Synthesis and antimicrobial evaluation of cationic low molecular weight amphipathic 1,2,3-triazoles

Article abstract of DOI:10.1016/j.bmcl.2017.01.092

A library of 28 small cationic 1,4-substituted 1,2,3-triazoles was prepared for studies of antimicrobial activity. The structures addressed the pharmacophore model of small antimicrobial peptides and an amphipathic motif found in marine antimicrobials. Eight compounds showed promising antimicrobial activity, of which the most potent compound 10b displayed minimum inhibitory concentrations of 4–8?μg/mL against Streptococcus agalacticae, Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Enterococcus faecalis. The simple syntheses and low degree of functionalization make these 1,4-substituted 1,2,3-triazoles interesting for further optimizations.

Full text of DOI:10.1016/j.bmcl.2017.01.092

Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and antimicrobial evaluation of cationic low molecular weight
amphipathic 1,2,3-triazoles
Thomas A. Bakka a, Morten B. Strøm b, Jeanette H. Andersen c, Odd R. Gautun a,
⇑
a Department of Chemistry, Norwegian University of Science and Technology (NTNU), NO-7491 Trondheim, Norway
b Department of Pharmacy, Faculty of Health Sciences, University of Tromsø, NO-9037 Tromsø, Norway
c Marbio, Faculty of Biosciences, Fisheries and Economics, University of Tromsø, NO-9037 Tromsø, Norway
a r t i c l e i n f o
a b s t r a c t
Article history:
A library of 28 small cationic 1,4-substituted 1,2,3-triazoles was prepared for studies of antimicrobial
activity. The structures addressed the pharmacophore model of small antimicrobial peptides and an
amphipathic motif found in marine antimicrobials. Eight compounds showed promising antimicrobial
activity, of which the most potent compound 10b displayed minimum inhibitory concentrations of
Received 27 January 2017
Accepted 28 January 2017
Available online xxxx
4–8 lg/mL against Streptococcus agalacticae, Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia
Keywords:
Antibacterial
Click chemistry
Marine natural product mimics
Peptidomimetics
1,2,3-Triazoles
coli, and Enterococcus faecalis. The simple syntheses and low degree of functionalization make these
1,4-substituted 1,2,3-triazoles interesting for further optimizations.
Ó 2017 Elsevier Ltd. All rights reserved.
Antimicrobial resistance to conventional antibiotic treatment is
rapidly increasing, combined with lackluster efforts to develop
novel classes of antibiotics by major pharmaceutical compa-
nies.1–3 Infections caused by multi resistant bacteria is therefore
one of the fastest growing medical threats to modern society.4
Disturbingly, resistant bacteria have existed since the discovery
of the first antibiotics. In recent years the race between growing
resistance and progress of new antibiotics has intensified in favor
of the bacteria. Unfortunately, no antibiotic has yet passed clinical
trials for which there has not been reported cases of resistance.5,6
Most antibiotics applied today work through specific interac-
tions with key intra- and extra-cellular targets in bacteria, and in
a highly specific manner.7 Due to the high target specificity, uncrit-
ical use of antibiotics easily selects for mutated bacteria to prolif-
erate. A well known mechanism of resistance is expression of
beta-lactamases that metabolizes beta-lactam based antibiotics.8
An expanding field within antibiotic research in academia focuses
on structures working through less specific mechanisms, like
interactions with the bacterial cell membrane and non-specific
interactions with intracellular targets.9–12 The interest in these
mechanisms of action comes from antimicrobial peptides (AMPs),
that are important constituents of innate immunity in most living
organisms. AMPs have a net positive charge (+2 to +9), consist of
12–50 residues, and fold into secondary structures with bacterici-
dal properties.13 These amphipathic structures, having a positively
charged hydrophilic face and a lipophilic face, interact with anionic
phospholipids on the surface of bacterial cell membranes. This is
followed by membrane permeabilization by the lipophilic residues,
leading to cell membrane disruption and ultimately cell lysis.14,15
Even though AMPs are considered to be highly active therapeu-
tic compounds, there are some major issues in utilizing them on a
large scale. Important drawbacks include low oral bio-availability,
low metabolic stability, high manufacturer costs, and lack of
patient-friendly administration methods aside from topical
treatments.16 Due to these obstacles, only a small number of
antimicrobial agents utilized today are AMPs.17 A way to circum-
vent the practical challenges associated with AMPs is to make
smaller peptides and scaffold-based peptidomimetics that main-
tain the antimicrobial activity, but have improved pharmacokinetic
properties. This has been demonstrated by Strøm et al., who have
synthesized small beta-peptidomimetic structures (MW < 650)
with high activity against a variety of resistant bacteria and with
potential for per oral administration.18,19 Recently, the group of
Strøm20 has reported a series of small cationic aminobenzamides
(example shown as E23 in Fig. 1) that mimic amphipathic struc-
tures found in marine antimicrobials such as synoxazolidinone
A21 and ianthelline22 and display
a membranolytic effect
resembling many AMPs. The focus of this work was to further
develop such amphipathic structures addressing both small AMPs
and marine antimicrobials, and optimize these for antimicrobial
activity. The di-functionalized 1,2,3-triazole was chosen as the core
⇑
Corresponding author.
E-mail address: odd.r.gautun@ntnu.no (O.R. Gautun).
http://dx.doi.org/10.1016/j.bmcl.2017.01.092
0960-894X/Ó 2017 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Bakka T.A., et al. Bioorg. Med. Chem. Lett. (2017), http://dx.doi.org/10.1016/j.bmcl.2017.01.092

2
T.A. Bakka et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
Scheme 1. (i) CuSO4Á5H2O (5 mol %), Sodium ascorbate (10 mol%), Benzoic acid
(10 mol%), tBuOH:H2O (1:2), rt, 10 min – over night (Ar; 5a = Ph, 5b = naphthyl,
5c = 3,5-di-t-Bu-Ph and 5d = 3,5-CF3-Ph).
1,4-substitution pattern was chosen here, due to the fact that the
copper(I) catalysts used in these reactions are water insensitive
(unlike their ruthenium counterparts), excluding the need for
working under inert conditions. Thus, the first target compounds
given in Fig. 2 (1a–4f) were prepared in order to screen the effects
of different lipophilic aromatic groups and hydrophilic cationic
nitrogen groups.
The ‘‘click” chemistry protocol requires two coupling partners
carrying an azide and a terminal alkyne. It was found most conve-
nient to insert the azide on the lipophilic moiety and the terminal
alkyne on the nitrogen carrying functionality. The azides (5a-d,
shown in Scheme 1) were synthesized from the respective com-
mercially available bromides and alcohols, by well-established
reactions (details are shown in the supporting information).31–34
The alkynes carrying a handle for N-functionalization, were pre-
pared from 3-butyn-1-ol and 1-chloropent-4-yne respectively,
under Mitsunobu- or Finkelstein modified Gabriel-conditions
(details shown in the supporting information).35,36 This yielded
6a and 6b (shown in Scheme 1) with the same masked N-function-
ality and a difference of one methylene group in the carbon chain.
The alkynes (6a and 6b) and azides (5a-d) were then combined
to form [1,4]-1,2,3-triazoles (7a-h) using copper catalyzed ‘‘click”-
chemistry conditions as shown in Scheme 1.37 Thus, by using four
different azides and two lipophiles, eight different ‘‘core” 1,2,3-tri-
azoles ready for N-functionalization (7a-h) were prepared (see
Scheme 1).
Fig. 1. Synoxazolidone A21 (MRSA (MIC); 10
l
g/mL), Ianthelline22 (MRSA (MIC);
g/
20
l
g/mL), and E23; a natural product mimic by Strøm et al.20 (MRSA (MIC); 4
l
mL).
scaffold, due to the known biochemical properties of this type of
structures.23,24 Of importance was that triazoles are bioisosteres
of amide bonds, which are more stable against proteolytic degrada-
tion than amides in AMPs.25–27 The study included initial synthesis
of 24 compounds to investigate the effects of varying between four
lipophilic groups and three cationic groups, and including chain
length variations. These results were followed up by synthesis of
four optimized compounds based on the results from the initial
series of di-functionalized 1,2,3-triazoles.
In order to synthesize a collection of disubstituted 1,2,3-triazole
amphiphiles with the desired lipophilic- and cationic hydrophilic
functionalities, the ‘‘click” chemistry protocol developed by Sharp-
less28 and Meldal29 was chosen. By using different catalysts for the
‘‘click” chemistry step, 1,2,3-triazoles with different substitution
patterns can be prepared, i.e., 1,4-substitution when using copper
(I) and 1,5-substitution when using ruthenium(II)-catalysis.30 The
Three different cationic groups were evaluated; a primary
amine (a and d), a tertiary amine (b and e) and a guanidine group
(c and f) as shown in Scheme 2. The interest for the primary amine
and the guanidine came from the functionalities found in AMPs,
Fig. 2. Initial target [1,4]-1,2,3-triazoles 1a–4f to be screened for antimicrobial effects. Counter-ion: ClÀ.
Please cite this article in press as: Bakka T.A., et al. Bioorg. Med. Chem. Lett. (2017), http://dx.doi.org/10.1016/j.bmcl.2017.01.092

T.A. Bakka et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
3
potent than the tertiary dimethyl- and primary amines (3a and
3e, Fig. 2), with the exception of 3a against E. coli. The difference
in activity was most pronounced against the gram-positive
S. aureus and Streptococcus gr. B (S. agalacticae) bacteria, where a
2- to 4-fold increase in activity was observed for the guanidine
compared to the other two cationic nitrogen groups.
So far, we have determined which lipophilic and cationic group
that most likely promoted the highest activity against the five
strains of bacteria tested. The third varying factor in the series of
amphipathic 1,2,3-triazoles tested was the two or three carbon
chain of the hydrophilic end of the triazole ring. A small increase
in efficacy was observed for the longer 3f compared to 3c against
S. aureus and E. coli. Furthermore, 3f showed the overall highest
activity against the gram-positive S. aureus and S. agalacticae
(10
against gram-positive E. faecalis and the gram-negative E. coli and
P. aeruginosa (40 g/mL). Lowered activity against gram-negative
lg/mL), while there was a 4-fold decrease in the activity
Scheme 2. (i) Hydrazine hydrate, toluene, reflux, (ii) HCl (conc. aq. or 2 M in Et2O),
iPrOH, MeCN or DCM, (iii) Formaldehyde, formic acid, MeCN, reflux, 1 h, acidic work
up and (iv) 1H-pyrazole carboxamidine hydrochloride, MeCN, reflux 2–4 h.
l
compared to gram-positive bacteria is commonly observed, due
to different outer membrane compositions.44 However, it was sur-
prising that the activity against E. faecalis was in the range of the
gram-negative strains. In addition to the antimicrobial effects,
some biofilm inhibition was observed in single concentration
assays of these structures. The amphiphiles 2f and 3 (except 3d)
were lysine and arginine residues contribute these groups to the
amphiphile, thus taking a vital part in induction of antimicrobial
activity.13 A tertiary amine was expected to be a steric and elec-
tronic mid-point between the two naturally occurring cationic
groups. In order to increase the steric bulk, without introducing
additional nitrogens and resonance possibilities, the tertiary
dimethylamino group was chosen as a mid-point between primary
amines and guanidines. The eight protected 1,2,3-triazoles were
deprotected using hydrazine hydrate according to a protocol
developed by Gabriel.38–40 The primary amines (8a-h) were subse-
quently functionalized in order to introduce the chosen functional-
ity, and the primary amine HCl-salts 1–4 (a, d) (Scheme 2) were
obtained by treatment with hydrochloric acid. The Eschweiler-
Clarke reductive amination was utilized to create the tertiary
amines 1–4 (b, e) (Scheme 2),41,42 and an electrophilic guanidine
reagent to create the guanidines 1–4 (c, f) (Scheme 2).43 Perform-
ing the given transformations on all eight protected triazoles
yielded the 24 different compounds depicted in Fig. 2 (1a-4f), in
sufficient purity (>95% HPLC) for biological evaluation.
showed biofilm inhibition at 50 lg/mL.
It was assumed from the pharmacological model18–20 that a
rather large lipophilic contribution would be important for achiev-
ing the desired antimicrobial effects. In order to rationalize our
findings we attempted to use calculated pKa adjusted partition
coefficients (ClogD) as an indicator for lipophilicity. The ClogD val-
ues were calculated (using the Marvinsketch software45) at physi-
ological pH (pH = 7.40), showing the guanidines (c and f, Fig. 2) to
be mostly protonated and the primary (a and d, Fig. 2) and tertiary
amines (b and e, Fig. 2) to exist in more partitioned equilibria.
However, when plotted against the values from the antimicrobial
MIC-assays, no apparent connection was found between the ClogD
and MIC-values. On the other hand, plotting all structures accord-
ing to their retention times (Rt) from C18-HPLC as shown in Fig. 3,
gave a more accurate picture of the effective lipophilic contribu-
tions. As the HPLC analyses were performed with an acid additive
(0.1% TFA) in order to inhibit peak broadening, all of the com-
pounds were assumed to exist mainly in their positively charged
state. This indicated that the lipophilic nature of the charged struc-
tures is an important parameter for biological activity; e.g. 3f is
more active than 3e, even though the calculated ClogD (displayed
in Fig. 3) of 3e is nearly the double of the one for 3f. The fact that
the Rts may be used as a rough indicator of antimicrobial activity
may prove useful when targeting new potential candidates for
optimization.
The 24 amphiphilic triazoles 1a-4f were tested against three
gram-positive and two gram-negative bacterial strains. In addition,
all 24 compounds were subjected to toxicity studies against
human fibroblasts (MRC-5). No activity was detected below
50 lg/mL, indicating low toxicity of the structures towards this
type of human cells. Four of the 24 structures (3a, 3c, 3e and 3f)
showed promising activities against several cell lines. These were
subjected to dilution assays in order to determine the minimum
inhibitory concentrations (MICs) against the chosen bacteria. The
MIC-values for the active compounds are shown in Table 1.
All the active compounds (3a, 3c, 3e and 3f) contained the heav-
ily hindered and non-polar 3,5-di-tert-butyl-phenyl functionality.
This indicated that a bulky and non-polar lipophilic contribution
was important for the activities in these structures. Furthermore,
the guanidine hydrochloride functionality appeared to be related
to the observed activities. As they (3c and 3f, Fig. 2) were more
Compound 3f from the initial screening and dose response
assessments showed the highest antimicrobial activities, with
MICs ranging from 10 to 40 lg/mL. In order to optimize the activ-
ities towards the target bacteria, a small and focused set of com-
pounds was prepared based on the structure of 3f. The first
change was inspired by the planar benzamide peptide mimics
Table 1
Antimicrobial activity (MIC in
highest tested concentration (50
l
g/mL) for the 1,2,3-triazoles that showed any activity in the antibacterial assays. The ‘‘À”-sign in the table indicates no activity in the assay at the
g/mL).
l
Entry
3a
3c
3e
3f
Ref.a
E. faecalisb
S. aureusb
–
40
20
10
50
40
–
40
10
10
40
40
10
0.13
4
0.5
0.5
40
40
40
50
40
50
–
S. agalacticaeb
E. colib
P. aeruginosab
–
a
Ref.: Gentamicin.
b
E. faecalis (ATCC 29212), S. aureus (ATCC 25923), S. agalacticae (ATCC 12386), E. coli (ATCC 25922), P. aeruginosa (ATCC 27853).
Please cite this article in press as: Bakka T.A., et al. Bioorg. Med. Chem. Lett. (2017), http://dx.doi.org/10.1016/j.bmcl.2017.01.092

4
T.A. Bakka et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
ture of the lipophilic group. By going from the bulky t-Bu-functions
to a linear alkyl chain with similar surface area and molecular
weight, we hoped to mimic the membrane snorkeling effect of
lysine and arginine rich proteins.46 Thus, the four amphiphiles
shown in Scheme 3 (9a-b and 10a-b) were prepared for further
studies.
The synthesis of 9a-b and 10a-b from the azides 5e and 5f were
performed according to the methods presented in Schemes 1 and 2
with total yields ranging from 19 to 71% over three steps. The
azides (5e and 5f) were synthesized from commercially available
iodophenol and 3,5-t-Bu-bromobenzene using a copper catalyzed
synthesis presented by Zhu et al.47 (experimental details are found
in the supplementary information). The four triazole amphiphiles
were subjected to the same bacterial strains as the initial 24
amphiphiles. The obtained MIC values are displayed in Table 2
together with the reference antibiotic gentamicin.
Fig. 3. Top plot: C18-Rt (MeOH/water, 1:1 + 0.1% TFA) for 1a-f, 2a-f and 4a-f
plotted against calculated ClogD at pH 7.40 (using the MarvinSketch suite). Bottom
plot: C18-Rt (MeOH/water, 5:3 + 0.1% TFA) for 3a-f plotted against calculated ClogD
at pH 7.40 (using the MarvinSketch suite).
Removal of the benzylic methylene group and introduction of a
more rigid and planar structure with possibility for conjugation
lead to an approximate two-fold increase in activity against all
the tested bacteria (9b compared to 3f), and MIC-values as low
as 4 lg/mL against S. aureus and S. agalacticae. Again, the guanidine
hydrochloride (9b and 10b) proved to be the most active hydro-
phile (compared to NH+3), as it led to a two-fold increase in activity
against all bacteria (except for E. coli) compared to the ammonium
hydrochlorides (9a and 10a). Substituting the bulky 3,5-t-Bu group
with a heptyl ether chain (10a and 10b) led to a further two-fold
increase in the activity against the gram-negative strains and the
gram-positive E. faecalis. This may in turn be attributed to the hep-
thyl chain’s (10b) ability to penetrate deeper into the membrane
compared to the t-Bu groups in 9b. However, the exact mechanism
of action for these compounds has not been investigated yet. It
should also be noted that the activity of 10b surpassed that of
Gentamicin against E. faecalis and matched the activity against
S. agalacticae.
As for the initial 24 amphiphiles, there was no evident correla-
tion between the MIC-values and calculated CLogD. The most
active compound (10b) had the lowest calculated CLogD of the four
structures (Table 2). However, the retention times from C18-HPLC
showed a better correlation, where the most active structure had
the highest retention time on the C18-column (Table 2).
Scheme 3. Improved structures 9a-b and 10a-b based on 3f, synthesized in three
steps from 5e and 5f utilizing the chemistry displayed in Schemes 1 and 2. Counter
ion: ClÀ.
We have successfully synthesized 28 low molecular weight
cationic triazole-based amphiphiles with different lipophilic and
hydrophilic functionalities, and screened for antimicrobial effects
against S. agalacticae, S. aureus, P. aeruginosa, E. coli, and E. faecalis.
The most potent compound in our library (10b) displayed MIC-val-
presented by Strøm et al.,20 where they achieved <10
lg/mL
against the same bacteria. By using an aromatic azide instead of
a benzylic azide in the ‘‘click”-coupling, the obtained amphiphiles
would be more planar and rigid compared to 1a-4f (Fig. 2). This
was expected since the benzylic methylene group create an angle
between the triazole ring and the lipophilic aromatic group and
give more rotational freedom, which might be disfavourable for
antibacterial activity. A second modification of 3f in addition to
removing the benzylic methylene group, was to change the struc-
ues between 4 and 8 lg/mL, which either matched or surpassed
the activity of the marine natural product peptide mimics Synoxa-
zolidone A and Ianthelline. The activity of 10b also matched the
activity against gram-negative bacteria for the benzamides pre-
sented by Strøm et al.20 Thus, bioisosteres of amide bonds can be
Table 2
Antimicrobial activity (MIC in
H2O + 0.1% TFA).
lg/mL) for the improved amphiphilic triazoles based on 3f, calculated ClogD (pH = 7.4) and reverse phase HPLC retention times (min in 5:3 MeOH:
Entry
9a
9b
10a
10b
Ref.a
E. faecalisb
32
16
16
16
32
2.00
22.9
16
4
4
16
16
1.75
28.0
16
16
8
8
4
4
8
8
1.16
34.6
10
0.13
4
0.5
0.5
–
S. aureusb
S. agalacticaeb
E. colib
8
P. aeruginosab
ClogD (calc.)c
RT (C18-HPLC)d
16
1.41
20.4
–
a
Ref.: Gentamicin.
b
E. faecalis (ATCC 29212), S. aureus (ATCC 25923), S. agalacticae (ATCC 12386), E. coli (ATCC 25922), P. aeruginosa (ATCC 27853).
Calculated at pH = 7.40 using the MarvinSketch suite.
In 5:3 MeOH:H2O + 0.1% TFA with 0.75 mL/min.
c
d
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T.A. Bakka et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
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