Structure–Activity Relationship Studies of Indolglyoxyl-Polyamine Conjugates as Antimicrobials and Antibiotic Potentiators

Antibiotic resistance is a growing global health threat, requiring urgent attention. One approach to overcome antibiotic resistance is to discover and develop new antibiotic enhancers, molecules that work with legacy antibiotics to enhance their efficacy against resistant bacteria. Our previous screening of a library of purified marine natural products and their synthetic analogues led to the discovery of an indolglyoxyl-spermine derivative that exhibited intrinsic antimicrobial properties and was also able to potentiate the action of doxycycline towards the difficult to treat, Gram-negative bacterium Pseudomonas aeruginosa. A set of analogues have now been prepared, exploring the influence of indole substitution at the 5- and 7- positions and length of the polyamine chain on biological activity. While limiting cytotoxicity and/or hemolytic activities were observed for many analogues, two 7-methyl substituted analogues (23b and 23c) were found to exhibit strong activity towards Gram-positive bacteria with no detectable cytotoxicity or hemolytic properties. Different molecular attributes were required for antibiotic enhancing properties, with one example identified, a 5-methoxy-substitiuted analogue (19a), as being a non-toxic, non-hemolytic enhancer of the action of two tetracycline antibiotics, doxycycline and minocycline, towards P. aeruginosa. These results provide further stimulation for the search for novel antimicrobials and antibiotic enhancers amongst marine natural products and related synthetic analogues.


Introduction
The global increase in microbial antibiotic resistance is a growing health threat, requiring urgent attention. With only limited numbers of new antibiotics being approved for clinical use [1][2][3] the search is on for novel strategies that can prove effective against drug-resistant pathogens. One option for treatment is to restore the antibiotic action of legacy antibiotics, requiring the discovery of antibiotic adjuvants or enhancers [4][5][6][7][8]. Marine natural products represent an excellent reservoir of small drug-like molecules from which to discover both new classes of antimicrobial agents [9][10][11] as well as antibiotic enhancers [8,[12][13][14].
Our screening of a library of marine natural product-related α,ω-disubstituted spermine analogues for antimicrobial and antibiotic enhancing properties identified the 6-bromoindolglyoxyl derivative 1 ( Figure 1) as a moderately active antimicrobial towards the Gram-positive bacteria Staphylococcus aureus ATCC 25923 (MIC 6.25 µM) and the fungus Our screening of a library of marine natural product-related α,ω-disubstituted spermine analogues for antimicrobial and antibiotic enhancing properties identified the 6-bromoindolglyoxyl derivative 1 (Figure 1) as a moderately active antimicrobial towards the Gram-positive bacteria Staphylococcus aureus ATCC 25923 (MIC 6.25 µM) and the fungus Cryptococcus neoformans (MIC 1.1 µM). In addition, the combination of 1 with doxycycline exhibited a strong antibiotic enhancement effect towards the Gram-negative bacterium Pseudomonas aeruginosa [15]. Interest in these activities was somewhat tempered by the observation of associated cytotoxicity (human embryonic kidney cell line HEK293, IC50 5.1 µM; rat skeletal myoblast cell line L6, IC50 7.7 µM), prompting the search for less toxic analogues. Subsequent studies identified the requirement of substitution on the indole ring for activity, with 2 being inactive as an antimicrobial or antibiotic enhancer, and that some examples of 5-and 7-substituted analogues (3)(4)(5)(6)(7)(8), notably including halogen, methoxy or methyl functionality, exhibited more modest antimicrobial activities (Table 1), were moderate to excellent antibiotic enhancers ( Table 2) and were generally less cytotoxic and non-hemolytic (Table 3) [16]. Taken together, these studies enabled the identification of the structural requirements for antibiotic enhancement properties amongst a limited set of indolglyoxyl-spermine conjugates, summarized in Figure 2.  Taken together, these studies enabled the identification of the structural requirements for antibiotic enhancement properties amongst a limited set of indolglyoxyl-spermine conjugates, summarized in Figure 2. Our screening of a library of marine natural product-related α,ω-disubstituted spermine analogues for antimicrobial and antibiotic enhancing properties identified the 6-bromoindolglyoxyl derivative 1 (Figure 1) as a moderately active antimicrobial towards the Gram-positive bacteria Staphylococcus aureus ATCC 25923 (MIC 6.25 µM) and the fungus Cryptococcus neoformans (MIC 1.1 µM). In addition, the combination of 1 with doxycycline exhibited a strong antibiotic enhancement effect towards the Gram-negative bacterium Pseudomonas aeruginosa [15]. Interest in these activities was somewhat tempered by the observation of associated cytotoxicity (human embryonic kidney cell line HEK293, IC50 5.1 µM; rat skeletal myoblast cell line L6, IC50 7.7 µM), prompting the search for less toxic analogues. Subsequent studies identified the requirement of substitution on the indole ring for activity, with 2 being inactive as an antimicrobial or antibiotic enhancer, and that some examples of 5-and 7-substituted analogues (3)(4)(5)(6)(7)(8), notably including halogen, methoxy or methyl functionality, exhibited more modest antimicrobial activities (Table 1), were moderate to excellent antibiotic enhancers ( Table 2) and were generally less cytotoxic and non-hemolytic (Table 3) [16]. Taken together, these studies enabled the identification of the structural requirements for antibiotic enhancement properties amongst a limited set of indolglyoxyl-spermine conjugates, summarized in Figure 2.  A component of the structure-activity relationship yet to be addressed in this compound series is the effect, if any, of variation in the polyamine (PA) chain length on intrinsic antimicrobial, antibiotic enhancement and cytotoxicity/hemolysis biological activities. Previous studies investigating disubstituted polyamine-bearing arylacyl [17] head groups The indole-3-glyoxyl head groups used in the current study (10)(11)(12)(13)(14)(15)(16) (Figure 4) were the same set previously explored in analogues 2-8 [16]. Syntheses of 10-15, as either the glyoxylic acid or glyoxylchloride, have been previously reported [22][23][24][25]. In the case of the 7-methyl analogue 16, it was prepared using the two-step protocol shown in Scheme 1. Reaction of 7-methyl-1H-indole with excess oxalyl chloride afforded the oxalylchloride intermediate which was not isolated but hydrolyzed by heating with saturated aq. NaHCO3 solution to afford 2-(7-methyl-1H-indol-3-yl)-2-oxoacetic acid (16) ( Figure S1) in 95% yield over two steps. The indole-3-glyoxyl head groups used in the current study (10)(11)(12)(13)(14)(15)(16) (Figure 4) were the same set previously explored in analogues 2-8 [16]. Syntheses of 10-15, as either the glyoxylic acid or glyoxylchloride, have been previously reported [22][23][24][25].
A component of the structure-activity relationship yet to be addressed in this compound series is the effect, if any, of variation in the polyamine (PA) chain length on intrinsic antimicrobial, antibiotic enhancement and cytotoxicity/hemolysis biological activities. Previous studies investigating disubstituted polyamine-bearing arylacyl [17] head groups identified that changes in the chain length of the core polyamine fragment can lead to wide variation in antimicrobial and/or antibiotic enhancing properties. Herein we report details on the synthesis of a new set of indolglyoxyl-polyamine conjugates that vary in substitution at the 5-and 7-positions on the indole ring and that vary in polyamine chain length, and the abilities of these analogues to exhibit intrinsic antimicrobial properties and to potentiate the activity of doxycycline towards the Gram-negative bacteria Pseudomonas aeruginosa.

Biological Evaluation
The antimicrobial activity of the series was evaluated against a range of bacterial strains (S. aureus, MRSA, P. aeruginosa and Escherichia coli) and the fungus Candida albicans (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. . In many cases, those analogues that exhibited good levels of antimicrobial activity also unfortunately demonstrated cytotoxicity and/or hemolytic activity. There were some examples identified, however, that were devoid of these detrimental properties including the 7-methyl substituted analogues 23b (MIC MRSA ≤ 0.30 µM, cytotoxicity IC 50 > 38 µM, hemolysis HC 10 > 38 µM) and 23c (MIC MRSA ≤ 0.29 µM, cytotoxicity IC 50 > 37 µM, hemolysis HC 10 > 37 µM). Overall, the discovery of Gram-positive antibacterial activity for 23b and 23c with low to no cytotoxicity and hemolytic activity suggests a narrow structure-activity requirement of 7-methyl substitution and polyamine mid-chain length of 7 (PA3-7-3) or 8 (PA3-8-3) carbons for optimal activity.
The set of compounds were next evaluated for the ability to potentiate the activity of the antibiotic doxycycline towards the Gram-negative bacteria P. aeruginosa ATCC 27853 ( Table 2). In these assays, doxycycline is present at a concentration of 2 µg/mL (4.5 µM), well below the observed MIC of 20 µg/mL (50 µM) towards this drug-resistant human pathogen.  19e >200 a Concentration (µM) required to restore doxycycline activity at 2 µg/mL (4.5 µM) against P. aeruginosa ATCC 27853; b Data taken from Cadelis et al. [16].
A closer investigation of the ability of the 5-methoxy-indolglyoxyl-PA3-6-3 analogue 19a to enhance the action of other antibiotics towards P. aeruginosa identified it to be capable of reactivating another tetracycline antibiotic minocycline (MIC 14.5 µM), was only a weak activator of chloramphenicol (MIC 58 µM) and could not restore the activity of erythromycin or nalidixic acid (Table 3). All values presented as the mean (n = 3). a Concentration (µM) of compound 19a required to restore antibiotic activity at 2 µg/mL concentration of antibiotic. P. aeruginosa ATCC 27853 against minocycline (MIC 70 µM), erythromycin (MIC >200 µM), chloramphenicol (MIC >200 µM) and nalidixic acid (MIC >200 µM).
The spectrum of antibiotic potentiating activity of the 7-fluoro analogue 21a was also investigated, evaluating its ability to enhance other antibiotics against other Gram-negative bacteria ( Table 4). The polyamine-conjugate was able to restore the action of doxycycline against E. coli (MIC 1.56 µM) and to a lesser degree against Acinetobacter baumannii (MIC 12.5 µM). Of the other combinations examined, 21a was also found to weakly enhance the action of chloramphenicol and nalidixic acid towards P. aeruginosa. We have observed similar levels of drug-organism antibiotic enhancement for other examples of indolglyoxylpolyamines [16]. All values presented as the mean (n = 3). a Concentration (µM) of compound 21a required to restore antibiotic activity at 2 µg/mL concentration of antibiotic; b P. aeruginosa

Chemistry: General Remarks
Infrared spectra were recorded on a Perkin-Elmer spectrometer 100 Fourier-transform infrared spectrometer (Perkin-Elmer, MA, USA) equipped with a universal ATR accessory. Mass spectra were acquired on a Bruker micrOTOF Q II spectrometer. 1 H and 13 C NMR spectra were recorded at 298 K on a Bruker (Karlsruhe, Germany) AVANCE 400 spectrometer using standard pulse sequences. Proto-deutero solvent signals were used as internal references (DMSO-d 6 : δ H 2.50, δ C 39.52). For 1 H NMR, the data are quoted as position (δ), relative integral, multiplicity (s = singlet, d = doublet, t = triplet, dt = doublet of triplet, tt = triplet of triplet, m = multiplet), coupling constant (J, Hz), and assignment to the atom. 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 Merck LiChroprep RP-8 (40-63 µm) (Merck Millipore, Darmstadt, Germany). Thin-layer chromatography was conducted on Merck DC Kieselgel 60 RP-18 F254S plates. 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. Protected (15) [25], and polyamine conjugates (17c/17e/19c/19e/22c/22e) [25] were synthesized using procedures from the literature.

General Procedure B-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 followed by solvent removal under reduced pressure. The crude product was purified using C 8 reversed-phase flash column chromatography (0%-50% MeOH/H 2 O (+0.05% TFA)) to afford the product as a di-TFA salt.

Conclusions
Our original screening for antimicrobial and antibiotic enhancing compounds from a library of marine natural products and their synthetic analogues identified a 6-bromoindolglyoxylamido-spermine conjugate as an active lead compound. Due to associated cytotoxicity and hemolytic properties, further efforts to explore the structure-activity relationship have investigated variation of substitution on the indole ring, and changes in the chain length of the polyamine fragment. While many analogues that were active as Gram-positive antibacterials were also associated with variable levels of cytotoxicity and/or hemolytic properties, the current study has identified two 7-methyl substituted analogues (23b and 23c) with excellent anti-MRSA activity that are non-cytotoxic and non-hemolytic. This result defines a very narrow range of structural features required for optimal antibacterial properties. From the same set of analogues, only one example (19a), a 5-methoxy-PA3-6-3 conjugate, was non-toxic while also exhibiting strong tetracycline antibiotic enhancing activity towards P. aeruginosa. Further studies will be required to refine the mechanism of antibiotic enhancement.