Natural product-derived quaternary ammonium compounds with potent antimicrobial activity

Full text of DOI:10.1038/ja.2015.107

The Journal of Antibiotics (2015), 1–4
2015 Japan Antibiotics Research Association All rights reserved 0021-8820/15
www.nature.com/ja
&
NOTE
Natural product-derived quaternary ammonium
compounds with potent antimicrobial activity
Maureen D Joyce1, Megan C Jennings2, Celina N Santiago1, Madison H Fletcher2, William M Wuest2 and
Kevin PC Minbiole1
The Journal of Antibiotics advance online publication, 18 November 2015; doi:10.1038/ja.2015.107
Dedicated to Professor Amos B. Smith, III, in celebration of his products that are not, in their own right, antibacterial agents. Further,
seminal contributions to basic science and human health.
Quaternary ammonium compounds (QACs) bearing long alkyl
we aimed to generate analogous series of structures that could bear
one or two cationic residues, as multicationic QACs (multiQACs) have
chains are classical examples of amphiphiles, displaying a variety of been recently shown to exhibit strong antibacterial activity, and
interesting physical properties, such as the capacity for micelle
formation and gelation.1 QACs also enjoy extensive precedent and
importantly, be able to evade the QAC resistance traits observed due
to efflux pumps. The function of bisQACs in particular still requires
applications in bacterial cell membrane disruption, leading to their investigation, as aromatic derivatives seem to show more susceptibility
widespread use as antiseptics.2 Both synthetic QACs and peptide-based to bacterial resistance than their alkyl counterparts.11
amphiphiles (notably, antimicrobial peptides or AMPs3) are prevalent.
Two natural products that presented themselves as ideal core
However, aside from modified peptides, there are relatively few QACs structures for this investigation were quinine and nicotine
in scaffolds of natural products.
(Figure 1b). Each presents a pyridine nitrogen, as well as a separate
Amongst the examples of natural products with permanent cationic tertiary amine with only modest steric hindrance. Each compound is
charges based at nitrogen are a series of tetrahydroisoquinolinium naturally abundant, therefore, making it inexpensive, and presents no
structures isolated from the Chinese vine Gnetum montanum, includ- significant reported antibacterial property as the unmodified natural
ing magnocurarine, cyclized derivatives thereof, and the latifolians,4 as product. Furthermore, although each has precedent to react as chiral
illustrated in Figure 1a. Latifolian A demonstrated modest antimicro- bases12 and nucleophiles, neither has been systematically investigated
bial activity, with a MIC of 35 μM against Pseudomonas aeruginosa.5 regarding their alkylation to form antimicrobial amphiphiles.13,14
However, it only demonstrated 55% inhibition of methicillin-resistant
Interestingly, these two natural products present opposite trends in
Staphyloccocus aureus (MRSA) at 350 μM while magnocuraine and its their reported alkylation chemistry; nicotine is preferably alkylated at
tetracyclic derivatives showed no effectiveness at this concentration, its pyridine nitrogen,15 and quinine is precedented to react at its
which perhaps correlates to the lack of an alkyl chain.
tertiary aliphatic amine (Figure 1b).16 For both compounds, however,
Related isoquinolinium structures bearing additional aromatic rings little is known about the amphiphilic properties of long-chain
include chelerythrine, sanguinarine and berberine. Berberine, also alkylated derivatives, though some nicotine-based amphiphiles have
identified from a Chinese herb, has shown micromolar activity against been investigated for their affects on the central nervous system17 and
P. aeruginosa.6 Other quinolinium natural products with a quaternary applications as ionic liquids;18 quinine derivatives have served as
ammonium center include tabouensinium chloride7 and the phase-transfer catalysts19 and substrates for phosphorylation.20 We
quinocitrines.8 Finally, ageloxime D9 and dehydroevodiamine10 diver- thus set out to prepare a series of mono- and bis-alkylated derivatives
sify this structural class and present a positive charge delocalized over of quinine and nicotine for the purpose of evaluating their anti-
two nitrogens. In sum, a trend emerges; half of the illustrated natural microbial potential.
products that possess quaternized nitrogen atoms simply represent
N-methylated alkaloids, and overall structural variety is modest.
To this end, we first explored the alkylation of quinine, which
proceeded under high-concentration conditions (~1 M, CH3CN) using
Inspired by these natural precedents, we set out to address a a variety of alkyl bromide electrophiles (Figure 2a). Yields were
question: could natural products lacking a quaternary ammonium uniformly high, and led to a simple production of the monocationic
group be converted into QACs, and thus gain antimicrobial activity? compounds abbreviated as Q-n,0 (Figure 2c). Subsequent alkylation
We were determined to confer amphiphilic properties to natural proved to be limited in scope; we found that a second alkylation with a
1Department of Chemistry, Villanova University, Villanova, PA, USA and 2Department of Chemistry, Temple University, Philadelphia, PA, USA
Correspondence: Professor WM Wuest, Department of Chemistry, Temple University, 1801N Broad St, Villanova, PA 19085, USA.
E-mail: wwuest@temple.edu
or Professor KPC Minbiole, Department of Chemistry, Villanova University, Chemistry, 800 E Lancaster Ave, Villanova, PA 19085, USA.
E-mail: kevin.minbiole@villanova.edu
Received 13 August 2015; revised 10 September 2015; accepted 24 September 2015

QACs with potent antimicrobial activity
MD Joyce et al
2
Figure 1 Natural products and their derivatives. (a) Examples of quaternary ammonium natural products. (b) Quinine and nicotine, and alkylation thereof.
long-chained alkyl bromide or iodide was difficult to complete, Gram-negative strains. Control compounds, including the parent
resulting in a mixture of compounds. However, exposure to neat natural products as well as their bis-methylated derivatives (Q-1,1
methyl iodide led to nearly quantitative alkylation overnight, affording and N-1,1) showed essentially no antimicrobial activity in our hands.
the Q-n,1 series after simple evaporation. Characterization data and Longer chain alkyl derivatives of these natural products showed clear
experimental details for all synthesized compounds are provided in the correlations between alkyl chain length and antimicrobial activity. In
Supplementary Information.
the nicotine series, the strongest activity was observed for N-16,0 and
Nicotine derivatives were alkylated in a highly analogous manner, N-18,1 for the mono- and bisQACs, respectively. Each showed low
again leading to alkylation in high yields (1 equiv RBr, CH3CN, Δ, micromolar activity (⩽ 4 μM) for all bacteria except for P. aeruginosa.
o/n), as illustrated in Figure 2b. As predicted by literature examples, For the monocationic quinine derivatives, the strongest activity was
we saw exclusive alkylation at the pyridine nitrogen. Subsequent observed for Q-14,0; however, longer chains proved optimal for the
alkylation, converting the N-n,0 series to the N-n,1 series, proceeded bisQACs (Q-16,1 and Q-18,1), and in fact provided two amphiphiles
somewhat more slowly, completing in 48 h at high yield. Evaporation with single-digit micromolar activity against all bacteria tested. The
provided the final set of compounds abbreviated as N-n,1, setting the modest inconsistencies in optimal chain length are somewhat unex-
stage for biological investigation.
pected, and may reflect an optimum balance between polar and non-
MIC values against Gram-positive Staphylococcus aureus (methicil- polar sections of the amphiphiles, as well as the markedly different
lin-susceptible Staphyloccocus aureus (MSSA) and two MRSA strains— core natural products.
USA300-0114 and ATCC33591) and Enterococcus faecalis and Gram-
We were surprised to observe roughly comparable activity when
negative Escherichia coli and P. aeruginosa were determined according comparing the mono- and bis-cationic compounds for most of this
to standard methods (Supplementary Information), and results appear data set. BisQACs did not prove uniformly more potent than the
in Figure 2c.
singly cationic analogs, and in many cases proved inferior to the
The amphiphiles derived from both quinine and nicotine displayed analogous monoQACs (for example, N-12,0 vs N-12,1). When
strong antibacterial activity against a number of Gram-positive and comparing activity against MSSA and MRSA (shaded entries in
The Journal of Antibiotics

QACs with potent antimicrobial activity
MD Joyce et al
3
Figure 2 Synthesis of (a) quinine- and (b) nicotine-derived quaternary ammonium compounds, with (c) synthetic and biological data. Results from two MRSA
strains are shaded. MRSA 1 = USA300-0114; MRSA 2 = ATCC33591; BAC, benzalkonium chloride.
Figure 2c), nearly every natural product-derived QAC showed MRSA resistance seems to be associated with monoQACs as well as
significant levels of bacterial resistance, with up to a 32-fold higher bisQACs based on aromatic substrates,11 but it is possible that alkyl
MIC for MRSA strains. In fact, in only two of the 28 compounds chain length should also be a consideration.
prepared did we observe comparable activity against MSSA and MRSA
In summary, we have demonstrated that selected natural products
—only Q-16,1 and Q-18,1 were unaffected by QAC resistance. This can serve as the platform for amphiphile construction, and that such
stands in stark contrast to Q-18,0, which showed strong susceptibility derivatization is capable of imparting significant levels of antibacterial
to MRSA resistance, resulting in a 32-fold increase in MIC of MRSA as activity. Further, we have corroborated the observation that mono-
compared to MSSA. This largely supports previous observations that QACs are susceptible to MRSA resistance, presumably through the
The Journal of Antibiotics

QACs with potent antimicrobial activity
MD Joyce et al
4
effect of efflux pumps.20 Such observations were not as clear for
bisQACs, wherein we observed MRSA resistance for the majority of
compounds, yet two longer chained bisQACs derived from quinine
showed no resistance at all to two MRSA strains. In light of the
simplicity of synthesis, and potency of many of the compounds
presented herein, we suggest natural product derivatization as a viable
strategy for antiseptic discovery, and perhaps as an inspiration for
future antibiotics.
6
7
8
Čerňáková, M. & Košťálová, D. Antimicrobial activity of berberine—a constituent of
Mahonia aquifolium. Folia Microbiol. 47, 375–378 (2002).
Wabo, H. K. et al. Tabouensinium chloride, a novel quaternary pyranoquinoline alkaloid
from Araliopsis tabouensis. Nat. Prod. Res. 19, 591–595 (2005).
Kozlovsky, A. G., Zhelifonova, V. P. & Antipova, T. V. The fungus Penicillium citrinum,
isolated from permafrost sediments, as a producer of ergot alkloids and new quinoline
alkalods quinocitrinines. Appl. Biochem. Microbiol. 41, 499–502 (2005).
Hertiani, T. et al. From anti-biofouling to biofilm inhibition: new cytotoxic secondary
metabolites from two Indonesian Agelas sponges. Bioorg. Med. Chem. 18,
1297–1311 (2010).
9
10 Park, C. H. et al. Novel anticholinesterase and antiamnesic activities of
dehydroevodiamine,
a
constituent of Evodia rutaecarpa. Planta Med. 62,
405–409 (1996).
CONFLICT OF INTEREST
The authors declare no conflict of interest.
11 Jennings, M. C., Minbiole, K. P. C. & Wuest, W. M. Quaternary ammonium compounds:
an antimicrobial mainstay and platform for innovation to address bacterial resistance.
ACS Infect. Dis. 1, 288–303 (2015).
12 Palomo, C., Oiarbide, M. & López, R. Asymmetric organocatalysis by chiral Bronsted
bases: implications and applications. Chem. Soc. Rev. 38, 632–653 (2009).
13 Lv, J., Qian, Y., Liu, T. & Wang, Y. Synthesis and evaluation of amphiphilic cationic
quinine-derived for antibacterial activity against methicillin-resistance Staphylococcus
aureus. Bioorg. Med. Chem. Lett. 17, 4102–4106 (2007).
14 Coates, W. J. et al. The antimicrobial activity of Q-7,0 appeared in a patent. Preparation
of piperidinylalkylquinolines as antibacterials. WO 9937635 (1999)
15 Shibagaki, M. et al. The selectivity in N-alkylation of nicotine. Heterocycles 19,
1641–1645 (1982).
ACKNOWLEDGEMENTS
This work was funded by a University City Science Center QED Proof of
Concept Grant (Grant S1403 to WMW), as well as by Temple and Villanova
Universities. MCJ acknowledges a National Science Foundation Pre-Doctoral
Fellowship (DGE1144462). The authors thank CW Ross (Temple Mass
Spectrometry Facility) for obtaining MS data.
16 Baidya, M., Horn, M., Zipse, H. & Mayr, H. Organocatalytic activity of cinchona
alkaloids: which nitrogren is more nucleophilic? J. Org. Chem. 74, 7157–7164
(2009).
17 Crooks, P. A., Dwoshin, L., Xu, R. & Ayers, J. Chain-modified pyridine-N substituted
nicotine compounds for use in the treatment of CNS pathologies. US Pat. Appl. Publ.
20030225142 (2003).
18 Crooks, P. A. et al. Development of subtype-selective ligands as antagonists at nicotinic
receptors mediating nicotine-evoked dopamine release. Bioorg. Med. Chem. Lett. 14,
1869–1874 (2004).
1
2
3
Steichen, D. S. in Handbook of Applied Surface and Colloid Chemistry (ed. Holmberg K.)
310–347 (John Wiley & Sons, Ltd., New York, 2002).
Walker, E. B. & Paulson, D. Quaternary Ammonium Compounds (Marcel Dekker,
New York, 2002).
Guani-Guerra, E., Santos-Mendoza, T., Lugo-Reyes, S. O. & Teran, L. M. Antimicrobial
peptides: general overview and clinical implications in human health and disease. Clin.
Immun. 135, 1–11 (2010).
Rochfort, S. J. et al. Latifolians A and B, novel JNK3 kinase inhibitors from the papua
new guinean plant Gnetum latifolium. J. Nat. Prod. 68, 1080–1082 (2005).
Martin, F. et al. Alkaloids from the Chinese Vine Gnetum montanum. J. Nat. Prod. 74,
2425–2430 (2011).
19 Singh, P.
& Arora, G. Organic synthesis under PTC: part 6—preparation and
decomposition of chiral phase transfer catalysts derived from quinine. Indian J. Chem.
Sect. B 25B, 1034–1037 (1986).
4
5
20 Gavrilov, K. N., Mikhel, I. S., Lechkin, D. V.
& Timofeeva, G. I. Quinine-based
dioxaphospholane with N-decylquinuclidine fragment. Phosphorus Sulfur Silicon Relat.
Elem. 108, 285–287 (1996).
Supplementary Information accompanies the paper on The Journal of Antibiotics website (http://www.nature.com/ja)
The Journal of Antibiotics