity towards HEK293 (human kidney epithelial cell line, IC50) and hemolytic activity against human red blood cells (HC10) were also determined ( Table 2). The thiourea analogues 6a-f uniformly exhibited strong growth inhibition of bacteria MRSA (MIC ≤ 0.34 µM) and E. coli (MIC < 2.8 µM) and fungus C. albicans (MIC ≤ 0.3 µM). The S. aureus and E. coli inhibition results for 6a were consistent with previously reported data for the same compound, although, in our case, less pronounced activity was observed towards P. aeruginosa [10]. There was no apparent effect of polyamine chain length on antimicrobial activity. Variable levels of cytotoxicity and hemolytic properties were observed for 6a-f, with the PA-3-10-3 analogue (6e) identified as having the most favorable (least toxic) profile with IC50 > 39.5 µM and HC10 > 39.5 µM. The observation of cytotoxicity/hemolytic properties prompted our investigation of a further set of analogues that explored replacement of the thiourea linking group present in 6a-f with more simplified amide-based linkers. Further, 3-phenylpropionic acid (13a-f) and 3-phenylpropylamine-succinic acid (14a-f) -based linkers were almost universally devoid of antimicrobial properties except for the longer chain PA-3-10-3 and PA-3-12-3 variants, which exhibited weak (13e, 13f, 14e) to potent (14f) activity towards MRSA. When compounds in these sets were evaluated for detrimental cellular effects, none exhibited cytotoxicity or hemolytic properties, identifying PA-3-12-3 analogue 14f as a non-toxic, non-hemolytic, strongly active anti-MRSA molecule. Increasing the lipophilicity by inclusion of an additional phenyl ring in the capping acid provided two sets of analogues (15a-f, 16a-f) that exhibited enhanced anti-MRSA activity (all examples MIC ≤ 0.29 µM) and with some examples exhibiting activity towards the Gram-negative bacteria E. coli (15a-f, MIC ≤ 0.27 to 4.6 µM) and fungus C. neoformans (15c, 15f, 16a, 16e, MIC ≤ 0.28 µM). Of these two compound sets, only the longer chain variants 15f, 16e and 16f, exhibited cytotoxicity and/or hemolytic properties, identifying, in particular, the majority

Results and Discussion
The intrinsic antimicrobial activity of the series was evaluated against a range of Grampositive (S. aureus and MRSA) and Gram-negative (Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae and Acinetobacter baumannii) bacteria and two fungal strains (Candida albicans and Cryptococcus neoformans) ( Table 1). Cytotoxicity towards HEK293 (human kidney epithelial cell line, IC 50 ) and hemolytic activity against human red blood cells (HC 10 ) were also determined ( Table 2). The thiourea analogues 6a-f uniformly exhibited strong growth inhibition of bacteria MRSA (MIC ≤ 0.34 µM) and E. coli (MIC < 2.8 µM) and fungus C. albicans (MIC ≤ 0.3 µM). The S. aureus and E. coli inhibition results for 6a were consistent with previously reported data for the same compound, although, in our case, less pronounced activity was observed towards P. aeruginosa [10]. There was no apparent effect of polyamine chain length on antimicrobial activity. Variable levels of cytotoxicity and hemolytic properties were observed for 6a-f, with the PA-3-10-3 analogue (6e) identified as having the most favorable (least toxic) profile with IC 50 > 39.5 µM and HC 10 > 39.5 µM. The observation of cytotoxicity/hemolytic properties prompted our investigation of a further set of analogues that explored replacement of the thiourea linking group present in 6a-f with more simplified amide-based linkers. Further, 3-phenylpropionic acid (13a-f) and 3-phenylpropylamine-succinic acid (14a-f) -based linkers were almost universally devoid of antimicrobial properties except for the longer chain PA-3-10-3 and PA-3-12-3 variants, which exhibited weak (13e, 13f, 14e) to potent (14f) activity towards MRSA. When compounds in these sets were evaluated for detrimental cellular effects, none exhibited cytotoxicity or hemolytic properties, identifying PA-3-12-3 analogue 14f as a non-toxic, non-hemolytic, strongly active anti-MRSA molecule. Increasing the lipophilicity by inclusion of an additional phenyl ring in the capping acid provided two sets of analogues (15a-f, 16a-f) that exhibited enhanced anti-MRSA activity (all examples MIC ≤ 0.29 µM) and with some examples exhibiting activity towards the Gram-negative bacteria E. coli (15a-f, MIC ≤ 0.27 to 4.6 µM) and fungus C. neoformans (15c, 15f, 16a, 16e, MIC ≤ 0.28 µM). Of these two compound sets, only the longer chain variants 15f, 16e and 16f, exhibited cytotoxicity and/or hemolytic properties, identifying, in particular, the majority of the diphenylpropyl analogue set (15a-e) as being of further interest as antimicrobial agents.   The observation of increased cytotoxicity/hemolytic activity for the longer chain variants identified that toxicity, likely arising from enhanced penetration/disruption of mammalian membranes, was not solely dependent upon the lipophilicity of just the aromatic head group but was a function of the whole molecule. Calculated logP values (cLogP) were generated for free base structures using DataWarrior [32] and are included in Table 2. For the two sets of analogues bearing diphenyl groups at each end of the polyamine, the 'second hydrophobicity threshold' [33] appears to be in the order of cLogP 9-10. The concept of a second hydrophobicity threshold was proposed to explain the observed ability of cationic antimicrobial peptides (CAPs) to insert into and ultimately disrupt bacterial versus mammalian membranes, with higher hydrophobicity/lipophilicity associated with hemolytic activity in erythrocytes. A nuance to the hydrophobicity threshold model of mammalian cell toxicity in the current context was observed for the final two sets of analogues (17a-f, 18a-f) bearing three phenyl ring-containing substituents at each end of the polyamine chain. Although both sets of analogues exhibited broad spectrum activity towards a range of microbes in the screening panel, including MRSA, E. coli, A. baumannii, C. albicans and C. neoformans, interest in these compounds was abrogated by their moderate to strong cytotoxic/hemolytic properties. The calculated LogP values for these last two sets of analogues (Table 2) covered the range of 7. 8-12.4, with the lower values of 7.8-9.2 (17a-c, 18a) being of similar magnitude to those calculated for non-cytotoxic/non-hemolytic diphenyl variants 15c-e and 16c-d, suggesting that, in the cases of 17a-f and 18a-f, the presence of the triphenyl aromatic head group was itself enough to cause mammalian toxicity.
Analogue 15d was chosen for closer examination of antibacterial activity and preliminary mechanism of action evaluation as it exhibited potent antibacterial properties with no detectable cytotoxicity or hemolytic activities. The kinetics of antibacterial activity of 15d towards Gram-positive bacteria were undertaken by measuring real-time growth inhibition curves against S. aureus ATCC 25923, MRSA (CF-Marseille) [34] and Bacillus cereus ATCC 11778. The test compound completely inhibited all three strains at 4.4 µM (4 µg/mL) and 17 µM (16 µg/mL) concentration, whereas, at the lowest tested concentration, 2.2 µM (2 µg/mL), bacterial growth was detected after 6 h for S. aureus, 4 h for MRSA and 12 h for B. cereus ( Figure 6). Classical microdilution methodology determined an MIC value of 4.4 µM (4 µg/mL) for 15d towards these three microorganisms, with the values matching those observed at 18 h in the real-time growth inhibition curve plots. The same values were observed for the minimum bactericidal concentration (MBC) for 15d against the three organisms, identifying this analogue as being bactericidal. mammalian cell toxicity in the current context was observed for the final two sets of analogues (17a-f, 18a-f) bearing three phenyl ring-containing substituents at each end of the polyamine chain. Although both sets of analogues exhibited broad spectrum activity towards a range of microbes in the screening panel, including MRSA, E. coli, A. baumannii, C. albicans and C. neoformans, interest in these compounds was abrogated by their moderate to strong cytotoxic/hemolytic properties. The calculated LogP values for these last two sets of analogues (Table 2) covered the range of 7. 8-12.4, with the lower values of 7.8-9.2 (17a-c, 18a) being of similar magnitude to those calculated for non-cytotoxic/non-hemolytic diphenyl variants 15c-e and 16c-d, suggesting that, in the cases of 17a-f and 18a-f, the presence of the triphenyl aromatic head group was itself enough to cause mammalian toxicity.
Analogue 15d was chosen for closer examination of antibacterial activity and preliminary mechanism of action evaluation as it exhibited potent antibacterial properties with no detectable cytotoxicity or hemolytic activities. The kinetics of antibacterial activity of 15d towards Gram-positive bacteria were undertaken by measuring real-time growth inhibition curves against S. aureus ATCC 25923, MRSA (CF-Marseille) [34] and Bacillus cereus ATCC 11778. The test compound completely inhibited all three strains at 4.4 µM (4 µg/mL) and 17 µM (16 µg/mL) concentration, whereas, at the lowest tested concentration, 2.2 µM (2 µg/mL), bacterial growth was detected after 6 h for S. aureus, 4 h for MRSA and 12 h for B. cereus ( Figure 6). Classical microdilution methodology determined an MIC value of 4.4 µM (4 µg/mL) for 15d towards these three microorganisms, with the values matching those observed at 18 h in the real-time growth inhibition curve plots. The same values were observed for the minimum bactericidal concentration (MBC) for 15d against the three organisms, identifying this analogue as being bactericidal. The mechanism of action of antibacterial activity observed for 15d was attributed to the ability to disrupt the bacterial cell membrane. Brief (1 s) exposure of S. aureus ATCC 25923 cells to the test compound led to rapid dose-dependent leakage of intracellular ATP, as determined by a bioluminescence assay ( Figure 7) [8]. The higher compound doses (62.5 to 125 µM) provided leakage comparable in magnitude to that observed for the positive control, a 1% solution of cationic detergent cetyltrimethylammonium bromide (CTAB). The mechanism of action of antibacterial activity observed for 15d was attributed to the ability to disrupt the bacterial cell membrane. Brief (1 s) exposure of S. aureus ATCC 25923 cells to the test compound led to rapid dose-dependent leakage of intracellular ATP, as determined by a bioluminescence assay ( Figure 7) [8]. The higher compound doses (62.5 to 125 µM) provided leakage comparable in magnitude to that observed for the positive control, a 1% solution of cationic detergent cetyltrimethylammonium bromide (CTAB). The original report describing (bis)arylthioureido analogues 3 and 4 identified the latter as being synergistic with kanamycin, causing 8-fold reduction in MIC against S. aureus and P. aeruginosa [10]. No synergism was observed in combination with ampicillin or norfloxacin. We have evaluated the set of analogues for the ability to enhance the antibiotic activity of doxycycline against P. aeruginosa ATCC 27853 (Table 3). In this assay, a fixed concentration of doxycycline of 2 µg/mL (4.5 µM), which is twenty-fold lower than the intrinsic MIC [40 µg/mL (90 µM)] against this organism, is used, with each of the test compounds evaluated at a range of concentrations varying from 3.125 to 50-100 µM, with the upper concentration dependent upon compounds' intrinsic MIC towards P. aeruginosa. All but the longest chain variant of the thiourea analogues exhibited antibiotic enhancement, with 6a-c and 6e being particularly strong enhancers. Of the remaining thirtysix compounds (13a-f to 18a-f), only modest potency of antibiotic enhancement was observed, with 16a (8-fold increase to 25 µM) and 15a (4-fold increase to 12.5 µM) being the most active. Although disappointing, these results highlight that further research is required to fine-tune the attributes of the aromatic head groups and their linker unit of α,ωdisubstituted polyamines to develop non-toxic drug candidates that can enhance action of antibiotics towards drug-resistant bacteria. The current results have identified the diaryl head group series 15a-e and 16a-d as good starting points for further optimization as antimicrobial agents, details of which will be reported in due course.  The original report describing (bis)arylthioureido analogues 3 and 4 identified the latter as being synergistic with kanamycin, causing 8-fold reduction in MIC against S. aureus and P. aeruginosa [10]. No synergism was observed in combination with ampicillin or norfloxacin. We have evaluated the set of analogues for the ability to enhance the antibiotic activity of doxycycline against P. aeruginosa ATCC 27853 (Table 3). In this assay, a fixed concentration of doxycycline of 2 µg/mL (4.5 µM), which is twenty-fold lower than the intrinsic MIC [40 µg/mL (90 µM)] against this organism, is used, with each of the test compounds evaluated at a range of concentrations varying from 3.125 to 50-100 µM, with the upper concentration dependent upon compounds' intrinsic MIC towards P. aeruginosa. All but the longest chain variant of the thiourea analogues exhibited antibiotic enhancement, with 6a-c and 6e being particularly strong enhancers. Of the remaining thirty-six compounds (13a-f to 18a-f), only modest potency of antibiotic enhancement was observed, with 16a (8-fold increase to 25 µM) and 15a (4-fold increase to 12.5 µM) being the most active. Although disappointing, these results highlight that further research is required to fine-tune the attributes of the aromatic head groups and their linker unit of α,ω-disubstituted polyamines to develop non-toxic drug candidates that can enhance action of antibiotics towards drug-resistant bacteria. The current results have identified the diaryl head group series 15a-e and 16a-d as good starting points for further optimization as antimicrobial agents, details of which will be reported in due course.

General Procedure A: Reaction of Benzhydryl Isothiocyanate with Boc-Protected Polyamine
To a stirred solution of Boc-protected polyamine (5a-f) (1 equiv.) in anhydrous CH 2 Cl 2 (5 mL) at 0 • C was added benzhydryl isothiocyanate (2.2 eq.) in CH 2 Cl 2 (5 mL) dropwise. The reaction mixture was stirred for 18 h before the solvent was removed under reduced pressure and the crude product purified by silica gel column chromatography (hexane:EtOAc, 60:40 to 25:75).

General Procedure B: Boc Deprotection Using 1% HCl/EtOAc
A solution of Boc-protected thiourea polyamine (20-50 mg) was dissolved in 1% HCl in EtOAc (5-12.5 mL) and stirred at rt under N 2 for 12 h. Additional EtOAc (2 × 20 mL) was added and the mixture stirred for 15 min before the liquid was decanted and the solid product dried under reduced pressure.
Colistin and vancomycin were used as positive bacterial inhibitor standards for Gramnegative and Gram-positive bacteria, respectively. Fluconazole was used as a positive fungal inhibitor standard for C. albicans and C. neoformans. The antibiotics were provided in 4 concentrations, with 2 above and 2 below its MIC value, and plated into the first 8 wells of column 23 of 384-well NBS plates. Quality control (QC) of the assays was determined by antimicrobial controls and Z'-factor (using positive and negative controls). Each plate was deemed to fulfil the quality criteria (pass QC) if the Z'-factor was above 0.4 and the antimicrobial standards showed full range of activity, with full growth inhibition at their highest concentration and no growth inhibition at their lowest concentration.

Determination of the MICs of Antibiotics in the Presence of Synergizing Compounds
Briefly, restoring enhancer concentrations were determined with an inoculum of 5 × 10 5 CFU in 200 µL of MHB containing two-fold serial dilutions of each derivative in the presence of doxycycline at 2 µg/mL. The lowest concentration of the polyamine adjuvant that completely inhibited visible growth after incubation for 18 h at 37 • C was determined. These measurements were independently repeated in triplicate.

Cytotoxicity Assays
HEK293 cells were counted manually in a Neubauer hemocytometer and plated at a density of 5000 cells/well into each well of the 384-well plates containing the 25× (2 µL) concentrated compounds. The medium used was Dulbecco's modified eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Cells were incubated together with the compounds for 20 h at 37 • C, 5% CO 2 . To measure cytotoxicity, 5 µL (equals 100 µM final) of resazurin was added to each well after incubation and incubated for further 3 h at 37 • C with 5% CO 2 . After final incubation, fluorescence intensity was measured as Fex 560/10 nm, em 590/10 nm (F 560/590 ) using a Tecan M1000 Pro monochromator plate reader. CC 50 values (concentration at 50% cytotoxicity) were calculated by normalizing the fluorescence readout, with 74 µg/mL tamoxifen as negative control (0%) and normal cell growth as positive control (100%). The concentration-dependent percentage cytotoxicity was fitted to a dose-response function (using Pipeline Pilot) and CC 50 values determined.

Hemolytic Assays
Human whole blood was washed three times with 3 volumes of 0.9% NaCl and then resuspended in same to a concentration of 0.5 × 10 8 cells/mL, as determined by manual cell count in a Neubauer hemocytometer. The washed cells were then added to the 384-well compound-containing plates for a final volume of 50 µL. After a 10 min shake on a plate shaker, the plates were then incubated for 1 h at 37 • C. After incubation, the plates were centrifuged at 1000× g for 10 min to pellet cells and debris; 25 µL of the supernatant was then transferred to a polystyrene 384-well assay plate. Hemolysis was determined by measuring the supernatant absorbance at 405 mm (OD 405 ). The absorbance was measured using a Tecan M1000 Pro monochromator plate reader. HC 10 and HC 50 (concentration at 10% and 50% hemolysis, respectively) were calculated by curve fitting the inhibition values vs. log (concentration) using a sigmoidal dose-response function with variable fitting values for top, bottom and slope.

Real-Time Growth Curves
Solutions of compound 15d at concentrations of 2, 4 and 16 µg/mL were tested, each in triplicate, against S. aureus ATCC 25923, MRSA (CF-Marseille) and Bacillus cereus ATCC 11778. Typically, in a 96-well plate, 10 µL of 40, 80 and 320 µg/mL stock solutions of compound 15d were placed, as well as 190 µL of a 5 × 10 5 CFU/mL of the selected bacterial suspension in brain heart infusion (BHI) broth. Positive controls containing only 200 µL of a 5 × 10 5 CFU/mL of bacterial suspension in BHI and negative controls containing only 200 µL of BHI broth were added. The plate was incubated at 37 • C in a TECAN Spark Reader (Roche Diagnostic), and real-time bacterial growth was followed by OD 590 nm measurement every 10 min during 19 h.

Minimum Bactericidal Concentration Test
A pure culture of a specified microorganism was grown overnight, then diluted in growth-supporting broth (typically Mueller-Hinton II broth) to a concentration between 1 × 10 5 and 1 × 10 6 CFU/mL. A stock dilution of the antimicrobial test compound was created at approximately 100 times the expected previously determined MIC. Further 1:1 dilution was made in 96-well microtiter plates. All dilutions of the test compound were inoculated with equal volumes of the specified microorganism (typically 100 µL). A positive and negative control tube or well is included to demonstrate adequate microbial growth over the course of the incubation period and media sterility, respectively. An aliquot of the positive control is plated and used to establish a baseline concentration of the microorganism used. The microtiter plates were then incubated at 37 • C for 24 h. Turbidity indicates growth of the microorganism, and the MIC is the lowest concentration where no growth is visually observed. To determine the minimum bactericidal concentration (MBC), the dilution representing the MIC and at least two of the more concentrated test product dilutions are plated on a solidified agar plate to determine the bacterial viability. The MBC is the lowest concentration where no growth is encountered when compared to the MIC dilution.

ATP Release Assay
Solutions of test compound 15d were prepared in DMSO at various concentrations. A suspension of growing S. aureus to be studied in Muller-Hinton II broth was prepared and incubated at 37 • C. An aliquot (90 µL) of this suspension was added to 10 µL of test compound solution and vortexed for 10 s. Luciferin-luciferase reagent (Yelen, France; 50 µL) was immediately added to this mixture and luminescent signal quantified with an Infinite M200 microplate reader (Tecan) over a 30 min period. ATP concentration was quantified by internal sample addition. A similar procedure was used for the CTAB positive control.

Conclusions
In this study, α,ω-disubstituted polyamines exhibit promising antimicrobial properties and can also enhance action of other antibiotics towards drug-resistant Gram-negative bacteria. The present study explored variation in polyamine chain length, aromatic head group lipophilicity and linker chemistry on intrinsic antimicrobial and antibiotic enhancement biological activities. Favorable antimicrobial and antibiotic enhancement activities were observed for thiourea-linked examples, supporting previously reported interest in this class of polyamine derivative. The observation of cytotoxicity and/or hemolytic properties prompted investigation of alternative amide-linked alternatives. Of note was the discovery of diaryl-aromatic-head-group-substituted examples that exhibited growth inhibition of MRSA and E. coli with no cytotoxic or red blood cell hemolytic effects. Presence of diaryl substitution at each end of the polyamine chain was found to be optimal for antimicrobial selectivity, with mono-aryl examples being essentially inactive and triaryl variants being cytotoxic and/or hemolytic. While antibiotic enhancement was observed for the majority of the thiourea-linked examples, little to no enhancement was observed for the amide-linked analogues, highlighting the need for further research to define the attributes and influence of end group and linker chemistry on antimicrobial and antibiotic enhancement properties of substituted polyamines.