Methyl propiolate and 3-butynone: Starting points for synthesis of amphiphilic 1,2,3-triazole peptidomimetics for antimicrobial evaluation

Article abstract of DOI:10.1016/j.bmc.2017.07.060

A library of 29 small 1,4-substituted 1,2,3-triazoles was prepared for studies of antimicrobial activity. The pharmacophore model investigated with these substrates was based on small peptidomimetics of antimicrobial peptides and antimicrobials isolated from marine organisms from sub-arctic regions. Using methyl 1,2,3-triazole-carboxylates and 1,2,3-triazole methyl ketones prepared through “click” chemistry we were able to synthesize the different cationic amphiphiles through three steps or less. Several structural modifications to the lipopohilic side and hydrophilic sides of the amphiphiles were investigated and compared with regards to antimicrobial activity and cytotoxicity in particular. The most promising amphiphile 10f displayed minimum inhibitory concentrations (MICs) between 4–16 μg/mL against Gram-positive Enterococcus faecalis, Staphylococcus aureus, Streptococcus agalacticae, and Gram-negative Escherichia coli and Pseudomonas aeruginosa. The decent level of antimicrobial activity and biofilm inhibition, short synthesis, and accessible reagents, makes this type of amphiphilic mimics interesting leads for further development.

Full text of DOI:10.1016/j.bmc.2017.07.060

Accepted Manuscript
Methyl propiolate and 3-butynone: starting points for synthesis of amphiphilic
1,2,3-triazole peptidomimetics for antimicrobial evaluation
Thomas A. Bakka, Morten B. Strøm, Jeanette H. Andersen, Odd R. Gautun
PII:
S0968-0896(17)31284-1
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.07.060
BMC 13897
Reference:
Bioorganic & Medicinal Chemistry
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Received Date:
Accepted Date:
27 June 2017
28 July 2017
Please cite this article as: Bakka, T.A., Strøm, M.B., Andersen, J.H., Gautun, O.R., Methyl propiolate and 3-
butynone: starting points for synthesis of amphiphilic 1,2,3-triazole peptidomimetics for antimicrobial evaluation,
Bioorganic & Medicinal Chemistry (2017), doi: http://dx.doi.org/10.1016/j.bmc.2017.07.060
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Methyl propiolate and 3-butynone: starting points for synthesis of
amphiphilic 1,2,3-triazole peptidomimetics for antimicrobial
evaluation
Thomas A. Bakkaa, Morten B. Strømb, Jeanette H. Andersenc, and Odd R. Gautuna,
aDepartment of Chemistry, Norwegian University of Science and Technology (NTNU), NO-7491 Trondheim, Norway
bDepartment of Pharmacy, Faculty of Health Sciences, UiT – the arctic university of Norway, NO-9037 Tromsø, Norway
cMarbio, Faculty of Biosciences, Fisheries and Economics, UiT – the arctic university of Norway, NO-9037 Tromsø Norway
ABSTRACT
A library of 29 small 1,4-substituted 1,2,3-triazoles was prepared for studies of antimicrobial
activity. The pharmacophore model investigated with these substrates was based on small
peptidomimetics of antimicrobial peptides and antimicrobials isolated from marine organisms from
sub-arctic regions. Using methyl 1,2,3-triazole-carboxylates and 1,2,3-triazole methyl ketones
prepared through “click” chemistry we were able to synthesize the different cationic amphiphiles
through three steps or less. Several structural modifications to the lipopohilic side and hydrophilic
sides of the amphiphiles were investigated and compared with regards to antimicrobial activity and
cytotoxicity in particular. The most promising amphiphile 10f displayed minimum inhibitory
concentrations (MICs) between 4 - 16 µg/mL against Gram-positive Enterococcus faecalis,
Staphylococcus aureus, Streptococcus agalacticae, and Gram-negative Escherichia coli and
Pseudomonas aeruginosa. The decent level of antimicrobial activity and biofilm inhibition, short
synthesis, and accessible reagents, makes this type of amphiphilic mimics interesting leads for
further development.
Keywords: Antibacterial; Click chemistry; Marine natural product mimics; 1,2,3-Triazoles
1. Introduction
The ability to treat bacterial infections with antibiotics is one of the major constituents in any basic
health care system.1-3 However, increased consumption of antibiotics, both through agriculture and
health services is causing rapid proliferation of resistant bacteria.3 Combined with reduced focus on
development of novel antibiotics, has made antimicrobial resistance one of the fastest growing
threats to human health.2,4 It is estimated that 700 000 people die each year due to events related to
antimicrobial resistance.3 Moreover, if resistance is allowed to develop without countermeasures, as
many as 10 million people may die annually by the year 2050. This means that deaths related to
antimicrobial resistance will surpass the number of deaths caused by cancer.3
Some of the first antibiotics were natural products and many important antibiotics today are based
on natural or semi-synthetic compounds.5 Antimicrobial natural products are found in animals and
plants, and have through evolution evolved in eukaryotes living in a world inhabited by potential
pathogenic prokaryotes. The ability to prevent and overcome infections has always been important
for survival.6 Natural product antimicrobials therefore form an important starting platform when
searching for novel antimicrobial scaffolds. One natural product class of particular interest is
antimicrobial peptides (AMPs).7 These are small peptides between 12-50 residues that take part in
the primary immune response system of all living organisms. AMPs have an overall net positive
 Corresponding author. Tel.: +47 73594101; e-mail: odd.r.gautun@ntnu.no
1

charge (+2 to +9) and fold into amphiphilic secondary structures with one lipophilic face and one
hydrophilic face. These amphiphilic secondary structures interact more or less selectively with
bacterial cell membranes, and through various mechanisms of membrane disruption processes
cause inhibition of growth or lysis of the bacteria. Several mechanisms of action are suggested for
the membranolytic effect of AMPs and for an excellent review on the topic see Giuliani et al.7 Even
though AMPs are highly active against bacteria, there may be some drawbacks to their use as drugs.
Most of these problems are related to poor pharmacokinetic properties,8 such as low bioavailability,
low metabolic stability and lack of patient-friendly administration routes. This, in addition to high
manufacturing costs, makes AMPs less desirable for clinical development. There are however some
AMP-based drugs in clinical use today, but they are usually based on topical use, since
pharmacokinetic issues make them unfit for systemic use.9
AMPs may however provide a starting point for investigations of smaller drugs with improved
pharmacokinetic properties. It has been shown by the research group of Svendsen et al. that AMP-
like oligopeptides consisting solely of arginine and tryptophan have similar antimicrobial activities as
the native AMPs.10 This work eventually led to the synthetic antimicrobial peptidomimetic LTX 109,
which is currently undergoing clinical trials. Furthermore, the research group of Strøm et al. have
synthesized a library of tri-functionalized β2,2-amino acid derivatives based on AMPs without
compromising the activity against resistant bacteria.11,12 Assuming that these structures display
membranolytic effects similar to that of AMPs, Strøm et al. also developed a library of antimicrobial
aminobenzamide amphiphiles13 (E23, Fig. 1) mimicking the structures of the antimicrobial marine
Figure 1. Synoxazolidone A14 (methicillin-resistant S.
aureus MIC: 10 µg/mL), ianthelline15 (methicillin-resistant
S. aureus MIC: 20 µg/mL), and E23: example of
aminobenzamide peptidomimetic13 based on marine
antimicrobials synoxazolidinone A14 and ianthelline.15
Based on the pharmacophore model of small AMPs and marine peptide mimics we have created a
library of 1,2,3-triazole amphiphiles based on the structural motifs shown in Fig. 1. The nature of the
lipophilic part and the rigidity at the hydrophilic cationic nitrogen functionalities were varied as
shown in Fig. 2. The initial library was followed by optimization of activity by a more focused set of
compounds shown in Fig. 3. The 1,2,3-triazole was chosen as a link between the lipophilic and
hydrophilic side due to the simple synthesis16,17 and accessible starting materials. Furthermore,
triazoles are bioisosteres of amide bonds and stable against proteolytic degradation.18-20 The library
2

presented in this publication was prepared in parallel to a similar library of amphiphiles based on
1,2,3-triazole phthalimides.21
Figure 2. Compounds 4a-8d synthesized and evaluated for antimicrobial activity. Amphiphile 7c was
not prepared, as discussed in the synthesis section. Counter ion: Cl-.
2. Results and discussion
2.1. Synthesis of the initial library
The initial 19 compounds evaluated for antimicrobial activity in this study (Fig. 2) were prepared
according to Scheme 1. The chosen core molecules for this library of amphiphilic amido 1,2,3-
triazoles were the methyl 1,2,3-triazole carboxylates 3a-d. These carboxylates were obtained from
copper catalyzed “click” chemistry between the organic azides 1a-d and methyl propiolate (2), using
a method22 based on the established procedures by Sharpless16 and Meldal17 as displayed in Scheme
1. The ester group on 3 was then amidated with either piperazine (n4: R2 = H), N-methyl-piperazine
(n5: R2 = Me),23 or ethylene diamine (EDA) (n6).24 Preparation of the piperazine amides n4 and n5
were performed with stoichiometric NaOMe in addition to piperazine under dry conditions in order
to give the desired amides n4 and n5 in sufficient to good yields (39-74%). The reactions with N-
methylpiperazine for preparation of n5 afforded lower yields than the synthesis of n4, and generally
required substantially longer reaction times (63 – 115 h for n5 compared to 24-68 h for n4). Several
attempts at preparing n5d through this route failed, and 5d was eventually managed prepared
through a reductive amination of formaldehyde in acetic acid from n4d.25,26 The amides n6a-d were
obtained through addition of a large excess of EDA (typically 15 equiv) in MeOH and heating from
room temperature to reflux.24 The neutral (n) C-carbamoyl-1,2,3-triazole amines (n4, n5, and n6)
were then turned into their corresponding HCl-salts 4, 5, and 6 using aqueous HCl in MeCN. The
guanidinium salts (7 and 8) were prepared by reacting n4 and n6 with the electrophilic guanylation
reagent 1H-pyrazole carboxamidine hydrochloride in refluxing MeCN.27,28 All structures of n4 and n6
3

Scheme 1. i) 1a-d (1 – 1.05 equiv), CuSO4 x 5H2O (5% mol), sodium ascorbate (10% mol), benzoic acid (10% mol), t-
BuOH:H2O (1:2), rt, 10-18 h. ii) Piperazine (R2 = H) or 1-methyl piperazine (R2 = Me) (3 equiv), NaOMe (1 equiv), MS 4 Å,
MeOH, rt, 24 - 115 h. iii) EDA (15 equiv), MeOH, rt - reflux, 18 h. iv) 1H-Pyrazole carboxamidine hydrochloride (0.9 – 1.0
equiv), MeCN, reflux, 3-18 h. v) for 4d: HCHO (approx. 20 equiv), HCOOH (approx. 20 equiv), MeCN, reflux, 1.5 h. (R1; a =
heptyl, b = adamantyl, c = 4-t-Bu-benzyl, and d = 3,5-t-Bu-benzyl). Counter ions for charged species: Cl-. Free amine
versions of the HCl-salts were given the prefix “n” for neutral, in order to distinguish them from their ionic versions.
Amphiphile 7c was not successfully prepared using these reaction conditions.
underwent guanylation into 7a, 7d, and 8a-d in moderate to good yields (39 – 76%) except for
conversion of n4b and n4c. Concerning n4b solubility issues probably inhibited the conversion. Thus,
7b was prepared using DMF at room temperature instead of MeCN at reflux.27 This gave 7b in 64%
yield after 93 h at room temperature. The underlying cause for the unsuccessful preparation of 7c
was not further investigated.
2.2. Antimicrobial activity of the initial library
The 19 amphiphiles (4a-8d, Fig. 2) were tested for antimicrobial activity against Gram-positive
Enterococcus faecalis, Staphylococcus aureus, and Streptococcus agalacticae, and Gram-negative
Escherichia coli and Pseudomonas aeruginosa. The minimum inhibitory concentrations (MIC) are
shown in Table 1 together with the MIC value for the reference antibiotic gentamicin.
The only amphiphiles from Fig. 2 displaying antimicrobial activity were 4d, 6d, 7d, and 8d (MIC 16 –
64 µg/mL), and all contained the bulky 3,5-di-t-Bu-benzyl group as the lipophile. No antimicrobial
Table 1. Antimicrobial activity (MIC in µg/mL) for the 1,2,3-triazoles in Fig. 2 that were antimicrobially active
at ≤ 64 µg/mL.
Entry
E. faecalisa
S. aureusa
S. agalacticaea
E. colia
P. aeruginosaa
b
4d
-
64
64
64
64
32
16
4
64
32
-
-
6d
-
64
64
32
0.5
7d
-
64
8d
64
32
64
Ref.c
10
0.13
0.5
a E. faecalis (ATCC 29212), S. aureus (ATCC 25923), S. agalacticae (ATCC 12386), E. coli (ATCC 25922), P.
aeruginosa (ATCC 27853).
b The “-“-sign in the table indicates no antimicrobial activity at or below 64 µg/mL.
c Ref.: Gentamicin.
4

activity was observed for the other amphiphilies in Fig. 2. These observations were in line with
results presented by Strøm et al.,12 in which the most potent compounds contained the same 3,5-di-
t-Bu-benzyl group. The other lipophiles introduced in this initial library were an attempt to either
reduce the amount of lipophilic bulk (4-t-Bu-benzyl, c) or change the structure of the lipophilic
contribution with an aliphatic heptyl chain (a) or an adamantyl box-like structure (b). The heptyl
chain (a) was also inspired by the successful use of alkyl chains in antimicrobial peptide mimics by
Ghosh et al.,29 whereas introduction of the adamantyl group (b) was done to investigate the effects
of increasing the three-dimensional bulk of the lipophile. However, this initial screening showed that
the presence of an aromatic ring on the lipophile and a large lipophilic contribution was important
for achieving antimicrobial activity. The differences in lipophilicity related to antimicrobial activity
was supported by measuring C18-HPLC retention times (Rts), in which 4a-8a, 4b-8b, and 4c-8c all
had Rts below 10 min, while the active amphiphiles 4d-8d had Rts of approx. 30 min (results shown
in experimental section).
For the active amphiphiles, highest overall antimicrobial potency was observed for the guanidine
derivative 8d with a guanylated EDA link. Both the guanylated piperazine 7d and the guanidine 8d
showed higher antimicrobial activity than their corresponding piperazine 4d and EDA 6d derivatives,
except against E. coli where the EDA 6d was most potent. The N-methyl-piperazine derivative 5d was
inactive within the concentration range tested. The results indicated that having a piperazine group
(4d and 5d) was less beneficial for antimicrobial activity compared to a cationic EDA group (6d),
guanylated piperazine (7d), or guanylated EDA group (8d). However, the low activity of the N-
methyl-piperazine derivative 5d could also be attributed to increased steric hindrance around the
cationic nitrogen. In conclusion, highest antimicrobial activity was observed for the amphiphile 8d
prepared with the 3,5-t-Bu-benzyl group (d), the ethylene diamine chain (EDA) and a cationic
guanidine hydrochloride group.
2.3. Design and synthesis of a focused library based on 8d
The cationic amphiphile 8d was the most potent structure of all the amphiphiles shown in Fig. 2 and
active against all five bacteria tested (Table 1). However, the MIC-values for 8d were somewhat
disappointing as the level of activity was not close to that of the reference antibiotic gentamicin.
Thus, a more focused library based on 8d was prepared. Several changes to the structure of 8d were
included in the synthesis of the optimized structures 9e-11g shown in Fig 3. In all amphiphiles except
11d, the benzylic methylene group on the lipophile was removed to give a more rigid system
between the phenyl group and the 1,2,3-triazole ring. Repulsion between the ortho-protons of the
phenyl group and the 1,2,3-triazole ring was thought to reduce rotational freedom and give the
resulting molecules a “twisted” conformation. This kind of rigidification was also inspired by the
aminobenzamides prepared by Strøm et al. (E23, Fig. 1).13 The first resulting amphiphiles (9e-11e) all
contained a 3,5-di-t-Bu-phenyl group instead of the 3,5-di-t-Bu-benzyl group in 8d.
The second improvement, was to introduce a linear ether chain instead of the 3,5-di-t-Bu-groups on
the aromatic ring (9f-11f). The initial screening indicated that having a heptyl chain alone (4a-8a, Fig.
2) was not sufficient for achieving antimicrobial activity. However, an alkyl ether chain combined
with a phenyl group might be beneficial for antimicrobial efficacy. Conclusively, a heptyl ether chain
was introduced in the 4-position on the benzene ring giving 9f-11f. This was also based on the heptyl
ether group providing comparable aliphatic contribution as two tert-Butyl groups and also differing
marginally in molecular weight.
5

Figure 3. Improved structures 9e-11g based on 8d, for antimicrobial evaluation. Counter ion: Cl-
(CF3COO- for 11e and 11f, from preparative HPLC).
The last change on the lipophilic side of 8d was to introduce an additional aromatic ring to increase
the lipophilicity of the amphiphiles (9g-11g). The β2,2-amino acid derivatives prepared by Strøm et
al11,12 all contained two aromatic lipophilic groups in order to mimic the functionality of two
lipophilic amino acids (e.g. tryptophan). Thus, a 3,5-di-t-Bu-benzyl ether group was introduced in the
4-position of the benzene ring (9g-11g), analogously to the placement of the heptyl ether chain in 9f-
11f.
At the cationic end an iminoguanidine group (11d-g) was introduced in addition to compounds with
a primary EDA amine and guanidine functionality. The iminoguanidine functional group is reported
to improve antimicrobial effects against resistant strains of Gram-positive bacteria such as
methicillin-resistant S. aureus and vancomycin-resistant S. aureus.30 The iminoguanidine group was
also introduced in 8d and thereby resulting in the analogue 11d.
The amphiphiles 9e-g and 10e-g were obtained in two or three steps (21 - 76%) from azides 1e-g and
methyl propiolate (2) similarly to the synthesis of 6a-d and 8a-d as shown in Scheme 1. The azides
1e-g were prepared utilizing a copper catalyzed procedure by Zhu et al.31 Compund 1e was prepared
directly from commercially available 3,5-di-t-Bu-bromobenzene, whereas 1f-g were prepared with
one extra step from iodophenol. Furthermore, the methyl ketone analogues of the methyl-1,2,3-
triazole carboxylates 13d-g were prepared from 3-butynone (12) and the azides 1d-g. Compound
13d was prepared using the “click” chemistry conditions shown in Scheme 1, while 13e-g were
obtained through a modified procedure32 as shown in Scheme 2. DCM was added in addition to t-
BuOH and water to reduce the polarity of the solvent, which seemed to enhance the conversion.
Also, the amount of added 3-butynone (12) was increased since it seemed to be unstable over time
under the current reaction conditions.
6

Scheme 2. Synthesis of improved structures 11d-g based on 12: i) 1d (1 equiv), CuSO4 x 5H2O (5 mol
%), Na-ascorbate (10 mol %), benzoic acid (10 mol %), t-BuOH/H2O (1:2), rt, 18 h. ii) 12 (2-3 equiv),
1e-g (1 equiv), CuSO4 x 5H2O (5 mol %), Na-ascorbate (10 mol %), benzoic acid (10 mol %), t-
BuOH/H2O/DCM (1:1:1), rt, 44-70 h. iii) Aminoguanidine hydrochloride (1.2 – 1.3 equiv), LiCl (0.3 –
0.7 equiv), EtOH, reflux – 90 oC (pressure tube), 25-54 h. iv) Aminoguanidine hydrochloride (1.2
The amphiphilic iminoguanidines 11d-g were attempted prepared from 13d-g and aminoguanidine
hydrochloride according to a LiCl-catalyzed method presented by Seleem et al.30 However, these
conditions gave rather slow conversion of 13 to 11, and not even elevation of the temperature to 90
oC in a pressure tube for 51 h gave full conversion to 11d-g. In an attempt to furnish full conversion,
catalytic amounts of LiCl was replaced with an excess of aqueous HCl33 (6 equiv). This improved the
reaction considerably and gave full conversion of 13d to 11d within 22 h, with minimal formation of
byproducts. However, when attempting the same conditions for synthesis of 11e-g, multiple
additional signals appeared in the 1H NMR spectra after workup, indicating formation of various
unidentified byproducts. Iminoguanidines 11e-g were instead prepared through the LiCl-catalyzed
procedure, which also provided easier purification using HPLC or crystallization. The final purification
of 11e and 11f was achieved with preparative C18-HPLC while 11g was purified through
crystallization (giving a poor isolated yield). 1H NMR spectra of 11d-g and HPLC analysis displayed a
mixture of two compounds, both of which were confirmed to have molecular weight corresponding
to the wanted products after MS analysis of analytical HPLC elute. Thus, the final products of 11d-g
were assumed to consist of a mixture of E- and Z-isomers of the imine. No separation was attempted
and the antimicrobial evaluation was performed on the mixtures of 11d-g (1:9 – 4:6 isomeric ratios
as determined from 1H NMR spectra of 11d-g).
2.4. Antimicrobial activity and cytotoxicity of the improved structures 9e-11g
All of the 10 amphiphiles in Fig. 3 (9e-11g) were tested for antimicrobial activity against the same
panel of bacteria as the 19 initial compounds (Fig. 2), in addition to investigation of inhibition of
Staphylococcus epidermis biofilm formation. The amphiphiles were also tested for cytotoxic
properties against human hepatic cells in the HepG2-assay in order to investigate the selectivity of
the structures for bacteria. All the data from these assays are displayed in Table 2.
2.5. Evaluation of antimicrobial activities of 9e-11g
Removal of the benzylic methylene group on the lipophile led immediately to an increase in
antimicrobial activity, and the 3,5-di-t-Bu-phenyl derivatives 9e and 10e were 2- to 4-fold more
potent against the five test bacteria than their previous 3,5-di-t-Bu-benzyl counterparts 6d and 8d.
7

Linking the 3,5-di-t-Bu-phenyl group directly to the 1,2,3-triazole in 9e-11e and restricting rotational
freedom of the lipophile was thereby shown beneficial for antimicrobial activity. Improved
antimicrobial activity by removing the benzylic methylene group was also seen for the
iminoguanidines, in which 11e with a 3,5-di-t-Bu-phenyl was overall more potent than the more
Table 2. Antimicrobial activity (MIC in µg/mL), actitivity against HepG2 cells (EC50 in µg/mL), inhibition of S. epidermis
biofilms (MIC in µg/mL), and the selectivity index (SI) for amphiphiles 9e-11g.
Entry
E. faecalisa
S. aureusa
S. agalacticaea
E. colia
P. aeruginosaa
HepG2
(EC50)b
8.0
SIc
S. epidermisd
9e
32
16
16
8
16
-
32
16
16
16
8
0.50
0.44
1.44
3.91
2.97
4.04
0.57
1.32
0.25
3.86
N.d.
-
4
e
9f
-
4
3.5
2.9
9g
8
-
2
-
8
10e
10f
10g
11d
11e
11f
11g
Ref.f
16
16
16
8
8
8
16
8
31.3
23.8
16.2
2.3
4
4
8
4
8
4
-
-
2
4
4
8
8
4
4
4
2
4
16
64
-
2.6
4
32
64
10
-
-
8
16
-
2.0
8
0.5
1.9
4
0.13
4
0.5
0.5
N.d. g
N.d.
a E. faecalis (ATCC 29212), S. aureus (ATCC 25923), S. agalacticae (ATCC 12386), E. coli (ATCC 25922), P. aeruginosa (ATCC
27853).
b EC50-value shown, not MIC.
c SI; selectivity index (EC50 HepG2 / MICS. agalacticae)
d S. epidermis biofilm inhibition
e The “-“-sign in the table indicates no activity at or below 64 µg/mL.
f Ref.: gentamicin.
g N.d.: not determined.
flexible 11d with a 3,5-di-t-Bu-benzyl group.
Introducing a heptyl ether chain together with the benzene ring in 9f-11f also improved
antimicrobial activity compared to the previous inactive compounds 4a-8a with only a heptyl chain
(Fig. 2). This series of compounds showed that having an aromatic phenyl group as part of the
lipophile together with the heptyl ether chain was clearly of importance for achieving high
antimicrobial activity. Antimicrobial activity of the amine 9f and guanidine 10f with a 4-heptyloxy-
phenyl lipophile was also improved against certain test bacteria compared to the corresponding
amine 9e and guanidine 10e with the 3,5-di-t-Bu-phenyl lipophile. The improvement in antimicrobial
potency by changing lipophile was most obvious for 10f (4-heptyloxy-phenyl) compared to 10e (3,5-
di-t-Bu-phenyl), where a two-fold increase in antimicrobial activity was observed for 10f against
three strains of bacteria (S. aureus, E. coli, and P. aeruginosa). The improved activity of 10f against
the Gram-negative bacteria was particularly fascinating since Gram-negative bacteria are considered
as more difficult targets.34 When comparing the amines 9e and 9f, introduction of the 4-heptyloxy-
phenyl-group in 9f gave a two- to four-fold increase in antimicrobial activity (4-16 µg/mL) against S.
aureus, S. agalacticae, and P. aeruginosa compared to the 3,5-di-t-Bu-phenyl in 9e. However, we
also observed reduced antimicrobial activity for 9f (4-heptyloxy-phenyl) against E. faecalis and E. coli
(>64 µg/mL) compared to 9e (3,5-di-t-Bu-phenyl), showing strain variation against the present
8

amphiphiles. For the iminoguanidines a different tendency was observed, in which 11e (3,5-di-t-Bu-
phenyl) was overall more potent than 11f (4-heptyloxy-phenyl) against all test bacteria.
Introduction of an additional phenyl group in addition to the 3,5-di-t-Bu benzyl group in 9g-11g
improved the antimicrobial activity further, and especially against S. agalacticae. The most potent
amphiphile (11g) displayed a MIC-value of 0.5 µg/mL, which was eight times lower than the MIC-
value of gentamicin against S. agalacticae. The profound selectivity and high antimicrobial potency
of 11g against S. agalacticae could be of interest for developing antibiotics for prevention of neo-
natal infections, since S. agalacticae is one of the leading causes of infections in newborns.35 It
should also be noted that 10g displayed high potency against the Gram-positive bacteria (4-16
µg/mL), but no activity against the Gram-negative strains. Compound 9g was highly potent against S.
agalacticae (MIC 2 µg/mL), but displayed otherwise only antimicrobial activity against E. faecalis and
P. aeruginosa (MIC 8-16 µg/mL).
The iminoguanidine group was the most efficient cationic group in the library, and resulting in 11d
and 11e being the most potent amphiphiles with broad-spectrum activity and MIC-values ≤ 10
µg/mL against all five bacteria (only exception: 11e MIC 16 µg/mL against P. aeruginosa). The high
potency of the iminoguanidine compounds was particularly pronounced for 11d, which had a 3,5-di-
t-Bu-benzyl lipophile as in 8d from the first series of compounds (Fig. 2). The MIC-values of the
iminoguanidine 11d were however 4- to 8-fold improved compared to the guanidine 8d. We also
observed a 2- to 4-fold improvement in antimicrobial activity for iminoguanidine 11e compared to
the corresponding guanidine 10e, except for against P. aeruginosa where they both had MIC-values
of 16 µg/mL.
The amphiphiles 10e and 10f with a cationic guanidine group were in general more potent than the
similar amine derivatives 9e and 9f. However, an exception to the superiority of the guanidines was
observed for the amine 9g that was more potent than the guanidine 10g against the three strains E.
faecalis, S. agalacticae, and P. aeruginosa. Against S. agalacticae the amine 9g was the second most
potent compound prepared, displaying a MIC-value of 2 µg/mL.
Following the increased antimicrobial activity, the in vitro toxicity of the compounds also increased.
The in vitro toxicity was determined against HepG2 cells and dose-response curves are shown in Fig.
4. The EC50-values determined from the generated dose-response curves are shown in Table 2. The
selectivity index (SI) in Table 2 was furthermore calculated from the EC50-values against HepG2 cells
divided by the MIC-value against S. agalacticae, and showed that the structures displayed rather
poor selectivity with exception of 10e-g and 11g. The guanidines 10e-g were least toxic and the only
amphiphiles displaying EC50-values against HepG2 above 16 µg/mL. The highest SI achieved for the
10 amphiphiles in Table 2 was 4.04 for 10g, meaning that the MIC-value against S. agalacticae was 4
times lower than the EC50-value against HepG2. The cytotoxicity was particularly pronounced for the
9
Figure 4. Anti-proliferative activities of 9e-11g
against human hepatic cells (HepG2) after 24 h of
incubation. Graphs were plotted using a four-
variable slope normalized nonlinear regression

iminoguanidines 11d-g, where all amphiphiles displayed an EC50-value ≤ 2.6 µg/mL. The high toxicity
may be attributed to the relatively high overall lipophilicity of the iminoguanidines 11d-g, as their
calculated ClogD-values (pH = 7.40)36 shown in Fig. 5 were generally higher than for the
corresponding amines (9e-g) and guanidines (10e-g).
Greene et al. have reported that compounds with a ClogP exceeding 3 are more likely to be active
against human cells in <10 µM concentrations.37 Thus, the toxicity observed for 11d-g may be due to
nonspecific toxic interactions arising from a too large lipophilic bulk. However, this does not
completely explain why 11f (ClogD = 2.91) was among the most toxic compound of the series (EC50
=
2.0 µg/mL). It was also observed that the guanidines 10e-g were notably less toxic than their amine
9e-g and iminoguanidine 11d-g counterparts. The guanidine group thereby remains the main
cationic group of choice for future target compounds. The lowered toxicity of the guanidines 10e-g
compared to the iminoguanidines 11d-g also corresponded well with the calculated ClogD-values
shown in Fig. 5.
10f 10e 9f
9e
10g
11f 9g 11e
11g
11d
0
1
2
3
4
5
Figure 5. Calculated ClogD at pH = 7.4 for amphiphiles 9e-
11g. Calculated using MarvinSketch 16.11.7 from
36
2.6. Biofilm inhibiting activities of 9e-11g
The 10 amphiphiles in Fig. 3 were also investigated for biofilm inhibiting effects against S. epidermis,
and the obtained MIC-values are shown in Table 2. All of the amphiphiles displayed good biofilm
inhibiting effects (2-8 µg/mL), with the exception of 9e (MIC >64 µg/mL). The compound displaying
the highest activity for inhibition of biofilms was 10g with a MIC-value of 2 µg/mL. This was
remarkable considering that 10g was among the least toxic amphiphiles tested with an EC50-value of
16.2 µg/mL against HepG2 cells. If the biofilm inhibition were due to general toxicity, one would
expect the most toxic structures to display highest activity towards biofilm inhibition. Thus, the
biofilm inhibition may arise from more specific inhibition mechanisms. However, as the amphiphiles
have not been tested in an antimicrobial assay against S. epidermis, the observed values from biofilm
inhibition assays may be caused by general antimicrobial properties and not specific biofilm-
targeting mechanisms.
3. Conclusion
This study describes the synthesis of a library of 29 novel low molecular weight amphiphilic 1,2,3-
triazoles. The library was prepared using the “click” chemistry products 3 and 13 as key
intermediates, followed by functionalization leading to various cationic nitrogen hydrophiles, i. e.
primary amines, tertiary amines, guanidines, and iminoguanidines. The 1,2,3-triazole amphiphiles
were then assessed for antimicrobial activities against three Gram-positive and two Gram-negative
bacteria, in addition to their ability to inhibit S. epidermis biofilm formation. The in vitro toxicities
against human hepatic cells (HepG2) were also measured for the ten most active structures. The
amphiphiles 10e and 10f displayed the most promising broad-spectrum antimicrobial activities, with
10

MIC-values < 16 µg/mL against all five test bacteria. It should also be noted that the guanidine
amphiphile 10g was shown to display selective activity against the Gram-positive bacteria and with
MIC-values of 4-16 µg/mL. Furthermore, the amphiphiles with the iminoguanidine cationic group
(11d-g) displayed increased potency compared to the corresponding guanidines (10e-g) in the
antimicrobial assays, but this also led to enhanced toxicity in the HepG2-assay. The iminoguanidines
11d-g therefore gave lower bacterial selectivity (except for 11g against S. agalalcticae) compared to
the guanidine amphiphiles 10e-g. The guanidine 10f was 2.5 times more potent against S. aureus
than synoxazolidinone A whereas 10e was comparable to the marine natural product (8 µg/mL vs. 10
µg/mL). Furthermore, it was shown that structures functionalized with an additional phenyl ring
displayed more selective activity, particularly against S. agalacticae. The overall most potent
structure 11g against S. agalacticae – displayed a MIC-value of 0.5 µg/mL, which was 8 times lower
than the reference antibiotic gentamicin. The presented structures also displayed promising activity
towards biofilm inhibition, where 10g was the most potent compound against S. epidermis biofilm
formation with a MIC-value of 2 µg/mL. Based on broad-spectrum activity against all five test strains
and good antibiofilm activity, 10f was one of the most promising compounds prepared and with
second lowest toxicity against HepG2 cells. Further studies on this type of amphiphilic 1,2,3-triazoles
will revolve around further reducing HepG2 toxicity, whilst retaining a high antimicrobial activity.
4. Experimental
4.1. General information
Chemicals were purchased from Sigma Aldrich and used without further purification. All reactions
sensitive to air or moisture were performed under nitrogen atmosphere with dried solvents and
reagents. Melting points were determined on a Buchi 535 apparatus and are uncorrected. TLC was
performed on Merck silica gel 60 F254 plates, using UV light at 312 nm and a 5% solution of
molybdophosphoric acid in 96% EtOH for detection. Column chromatography was performed with
Silica gel (pore size 60 A, 230 - 400 mesh particle size) from Fluka. HPLC analyses were performed on
an Agilent 1290 chromatograph equipped with a Zorbax Eclipse C18 5 µm (150 x 4.6 mm) column
and a diode array detector (main detection region 214 nm). Preparative HPLC purifications were
performed on an Agilent 1260 Infinity equipped with a Zorbax XDB-C18 5 µm (150 x 21.2 mm) and a
diode array detector (main detection region 214 nm). NMR spectra were recorded on a Bruker 600
MHz Avance III HD or a Bruker 400 MHz Avance III HD instrument. Chemical shifts (δ) are reported in
parts per million. Where CDCl3 has been used, shift values for proton are reported with reference to
TMS (0.00) via the lock signal of the solvent. Reference values for other NMR-solvents are taken
from Fulmer et al.38 (1H NMR: DMSO-d6: 2.49, MeOD-d4: 3.31; 13C NMR: DMSO-d6: 39.5, CDCl3: 77.0,
MeOD-d4: 49.15). Signal patterns are indicated as s (singlet), d (doublet), t (triplet), q (quartet), p
(pentet), m (multiplet) or bs (broad singlet). 1H and 13C NMR signals were assigned by 2D correlation
techniques (COSY, HSQC, HMBC). IR spectra were recorded from a Thermo Nicolet FT-IR NEXUS
instrument (only the strongest/structurally most important peaks are listed as either weak (w),
medium (m) or strong (s) (cm-1)). Accurate mass determination in positive and negative mode was
performed on a "Synapt G2-S" Q-TOF instrument from Waters™. Samples were ionized by the use of
ASAP probe (APCI) or ESI probe.
4.2. 1-Azidoheptane (1a). The title compound 1a was prepared according to a published
procedure.39,40 A mixture of 1-bromoheptane (5.00 g, 27.9 mmol) and NaN3 (2.72 g, 41.9 mmol) in
DMF (50 mL) was heated to 50 oC for 19 hours. The suspension was then added DCM (80 mL) and
washed with water (3 x 100 mL), before it was dried over MgSO4 and evaporated. Yielding 1a as a
lightly yellow oil (3.06 g, 21.7 mmol, 78%). 1H NMR analyses corresponded with previously reported
11

spectra for 1a.41 1H NMR (400 MHz, CDCl3): δ 3.25 (t, 2H, J = 6.9 Hz, azide-CH2), 1.60 (p, 2H, J = 7.4 Hz,
CH2), 1.41 – 1.25 (m, 8H, 4x CH2), 0.92 – 0.86 (m, 3H, CH3).
4.3. 1-(Azidomethyl)-4-(tert-butyl)benzene (1c). The title compound 1c was prepared according to
the procedure for 1a from 1-(bromomethyl)-4-(tert-butyl)benzene (3.00 g, 13.2 mmol), affording 1c
as a yellow oil (2.08 g, 11.0 mmol, 83%). 1H NMR analyses corresponded with previously reported
spectra for 1c.42 1H NMR (400 MHz, CDCl3): δ 7.42 – 7.37 (m, 2H, Ph), 7.27 – 7.22 (m, 2H, Ph), 4.29 (s,
2H, CH2), 1.32 (s, 9H, t-Bu).
4.4. 1-(Azidomethyl)-3,5-di-tert-butylbenzene (1d). The title compound 1d was prepared according
to the procedure for 1a from 1-(bromomethyl)-3,5-di-(tert-butyl)benzene (0.80 g, 2.82 mmol),
affording 1d as a clear oil (0.620 g, 2.53 mmol, 90%). 1H NMR analyses corresponded with previously
reported spectra for 1d.43 1H NMR (400 MHz, CDCl3): δ 7.40 (s, 1H, HPh-4), 7.13 (d, J = 1.3 Hz, 2H, HPh-
2 and HPh-6), 4.32 (s, 2H, CH2), 1.33 (s, 18H, 2x t-Bu) ppm.
4.5. 1-Azido-3,5-di-tert-butylbenzene (1e). The title compound 1e was prepared according to a
procedure described by Zhu et al.31 Where 1-bromo-3,5-di-tert-butylbenzene (2.50 g, 9.30 mmol),
CuI (0.177 g, 0.93 mmol), NaN3 (1.21 g, 18.57 mmol), L-proline (0.321 g, 2.74 mmol) and NaOH (0.11
g, 2.79 mmol) were added to EtOH:H2O (7:3, 20 mL) and heated to 95 oC in a sealed tube for 23
hours. The reaction mixture was then added water (30 mL) and extracted with EtOAc (3 x 30 mL).
Drying over MgSO4 and evaporation under reduced pressure yielded a yellow oil, which then was
purified using flash column chromatography (pentane), affording 1e as a colorless oil (0.725 g, 3.13
mmol, 34%). 1H NMR spectra coincided with previously reported data.44 1H NMR (400 MHz, CDCl3): δ
7.20 (t, 1H, J = 1.5 Hz, HPh-4), 6.86 (d, 2H, J = 1.6 Hz, HPh-2 and HPh-6), 1.31 (s, 18H, 2x t-Bu).
4.6. 1-Azido-4-(heptyloxy)benzene (1f). The iodo-precursor (1-(heptyloxy)-4-iodobenzene) to 1f
was prepared using 4-iodophenol (2.00 g, 9.09 mmol), heptyl bromide (1.57 mL, 10.00 mmol), and
K2CO3 (1.62 g, 11.7 mmol) in DMF (12 mL) at rt, as reported by Ban et al45 in 69% yield (1.99 g, 6.24
mmol). The spectra coincided with previously reported data.46 This aromatic iodide was turned into
its corresponding azide (1f) using a procedure described by Zhu et al.31 Where 1-(Heptyloxy)-4-
iodobenzene (1.50 g, 4.71 mmol), CuI (0.09 g, 0.47 mmol), NaN3 (0.37 g, 5.66 mmol), L-proline (0.11
g, 0.94 mmol) and NaOH (0.04 g, 0.94 mmol) were added to DMSO (10 mL) and heated to 60 oC in a
sealed tube for 14 hours. After which the mixture was added water (35 mL), extracted with EtOAc (3
x 40 mL), and dried over MgSO4. Evaporation under reduced pressure yielded a brown oil, which
then was purified using flash column chromatography (pentane), affording 1f as a yellow oil (0.856 g,
3.67 mmol, 78%). 1H NMR (400 MHz, CDCl3): δ 6.96 – 6.91 (m, 2H, HPh-3 and HPh-5), 6.90 – 6.85 (m,
2H, HPh-2 and HPh-6), 3.92 (t, 2H, J = 6.8 Hz, O-CH2), 1.77 (p, 2H, J = 7.3 Hz, O-CH2-CH2), 1.49 – 1.23 (m,
8H, 4x CH2), 0.92 – 0.85 (m, 3H, CH3). 13C NMR (100 MHz, CDCl3): δ 156.6 (CPh-4), 132.1 (CPh-1), 120.0
(CPh-3 and CPh-5), 115.7 (CPh-2 and CPh-6), 68.4 (CH2), 31.8 (CH2), 29.3 (CH2), 29.1 (CH2), 26.0 (CH2),
22.6 (CH2), 14.1 (CH3). IR: 2927 (w), 2857 (w), 2105 (s), 1503 (s), 1470 (w), 1280 (m), 1239 (s), 822 (s)
cm-1. HRMS (APCI/ASAP, m/z): 233.1531 (Calcd. C13H19N3O, 233.1528, [M]-).
4.7. 1-((4-Azidophenoxy)methyl)-3,5-di-tert-butylbenzene (1g). The iodo-precursor (1,3-di-tert-
butyl-5-((4-iodophenoxy)methyl)benzene) to 1g was prepared according to the procedure described
for the iodo-precursor of 1f, using 4-iodophenol (1.00 g, 4.55 mmol), 1-(bromomethyl)-3,5-di-t-Bu-
benzene (1.17 g, 4.13 mmol), and K2CO3 (0.74 g, 5.37 mmol).45 This afforded 1,3-di-tert-butyl-5-((4-
iodophenoxy)methyl)benzene as a white solid (1.55 g, 3.67 mmol, 89%, mp 147.1 – 148.2 oC). 1H
NMR (400 MHz, CDCl3): δ 7.60 – 7.53 (m, 2H, HPhenox-2 and -6), 7.40 (t, 1H, J = 1.8 Hz, HPh-4), 7.23 –
7.28 (m, 2H, HPh-2 and HPh-6), 6.82 – 6.74 (m, 2H, HPhenox-3 and -5), 4.98 (s, 2H, CH2), 1.33 (s, 18H, 2x
tBu). 13C NMR (100 MHz, CDCl3): δ 158.9 (CPhenox-1), 151.2 (CPh-3 and CPh-5), 138.2 (CPhenox-2 and -6),
135.4 (CPh-1), 122.4 (CPh-4), 122.1 (CPh-2 and CPh-6), 117.3 (CPhenox-3 and -5), 82.9 (CPhenox-1), 71.0 (CBn),
34.9 (Cq-tBu), 31.5 (tBu). IR: 2958 (w), 1585 (w), 1485 (m), 1232 (s), 1006 (m), 895 (m), 831 (m), 803
(w), 714 (w), 681 (w) cm-1. HRMS (APCI/ASAP, m/z): 421.1022 (Calcd. C21H26OI, 421.1028, [M-H]-).
12

1,3-Di-tert-butyl-5-((4-iodophenoxy)methyl)benzene (0.40 g, 0.947 mmol) was turned into its
corresponding azide 1g using the procedure decribed for preparation of 1f with CuI (18 mg, 0.095
mmol), NaN3 (0.074 g, 1.137 mmol), L-proline (22 mg, 0.189 mmol), and NaOH (7.6 mg, 0.189 mmol).
This afforded 1g as a yellow solid (0.210 g, 0.62 mmol, 66%, mp 119.0 – 120.2 oC) after purification
with flash column chromatography (pentane). 1H NMR (400 MHz, CDCl3): δ 7.41 (t, 1H, J = 1.4 Hz,
HPh-4), 7.26 (s, 2H, HPh-2 and HPh-6), 7.02 – 6.93 (m, 4H, HPhenox), 5.00 (s, 2H, CH2), 1.33 (s, 18H, 2x
tBu). 13C NMR (100 MHz, CDCl3): δ 156.5 (Cphenox-1), 151.1 (CPh-3 and CPh-5), 132.5 (Cphenox-4), 122.3
(CPh-4), 122.1 (CPh-2 and CPh-6), 120.0 (Cphenox-3 and -5), 116.2 (Cphenox-2 and -6), 71.3 (CH2), 34.5 (Cq-
tBu), 31.5 (tBu). IR: 2961 (w), 2112 (s), 2079 (w), 1504 (s), 1307 (s), 1011 (w) cm-1. HRMS (APCI/ASAP,
m/z): 308.2015 (Calcd. C21H26NO, 308.2014, [M-N2-H]-). Ph = 3,5-di-tert-butylbenzyl.
4.8.1. Method A, “Click” reactions with methyl propiolate: synthesis of methyl 1-heptyl-1H-1,2,3-
triazole-4-carboxylate (3a). The title compound 3a was prepared according to a general procedure
described by Shao et al.22 Where a suspension of methyl propiolate (2) (0.57 g, 6.74 mmol), CuSO4 x
5H2O (0.34 mL, 1 M in H2O, 5 mol %), sodium ascorbate (0.34 mL, 2 M in H2O, 10 mol %) and benzoic
acid (82 mg, 10 mol %) in H2O/t-BuOH (9 mL, 2:1) was added 1a (1.00 g, 7.08 mmol) and stirred for
23 hours at room temperature. The suspension was then added H2O (20 mL), filtered and the
precipitate washed with H2O. Before being dissolved in DCM (30 mL), dried over MgSO4 and partially
evaporated. Crystallization with pentane afforded 3a as a lightly yellow solid (1.35 g, 5.59 mmol,
89%, mp 80.2 – 81.6 oC). 1H NMR (400 MHz, CDCl3): δ 8.07 (s, 1H, Htriazole-5), 4.41 (t, 2H, J = 7.2 Hz,
triazole-CH2), 3.96 (s, 3H, OMe), 1.93 (p, 2H, J = 7.0 Hz, triazole-CH2-CH2), 1.39 – 1.21 (m, 8H, 4x CH2),
0.88 (t, 3H, J = 7.1 Hz, CH3). 13C NMR (100 MHz, CDCl3): δ 161.3 (CC=O, from HMBC), 140.0 (Ctriazole-4,
from HMBC), 127.2 (Ctriazole-5), 52.2 (OMe), 50.7 (triazole-CH2), 31.5 (CH2), 30.1 (triazole-CH2-CH2),
28.6 (CH2), 26.3 (CH2), 22.5 (CH2), 14.0 (CH3). IR: 3123 (w), 2953 (w), 2915 (w), 2850 (w), 1728 (s),
1542 (m), 1239 (s), 1048 (m), 1019 (m), 777 (m) cm-1. 226.1553 (Calcd. C11H20N3O2, 226.1556,
[M+H]+).
4.8.2. Methyl 1-(adamantan-1-yl)-1H-1,2,3-triazole-4-carboxylate (3b). The title compound 3b was
prepared according to Method A from 2 (0.14 mL, 1.61 mmol) and azidoadamantane (0.30 g, 1.69
mmol), affording 3b as a lightly yellow solid (0.231 g, 0.88 mmol, 55%, mp 110.1 – 111.7 oC). 1H NMR
(400 MHz, CDCl3): δ 8.15 (s, 1H, Htriazole-5), 3.95 (s, 3H, OMe), 2.33 – 2.22 (m, 9H, HAda-CH/CH2), 1.86 –
1.75 (m, 6H, 3x HAda-CH2). 13C NMR (100 MHz, CDCl3): δ 161.6 (CC=O), 139.1 (Ctriazole-4, from HMBC),
124.2 (Ctriazole-5), 60.5 (Cq-Ada), 52.1 (OMe), 42.9, 35.8, 29.4. IR: 2928 (w), 2894 (w), 1731 (s), 1366
(m), 1205 (s), 1037 (s), 781 (s) cm-1. HRMS (APCI/ASAP, m/z): 262.1553 (Calcd. C14H20N3O2, 262.1556,
[M+H]+).
4.8.3. Methyl 1-(4-(tert-butyl)benzyl)-1H-1,2,3-triazole-4-carboxylate (3c). The title compound 3c
was prepared according to Method A from 2 (0.571 g, 6.79 mmol) and 1c (1.50 g, 7.13 mmol),
affording 3c as a light blue solid (1.23 g, 4.50 mmol, 66%). 1H NMR analyses corresponded with
previously reported spectra for 3c.47 1H NMR (400 MHz, CDCl3): δ 7.97 (s, 1H, Htriazole-5), 7.42 (d, 2H, J
= 8.4 Hz, HPh), 7.23 (d, 2H, J = 7.8 Hz, HPh), 5.54 (s, 2H, CH2), 3.93 (s, 3H, OMe), 1.32 (s, 9H, t-Bu).
4.8.4. Methyl 1-(3,5-di-tert-butylbenzyl)-1H-1,2,3-triazole-4-carboxylate (3d). The title compound
3d was prepared according to Method A from 2 (0.294 g, 3.49 mmol) and 1d (1.00 g, 3.67 mmol),
affording 3d as a white solid (0.818 g, 2.48 mmol, 71%, mp 172.8 – 174.4 oC). 1H NMR (400 MHz,
CDCl3): δ 7.96 (s, 1H, Htriazole-5), 7.44 (t, 1H, J = 1.8 Hz, HPh-4), 7.12 (d, 2H, J = 1.8 Hz, HPh-2 and HPh-6),
5.55 (s, 2H, CH2), 3.94 (s, 3H, OMe), 1.30 (s, 18H, 2x t-Bu). 13C NMR (100 MHz, CDCl3): δ 161.2 (CC=O),
152.1 (CPh-3 and CPh-5), 140.2 (Ctriazole-4), 132.7 (CPh-1), 127.3 (Ctriazole-5), 123.3 (CPh-4), 122.6 (CPh-2
and CPh-6), 55.2 (CBn), 52.2 (OMe), 34.9 (Cq-t-Bu), 31.4 (t-Bu). IR: 2957 (w), 1713 (s), 1540 (m), 1234
(s), 1045 (s), 1017 (m), 782 (m) cm-1. HRMS (APCI/ASAP, m/z): 330.2179 (Calcd. C19H28N3O2,
330.2182, [M+H]+).
13

4.8.5. Methyl 1-(3,5-di-tert-butylphenyl)-1H-1,2,3-triazole-4-carboxylate (3e). The title compound
3e was prepared according to Method A from 2 (0.182 g, 2.16 mmol) and 1e (0.50 g, 2.16 mmol),
with a different workup: after complete conversion (17 hours), the suspension was added H2O (25
mL) and extracted with DCM (3 x 25 mL). The organic phase was then dried over MgSO4 and partially
evaporated, before it was crystallized with pentane affording 3e as white solid (0.580 g, 1.84 mmol,
85%, mp 105.1 – 107.1 oC). 1H NMR (400 MHz, CDCl3): δ 8.51 (s, 1H, Htriazole-5), 7.55 (t, 1H, J = 1.7 Hz,
HPh-4), 7.53 (d, 2H, J = 1.7 Hz, HPh-2 and HPh-6), 4.01 (s, 3H, OMe), 1.38 (s, 18H, 2x t-Bu). 13C NMR (100
MHz, CDCl3): δ 161.3 (CC=O), 153.2 (CPh-3 and CPh-5), 140.3 (Ctriazole-4), 136.1 (CPh-1), 126.0 (Ctriazole-5),
123.8 (CPh-4), 115.6 (CPh-2 and CPh-6), 52.4 (OMe), 35.2 (Cq-t-Bu), 31.3 (t-Bu). IR: 2952 (w), 1746 (s),
1533 (m), 1361 (s), 1211 (s), 1182 (w), 1146 (s), 1035 (s), 879 (m), 770 (s), 709 (m) cm-1. HRMS
(APCI/ASAP, m/z): 316.2019 (Calcd. C18H26N3O2, 316.2025, [M+H]+).
4.8.6. Methyl 1-(4-(heptyloxy)phenyl)-1H-1,2,3-triazole-4-carboxylate (3f). The title compound 3f
was prepared according to the procedure described for 3e using 2 (0.119 g, 1.41 mmol) and 1f (0.30
g, 1.29 mmol), affording 3f as an off-white solid (0.341 g, 1.07 mmol, 84%, mp 120.1 – 121.5 oC). 1H
NMR (400 MHz, CDCl3): δ 8.42 (s, 1H, Htriazole-5), 7.66 – 7.60 (m, 2H, HPh-3 and HPh-5), 7.06 – 7.00 (m,
2H, HPh-2 and HPh-6), 4.04 – 3.98 (m, 5H, OMe + O-CH2), 1.87 – 1.76 (m, 2H, O-CH2-CH2), 1.53 – 1.26
(m, 8H, 4x CH2), 0.93 – 0.87 (m, 3H, CH3). 13C NMR (100 MHz, CDCl3): δ 161.2 (CC=C), 160.0 (CPh-4),
140.4 (Ctriazole-4), 129.5, 125.6 (Ctriazole-5), 122.4 (CPh-3 and CPh-5), 115.5 (CPh-2 and CPh-6), 68.6 (O-CH2),
52.3 (OMe), 31.8 (CH2), 29.1 (O-CH2-CH2), 29.0 (CH2), 25.9 (CH2), 22.6 (CH2), 14.1 (CH3). IR: 2926 (w),
1711 (s), 1540 (w), 1520 (m), 1269 (m), 1252 (s), 1135 (s), 831 (s), 775 (m) cm-1. HRMS (APCI/ASAP,
m/z): 318.1812 (Calcd. C17H24N3O3, 318.1818, [M+H]+).
4.8.7. Methyl 1-(4-((3,5-di-tert-butylbenzyl)oxy)phenyl)-1H-1,2,3-triazole-4-carboxylate (3g). The
title compound 3g was prepared according to the procedure described for 3e using 2 (53 mg, 0.63
mmol) and 1g (0.20 g, 0.57 mmol), followed by purification with flash column chromatography (DCM
– 10% EtOAc in DCM). Affording 3g as a white solid (0.227 g, 0.54 mmol, 95%, mp 199.3 – 201.1 oC).
1H NMR (400 MHz, CDCl3): δ 8.43 (s, 1H, Htriazole-5), 7.69 – 7.62 (m, 2H, Hphenox-3 and -5), 7.43 (t, 1H, J
= 1.8 Hz, HPh-4), 7.29 (d, 2H, J = 1.8 Hz, HPh-2 and HPh-6), 7.17 – 7.12 (m, 2H, Hphenox-2 and -6), 5.10 (s,
2H, HBn), 4.00 (s, 3H, OMe), 1.35 (s, 18H, 2x t-Bu). 13C NMR (100 MHz, CDCl3): δ 161.3 (CC=O), 159.9
(Cphenox-4), 151.4 (CPh-3 and CPh-5), 140.5 (Ctriazole-4), 135.2 (CPh-1), 129.9 (Cphenox-1), 125.7 (Ctriazole-5),
122.7 (CPh-4), 122.6 (Cphenox-3 and -5), 122.2 (CPh-2 and CPh-6), 116.0 (Cphenox-2 and -6), 71.5 (OMe),
52.5 (CBn), 35.0 (Cq-t-Bu), 31.6 (t-Bu). IR: 2959 (w), 1729 (s), 1518 (s), 1237 (s), 1152 (m), 1042 (s),
1006 (s), 881 (w), 847 (m), 778 (w), 695 (w) cm-1. HRMS (APCI/ASAP, m/z): 422.2436 (Calcd.
C25H32N3O3, 422.2444, [M+H]+). Ph = 3,5-di-tert-butylbenzyl.
4.8.8. 1-(1-(3,5-Di-tert-butylbenzyl)-1H-1,2,3-triazol-4-yl)ethan-1-one (13d). The title compound
13d was prepared according to the procedure described for 3e using 3-butynone (12) (0.139 g, 1.94
mmol) and 1d (0.50, 1.94 mmol), affording 13d as an off-white solid (0.531 g, 1.69 mmol, 88%, mp
145.0 – 146.8 oC). 1H NMR (400 MHz, CDCl3): δ 7.93 (s, 1H, Htriazole-5), 7.45 (t, 1H, J = 1.8 Hz, HPh-4),
7.13 (d, 2H, J = 1.8 Hz, HPh-2 and HPh-6), 5.53 (s, 2H, HBn), 2.68 (s, 3H, CH3), 1.30 (s, 18H, 2x t-Bu). 13
C
NMR (100 MHz, CDCl3): δ 193.0 (CC=O), 152.1 (CPh-3 and CPh-5), 148.3 (Ctriazole-4), 132.7 (CPh-1), 125.2
(Ctriazole-5), 123.3 (CPh-4), 122.7 (CPh-2 and CPh-6), 55.2 (CBn), 34.9 (Cq-t-Bu), 31.4 (t-Bu), 27.1 (CH3). IR:
2953 (w), 1684 (s), 1528 (m), 1360 (m), 1238 (w), 1200 (s), 1045 (m), 756 (s), 676 (w) cm-1. HRMS
(APCI/ASAP, m/z): 314.2227 (Calcd. C19H28N3O, 314.2232, [M+H]+).
4.9.1. Method B, “Click” reactions with 3-butynone (12): synthesis of 1-(1-(3,5-di-tert-butylphenyl)-
1H-1,2,3-triazol-4-yl)ethan-1-one (13e). The title compound 13e was prepared according to a
published procedure,32 where a suspension of 3-butynone (12) (68 mg, 1.00 mmol), CuSO4 x 5H2O
(17 µL, 1 M in H2O, 5 mol %), sodium ascorbate (17 µL mL, 2 M in H2O, 10 mol %) and benzoic acid (3
mg, 10 mol %) in H2O/t-BuOH/DCM (1.5 mL, 1:1:1) was added 1e (77 mg, 0.33 mmol) and stirred for
45 hours at room temperature (with addition of additional 2 eq of 12 after 6 hours). The reaction
mixture was then added H2O (10 mL) and extracted with DCM (3x 15 mL), before the organic phase
14

was dried over MgSO4 and evaporated. Purification with flash column chromatography (DCM)
afforded 13e as a white solid (52 mg, 0.17 mmol, 52%, mp 140.5 – 145.3 oC). 1H NMR (400 MHz,
CDCl3): δ 8.48 (s, 1H, Htriazole-5), 7.56 – 7.54 (m, 1H, HPh-4), 7.54 – 7.52 (m, 2H, HPh-2 and HPh-6), 2.77
(s, 3H, CH3), 1.38 (s, 18H, 2x t-Bu). 13C NMR (100 MHz, CDCl3): δ 193.1 (CC=O), 153.2 (CPh-3 and CPh-5),
148.4 (Ctriazole-4), 136.1 (CPh-1), 123.8 (CPh-4), 123.7 (Ctriazole-5), 115.6 (CPh-2 and CPh-6), 35.2 (Cq-t-Bu),
31.3 (t-Bu), 27.3 (CH3). IR: 2958 (w), 1683 (s), 1532 (m), 1236 (m), 1028 (w), 990 (w), 878 (w) cm-1.
HRMS (APCI/ASAP, m/z): 300.2070 (Calcd. C18H26N3O, 300.2070, [M+H]+).
4.9.2. 1-(1-(4-(Heptyloxy)phenyl)-1H-1,2,3-triazol-4-yl)ethan-1-one (13f). The title compound 13f
was prepared according to Method B from 12 (0.123 g, 1.80 mmol) and 1f (0.20 g, 0.86 mmol).
Affording 3f as a white solid (0.172 g, 0.57 mmol, 67%, mp 112.7 – 115.5 oC) after purification with
flash column chromatography (DCM). 1H NMR (400 MHz, CDCl3): δ 8.39 (s, 1H, Htriazole-5), 7.66 – 7.59
(m, 2H, HPh-3 and HPh-5), 7.06 – 7.00 (m, 2H, HPh-2 and HPh-6), 4.02 (t, 2H, J = 6.9 Hz, O-CH2), 2.75 (s,
3H, ketone-CH3), 1.82 (p, 2H, J = 7.3 Hz, O-CH2-CH2), 1.53 – 1.27 (m, 8H, 4x CH2), 0.94 – 0.86 (m, 3H,
CH3). 13C NMR (100 MHz, CDCl3): δ 193.0 (CC=O), 160.0 (CPh-4), 148.4 (Ctriazole-4), 129.5 (CPh-1), 123.3
(Ctriazole-5), 122.4 (CPh-3 and CPh-5), 115.5 (CPh-2 and CPh-6), 68.6 (O-CH2), 31.8 (CH2), 29.1 (O-CH2-CH2),
29.0 (CH2), 27.3 (ketone-CH3), 26.0 (CH2), 22.6 (CH2), 14.1 (CH3). IR: 3131 (w), 2923 (w), 1682 (s),
1516 (s), 1253 (s), 1241 (s), 1171 (m), 823 (s), 678 (m) cm-1. HRMS (APCI/ASAP, m/z): 302.1863
(Calcd. C17H24N3O2, 302.1869, [M+H]+).
4.9.3. 1-(1-(4-((3,5-Di-tert-butylbenzyl)oxy)phenyl)-1H-1,2,3-triazol-4-yl)ethan-1-one (13g). The
title compound 13g was prepared according Method B in two reactions. Firstly with 12 (30 mg, 0.41
mmol) and 1g (0.15 g, 0.41 mmol) for 24 hours then followed by addition of 12 (60 mg, 0.83 mmol)
to the extracted crude (0.26 g, 1g/13g 3:1) followed by stirring for 47 hours at room temperature.
Purification with flash column chromatography (DCM) afforded 13g as a white solid (98 mg, 0.24
mmol, 58%, mp 179.4 – 181.1 oC). 1H NMR (400 MHz, CDCl3): δ 8.40 (s, 1H, Htriazole-5), 7.69 – 7.63 (m,
2H, Hphenox-3 and -5), 7.44 (t, 1H, J = 1.7 Hz, HPh-4), 7.28 (d, 2H, J = 2.0 Hz, HPh-3 and HPh-5), 7.18 – 7.12
(m, 2H, Hphenox-2 and -6), 5.10 (s, 2H, HBn), 2.75 (s, 3H, ketone-CH3), 1.35 (s, 18H, 2x t-Bu). 13C NMR
(100 MHz, CDCl3): δ 192.9 (CC=O), 159.8 (Cphenox-4), 151.3 (CPh-3 and CPh-5), 148.4 (Ctriazole-4), 135.1 (CPh-
1), 123.3 (Ctriazole-5), 122.5 (CPh-4), 122.4 (Cphenox-3 and Cphenox -5), 122.1 (CPh-2 and CPh-6), 116.0
(Cphenox-2 and Cphenox -6), 71.4 (CBn), 34.9 (Cq-t-Bu), 31.5 (t-Bu), 27.3 (ketone-CH3). IR: 2955 (w), 1693
(s), 1517 (s), 1248 (s), 985 (m), 882 (w), 829 (s), 696 (m) cm-1. HRMS (APCI/ASAP, m/z): 406.2490
(Calcd. C25H32N3O2, 406.2495, [M+H]+). Ph = 3,5-di-tert-butylbenzyl.
4.10.1. Method C, Piperazine amidation reactions: synthesis of (1-heptyl-1H-1,2,3-triazol-4-
yl)(piperazin-1-yl)methanone (n4a) and 4-(1-heptyl-1H-1,2,3-triazole-4-carbonyl)piperazin-1-ium
chloride (4a). The title compound n4a was prepared according to a general procedure described by
Oshima et al.23 with some modifications. Where a suspension of 3a (0.30 g, 1.33 mmol), piperazine
(0.344 g, 3.99 mmol), NaOMe (0.07 g, 1.33 mmol), mol. sieves (0.5 – 1.0 g, activated, 4 Å) and MeOH
(6 mL) was stirred under N2-atmosphere for 43 hours. After completed stirring, the reaction mixture
was evaporated and dissolved in DCM before it was filtered through celite. Subsequent purification
with flash column chromatography (SiO2 pre-deactivated with 1% TEA in eluent, eluent: CHCl3/MeOH
95:5) afforded n4a as a white solid (0.201 g, 0.72 mmol, 54%). 1H NMR (400 MHz, CDCl3): δ 8.06 (s,
1H, Htriazole-5), 4.37 (t, 2H, J = 7.1 Hz, triazole-CH2), 4.30 (t, 2H, J = 4.8 Hz, HPip-2 and HPip-6), 3.77 (t,
2H, J = 5.0 Hz, HPip-2 and HPip-6), 3.01 – 2.94 (m, 4H, HPip-3 and HPip-5), 1.92 (t, 2H, J = 7.0 Hz, CH2),
1.41 – 1.20 (m, 8H, 4x CH2), 0.91 – 0.84 (m, 3H, CH3). 13C NMR (100 MHz, CDCl3): δ 159.9 (CC=O), 144.4
(Ctriazole-4), 128.0 (Ctriazole-5), 50.5 (CH2), 48.0 (CPip-2 or CPip-6), 46.7 (CPip-3 and CPip-5), 46.0 (CPip-3 and
CPip-5), 43.8 (CPip-2 or CPip-6), 31.5 (CH2), 30.1 (CH2), 28.6 (CH2), 26.4 (CH2), 22.5 (CH2), 14.0 (CH3). The
free amine n4a was then turned into its HCl-salt, by mixing n4a (40 mg, 0.14 mmol) in MeCN (3 mL)
and adding HCl (0.1 mL, 1.22 mmol, 37%, aq.). The suspension was evaporated, washed with MeCN
(3 x 1 mL) and dried, affording 4a as a white solid (27 mg, 0.09 mmol, 60%, mp 228.8 – 230.7 oC).
HPLC (C18, 3:5 H2O/MeOH + 0.1% TFA, 0.75 mL/min, 214 nm): 5.1 min, 99% pure. 1H NMR (400 MHz,
d4-MeOD): δ 8.46 (s, 1H, Htriazole-5), 4.61 – 4.39 (m, 4H, HPip-2 and HPip-6 + CH2), 4.01 (bs, 2H, HPip-2
15

and HPip-6), 3.40 – 3.35 (m, 4H, HPip-3 and HPip-5), 1.95 (p, 2H, J = 7.3 Hz, CH2), 1.44 – 1.25 (m, 8H, 4x
CH2), 0.92 (t, 3H, J = 6.9 Hz, CH3). IR: 2931 (w), 2730 (w), 1625 (s), 1594 (w), 1429 (m), 1248 (m), 1049
(m), 988 (m), 759 (m) cm-1. HRMS (APCI/ASAP, m/z): 280.2139 (Calcd. C14H26N5O, 280.2137, [M-Cl]+).
4.10.2. (1-(Adamantan-1-yl)-1H-1,2,3-triazol-4-yl)(piperazin-1-yl)methanone (n4b) and 4-(1-
(adamantan-1-yl)-1H-1,2,3-triazole-4-carbonyl)piperazin-1-ium chloride (4b). The title compound
n4b was prepared according Method C from 3b (0.23 g, 0.88 mmol) and piperazine (0.227 g, 2.64
mmol), with 68 hours reaction time at room temperature. Affording n4b as a white solid (0.205 g,
0.65 mmol, 74%) after purification with flash column chromatography (CHCl3/MeOH 95:5). 1H NMR
(400 MHz, CDCl3): δ 8.15 (s, 1H, Htriazole-5), 4.31 (s, 2H, HPip-2 and HPip-6), 3.76 (s, 2H, HPip-2 and HPip-6),
2.98 – 2.93 (m, 4H, HPip-3 and HPip-5), 2.31 – 2.21 (m, 9H, HAda-CH/CH2), 1.86 – 1.75 (m, 6H, HAda-CH2).
13C NMR (100 MHz, CDCl3): δ 160.3 (CC=O), 143.5 (Ctriazole-4), 125.0 (Ctriazole-5), 60.1 (Cq-ada), 48.1 (CPip-2
or CPip-6), 46.7 (CPip-3 and CPip-5), 46.1 (CPip-3 and CPip-5), 43.8 (CPip-2 or CPip-6), 42.8 (CAda), 35.8 (CAda),
29.4 (CAda). The free amine n4b was then turned into its HCl-salt, by adding HCl (5 mL, 10 mmol, 2M
in Et2O) to a solution of n4b (30 mg, 0.09 mmol) followed by filtration. Drying afforded 4b as a white
solid (33 mg, 0.09 mmol, 99%, mp >280 oC decomp.). HPLC (C18, 3:5 H2O/MeOH + 0.1% TFA, 0.75
mL/min, 214 nm): 4.8 min, 99% pure. 1H NMR (400 MHz, d4-MeOD): δ 8.49 (s, 1H, Htriazole-5), 4.48
(bs, 2H, HPip-2 and HPip-6), 4.01 (bs, 2H, HPip-2 and HPip-6), 3.40 – 3.33 (m, 4H, HPip-3 and HPip-5), 2.33 –
2.23 (m, 9H, HAda-CH/CH2), 1.91 – 1.79 (m, 6H, HAda-CH2). 13C NMR (100 MHz, d4-MeOD, rotamers*):
δ 162.5 (CC=O), 143.1 (Ctriazole-4), 126.9 (Ctriazole-5), 62.0 (Cq-Ada), 44.9 (bs, CPip), 43.9*/43.8* (CAda), 40.8
(bs, CPip) 36.9*/36.9* (CAda), 31.1 (CAda). IR: 3376 (bw), 2912 (m), 1606 (s), 1547 (m), 1451 (m), 1423
(m), 1250 (m), 1235 (w), 1013 (m), 756 (m) cm-1. HRMS (APCI/ASAP, m/z): 316.2135 (Calcd.
C17H26N5O, 316.2137, [M-Cl]+).
4.10.3. (1-(4-(tert-Butyl)benzyl)-1H-1,2,3-triazol-4-yl)(piperazin-1-yl)methanone (n4c) and 4-(1-(4-
(tert-butyl)benzyl)-1H-1,2,3-triazole-4-carbonyl)piperazin-1-ium chloride (4c). The title compound
n4c was prepared according to Method C from 3c (0.10 g, 0.37 mmol) and piperazine (0.095 g, 1.10
mmol), with 44 hours reaction time at room temperature. Affording n4c as a clear oily solid (69 mg,
1
0.21 mmol, 58%) after purification with flash column chromatography (CHCl3/MeOH 9:1). H NMR
(400 MHz, CDCl3): δ 7.96 (s, 1H, Htriazole-5), 7.45 – 7.36 (m, 2H, HPh-2 and HPh-6), 7.29 – 7.19 (m, 2H,
HPh-3 and HPh-5), 5.50 (s, 2H, HBn), 4.29 (t, 2H, J = 4.9 Hz, HPip-2 and HPip-6), 3.74 (t, 2H, J = 5.0 Hz, HPip-
2 and HPip-6), 2.99 – 2.91 (m, 4H, HPip-3 and HPip-5), 1.31 (s, 9H, t-Bu). 13C NMR (100 MHz, CDCl3): δ
159.8 (CC=O), 152.4 (CPh-4), 144.6 (Ctriazole-4), 130.7 (CPh-1), 128.3 (CPh-3 and CPh-5), 128.2 (Ctriazole-5),
126.2 (CPh-2 and CPh-6), 54.1 (CBn), 47.9 (CPip-2 or CPip-6), 46.6 (CPip-3 and CPip-5), 46.0 (CPip-3 and CPip-
5), 43.7 (CPip-2 and CPip-6), 34.7 (Cq-t-Bu), 31.2 (t-Bu). The free amine n4c was turned into its HCl-salt
using the procedure for 4a with n4c (51 mg, 0.13 mmol), affording 4c as a white solid (42 mg, 0.12
mmol, 74%, mp >190 oC decomp.). HPLC (C18, 3:5 H2O/MeOH + 0.1% TFA, 0.75 mL/min, 214 nm): 6.5
min, 96% pure. 1H NMR (400 MHz, d4-MeOD): δ 8.42 (s, 1H, Htriazole-5), 7.50 – 7.41 (m, 2H, HPh-2 and
HPh-6), 7.36 – 7.29 (m, 2H, HPh-3 and HPh-5), 5.63 (s, 2H, HBn), 4.49 (bs, 2H, HPip-2 and HPip-6), 4.00 (bs,
2H, HPip-2 and HPip-6), 3.39 – 3.34 (m, 4H, HPip-3 and HPip-5), 1.32 (s, 9H, t-Bu). 13C NMR (100 MHz, d4-
MeOD): δ 153.3 (CPh-4), 144.2 (Ctriazole-4, from HMBC), 133.5 (CPh-1), 130.1 (Ctriazole-5), 129.3 (CPh-3 and
CPh-5), 127.2 (CPh-2 and CPh-6), 55.0 (CBn), 44.8 (CPip, from HSQC), 40.7 (CPip, from HSQC), 35.6 (Cq-t-
Bu), 31.8 (t-Bu). IR: 2951 (w), 2730 (w), 1616 (s), 1593 (w), 1540 (w), 1431 (m), 1248 (s), 1049 (s), 990
(s), 757 (s) cm-1. HRMS (APCI/ASAP, m/z): 328.2137 (Calcd. C18H26N5O, 328.2137, [M-Cl]+).
4.10.4. (1-(3,5-Di-tert-butylbenzyl)-1H-1,2,3-triazol-4-yl)(piperazin-1-yl)methanone (n4d) and 4-(1-
(3,5-di-tert-butylbenzyl)-1H-1,2,3-triazole-4-carbonyl)piperazin-1-ium chloride (4d). The title
compound n4d was prepared according to Method C from 3d (0.25 g, 0.76 mmol) and piperazine
(0.196 g, 2.28 mmol), affording n4d as an off-white solid (0.199 g, 0.52 mmol, 69%) after purification
with flash column chromatography (CHCl3/MeOH 95:5). 1H NMR (400 MHz, CDCl3): δ 7.96 (s, 1H,
Htriazole-5), 7.44 (s, 2H, HPh-4), 7.15 (d, 2H, J = 1.2 Hz, HPh-2 and HPh-6), 5.50 (s, 2H, HBn), 4.28 (bs, 2H,
16

HPip-2 and HPip-6), 3.74 (bs, 2H, HPip-2 and HPip-6), 2.99 – 2.92 (m, 4H, HPip-3 and HPip-5), 1.30 (s, 18H,
2x t-Bu). 13C NMR (100 MHz, CDCl3): δ 159.9 (CC=O), 152.0 (CPh-3 and CPh-5), 144.5 (Ctriazole-4), 132.8
(CPh-1), 128.1 (Ctriazole-5), 123.2 (CPh-4), 122.8 (CPh-2 and CPh-6), 55.1 (CBn), 48.0 (CPip-2 or CPip-6), 46.7
(CPip-3 and CPip-5), 46.0 (CPip-3 and CPip-5), 43.7 (CPip-2 or CPip-6), 34.9 (Cq-t-Bu), 31.4 (t-Bu). The free
amine n4d was turned into its HCl-salt by adding an excess of HCl (5 mL, 10 mmol, 2 M in Et2O) to
n4d (30 mg, 0.08 mmol) in DCM (3 mL). Drying afforded 4d as a white solid (30 mg, 0.07 mmol, 91%,
mp 221.2 – 226.6 oC). HPLC (C18, 3:5 H2O/MeOH + 0.1% TFA, 0.75 mL/min, 214 nm): 30.2 min, 99%
pure. 1H NMR (400 MHz, d4-MeOD): δ 8.44 (s, 1H, Htriazole-5), 7.47 (s, 1H, HPh-4), 7.27 (s, 2H, HPh-2 and
HPh-6), 5.64 (s, 2H, HBn), 4.52 (bs, 2H, HPip-2 and HPip-6), 4.00 (bs, 2H, HPip-2 and HPip-6), 3.56 (bs, 4H,
HPip-3 and HPip-5), 1.32 (s, 18H, 2x t-Bu). 13C NMR (100 MHz, d4-MeOD): δ 162.1 (CC=O), 153.1 (CPh-3
and CPh-5), 144.1 (Ctriazole-4), 135.7 (CPh-1), 130.2 (Ctriazole-5), 124.0 (CPh-4), 123.8 (CPh-2 and CPh-6), 55.8
(CBn), 45.0 (bs, CPip), 40.8 (bs, CPip) 35.9 (Cq-t-Bu), 31.9 (t-Bu). IR: 2953 (w), 1605 (s), 1443 (m), 1248
(s), 1053 (m), 994 (s), 758 (s) cm-1. HRMS (APCI/ASAP, m/z): 384.2759 (Calcd. C22H34N5O, 384.2763,
[M-Cl]+).
4.10.5. (1-Heptyl-1H-1,2,3-triazol-4-yl)(4-methylpiperazin-1-yl)methanone (n5a) and 4-(1-heptyl-
1H-1,2,3-triazole-4-carbonyl)-1-methylpiperazin-1-ium chloride (5a). The title compound n5a was
prepared according to Method C from 3a (0.15 g, 0.67 mmol) and 1-methylpiperazine (0.20 g, 2.00
mmol), with 76 hours reaction time at room temperature. Affording n5a as a white solid (99 mg,
0.34 mmol, 51%) after purification with flash column chromatography (CHCl3/MeOH 95:5). 1H NMR
(400 MHz, CDCl3): δ 8.06 (s, 1H, Htriazole-5), 4.33 – 4.28 (m, 4H, CH2 + HPip-2 and HPip-6), 3.77 (t, 2H, J =
5.0 Hz, HPip-2 and HPip-6), 2.54 – 2.47 (m, 4H, HPip-3 and HPip-5), 2.33 (s, 3H, N-CH3) 1.92 (p, 2H, J = 7.0
Hz, CH2), 1.40 – 1.21 (m, 8H, 4x CH2), 0.91 – 0.84 (m, 3H, CH3). 13C NMR (100 MHz, CDCl3): δ 159.9
(CC=O), 144.4 (Ctriazole-4), 128.1 (Ctriazole-5), 55.6 (CPip-3 and CPipe-5), 54.8 (CPip-3 and CPip-5), 50.2 (CH2),
46.6 (CPip-2 or CPip-6), 46.1 (N-CH3), 42.6 (CPip-2 or CPip-6), 31.5 (CH2), 30.1 (CH2), 28.6 (CH2), 26.4 (CH2),
22.5 (CH2), 14.0 (CH3). The free amine n5a was turned into its HCl-salt by adding HCl (0.15 mL, 1.83
mmol, 37%, aq) to n5a (40 mg, 0.136 mmol) dissolved in MeCN (4 mL), which upon drying afforded
5a as a white solid (43 mg, 0.130 mmol, 96%, mp 189.9 – 191.5 oC). HPLC (C18, 3:5 H2O/MeOH +
0.1% TFA, 0.75 mL/min, 214 nm): 5.0 min, 97% pure. 1H NMR (400 MHz, d4-MeOD, rotamers*): δ
8.57*/8.47* (s, 1H, Htriazole-5), 5.46 (bs, 1H, Pip), 4.82 (bs, 1H, Pip), 4.48 (t, 2H, J = 6.9 Hz, CH2), 3.81 –
3.48 (m, 4H, Pip), 3.26 (bs, 2H, Pip), 3.04*/2.99* (s, 3H, N-CH3), 1.98 – 1.90 (m, 2H, CH2), 1.43 – 1.25
(m, 8H, 4x CH2), 0.96 – 0.87 (m, 3H, CH3). 13C NMR (100 MHz, d4-MeOD, rotamers*): δ 162.0 (CC=O),
143.7 (Ctriazole-4), 130.3*/129.7* (Ctriazole-5), 54.5 (bs, CPip), 51.8*/51.7* (CH2), 51.3 (CPip), 43.9*/43.8*
(N-CH3), 42.2 (CPip), 32.9*/32.9* (CH2), 31.3*/31.3* (CH2), 29.9*/29.8* (CH2), 27.5*/27.5* (CH2), 23.7
(CH2), 14.5*/14.5* (Me). IR: 2919 (w), 1625 (s), 1539 (w), 1425 (m), 1246 (s), 1049 (m), 974 (s), 759
(m) cm-1. HRMS (APCI/ASAP, m/z): 294.2293 (Calcd. C15H28N5O, 294.2294, [M-Cl]+).
4.10.6. (1-(Adamantan-1-yl)-1H-1,2,3-triazol-4-yl)(4-methylpiperazin-1-yl)methanone (n5b) and 4-
(1-(adamantan-1-yl)-1H-1,2,3-triazole-4-carbonyl)-1-methylpiperazin-1-ium chloride (5b). The title
compound n5b was prepared according to Method C from 3b (0.15 g, 0.57 mmol) and 1-
methylpiperazine (0.172 g, 1.72 mmol), with 115 hours at room temperature. Affording n5b as a
white wax (99 mg, 0.30 mmol, 53%) after purification with flash column chromatography
(CHCl3/MeOH 95:5). 1H NMR (400 MHz, CDCl3): δ 8.14 (s, 1H, Htriazole-5), 4.37 (bs, 2H, HPip-2 and HPip-
6), 3.81 (bs, 2H, HPip-2 and HPip-6), 2.51 (bs, 4H, HPip-3 and HPip-5), 2.34 (s, 3H, N-CH3), 2.31 – 2.19 (m,
9H, HAda-CH/CH2), 1.86 – 1.74 (m, 6H, HAda-CH2). 13C NMR (100 MHz, CDCl3): δ 160.2 (CC=O), 143.5
(Ctriazole-4), 125.0 (Ctriazole-5), 60.1 (Cq-Ada), 55.6 (CPip-3 and CPip-5), 54.9 (CPip-3 and CPip-5), 46.6 (CPip-2
or CPip-6), 46.0 (N-CH3), 42.9 (CAda), 42.6 (CPip-2 or CPip-6), 35.8 (CAda), 29.4 (CAda). The free amine n5b
was turned into its HCl-salt according to the procedure for 5a with n5b (35 mg, 0.11 mmol) and HCl
(0.10 mL, 1.22 mmol, 37% aq), affording 5b as a white solid (39 mg, 0.11 mmol, quant., mp 250.3 –
17

253.9 oC). HPLC (C18, 3:5 H2O/MeOH + 0.1% TFA, 0.75 mL/min, 214 nm): 4.8 min, 98% pure. 1H NMR
(400 MHz, d4-MeOD, rotamers*): δ 8.64*/8.52* (s, 1H, Htriazole-5), 5.43 (bs, 1H, Pip), 4.80 (bs, 1H,
Pip), 3.92 – 3.45 (m, 4H, Pip), 3.26 (bs, 2H, Pip), 3.04*/2.99* (s, 3H, N-CH3), 2.35 – 2.25 (m, 9H, Ada),
1.93 – 1.81 (m, 6H, Ada). 13C NMR (100 MHz, d4-MeOD, rotamers*): δ 162.3 (CC=O), 143.1 (Ctriazole-4),
127.1 (Ctriazole-5), 62.2*/62.0* (Cq-Ada), 54.5 (bs, CPip), 51.3 (CPip), 43.9 (CAda), 43.8*/43.8* (N-CH3),
42.2 (CPip), 36.9*/36.9* (CAda), 31.1 (CAda). IR: 2909 (m), 1620 (s), 1539 (w), 1450 (m), 1422 (m), 1244
(m), 979 (m), 756 (m) cm-1. HRMS (APCI/ASAP, m/z): 330.2294 (Calcd. C18H28N5O, 330.2294, [M-Cl]+).
4.10.7. (1-(4-(tert-Butyl)benzyl)-1H-1,2,3-triazol-4-yl)(4-methylpiperazin-1-yl)methanone (n5c) and
4-(1-(4-(tert-butyl)benzyl)-1H-1,2,3-triazole-4-carbonyl)-1-methylpiperazin-1-ium chloride (5c). The
title compound n5c was prepared according to Method C from 3c (0.15 g, 0.55 mmol) and 1-
methylpiperazine (0.165 g, 1.65 mmol), with 63 hours reaction time at room temperature. Affording
n5c as an off-white solid (75 mg, 0.21 mmol, 39%) together with 3% of 3c (from 1H NMR) after
purification with flash column chromatography (SiO2 pre-deactivated with 1% TEA in eluent, eluent:
CHCl3/MeOH 95:5). 1H NMR (400 MHz, CDCl3): δ 7.95 (s, 1H, Htriazole-5), 7.43 – 7.38 (m, 2H, HPh-2 and
HPh-6), 7.26 – 7.22 (m, 2H, HPh-3 and HPh-5), 5.50 (s, 2H, HBn), 4.32 (t, 2H, J = 4.9 Hz, HPip-2 and HPip-6),
3.77 (t, 2H, J = 4.9 Hz, HPip-2 and HPip-6), 2.52 – 2.45 (m, 4H, HPip-3 and HPip-5), 2.32 (s, 3H, N-CH3),
1.31 (s, 9H, t-Bu). 13C NMR (100 MHz, CDCl3): δ 159.8 (CC=O), 152.4 (CPh-4, from HMBC), 144.6 (Ctriazole
-
4), 130.7 (CPh-1, from HMBC), 128.3 (CPh-3 and CPh-5), 128.2 (Ctriazole-5), 126.2 (CPh-2 and CPh-6), 55.5
(CPip-3 and CPip-5), 54.8 (CPip-3 and CPip-5), 54.1 (CBn), 46.5 (CPip-2 or CPip-6), 46.0 (N-CH3), 42.6 (CPip-2 or
CPip-6), 34.7 (Cq-t-Bu), 31.2 (t-Bu). The free amine n5c was turned into its HCl-salt according to the
procedure described for 4a with n5c (63 mg, 0.18 mmol) and HCl (0.25 mL, 3.04 mmol, 37% aq),
affording 5c as a white solid (43 mg, 0.11 mmol, 60%, mp 227.5 – 231.5 oC). HPLC (C18, 3:5
H2O/MeOH + 0.1% TFA, 0.75 mL/min, 214 nm): 6.3 min, 97% pure. 1H NMR (400 MHz, d4-MeOD): δ
8.43 (s, 1H, Htriazole-5), 7.47 – 7.42 (m, 2H, HPh-2 and HPh-6), 7.35 – 7.31 (m, 2H, HPh-3 and HPh-5), 5.63
(s, 2H, HBn), 5.45 (bs, 1H, Pip), 4.82 (bs, 1H, Pip), 3.90 – 3.40 (m, 4H, Pip), 3.26 (bs, 2H, Pip), 2.96 (s,
3H, N-CH3), 1.33 (s, 9H, t-Bu). IR: 2955 (w), 2434 (w), 1624 (s), 1540 (w), 1427 (w), 1247 (s), 1049 (s),
976 (s), 757 (m) cm-1. HRMS (APCI/ASAP, m/z): 342.2292 (Calcd. C19H28N5O, 342.2294, [M-Cl]+).
4.10.8. 4-(1-(3,5-Di-tert-butylbenzyl)-1H-1,2,3-triazole-4-carbonyl)-1-methylpiperazin-1-ium
chloride (5d). The title compound 5d was prepared through an Eschweiler-Clarke reductive
amination,25,26 where n4d (50 mg, 0.13 mmol), formaldehyde (0.20 mL, 37% aq, 2.69 mmol) and
formic acid (0.10 mL, 96% aq, 2.50 mmol) were refluxed in MeCN (2 mL) for 1.5 hours. After cooling
to room temperature, HCl (0.25 mL, 3.04 mmol, 37%, aq.) was added and the reaction mixture was
evaporated. The crude was then crystallized from DCM and Et2O, affording 5d as a white solid (46
mg, 0.11 mmol, 81%, mp 150.4 – 155.8 oC). HPLC (C18, 3:5 H2O/MeOH + 0.1% TFA, 0.75 mL/min, 214
nm): 29.3 min, 97% pure. 1H NMR (400 MHz, d4-MeOD): δ 8.45 (s, 1H, Htriazole-5), 7.47 (s, 1H, HPh-4),
7.27 (s, 2H, HPh-2 and HPh-6), 5.64 (s, 2H, HBn), 5.49 (bs, 1H, HPip), 4.74 (bs, 1H, HPip), 3.62 (bs, 3H, HPip),
3.28 (bs, 3H, HPip), 2.97 (s, 3H, N-CH3), 1.32 (s, 18H, 2x t-Bu). 13C NMR (100 MHz, d4-MeOD): δ 161.9
(CC=O), 153.1 (CPh-3 and CPh-5), 144.1 (Ctriazole-4), 135.7 (CPh-1), 130.2 (Ctriazole-4), 124.0 (CPh-4), 124.0
(CPh-2 and CPh-6), 67.0, 55.8 (CBn), 54.6 (broad, CPip-3 and CPip-5), 45.1 (broad, CPip-2 or CPip-6), 43.8 (N-
CH3), 41.1 (broad, CPip-2 or CPip-6), 35.9 (Cq-t-Bu), 31.9 (t-Bu), 15.6. IR: 2956 (w), 1634 (s), 1425 (m),
1240 (s), 1052 (s), 975 (m) cm-1. HRMS (APCI/ASAP, m/z): 398.2916 (Calcd. C23H36N5O, 398.2920, [M-
Cl]+).
4.11.1. Method D, Ethylene diamine amidation reactions: synthesis of N-(2-aminoethyl)-1-heptyl-
1H-1,2,3-triazole-4-carboxamide (n6a) and 2-(1-heptyl-1H-1,2,3-triazole-4-carboxamido)ethan-1-
aminium chloride (6a). The title compound n6a was prepared using a general procedure described
by Davis et al,24 where 3a (0.15 g, 0.67 mmol) and ethylene diamine (0.67 mL, 10 mmol) was heated
18

to reflux in MeOH (3 mL) for 17 hours. Evaporation of volatiles afforded n6a as a light green solid
(0.170 g, 0.67 mmol, quant.). 1H NMR (400 MHz, CDCl3): δ 8.03 (s, 1H, Htriazole-5), 7.46 (bs, 1H, NH),
4.39 (t, 2H, J = 7.2 Hz, CH2), 3.51 (q, 2H, J = 6.6 Hz, CH2), 2.94 (bs, 2H, CH2), 1.92 (p, 2H, J = 6.9 Hz,
CH2), 1.40 – 1.21 (m, 8H, 4x CH2), 0.88 (t, 3H, J = 6.9 Hz, CH3). The free amine n6a was turned into its
HCl-salt by adding HCl (0.5 mL, 6.1 mmol, 37%, aq) to n6a (74 mg, 0.29 mmol) in MeCN (4 mL). The
evaporated crude salt was then crystallized from EtOH, washed with MeCN (3 x 2 mL) and dried,
affording 6a as an off-white solid (23 mg, 0.08 mmol, 27%, mp 144.0 – 146.2 oC). HPLC (C18, 3:5
H2O/MeOH + 0.1% TFA, 0.75 mL/min, 214 nm): 5.1 min, 99% pure. 1H NMR (600 MHz, d4-MeOD): δ
8.42 (s, 1H, Htriazole-5), 4.48 (t, 2H, J = 6.9 Hz, CH2), 3.70 (t, 2H, J = 5.8 Hz, CH2), 3.19 (t, 2H, J = 5.7 Hz,
CH2), 1.94 (p, 2H, J = 7.4 Hz, CH2), 1.41 – 1.26 (m, 8H, 4x CH2), 0.91 (t, 3H, J = 7.3 Hz, CH3). 13C NMR
(150 MHz, d4-MeOD): δ 164.0 (CC=O), 143.6 (Ctriazole-4), 127.5 (Ctriazole-5), 51.7 (triazole-CH2), 41.2
(CH2), 38.2 (CH2), 32.9 (CH2), 31.4 (CH2), 29.9 (CH2), 27.5 (CH2), 23.7 (CH2), 14.5 (CH3). IR: 3280 (w),
1919 (m), 2855 (w), 1653 (s), 1570 (s), 1522 (m), 1509 (w), 1466 (w), 1256 (m), 1233 (m), 1169 (s),
1047 (s), 848 (s), 716 (m), 688 (s) cm-1. HRMS (APCI/ASAP, m/z): 254.1980 (Calcd. C12H24N5O,
254.1981, [M-Cl]+).
4.11.2. 1-(Adamantan-1-yl)-N-(2-aminoethyl)-1H-1,2,3-triazole-4-carboxamide (n6b) and 2-(1-
(adamantan-1-yl)-1H-1,2,3-triazole-4-carboxamido)ethan-1-aminium chloride (6b). The title
compound n6b was prepared according to Method D with 3b (70 mg, 0.27 mmol) and ethylene
diamine (0.338 g, 5.63 mmol), affording n6b as a white solid (82 mg*, 0.27 mmol, quant.). 1H NMR
(400 MHz, CDCl3): δ 8.13 (s, 1H, Htriazole-5), 7.45 (bs, 1H, NH), 3.51 (q, 2H, J = 6.1 Hz, CH2), 2.93 (t, 2H, J
= 6.1 Hz, CH2), 2.32 – 2.19 (m, 9H, HAda-CH/CH2), 1.86 – 1.73 (m, 6H, HAda-CH2). The free amine 6b was
turned into its HCl-salt by adding HCl (0.25 mL, 3.04 mmol, 37%, aq) to n6a (25 mg, 0.09 mmol) in
MeCN (4 mL). The evaporated crude salt was then washed with MeCN (3 x 2 mL) and DCM (3 x 2 mL)
and dried, affording 6b as a white solid (31 mg**, 0.09 mmol, quant., mp >195 oC decomp.). HPLC
(C18, 3:5 H2O/MeOH + 0.1% TFA, 0.75 mL/min, 214 nm): 5.0 min, 98%. 1H NMR (400 MHz, d4-
MeOD): δ 8.49 (s, 1H, Htriazole-5), 3.70 (t, 2H, J = 5.8 Hz, CH2), 2.93 (t, 2H, J = 5.6 Hz, CH2), 2.35 – 2.25
(m, 9H, HAda-CH/CH2), 1.94 – 1.81 (m, 6H, HAda-CH2). 13C NMR (100 MHz, d4-MeOD): δ 164.3 (CC=O
,
from HMBC), 143.1 (Ctriazole-4, from HMBC), 124.2 (Ctriazole-5), 61.9 (Cq-Ada), 44.0 (CAda), 41.1 (CH2),
38.1 (CH2), 37.0 (CAda), 31.1 (CAda). IR: 3349 (m), 2910 (s), 1556 (s), 1578 (s), 1510 (m), 1259 (w),
1237 (w), 1169 (w), 1035 (w), 1022 (w), 848 (m), 690 (s) cm-1. HRMS (APCI/ASAP, m/z): 290.1980
(Calcd. C15H24N5O, 290.1981, [M-Cl]+).
*) Theoretic 100% yield = 78 mg: 4 mg from unidentified byproducts and residual ethylene diamine.
**) The additional 3 mg were mostly water and solvent residues.
4.11.3. N-(2-Aminoethyl)-1-(4-(tert-butyl)benzyl)-1H-1,2,3-triazole-4-carboxamide (n6c) and 2-(1-
(4-(tert-butyl)benzyl)-1H-1,2,3-triazole-4-carboxamido)ethan-1-aminium chloride (6c). The title
compound was prepared according to Method D with 3c (0.20 g, 0.73 mmol) and ethylene diamine
(0.66 g, 10.98 mmol), for 24 hours at 50 oC. Affording n6c as a white solid (0.220 g, 0.73 mmol,
quant.). 1H NMR (400 MHz, CDCl3): δ 7.94 (s, 1H, Htriazole-5), 7.48 – 7.36 (m, 3H, HPh-2 and HPh-6 + NH),
7.25 – 7.19 (m, 2H, HPh-3 and HPh-5), 5.51 (s, 2H, HBn), 3.49 (q, 2H, J = 6.0 Hz, CH2), 2.92 (t, 2H, J = 5.7
Hz, CH2), 1.31 (s, 9H, t-Bu). The free amine n6c was turned into its HCl-salt by adding HCl (0.75 mL,
9.12 mmol, 37%, aq) to n6c (75 mg, 0.25 mmol) in i-PrOH (5 mL). The evaporated crude salt was then
crystallized in DCM and washed with MeCN (3 x 2 mL) and DCM (3 x 2 mL) and dried, affording 6c as
a white solid (50 mg, 0.15 mmol, 59 %, mp 218.0 – 220.4 oC). HPLC (C18, 3:5 H2O/MeOH + 0.1% TFA,
0.75 mL/min, 214 nm): 6.7 min, 99% pure. 1H NMR (600 MHz, d4-MeOD): δ 8.39 (s, 1H, Htriazole-5),
7.47 – 7.42 (m, 2H, HPh-2 and HPh-6), 7.34 – 7.28 (m, 2H, HPh-3 and HPh-5), 5.63 (s, 2H, HBn), 3.68 (t,
2H, J = 6.0 Hz, CH2), 3.17 (t, 2H, J = 5.7 Hz, CH2), 1.32 (s, 9H, t-Bu). 13C NMR (150 MHz, d4-MeOD): δ
163.7 (CC=O), 153.2 (CPh-4), 143.9 (Ctriazole-4), 133.6 (CPh-1), 129.2 (CPh-3 and CPh-5), 127.5 (Ctriazole-5),
19

127.2 (CPh-2 and CPh-6), 55.0 (CBn), 41.1 (CH2), 38.1 (CH2), 35.6 (Cq-t-Bu), 31.8 (t-Bu). IR: 2955 (w),
2904 (s), 1658 (s), 1571 (s), 1507 (s), 1236 (m), 1033 (m), 844 (m) cm-1. HRMS (APCI/ASAP, m/z):
302.1980 (Calcd. C16H24N5O, 302.1981, [M-Cl]+).
4.11.4. N-(2-Aminoethyl)-1-(3,5-di-tert-butylbenzyl)-1H-1,2,3-triazole-4-carboxamide (n6d) and 2-
(1-(3,5-di-tert-butylbenzyl)-1H-1,2,3-triazole-4-carboxamido)ethan-1-aminium chloride (6d). The
title compound n6d was prepared according Method D with 3d (0.05 g, 0.15 mmol) and ethylene
diamine (0.136 g, 2.28 mmol), for 18 hours at 50 oC. Affording n6d as a white solid (54 mg, 0.15
mmol, quant.). 1H NMR (400 MHz, CDCl3): δ 7.92 (s, 1H, Htriazole-5), 7.50 – 7.41 (m, 2H, HPh-4 + NH),
7.12 (d, 2H, J = 1.6 Hz, HPh-2 and HPh-6), 5.51 (s, 2H, HBn), 3.49 (q, 2H, J = 6.2 Hz, CH2), 2.92 (t, 2H, J =
6.2 Hz, CH2), 1.30 (s, 18H, 2x t-Bu). The free amine n6d was turned into its HCl-salt by adding HCl
(0.25 mL, 3.04 mmol, 37%, aq) to n6d (37 mg, 0.10 mmol) in i-PrOH (3 mL). Evaporation of volatiles
afforded 6d as an off-white solid (38 mg, 0.097 mmol, 93%, mp 228.7 – 231.2 oC). HPLC (C18, 3:5
H2O/MeOH + 0.1% TFA, 0.75 mL/min, 214 nm) 31.5 min, 97% pure. 1H NMR (400 MHz, d6-DMSO): δ
+
8.76 – 8.70 (m, 2H, Htriazole-5 + NH), 8.00 (bs, 3H, NH3 ), 7.37 (t, 1H, J = 1.9 Hz, HPh-4), 7.23 (d, 2H, J =
1.7 Hz, HPh-2 and HPh-6), 5.62 (s, 2H, HBn), 3.51 (q, 2H, J = 5.8 Hz, CH2), 2.96 (q, 2H, J = 5.8 Hz, CH2),
1.26 (s, 18H, 2x t-Bu). 13C NMR (100 MHz, d6-DMSO): δ 160.3 (CC=O), 150.9 (CPh-3 and CPh-5), 142.7
(Ctriazole-4), 134.8 (CPh-1), 126.6 (Ctriazole-5), 122.3 (CPh-2 and CPh-6), 121.9 (CPh-4), 53.7 (CBn), 38.6 (CH2),
36.5 (imp.), 36.3 (CH2), 34.5 (Cq-t-Bu), 31.2 (t-Bu). IR: 2953 (m), 2903 (w), 1666 (s), 1572 (s), 1503
(m), 1362 (m), 1248 (m), 1047 (m), 1031 (m), 878 (w), 837 (m), 713 (s) cm-1. HRMS (APCI/ASAP, m/z):
358.2608 (Calcd. C20H32N5O, 358.2607, [M-Cl]+).
4.12. Amino(4-(1-heptyl-1H-1,2,3-triazole-4-carbonyl)piperazin-1-yl)methaniminium chloride (7a).
The title compound 7a was prepared according to a general and modified28 procedure by
Bernatowicz.27 Where n4a (0.10 g, 0.36 mmol) and 1H-pyrazole carboxamidine hydrochloride (50
mg, 0.34 mmol) were refluxed in MeCN (5 mL) for 3.5 hours. Then, the reaction mixture was cooled
down, and the formed precipitate was filtered off and washed with MeCN and DCM. The crude was
then recrystallized twice in MeCN and dried, affording 7a as white crystals (51 mg, 0.14 mmol, 42%,
mp 170.7 – 173.0 oC). HPLC (C18, 3:5 H2O/MeOH + 0.1% TFA, 0.75 mL/min, 214 nm): 5.4 min, 96%
pure. 1H NMR (400 MHz, d4-MeOD): δ 8.42 (s, 1H, Htriazole-5), 4.46 (t, 2H, J = 6.4 Hz, CH2), 4.35 (bs, 2H,
HPip-2 and HPip-6), 3.87 (bs, 2H, HPip-2 and HPip-6), 3.67 – 3.60 (m, 4H, HPip-3 and HPip-5), 1.92 (p, 2H, J =
7.4 Hz, CH2), 1.42 – 1.23 (m, 8H, 4x CH2), 0.90 (t, 3H, J = 6.8 Hz, CH3). 13C NMR (100 MHz, d4-MeOD):
δ 162.5 (CC=O), 158.6 (CGuan), 144.0 (Ctriazole-4), 129.9 (Ctriazole-5), 51.7 (CH2), 46.9 (CPip-2 or CPip-6), 46.1
(CPip-3 and CPip-5), 43.0 (CPip-2 or CPip-6), 32.9 (CH2), 31.4 (CH2), 29.9 (CH2), 27.5 (CH2), 23.7 (CH2), 14.5
(CH3). IR: 3310 (w), 3122 (w), 1649 (w), 1598 (s), 1247 (m), 1229 (w), 1052 (w), 988 (s), 762 (m) cm-1.
HRMS (APCI/ASAP, m/z): 322.2354 (Calcd. C15H28N7O, 322.2355, [M-Cl]+).
4.13. (4-(1-(Adamantan-1-yl)-1H-1,2,3-triazole-4-carbonyl)piperazin-1-yl)(amino)methaniminium
chloride (7b). The title compound 7b was prepared according to a procedure described by
Bernatowicz et al.27 Where n4b (25 mg, 0.08 mmol) and 1H-pyrazole carboxamidine hydrochloride
(12 mg, 0.08 mmol) were stirred in DMF (2 mL) for 97 hours at room temperature. The crude
product was precipitated from the mixture with Et2O and filtered. The crude precipitate was
crystallized from MeOH and Et2O and washed with DCM (3 x 2 mL) before it was dried, affording 7b
as a white solid (20 mg, 0.05 mmol, 64%, mp >175 oC decomp.). HPLC (C18, 3:5 H2O/MeOH + 0.1%
TFA, 0.75 mL/min, 214 nm): 5.0 min, 95% pure. 1H NMR (400 MHz, d4-MeOD): δ 8.47 (s, 1H, Htriazole
-
5), 4.35 (bs, 2H, HPip-2 and HPip-6), 3.89 (bs, 2H, HPip-2 and HPip-6), 3.67 – 3.60 (m, 4H, HPip-3 and HPip-
5), 2.33 – 2.24 (m, 9H, HAda-CH/CH2), 1.91 – 1.80 (m, 6H, HAda-CH2). 13C NMR (150 MHz, d4-MeOD): δ
162.7 (CC=O), 158.6 (CGuan), 143.5 (Ctriazole-4), 126.7 (Ctriazole-5), 62.0 (Cq-Ada), 46.9 (broad, CPip-2 or CPip-
6), 46.1 (broad, CPip-3 and CPip-5), 44.0 (CAda), 43.0 (broad, CPip-2 or CPip-6), 37.0 (CAda), 31.1 (CAda). IR:
20

3323 (w), 3154 (w), 1658 (m), 1597 (s), 1529 (m), 1441 (m), 1239 (s), 1017 (m), 990 (s) cm-1. HRMS
(APCI/ASAP, m/z): 358.2353 (Calcd. C18H28N7O, 358.2355, [M-Cl]+).
4.14. Amino(4-(1-(3,5-di-tert-butylbenzyl)-1H-1,2,3-triazole-4-carbonyl)piperazin-1-
yl)methaniminium chloride (7d). The title compound 7d was prepared according to the conditions
described for 7a from n4d (0.05 g, 0.13 mmol) and 1H-pyrazole carboxamidine hydrochloride (19
mg, 0.128 mmol), with 22 hours reflux. Filtration of the cooled reaction mixture and washing the
precipitate with MeCN (3 x 3 mL), DCM (3 x 2 mL) and Et2O (3 x 5 mL) followed by drying, afforded
7d as a white solid (31 mg, 0.067 mmol, 52%, mp 230.1 – 232.2 oC). HPLC (C18, 3:5 H2O/MeOH +
0.1% TFA, 0.75 mL/min, 214 nm): 33.1 min, 94% pure. 1H NMR (400 MHz, d4-MeOD): δ 8.39 (s, 1H,
Htriazole-5), 7.45 (s, 1H, HPh-4), 7.25 (s, 2H, HPh-2 and HPh-6), 5.62 (s, 2H, HBn), 4.35 (bs, 2H, HPip-2 and
HPip-6), 3.85 (bs, 2H, HPip-2 and HPip-6), 3.67 – 3.58 (m, 4H, HPip-3 and HPip-5), 1.31 (s, 18H, 2x t-Bu). 13
NMR (100 MHz, d4-MeOD): δ 162.4 (CC=O), 158.6 (CGuan), 153.1 (CPh-3 and CPh-5), 144.4 (Ctriazole-4),
C
135.7 (CPh-1), 129.9 (Ctriazole-5), 124.0 (CPh-4), 123.8 (CPh-2 and CPh-6), 55.8 (CBn), 46.9 (broad, CPip-2 or
CPip-6), 46.1 (broad, CPip-3 and CPip-5), 43.0 (broad, CPip-2 or CPip-6), 35.9 (Cq-t-Bu), 31.9 (t-Bu). IR: 2954
(w), 1668 (w), 1588 (s), 1549 (m), 1433 (m), 1244 (m), 1055 (w), 992 (s) cm-1. HRMS (APCI/ASAP,
m/z): 426.2978 (Calcd. C23H36N7O, 426.2981, [M-Cl]+).
4.15. Amino((2-(1-heptyl-1H-1,2,3-triazole-4-carboxamido)ethyl)amino)methaniminium chloride
(8a). The title compound 8a was prepared according to the conditions described for 7a from n6a
(0.10 g, 0.39 mmol) and 1H-pyrazole carboxamidine hydrochloride (0.052 g, 0.36 mmol), with 4
hours reflux. Filtration upon cooling and careful washing of the precipitate with MeCN (3 x 3 mL)
afforded 8a as a red solid (89 mg, 0.27 mmol, 76%, mp 144.0 – 146.2 oC). HPLC (C18, 3:5 H2O/MeOH
+ 0.1% TFA, 0.75 mL/min, 214 nm): 5.6 min, 96% pure. 1H NMR (400 MHz, d4-MeOD): δ 8.39 (s, 1H,
Htriazole-5), 4.45 (t, 2H, J = 7.1 Hz, CH2), 3.58 (t, 2H, J = 6.0 Hz, CH2), 3.42 (t, 2H, J = 6.2 Hz, CH2), 1.92 (p,
2H, J = 6.9 Hz, CH2), 1.41 – 1.22 (m, 8H, 4x CH2), 0.89 (t, 3H, J = 6.7 Hz, CH3). 13C NMR (100 MHz, d4-
MeOD): δ 163.6 (CC=O), 159.1 (CGuan), 143.7 (Ctriazole-4), 127.4 (Ctriazole-5), 51.7 (triazole-CH2), 42.2 (CH2),
39.4 (CH2), 32.9 (CH2), 31.4 (CH2), 29.9 (CH2), 27.5 (CH2), 23.7 (CH2), 14.5 (CH3). IR: 3350 (w), 3108
(w), 2921 (w), 1651 (s), 1629 (s), 1575 (s), 1504 (w), 1450 (w), 1225 (m), 1048 (m), 774 (m) cm-1.
HRMS (APCI/ASAP, m/z): 296.2197 (Calcd. C13H26N7O, 296.2199, [M-Cl]+).
4.16. ((2-(1-(Adamantan-1-yl)-1H-1,2,3-triazole-4-
carboxamido)ethyl)amino)(amino)methaniminium chloride (8b). The title compound 8b was
prepared according to the procedure described for 8a from n6b (0.047 g, 0.16 mmol) and 1H-
pyrazole carboxamidine hydrochloride (24 mg, 0.16 mmol). Affording 8b as a red solid (30 mg, 0.08
mmol, 50%, mp 180.0 – 186.8 oC). HPLC (C18, 3:5 H2O/MeOH + 0.1% TFA, 0.75 mL/min, 214 nm): 5.1
min, 95% pure. 1H NMR (400 MHz, d6-DMSO): δ 8.68 (s, 1H, Htriazole-5), 8.59 (t, 1H, J = 6.0 Hz, NH),
7.57 (t, 1H, J = 6.0 Hz, NH), 3.45 – 3.37 (m, 2H, CH2), 3.34 – 3.27 (m, 5H, CH2 + H2O from d6-DMSO),
2.21 (bs, 9H, HAda-CH/CH2), 1.75 (bs, 6H, HAda-CH2). 13C NMR (100 MHz, d4-MeOD): δ 163.7 (CC=O),
159.1 (CGuan), 143.0 (Ctriazole-4), 124.1 (Ctriazole-5), 61.9 (Cq-Ada), 44.0 (CAda), 42.2 (CH2), 39.4 (CH2), 37.0
(CAda), 31.1 (CAda). IR: 3358 (w), 3110 (w), 1653 (s), 1628 (s), 1573 (m), 1498 (w), 1049 (w) cm-1. HRMS
(APCI/ASAP, m/z): 332.2198 (Calcd. C16H26N7O, 332.2199, [M-Cl]+).
4.17. Amino((2-(1-(4-(tert-butyl)benzyl)-1H-1,2,3-triazole-4-
carboxamido)ethyl)amino)methaniminium chloride (8c). The title compound 8c was prepared
according to the conditions described for 7a from n6c (0.073 g, 0.24 mmol) and 1H-pyrazole
carboxamidine hydrochloride (34 mg, 0.23 mmol), where filtration of the cooled reaction mixture
and careful washing with MeCN (3 x 2 mL) afforded 8c as a red solid (60 mg, 0.16 mmol, 69%, mp
131.2 – 136.1 oC). HPLC (C18, 3:5 H2O/MeOH + 0.1% TFA, 0.75 mL/min, 214 nm): 7.1 min, 95% pure.
1H NMR (400 MHz, d4-MeOD): δ 8.35 (s, 1H, Htriazole-5), 7.45 – 7.38 (m, 2H, HPh-2 and HPh-6), 7.32 –
21

7.25 (m, 2H, HPh-3 and HPh-5), 5.60 (s, 2H, HBn), 3.56 (t, 2H, J = 5.9 Hz, CH2), 3.41 (t, 2H, J = 5.9 hz,
CH2), 1.30 (s, 9H, t-Bu). 13C NMR (100 MHz, d4-MeOD): δ 163.4 (CC=O), 159.1 (CGuan), 153.2 (CPh-4),
144.0 (Ctriazole-4), 133.5 (CPh-1), 129.2 (CPh-3 and CPh-5), 127.3 (CPh-2 and CPh-6), 127.2 (Ctriazole-5), 55.0
(CBn), 41.2 (CH2), 39.3 (CH2), 35.6 (Cq-t-Bu), 31.8 (t-Bu). IR: 3352 (w), 3112 (w), 2959 (w), 1652 (s),
1631 (s), 1573 (s), 1253 (w), 1230 (w), 1044 (w) cm-1. HRMS (APCI/ASAP, m/z): 344.2199 (Calcd.
C17H26N7O, 344.2199, [M-Cl]+).
4.18. Amino((2-(1-(3,5-di-tert-butylbenzyl)-1H-1,2,3-triazole-4-
carboxamido)ethyl)amino)methaniminium chloride (8d). The title compound 8d was prepared
according to the protocol shown for 8c from n6d (0.103 g, 0.29 mmol) and 1H-pyrazole
carboxamidine hydrochloride (40 mg, 0.27 mmol), where the cooled reaction mixture was
evaporated and crystallized with MeCN and Et2O. The crude was then recrystallized in MeCN
affording 8d as a red solid (47 mg, 0.11 mmol, 39%, mp 205.9 – 209.8 oC). HPLC (C18, 3:5 H2O/MeOH
+ 0.1% TFA, 0.75 mL/min, 214 nm): 33.7 min, 97% pure. 1H NMR (400 MHz, d4-MeOD): δ 8.36 (s, 1H,
HTriazole-5), 7.44 (s, 1H, HPh-4), 7.23 (s, 2H, HPh-2 and HPh-6), 5.62 (s, 2H, HBn), 3.57 (t, 2H, J = 5.6 Hz,
CH2), 3.41 (t, 2H, J = 6.0 hz, CH2), 1.30 (s, 18H, 2x t-Bu). 13C NMR (100 MHz, d4-MeOD): δ 163.4 (CC=O),
159.1 (CGuan), 153.2 (CPh-3 and CPh-5), 143.9 (Ctriazole-4), 135.7 (CPh-1), 127.3 (Ctriazole-5), 123.9 (CPh-4),
123.7 (CPh-2 and CPh-6), 55.8 (CBn), 42.2 (CH2), 39.3 (CH2), 35.9 (Cq-t-Bu), 31.9 (t-Bu). IR: 2960 (w),
1679 (w), 1656 (s), 1641 (s), 1574 (s), 1223 (m), 1061 (w), 848 (m) cm-1. HRMS (APCI/ASAP, m/z):
400.2821 (Calcd. C21H34N7O, 400.2821, [M-Cl]+).
4.19. N-(2-Aminoethyl)-1-(3,5-di-tert-butylphenyl)-1H-1,2,3-triazole-4-carboxamide (n9e) and 2-(1-
(3,5-di-tert-butylphenyl)-1H-1,2,3-triazole-4-carboxamido)ethan-1-aminium chloride (9e). The title
compound n9e was prepared according to Method D from 3e (0.20 g, 0.63 mmol) and ethylene
diamine (0.57 g, 9.51 mmol), for 20 hours at room temperature. Affording n9e as a white solid
(0.196 g, 0.57 mmol, 90%). 1H NMR (400 MHz, CDCl3): δ 8.49 (s, 1H, Htriazole-5), 7.57 – 7.47 (m, 4H, HPh
+ NH), 3.56 (q, 2H, J = 6.2 Hz, CH2), 2.98 (t, 2H, J = 5.5 Hz, CH2), 1.38 (s, 18H, 2x t-Bu). The free amine
n9e was turned into its HCl-salt by adding HCl (0.10 mL, 1.22 mmol, 37%, aq.) to n9e (30 mg, 0.09
mmol) in MeCN (2 mL). Evaporation of volatiles afforded 9e as a white solid (37 mg, 0.09 mmol,
quant., mp 261.1 – 267.5 oC). HPLC (C18, 3:5 H2O/MeOH + 0.1% TFA, 0.75 mL/min, 214 nm): 37.7
min, 98% pure. 1H NMR (400 MHz, d4-MeOD): δ 8.96 (s, 1H, Htriazole-5), 7.68 (bs, 2H, HPh-2 and HPh-6),
7.63 (bs, 1H, HPh-4), 3.73 (t, 2H, J = 5.7 Hz, CH2), 3.21 (t, 2H, J = 5.5 Hz, CH2), 1.39 (s, 18H, 2x t-Bu). 13
C
NMR (100 MHz, d4-MeOD): 163.7 (CC=O), 154.6 (CPh-3 and CPh-5), 144.3 (Ctriazole-4), 137.9 (CPh-1), 126.0
(Ctriazole-5), 124.8 (CPh-4), 116.6 (CPh-2 and CPh-6), 41.2 (CH2), 38.2 (CH2), 36.3 (Cq-t-Bu), 31.8 (t-Bu). IR:
3284 (w), 2957 (w), 1665 (s), 1580 (s), 1490 (s), 1169 (m), 1051 (m), 875 (m), 703 (s) cm-1. HRMS
(APCI/ASAP, m/z): 344.2449 (Calcd. C19H30N5O, 344.2450, [M-Cl]+).
4.20. N-(2-Aminoethyl)-1-(4-(heptyloxy)phenyl)-1H-1,2,3-triazole-4-carboxamide (n9f) and 2-(1-(4-
(heptyloxy)phenyl)-1H-1,2,3-triazole-4-carboxamido)ethan-1-aminium chloride (9f). The title
compound n9f was prepared according to Method D from 3f (0.10 g, 0.32 mmol) and ethylene
diamine (0.28 g, 4.73 mmol), with 20 hours at reflux. Re-evaporation of the crude from DCM
afforded n9f as a white solid (0.100 g, 0.29 mmol, 92%). 1H NMR (400 MHz, d6-DMSO): δ 9.15 (s, 1H,
Htriazole-5), 8.53 (t, 1H, J = 6.1 Hz, NH), 7.88 – 7.82 (m, 2H, HPh-3 and HPh-5), 7.17 – 7.10 (m, 2H, HPh-2
and HPh-6), 4.05 (t, 2H, J = 6.5 Hz, O-CH2), 3.29 (q, 2H, J = 6.5 Hz, CH2), 2.70 (t, 2H, J = 6.5 Hz, CH2),
1.79 – 1.70 (m, 2H, CH2), 1.48 – 1.24 (m, 8H, 4x CH2), 0.91 – 0.85 (m, 3H, CH3). The free amine n9f
was turned into its HCl-salt by adding HCl (0.15 mL, 1.83 mmol, 37% aq) to n9f (30 mg, 0.09 mmol) in
MeCN (3 mL). Evaporation of volatiles, washing with MeCN (3 x 2 mL) and drying afforded 9f as an
off white solid (27 mg, 0.07 mmol, 81%, mp 240.0 – 246.0 oC). HPLC (C18, 1:3 H2O/MeOH + 0.1% TFA,
22

0.75 mL/min, 214 nm): 7.3 min, 95% pure. 1H NMR (400 MHz, d4-MeOD): δ 8.83 (s, 1H, Htriazole-5),
7.76 (d, 2H, J = 7.9 Hz, HPh-3 and HPh-5), 7.12 (d, 2H, J = 8.6 Hz, HPh-2 and HPh-6), 4.06 (t, 2H, J = 6.5 Hz,
O-CH2), 3.71 (t, 2H, J = 5.6 Hz, CH2), 3.20 (t, 2H, J = 6.1 Hz, CH2), 1.82 (p, 2H, J = 7.0 Hz, CH2), 1.56 –
1.28 (m, 8H, 4x CH2), 0.95 – 0.88 (m, 3H, CH3). 13C NMR (150 MHz, d4-MeOD): δ 163.7 (CC=O), 161.6
(CPh-4), 144.2 (Ctriazole-4), 131.3 (CPh-1), 125.7 (Ctriazole-5), 123.6 (CPh-3 and CPh-5), 116.7 (CPh-2 and CPh-
6), 69.7 (O-CH2), 41.2 (CH2), 38.2 (CH2), 33.1 (CH2), 30.5 (CH2), 30.3 (CH2), 27.3 (CH2), 23.8 (CH2), 14.6
(CH3). IR: 2914 (w), 1659 (m), 1599 (m), 1578 (m), 1515 (s), 1250 (s), 1215 (m), 1180 (w), 1166 (m),
1054 (m), 1037 (m), 987 (w), 831 (s) cm-1. HRMS (APCI/ASAP, m/z): 346.2242 (Calcd. C18H28N5O2,
346.2243, [M-Cl]+).
4.21. N-(2-Aminoethyl)-1-(4-((3,5-di-tert-butylbenzyl)oxy)phenyl)-1H-1,2,3-triazole-4-carboxamide
(n9g) and 2-(1-(4-((3,5-di-tert-butylbenzyl)oxy)phenyl)-1H-1,2,3-triazole-4-carboxamido)ethan-1-
aminium chloride (9g). The title compound n9g was prepared according to Method D from 3g (0.10
g, 0.24 mmol) and ethylene diamine (0.21 g, 3.56 mmol), with 28 hours at reflux. Affording n9g as a
white solid (0.103 g, 0.23 mmol, 96%). 1H NMR (400 MHz, CDCl3): δ 8.41 (s, 1H, Htriazole-5), 7.68 – 7.61
(m, 2H, Hphenox-3 and -5), 7.52 (t, 1H, J = 6.0 Hz, NH), 7.43 (t, 1H, J = 1.9 Hz, HPh-4), 7.29 (d, 2H, J = 1.6
Hz, HPh-2 and HPh-6), 7.17 – 7.11 (m, 2H, Hphenox-2 and -6), 5.09 (s, 2H, HBn), 3.55 (q, 2H, J = 6.0 Hz,
CH2), 2.97 (t, 2H, 6.0 Hz, CH2), 1.35 (s, 18H, 2x t-Bu). The free amine n9g was turned into its HCl-salt
by adding HCl (0.10 mL, 1.22 mmol, 37% aq) to a filtered solution of n9g (20 mg, 0.044 mmol) in THF
(3 mL). Evaporation of volatiles, washing with THF (3 x 2 mL) and drying afforded 9g as a white solid
(18 mg, 0.037 mmol, 83%, mp 250.0 – 254.5 oC). HPLC (C18, 1:3 H2O/MeOH + 0.1% TFA, 0.75
mL/min, 214 nm): 23.3 min, 97% pure. 1H NMR (400 MHz, d4-MeOD): δ 8.84 (s, 1H, Htriazole-5), 7.78
(d, 2H, J = 8.4 Hz, Hphenox-3 and -5), 7.43 (s, 1H, HPh-4), 7.31 (s, 2H, HPh-2 and HPh-6), 7.22 (d, 2H, J = 8.4
Hz, Hphenox-2 and -6), 5.16 (s, 2H, HBn), 3.71 (t, 2H, J = 5.7 Hz, CH2), 3.20 (t, 2H, J = 5.4 Hz, CH2), 1.33 (s,
18H, 2x t-Bu). 13C NMR (150 MHz, d4-MeOD): δ 163.7 (CC=O), 161.3 (Cphenoxyl-4), 152.4 (CPh-3 and CPh-
5), 144.3 (Ctriazole-4), 137.4 (CPh-1), 131.5 (Cphenox-1), 125.7 (Ctriazole-5), 123.6 (Cphenox-3 and -5), 123.2
(CPh-4), 123.19 (CPh-2 and CPh-6), 117.2 (Cphenox-2 and -6), 72.3 (CBn), 41.2 (CH2), 38.2 (CH2), 35.9 (Cq-t-
Bu), 32.0 (t-Bu). IR: 2957 (w), 1662 (m), 1602 (m), 1581 (m), 1517 (s), 1248 (m), 1167 (m), 1054 (m),
873 (w), 823 (m) cm-1. HRMS (APCI/ASAP, m/z): 450.2869 (Calcd. C26H36N5O2, 450.2869, [M-Cl]+). Ph =
3,5-di-tert-butylbenzyl.
4.22. Amino((2-(1-(3,5-di-tert-butylphenyl)-1H-1,2,3-triazole-4-
carboxamido)ethyl)amino)methaniminium chloride (10e). The title compound 10e was prepared
according to the conditions shown for 7a from n9e (0.05 g, 0.15 mmol) and 1H-pyrazole
carboxamidine hydrochloride (21 mg, 0.15 mmol), with 21 hours reflux. The cooled reaction mixture
was evaporated, dissolved in MeOH (1-2 mL), filtered and crystallized with Et2O. Washing of the
formed precipitate with MeCN (2 x 2 mL) and Et2O (3 x 10 mL) followed by drying, afforded 10e as a
pink solid (32 mg, 0.076 mmol, 52%, mp 272 - 276 oC). HPLC (C18, 3:5 H2O/MeOH + 0.1% TFA, 0.75
1
mL/min, 214 nm): 42.3 min, 95% pure. H NMR (600 MHz, d4-MeOD): δ 8.95 (s, 1H, Htriazole-5), 7.67
(d, 2H, J = 1.7 Hz, HPh-2 and HPh-6), 7.63 (t, 1H, J = 1.7 Hz, HPh-4), 3.62 (t, 2H, J = 6.2 Hz, NH-CH2), 3.46
(t, 2H, J = 6.2 Hz, guanidine-CH2), 1.40 (s, 18H, 2x t-Bu). 13C NMR (150 MHz, d4-MeOD): δ 163.3
(CC=O), 159.1 (CGuan), 154.6 (CPh-3 and CPh-5), 144.4 (Ctriazole-4), 137.9 (CPh-1), 125.9 (Ctriazole-5), 124.8
(CPh-4), 116.6 (CPh-2 and CPh-6), 42.2 (guanidine-CH2), 39.5 (NH-CH2), 36.3 (Cq-t-Bu), 31.8 (t-Bu). IR:
3287 (w), 3139 (w), 2954 (w), 1657 (m), 1620 (s), 1578 (s), 876 (w), 851 (w), 705 (m) cm-1. HRMS
(APCI/ASAP, m/z): 386.2668 (Calcd. C20H32N7O, 386.2668, [M-Cl]+).
4.23. Amino((2-(1-(4-(heptyloxy)phenyl)-1H-1,2,3-triazole-4-
carboxamido)ethyl)amino)methaniminium chloride (10f). The title compound 10f was prepared
according to a modified28 general procedure described by Bernatowicz et al.27 Where n9f (40 mg,
23

0.12 mmol) and 1H-pyrazole carboxamidine hydrochloride (26 mg, 0.07 mmol) was refluxed for 20
hours, added triethylamine (50 mg, 0.49 mmol) refluxed for 20 hours, added 1H-pyrazole
carboxamidine hydrochloride (10 mg, 0.17 mmol) and triethylamine (20 mg, 0.20 mmol) followed by
a third round of reflux for 20 hours. The cooled reaction mixture was evaporated, and the crude was
washed with MeCN (3 x 2 mL) and H2O (3 x 1 mL) before it was dried. The dried crude was dissolved
in MeOH and filtered, evaporation afforded 10f as a red solid (24 mg, 0.057 mmol, 49%, mp 196.0 –
200.4 oC). HPLC (C18, 3:5 H2O/MeOH + 0.1% TFA, 0.75 mL/min, 214 nm): 33.7 min, 95% pure. 1H
NMR (400 MHz, d6-DMSO): δ 9.21 (s, 1H, Htriazole-5), 8.74 (t, 1H, J = 5.5 Hz, NH), 7.89 – 7.83 (m, 2H,
HPh-3 and HPh-5), 7.53 (t, 1H, J = 5.5 Hz, NH), 7.17 – 7.10 (m, 2H, HPh-2 and HPh-6), 4.05 (t, 2H, J = 7.4
Hz, O-CH2), 3.45 (q, 2H, J = 5.8 Hz, CH2), 3.37 – 3.29 (m, CH2 + H2O from DMSO), 1.75 (p, 2H, J = 8.2
Hz, CH2), 1.48 – 1.22 (m, 8H, 4x CH2), 0.92 – 0.85 (m, 3H, CH3). 13C NMR (150 MHz, d6-DMSO): δ
159.9 (CC=O), 159.1 (CPh-4), 157.0 (CGuan), 143.3 (Ctriazole-4), 129.5 (CPh-1), 124.6 (Ctriazole-5), 122.1 (CPh-3
and CPh-5), 115.4 (CPh-2 and CPh-6), 68.0 (O-CH2), 40.3 (CH2), 37.8 (CH2), 31.2 (CH2), 28.6 (CH2), 28.4
(CH2), 25.4 (CH2), 22.0 (CH2), 13.9 (CH3). IR: 3346 (w), 3098 (w), 2923 (w), 1658 (s), 1630 (s), 1572 (s),
1518 (s), 1500 (s), 1242 (s), 1036 (m), 826 (s) cm-1. HRMS (APCI/ASAP, m/z): 388.2458 (Calcd.
C19H30N7O2, 388.2461, [M-Cl]+).
4.24. Amino((2-(1-(4-((3,5-di-tert-butylbenzyl)oxy)phenyl)-1H-1,2,3-triazole-4-
carboxamido)ethyl)amino) methaniminium chloride (10g). The title compound 10g was prepared
according to the procedure shown for 10f from n9g (44 mg, 0.098 mmol) and 1H-pyrazole
carboxamidine hydrochloride (16 mg + 5 mg + 5 mg, total: 26 mg, 0.177 mmol), with DMF at room
temperature for the third round of stirring. Affording 10g as a red solid (12 mg, 0.023 mmol, 23%,
mp 259.6 – 261.3 oC). HPLC (C18, 3:7 H2O/MeOH + 0.1% TFA, 0.75 mL/min, 214 nm): 40.2 min, 96%
pure. 1H NMR (600 MHz, d4-MeOD): δ 8.82 (s, 1H, Htriazole-5), 7.80 – 7.75 (m, 2H, Hphenox-3 and -5),
7.43 (t, 1H, J = 1.7 Hz, HPh-4), 7.31 (d, 2H, J = 1.5 Hz, HPh-2 and HPh-6), 7.23 – 7.19 (m, 2H, Hphenox-2 and
-6), 5.15 (s, 2H, HBn), 3.61 (t, 2H, J = 6.5 Hz, CH2), 3.45 (t, 2H, J = 6.1 Hz, CH2), 1.33 (s, 18H, 2x t-Bu).
13C NMR (150 MHz, d4-MeOD): δ 163.3 (CC=O), 161.3 (CGuan), 159.1 (Cphenox-4), 152.4 (CPh-3 and CPh-5),
144.3 (Ctriazole-4), 137.4 (CPh-1), 131.5 (Cphenox-1), 125.6 (Ctriazole-5), 123.6 (Cphenox-3 and -5), 123.3 (CPh-
4), 123.2 (CPh-2 and CPh-6), 117.2 (Cphenox-2 and -6), 72.3 (CBn), 42.3 (CH2), 39.4 (CH2), 35.9 (Cq-t-Bu),
32.0 (t-Bu). IR: 2952 (w), 1660 (s), 1574 (s), 1506 (s), 1245 (s), 829 (s) cm-1. HRMS (APCI/ASAP, m/z):
492.3090 (Calcd. C27H38N7O2, 492.3087, [M-Cl]+). Ph = 3,5-di-tert-butylbenzyl.
4.25. Amino(2-(1-(1-(3,5-di-tert-butylbenzyl)-1H-1,2,3-triazol-4-
yl)ethylidene)hydrazinyl)methaniminium chloride (11d). The title compound 11d was prepared
according to a general procedure described by Hu-Ri et al.33 Where 13d (40 mg, 0.13 mmol),
aminoguanidine hydrochloride (20 mg, 0.18 mmol) and HCl (75 mg, 0.76 mmol, 37% aq) in EtOH (2
mL, abs.) was heated to 90 oC in a sealed tube for 22 hours. The cooled reaction mixture was then
evaporated, washed with H2O (2 x 2 mL) and dried, affording 11d as a white solid isomer mixture (30
mg, 0.07 mmol, 58%, 4:6 isomer ratio from 1H NMR, mp 253.6 – 259.0 oC). HPLC (C18, 3:7
H2O/MeOH + 0.1% TFA, 0.75 mL/min, 214 nm): 11.2 min (major isomer), 13.0 min (minor isomer),
97% pure (both). IR: 3314 (bm), 2953 (m), 1674 (m), 1599 (s), 1362 (w), 1224 (w), 1047 (w) cm-1.
1
HRMS (APCI/ASAP, m/z): 370.2713 (Calcd. C20H32N7, 370.2713, [M-Cl]+). H NMR (600 MHz, d4-
MeOD, major isomer): δ 8.47 (s, 1H, Htriazole-5), 7.46 – 7.43 (m, 1H, HPh-4), 7.22 (d, 2H, J = 1.7 Hz, HPh-
2 and HPh-6), 5.62 (s, 2H, HBn), 2.38 (s, 3H, imine-CH3), 1.30 (s, 18H, 2x t-Bu). 13C NMR (150 MHz, d4-
MeOD, major isomer): δ 157.9 (CGuan), 153.1 (CPh-3 and CPh-5), 147.6 (Ctriazole-4), 147.0 (Cimine), 135.9
(CPh-1), 127.5 (Ctriazole-5), 123.9 (CPh-4), 123.5 (CPh-2 and CPh-6), 55.9 (HBn), 35.9 (Cq-t-Bu), 31.9 (t-Bu),
14.3 (imine-CH3). 1H NMR (600 MHz, d4-MeOD, minor isomer): δ 8.53 (s, 1H, Htriazole-5), 7.46 – 7.43
(m, 1H, HPh-4), 7.26 (d, 2H, J = 1.7 Hz, HPh-2 and HPh-6), 5.68 (s, 2H, HBn), 2.35 (s, 3H, imine-CH3), 1.30
(s, 18H, 2x t-Bu). 13C NMR (150 MHz, d4-MeOD, minor isomer): δ 157.5 (CGuan), 153.1 (CPh-3 and CPh-
24

5), 144.8 (Ctriazole-4), 141.0 (Cimine), 135.6 (CPh-1), 125.4 (Ctriazole-5), 124.0 (CPh-4), 123.7 (CPh-2 and CPh-
6), 55.9 (HBn), 35.9 (Cq-t-Bu), 31.9 (t-Bu), 21.6 (imine-CH3).
4.26. Amino(2-(1-(1-(3,5-di-tert-butylphenyl)-1H-1,2,3-triazol-4-
yl)ethylidene)hydrazinyl)methaniminium trifluoroacetate (11e). The title compound 11e was
prepared according to a procedure described by Mohammad et al.30 Where 13e (35 mg, 0.116
mmol), aminoguanidine hydrochloride (16 mg, 0.14 mmol) and LiCl (2 mg, 0.05 mmol) in EtOH (2 mL,
abs.) were heated to 90 oC in a sealed tube for 48 hours. After which, the reaction mixture was
evaporated and purified with preparative C18-HPLC (80:20 MeOH/H2O + 0.1% TFA, 20 mL min, Rt:
2.8 – 4.0 min), affording 11e as an off-white solid isomer mixture (25 mg, 0.053 mmol, 46%, 3:7
isomer ratio from 1H NMR, mp 106.0 – 110.1 oC). HPLC (C18, 3:5 H2O/MeOH + 0.1% TFA, 0.75
mL/min, 214 nm): 59.2 min (both isomers), 98% pure (both). IR: 2961 (w), 1681 (m), 1606 (m), 1592
(m), 1200 (m), 1182 (m), 1135 (s), 1045 (w), 800 (w), 703 (w) cm-1. HRMS (APCI/ASAP, m/z):
356.2560 (Calcd. C19H30N7, 356.2560, [M-TFA]+). 1H NMR (600 MHz, d4-MeOD, major isomer): δ 9.07
(s, 1H, Htriazole-5), 7.74 (s, 1H, HPh-4), 7.70 (s, 2H, HPh-2 and HPh-6), 2.47 (s, 3H, imine-CH3), 1.41 (s, 18H,
2x t-Bu). 13C NMR (150 MHz, d4-MeOD, major isomer): δ 162.5 (bs, TFA), 158.0 (CGuan), 154.6 (CPh-3
and CPh-5), 148.0 (Ctriazole-4), 147.0 (Cimine), 138.0 (CPh-1), 123.6 (Ctriazole-5), 116.8 (CPh-4), 116.4 (CPh-2
and CPh-6), 36.3 (Cq-t-Bu), 31.8 (t-Bu), 30.9 (TFA), 14.0 (imine-CH3). 1H NMR (600 MHz, d4-MeOD,
minor isomer): δ 9.11 (s, 1H, Htriazole-5), 7.65 (s, 1H, HPh-4), 7.63 (s, 2H, HPh-2 and HPh-6), 2.47 (s, 3H,
imine-CH3), 1.41 (s, 18H, 2x t-Bu). 13C NMR (150 MHz, d4-MeOD, minor isomer): δ 162.5 (bs, TFA),
157.6 (CGuan), 154.7 (CPh-3 and CPh-5), 145.1 (Ctriazole-4), 141.1 (Cimine), 137.7 (CPh-1), 126.2 (Ctriazole-5),
125.1 (CPh-4), 124.7 (CPh-2 and CPh-6), 36.32 (Cq-t-Bu), 31.8 (t-Bu), 30.9 (TFA), 21.7 (imine-CH3).
4.27. Amino(2-(1-(1-(4-(heptyloxy)phenyl)-1H-1,2,3-triazol-4-
yl)ethylidene)hydrazinyl)methaniminium trifluoroacetate (11f). The title compound 11f was
prepared according to the procedure shown for 11e from 13f (0.05 g, 0.166 mmol) and
aminoguanidine hydrochloride (22 mg, 0.20 mmol), affording 11f as an off-white solid isomer
mixture (24 mg, 0.051 mmol, 31%, 1:9 isomer ratio from 1H NMR, mp 139.7 – 144.3 oC). HPLC (C18,
3:5 H2O/MeOH + 0.1% TFA, 0.75 mL/min, 214 nm): 68.1 min (major), 65.4 min (minor), 95% pure
(both). IR: 2927 (w), 1672 (m), 1606 (s), 1517 (m), 1262 (w), 1198 (m), 1169 (m), 1131 (s), 825 (m),
797 (m), 720 (w) cm-1. HRMS (APCI/ASAP, m/z): 358.2354 (Calcd. C18H28N7O, 358.2355, [M-TFA]+). 1H
NMR (600 MHz, d4-MeOD, major isomer): δ 8.93 (s, 1H, Htriazole-5), 7.78 – 7.72 (m, 2H, HPh-3 and HPh-
5), 7.16 – 7.07 (m, 2H, HPh-2 and HPh-6), 4.05 (t, 2H, J = 6.4 Hz, O-CH2), 1.81 (p, 2H, J = 7.9 Hz, CH2),
1.50 (p, 2H, J = 7.6 Hz, CH2), 1.44 – 1.31 (m, 6H, 3 x CH2), 0.95 – 0.89 (m, 3H, CH3). 13C NMR (150 MHz,
d4-MeOD, major isomer): δ 163.0 (bs, TFA), 161.5 (CGuan), 157.9 (CPh-4), 147.9 (Ctriazole-4), 146.9
(Cimine), 131.4 (CPh-1), 123.4 (CPh-3 and CPh-5), 123.3 (Ctriazole-5), 116.65 (CPh-2 and CPh-6), 69.7 (O-CH2),
33.1 (CH2), 30.9 (TFA), 30.5 (CH2), 30.3 (CH2), 27.3 (CH2), 23.8 (CH2), 14.6 (CH3), 14.1 (imine-CH3). 1H
NMR (600 MHz, d4-MeOD, minor isomer): δ 8.95 (s, 1H, Htriazole-5), 7.82 – 7.78 (m, 2H, HPh-3 and HPh-
5), 7.14 – 7.10 (m, 2H, HPh-2 and HPh-6), 4.05 (t, 2H, J = 6.4 Hz, O-CH2), 1.81 (p, 2H, J = 7.9 Hz, CH2),
1.50 (p, 2H, J = 7.6 Hz, CH2), 1.44 – 1.31 (m, 6H, 3x CH2), 0.95 – 0.89 (m, 3H, CH3).
4.28. Amino(2-(1-(1-(4-((3,5-di-tert-butylbenzyl)oxy)phenyl)-1H-1,2,3-triazol-4-
yl)ethylidene)hydrazinyl)methaniminium chloride (11g). The title compound 11g was prepared
according to the procedure for 11e from 13g (0.04 g, 0.099 mmol) and aminoguanidine
hydrochloride (13 mg, 0.12 mmol), for 25 hours at reflux. The crude product was crystallized from
the partially evaporated reaction mixture with H2O. The crude was in turn crystallized from THF and
EtOAc, affording 11g as a white solid (7 mg, 0.014 mmol, 14%, 1:9 isomer ratio from 1H NMR, mp
230 - 232 oC). HPLC (C18, 1:4 H2O/MeOH + 0.1% TFA, 0.75 mL/min, 214 nm): 11.2 min (major), 10.5
25

(minor) min, 98% pure (both). IR: 2958 (w), 1672 (m), 1622 (m), 1595 (s), 1542 (s), 1253 (s), 1035
(m), 830 (m) cm-1. HRMS (APCI/ASAP, m/z): 462.2978 (Calcd. C26H36N7O, 462.2981, [M-Cl+). 1H NMR
(600 MHz, d6-DMSO, major isomer): δ 10.94 (s, 1H, NH), 9.29 (s, 1H, Htriazole-5), 7.83 – 7.79 (m, 2H,
Hphenox-3 and -5), 7.39 (t, 1H, J = 1.8 Hz, HPh-4), 7.33 – 7.26 (m, 4H, Hphenox-2 and -6 + HPh-2 and HPh-6),
5.17 (s, 2H, HBn), 2.42 (s, 3H, CH3), 1.31 (s, 18H, 2x t-Bu). 13C NMR (150 MHz, d6-DMSO, major
isomer): δ 158.9 (Cphenox-4), 150.5 (CPh-3 and CPh-5), 146.8 (Ctriazole-4, from HMBC), 146.0 (Cimine, from
HMBC) 135.6 (CPh-1), 129.8 (Cphenox-1), 122.2 (CPh-2 and CPh-6), 121.8 (Cphenox-3 and -5), 121.6 (CPh-4 +
Ctriazole-5), 115.9 (Cphenox-2 and -6), 70.5 (CBn), 34.5 (Cq-t-Bu), 31.3 (t-Bu), 13.6 (imine-CH3). 1H NMR
(400 MHz, d6-DMSO, minor isomer): δ 9.38 (s, 1H, Htriazole-5), 7.94 – 7.89 (m, 2H, Hphenox-3 and -5),
7.33 – 7.26 (m, 4H, Hphenox-2 and -6 + HPh-2 and HPh-6), 5.18 (s, 2H, HBn), 2.41 (s, 3H, CH3), 1.31 (s, 18H,
2x t-Bu). Ph = 3,5-di-tert-butylbenzyl.
4.29. Inhibition of bacterial growth
Growth medium with MilliQ H2O was used as a negative control, while sterile MilliQ H2O and
bacteria suspension was used as a positive control. Bacteria were transferred from a blood plate to
growth medium (MH-bullion, VL787693 717, Merck) for E. coli, P. aeruginosa and S. aureus and BHI-
bullion (CM1135, OXOID) for E. faecalis and S. agalactiae gr. B and incubated at 37°C overnight. The
following day part of the bacteria suspension was transferred to fresh medium and cultivated in a
shaker incubator at 37°C for 1.5 h (E. coli, E. faecalis and Streptococcus gr. B) or 2.5 h (S. aureus and
P. aeruginosa). The bacteria suspension was then diluted 1:100 in medium and added to all wells on
a 96-well microtiter plate (Nunc 167008), followed by sample aliquotes (and Gentamicin as a
reference antibiotic) in duplicates. The plates were incubated at 37ºC overnight before growth was
controlled visually and photometrically at 600 nm.
4.30. Inhibition of biofilm formation
S. epidermidis was used to assess the effect of the test compounds on biofilm formation. Growth
media: tryptic soy broth (TS; Merck, Darmstadt, Germany). An overnight culture of S. epidermidis
grown in TS was diluted with fresh TS containing 1 % glucose (1:100). Aliquots of 50 μL were
transferred to a 96-well microtiter plate, and 50 μL of test compounds, dissolved in water at ranging
concentrations, was added. After overnight incubation at 37 °C, the bacterial suspension was
carefully discarded and the wells washed with water. The plate was dried and the biofilm fixed by
incubation for 1 h at 55 °C before the surface attached cells were stained with 100 μL of 0.1 % crystal
violet for 5 min. The crystal violet solution was removed and the plate once more washed with water
and dried at 55 °C for 1 h. After adding 70 μL of 70 % ethanol, the plate was incubated at room
temperature for 10 min. Biofilm formation was observed by visual inspection of the plates. The MIC
was defined as the lowest concentration where no biofilm formation was visible. A S. epidermidis
suspension, diluted with 50 μL of water, was used as a positive control, and 50 μL Staphylococcus
haemolyticus suspension with 50 μL of water was employed as a negative control. A mixture of 50 μL
water and 50 μL TS was used as assay control.
4.31. Cytotoxicity to HepG2-cells
26

Cytotoxicity of the test compounds was evaluated after 24 h exposure in human hepatocellular liver
carcinoma (HepG2, ATCC HB-8065™) cells. HepG2 were grown overnight (20,000 cells/well), and
then incubated with test compound (range of concentrations) diluted in MEM Earle's supplemented
with gentamycin (10 μg/mL), non-essential amino acids (1%), sodium pyruvate (1 mM), L-alanyl-L-
glutamine (2 mM), but without FBS (total volume was 100 μl) for 24 hours. Ten μL of CellTiter 96®
AQueous One Solution Reagent (Promega, Madison, WI, USA) was added and plates were then
further incubated for 1 h. Absorbance was measured at 485 nm in a DTX 880 Multimode Detector.
Results were calculated as % survival compared to negative (assay media) and positive (Triton X-100;
Sigma-Aldrich) controls.
Acknowledgements
The authors acknowledge the NV-faculty at NTNU and the NRC (grant no. 224790/O30) for financial
support. The authors also thank engineer Julie Asmussen and Dr. Susana Villa Gonzalez at NTNU for
MS analyses, and engineers Marte Albrigtsen and Kirsti Helland at Marbio (UiT) for performing the
various bioassays.
Supporting information
Supporting information containing full characterization (1H- and 13C-NMR spectra) of novel
compounds is electronically available through the publisher’s website.
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Graphical abstract
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