Antimicrobial 1,3,4-trisubstituted-1,2,3-triazolium salts

Article abstract of DOI:10.1016/j.bmcl.2018.09.011

A series of 1,3,4-trisubstituted-1,2,3-triazolium bromide salts were prepared by efficient two-step sequences of azide-alkyne cycloaddition and benzylic substitution. The antimicrobial activity of each triazolium salt and correlating triazole precursor wa

Full text of DOI:10.1016/j.bmcl.2018.09.011

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Antimicrobial 1,3,4-trisubstituted-1,2,3-triazolium salts
James T. Fletcher, Jill M. Sobczyk, Sarah C. Gwazdacz, Aaron J. Blanck
PII:
S0960-894X(18)30742-X
https://doi.org/10.1016/j.bmcl.2018.09.011
BMCL 26032
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
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16 July 2018
6 September 2018
9 September 2018
Please cite this article as: Fletcher, J.T., Sobczyk, J.M., Gwazdacz, S.C., Blanck, A.J., Antimicrobial 1,3,4-
trisubstituted-1,2,3-triazolium salts, Bioorganic & Medicinal Chemistry Letters (2018), doi: https://doi.org/10.1016/
j.bmcl.2018.09.011
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Antimicrobial 1,3,4-trisubstituted-1,2,3-triazolium salts
James T. Fletcher,* Jill M. Sobczyk, Sarah C. Gwazdacz and Aaron J. Blanck
Address: Department of Chemistry, Creighton University, 2500 California Plaza, Omaha, NE
68178, U.S.A.
Email: James T. Fletcher - jamesfletcher@creighton.edu
Abstract - A series of 1,3,4-trisubstituted-1,2,3-triazolium bromide salts were prepared by
efficient two-step sequences of azide-alkyne cycloaddition and benzylic substitution. The
antimicrobial activity of each triazolium salt and correlating triazole precursor was
evaluated using a minimum inhibitory concentration (MIC) assay. MIC activities as low as 1
µM against Gram-positive bacteria, 8 µM against Gram-negative bacteria and 4 µM against
fungi were observed for salt analogs, while neutral triazoles were inactive. Analogs
representing selective and broad-spectrum antimicrobial activity were each identified.
MIC structure-activity relationships observed within this motif indicate that the presence
of cationic charge and balance of overall hydrophobicity are strongly impactful, while
benzyl vs. aryl substituent identity and variation of substituent regiochemistry are not.
Keywords
Antibacterial; antifungal; benzylation; cycloaddition; organic salt.
1

Quaternary ammonium compounds (QACs) are an exemplary class of widely
utilized, fully synthetic antibiotics.1 Representative analogs such as benzalkonium chloride
have been used as antiseptics for nearly a century. While such compounds take structural
inspiration from natural products such as berberine,2 norspermidine3 and cationic
antimicrobial peptides,4–8 the fully synthetic nature of QACs permit relatively simple
structural variations that are necessary to complete the structure-activity relationship
(SAR) profiling studies needed for optimizing therapeutic properties. The cationic nitrogen
atoms defining of this class of antimicrobial compounds include benzylic, aliphatic and
aromatic ammonium centers. Among the heterocycles representing aromatic ammonium
analogs are pyridinium,9 imidazolium10,11 and 1,2,4-triazolium10,12 units.
While modification of simple hydrophobic groups around central ammonium
center(s) in a small molecule motif may seem relatively simplistic from a structural
diversity perspective, particularly in comparison to the frequently elaborate structures
represented by natural product antibiotics, this general synthetic approach has been
successfully utilized to prepare many effective and widely used QAC compounds and
remains an active area of contemporary research.1,13,14 Analogs of the 1,3,4-trisubstituted-
1,2,3-triazolium salt motif have been shown to display ionic liquid,15,16 catalytic,17
antileishmanial,18 and antimalarial10 properties. Surprisingly, although 1,2,4-triazolium
salts have been demonstrated as effective heterocyclic components of QAC compounds10,12
and examples of antimicrobial 1,2,3-triazolium-functionalized biopolymers have been
recently reported,19–21 studies describing the general antimicrobial properties of aliphatic
1,2,3-triazolium salts themselves are lacking.
With the recent advent of the “click chemistry” era, founded upon the Sharpless-
2

Meldal copper-catalyzed azide-alkyne coupling (CuAAC) reaction first reported in 2002,22,23
the efficient preparation of 1,2,3-triazole compounds with diverse peripheral functionality
is now commonplace.24,25 Hence, preparing QAC analogs deriving from this heterocyclic
motif and defining general SAR antimicrobial trends stands as an attractive target for
developing new antimicrobial compounds. As an initial effort to define general SAR
profiles for this class of molecular organic salts, the synthesis and antimicrobial evaluation
of amphiphilic 1,3,4-trisubstituted-1,2,3-triazolium bromides with systematic variations in
hydrophobicity, regiochemistry and substituent attachment is presented herein.
A representative family of 1,4-disubstituted-1,2,3-triazole and 1,3,4-trisubstituted-
1,2,3-triazolium compounds was prepared using the three established reaction sequences
illustrated in Figure 1. Those 1,4-disubstituted-1,2,3-triazole analogs with aryl substitution
at the N1 position were prepared from direct copper-catalyzed alkyne-azide cycloaddition
(CuAAC)24 (method A) between commercially available alkynes and aryl azides prepared
using the Sandmeyer reaction.26 Those 1,4-disubstituted-1,2,3-triazole analogs with N1
benzylic substitution were prepared using a tandem CuAAC method,15,27 where
commercially available benzyl bromide and sodium azide were used to prepare the organic
azide reactant in the presence of the CuAAC reactant and catalyst (method B), achieving a
two-step reaction sequence in a one-pot reaction. The 1,5-disubstituted-1,2,3-triazole
analog was prepared from base-catalyzed conditions28 (method C) that promote 1,5-
regioselective acetylide-alkyne cycloaddition using commercially available alkyne and
prepared aryl azide reactants.
Each 1,2,3-triazole analog was successfully converted to a 1,3,4-trisubstituted-1,2,3-
triazolium salt by substitution reaction at the N3 position,15,17 resulting in organic salts
3

with quaternized ammonium centers. The majority of these quaternization reactions
proceeded in high yield when an excess of benzyl bromide or 4-tert-butylbenzyl bromide
reactant was used, with the exception of 7 having bulky C4 phenyl substitution that
significantly reduced benzylation yields due to steric inhibition. Following evaporation of
reaction solvent, pure triazolium bromide salts were isolated from a hexane trituration that
removed unreacted neutral organics with minimal loss of ionic salt products. Hence, the
target compounds in this study were accessible in good yield using only 2-3 overall
reaction steps.
Figure 1. Direct (A, C) and tandem (B) two-step methods to prepare 1,3,4-trisubstituted-
1,2,3-triazolium salts. The numbering scheme used for substituent identification is shown.
4

Figure 2. Identities of the neutral 1,2,3-triazole analogs in this study. Isolated yields are
noted.
The antimicrobial properties of these triazole compounds and triazolium salts were
evaluated using microdilution minimum inhibitory concentration assays as described in
the CLSI protocol for bacteria29 and yeasts.30 BSL1 strains of Gram-positive bacteria
(Bacillus subtilis, Staphylococcus epidermidis), Gram-negative bacteria (Escherichia coli,
Enterobacter aerogenes) and yeast (Candida albicans, Saccharomyces cerevisiae) were
selected as model organisms for the desired general antimicrobial profiling. Assays were
run in triplicate and minimum inhibitory concentration (MIC) values were defined by
observing the optical transparency of the most dilute well suppressing microbial growth.
Two commercial QAC class antibiotics, cetylpyridinium chloride and benzalkonium
chloride, were used as controls.
Assay results are summarized in Table 1. Among this limited family of compounds,
examples of analogs displaying both selective and broad-spectrum antimicrobial activity
were observed. While it was unsurprising that as representatives of the QAC structural
motif these 1,3,4-trisubstituted-1,2,3-triazolium bromide salts did display antibiotic
properties, the significant influence that simple substituent variations had on both
selectivity and overall antimicrobial activity among this group of analogs was unexpected.
5

None of the neutral 1,2,3-triazole compounds 1-8 showed measurable MIC values, and
charged analogs 9, 12, 15, 21 and 23 possessing minimal hydrophobicity among the salts
studied also lacked measurable MIC activity.
Analogs in the series possessing a moderate amount of peripheral hydrophobicity
were observed to exert the most selective antimicrobial activity. 16 and 18 displayed
greater than 50-fold selectivity towards the Gram-positive bacteria tested relative to the
Gram-negative bacteria and fungi, with 10, 22 and 24 also showing such selectivity at
slightly lower discrepancy. Analogs representing the maximum amount of hydrophobicity
in the series, 14 and 20, showed expanded bioactivity towards both Gram-positive bacteria
and fungi but not Gram-negative bacteria. An increased degree of broad-spectrum
antimicrobial activity against all three classes of pathogens was observed for 11, 13, 17
and 19, which possessed more hydrophobicity than the Gram-positive selective analogs
(10, 16, 18) but less hydrophobicity than those analogs that discriminated against Gram-
negative bacteria (14, 20).
6

Table 1. Survey of Triazolium Salt Antimicrobial Activity
Analog
R1 =
CH2Ph
Yield*
Minimum Inhibitory Concentration (µM)**
B.S. S.E. E.C. E.A. C.A. S.C.
96% >250 >250 >250 >250 >250 >250
R2 =
n-C4H9
R3 =
CH2Ph
9
10 CH2Ph
11 CH2Ph
12 CH2Ph
13 CH2Ph
n-C8H17
n-C12H25 CH2Ph
n-C4H9
n-C8H17
CH2Ph
98%
95%
98%
98%
95%
8
2
8
1
2
8
4
8
2
2
>250 >250 250
16 16 16
>250 >250 250
250
16
250
16
CH2Ph(4-tBu)
CH2Ph(4-tBu)
31
31
8
8
14 CH2Ph
n-C12H25 CH2Ph(4-tBu)
250
250
16
15 Ph(4-CH3)
16 Ph(4-CH3)
17 Ph(4-CH3)
18 Ph(4-CH3)
19 Ph(4-CH3)
20 Ph(4-CH3)
21 Ph(4-CH3)
22 Ph(4-CH3)
23 CH2Ph
n-C4H9
n-C8H17
n-C12H25 CH2Ph
n-C4H9
n-C8H17
n-C12H25 CH2Ph(4-tBu)
Ph
Ph
Ph
CH2Ph
CH2Ph
93% >250 >250 >250 >250 >250 >250
89%
97%
93%
95%
97%
4
1
4
2
2
8
2
4
2
2
>250 >250 250
250
8
250
8
8
8
8
CH2Ph(4-tBu)
CH2Ph(4-tBu)
250 >250 250
16
250
16
250
4
4
4
CH2Ph
48% >250 >250 >250 >250 >250 >250
49% >250 >250 >250 >250
94% >250 >250 >250 >250 >250 >250
CH2Ph(4-tBu)
Ph(4-CH3)
Ph(4-CH3)
8
8
24 CH2Ph(4-tBu) Ph
Cetylpyridinium chloride
Benzalkonium chloride
93%
16
1
4
16
0.2
1
>250 >250 >250 >250
25
62
16
62
4
62
1
12
*Isolated yields indicated. **Concentrations surveyed: Initial range was 250, 125, 62, 31, 16, 8, 4
and 2 µM, repeated at 10x dilution for potent analogs. B.S. = Bacillus subtilis (ATCC 6051); S.E. =
Staphylococcus epidermidis (ATCC 14990); E.C. = Escherichia coli (ATCC 25922); E.A. = Enterobacter
aerogenes (ATCC 13048); C.A. = Candida albicans (ATCC 90028); S.C. = Saccharomyces cerevisiae
(ATCC 9763).
MIC potency for this family of triazolium salts was observed as low as 1 µM (0.5
µg/ml) against Gram-positive bacteria, 4 µM (2 µg/ml) against fungi and 8 µM (4 µg/ml)
against Gram-negative bacteria. In comparison to the QAC controls cetylpyridinium
chloride and benzalkonium chloride, these MIC values were slightly more potent against
Gram-negative bacteria and slightly less potent against Gram-positive bacteria and fungus
under the conditions assayed. Benzyl vs. aryl substituent identity at the N1 position was
insignificant, as the MIC results of 9-14 largely mirrored 15-20. Regiochemistry of diaryl
7

attachment was also observed to be inconsequential, with the regioisomer pairs 21/23 and
22/24 displaying analogous potency against the set of organisms tested.
Evaluation of this data set enables preliminary structure-activity relationship trends
to be defined for this family of molecular 1,3,4-trisubstituted-1,2,3-triazolium bromide
salts: (1) the cationic nature of the 1,2,3-triazolium salt is necessary for antimicrobial
potency, as none of the neutral 1,2,3-triazole analogs are significantly active (MIC ≥250 µM
for each); (2) increasing hydrophobicity at the N3 and C4 positions generally leads to
increased potency against Gram-positive bacteria and fungi, while sufficient but not
excessive hydrophobicity is ideal for maximum potency against Gram-negative bacteria;
(3) no significant differences in potency are observed between aryl and benzyl substitution
at the N1 position; and (4) no significant differences in potency are observed for
regioisomer analogs with varying 1,4- vs. 1,5-diaryl substitution.
With the dominant driving force for potency being cationic amphiphilic properties,
it is hypothesized that this class of molecules exerts antimicrobial activity via interaction
with and destabilization of the cell membranes of target organisms.31,32 Against both
Gram-positive bacteria and fungi, antimicrobial activity correlated with increasing
hydrophobicity within the range of analogs examined. In contrast, against Gram-negative
bacteria maximum potency was observed for analogs possessing a combination of
moderate hydrophobicity at the triazole N3 and C4 positions relative to those displaying
broad-spectrum effects. A decrease in QAC toxicity towards Gram-negative bacteria
relative to Gram-positive bacteria is commonly observed13 due to the presence of their
outer membrane, which in this case may be preventing the larger sized triazolium analogs
from reaching and destabilizing the inner membrane of such organisms.
8

This investigation demonstrates that simple 1,3,4-trisubstituted-1,2,3-triazolium
bromide salts display significant antimicrobial activity against model Gram-positive
bacteria, Gram-negative bacteria and fungi in a substituent-dependent manner and
establishes a preliminary SAR profile for this class of molecular organic salts. Each analog
in this study was prepared efficiently from a sequence of cycloaddition and substitution
reaction steps. Analogs representing both selective and broad-spectrum activity were each
identified among this family of compounds. While this limited set of analogs allowed a
general antibiotic SAR to be defined, a more comprehensive survey of substituents is
warranted. Future studies will attempt to optimize both the potency and selectivity of
bioactivity by embracing the facile synthetic variation of substituents at the 1-, 3- and 4-
positions, as well as examine the antimicrobial properties of select analogs against drug-
resistant and clinically-relevant pathogen strains.
Acknowledgments
This publication was made possible by grants from the National Institute for General
Medical Science (NIGMS) (5P20GM103427), a component of the National Institutes of
Health (NIH), and its contents are the sole responsibility of the authors and do not
necessarily represent the official views of NIGMS or NIH.
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Highlights
• Aliphatic 1,3,4-trisubstituted-1,2,3-triazolium salts show antimicrobial properties
• Analogs representing selective and broad-spectrum antimicrobial activity identified
• Potency strongly influenced by hydrophobicity; regiochemistry less impactful
• Triazolium salts prepared efficiently using click and benzylation reaction steps
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16