Synthesis and antimicrobial activity of 4-substituted 1,2,3-triazole-coumarin derivatives

Article abstract of DOI:10.3390/molecules23010199

A new series of coumarin-1,2,3-triazole conjugates with varied alkyl, phenyl and heterocycle moieties at C-4 of the triazole nucleus were synthesized using a copper(I)-catalysed Huisgen 1,3-dipolar cycloaddition reaction of corresponding O-propargylated coumarin (3) or N-propargylated coumarin (6) with alkyl or aryl azides. Based on their minimal inhibitory concentrations (MICs) against selected microorganisms, six out of twenty-six compounds showed significant antibacterial activity towards Enterococcus faecalis (MIC = 12.5–50 μg/mL). Moreover, the synthesized triazoles show relatively low toxicity against human erythrocytes.

Full text of DOI:10.3390/molecules23010199

molecules
Article
Synthesis and Antimicrobial Activity of 4-Substituted
1,2,3-Triazole-Coumarin Derivatives
Priscila López-Rojas 1, Monika Janeczko 2, Konrad Kubin´ski 2 ID , Ángel Amesty 1,*,
Maciej Masłyk 2,* and Ana Estévez-Braun 1,
*
ID
1
Instituto Universitario de Bio-Orgánica Antonio González (CIBICAN), Departamento de Química Orgánica,
Universidad de La Laguna, Avda. Astrofísico Fco. Sánchez 2, 38206 La Laguna, Tenerife, Spain;
priscila.quim@gmail.com
Department of Molecular Biology, The John Paul II Catholic University of Lublin, ul. Konstantynów 1i,
20-708 Lublin, Poland; mjanec@kul.pl (M.J.); kubin@kul.pl (K.K.)
2
*
Correspondence: aarnesty@ull.es (Á.A.); maciekm@kul.pl (M.M.); aestebra@ull.es (A.E.-B.)
Received: 18 December 2017; Accepted: 15 January 2018; Published: 18 January 2018
Abstract: A new series of coumarin-1,2,3-triazole conjugates with varied alkyl, phenyl and heterocycle
moieties at C-4 of the triazole nucleus were synthesized using a copper(I)-catalysed Huisgen
1,3-dipolar cycloaddition reaction of corresponding O-propargylated coumarin (
coumarin ( ) with alkyl or aryl azides. Based on their minimal inhibitory concentrations (MICs)
against selected microorganisms, six out of twenty-six compounds showed significant antibacterial
activity towards Enterococcus faecalis (MIC = 12.5–50 g/mL). Moreover, the synthesized triazoles
show relatively low toxicity against human erythrocytes.
3) or N-propargylated
6
µ
Keywords: triazoles; coumarin; antibacterial activity; Enterococcus faecalis; biofilm
1. Introduction
Antimicrobial resistance has been listed by the World Health Organization (WHO) as one of
the biggest threats to global health today [1]. The antibiotic resistance crisis has been attributed
to the overuse and misuse of these medications, as well as a lack of new drug development
by the pharmaceutical industry due to reduced economic incentives and challenging regulatory
requirements [
bacterial pathogens have acquired clever mechanisms to negate the effectiveness of numerous
therapeutic agents [ ]. Staphylococcus aureus is one bacterial pathogen that has emerged as a significant
concern to healthcare professionals worldwide. In this sense, isolated strains of S. aureus have
exhibited resistance to several classes of antibacterial drugs, including -lactam antibiotics [ ],
macrolides [ ], fluoroquinolones [10 12], glycopeptides [13] and oxazolidinones [14]. Enterococci
2–6]. Over the past decade, it has become apparent that several highly resistant
7
β
8
9
–
were previously considered commensal organisms of little clinical importance but have emerged
as serious nosocomial pathogens responsible for e.g. endocarditis and infections of the urinary
tract, bloodstream, meninges, wounds and the biliary tract [15]. Recent surveillance data indicate
that Enterococcus is the third most commonly isolated nosocomial pathogen (12% of all hospital
infections), only behind coagulase-negative Staphylococcus and Staphylococcus aureus [16]. The clinical
importance of the genus Enterococcus is directly related to its antibiotic resistance, which contributes
to the risk of colonization and infection. Enterococci are intrinsically resistant to many commonly
used antimicrobial agents (penicillins, ampicillins, cephalosporins, clindamycin) and exhibit native
resistance to clinically achievable concentrations of aminoglycosides. Although E. faecalis is naturally
resistant to quinupristin-dalfopristin, this combination is highly active against E. faecium strains that
lack specific resistance determinants. Enterococci are tolerant to the (normally) bactericidal activity
of cell-wall active agents, such as β-lactam antibiotics and vancomycin. Tolerance implies that the
Molecules 2018, 23, 199; doi:10.3390/molecules23010199
www.mdpi.com/journal/molecules

Molecules 2018, 23, 199
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bacteria can be inhibited by clinically achievable concentrations of the antibiotic but will only be killed
by concentrations far in excess of the inhibitory concentration [17]. The emergence of multi-resistant
E. faecalis strains, complicating the treatment, means that it is important to search for and identify new
treatment strategies.
All the information mentioned above highlights the urgent need to develop novel antibacterial
agents devoid of cross-resistance to marketed antibiotics.
The use of privileged structures in drug discovery has proven to be an effective strategy allowing
the generation of innovative hits⁄leads and successful optimization processes [18,19]. Coumarins are
considered to be privileged structures due to their broad range of biological properties including
anticoagulant [20], anti-neurodegenerative [21], antioxidant [22], anticancer [23] and antimicrobial
activities [24
of the 2H-chromen-2-one core; its aromatic ring can establish a series of hydrophobic,
cation– interactions and the two oxygen atoms in the lactone ring can hydrogen-bond to a series of
–28]. These interesting properties of coumarins can be ascribed to the chemical attributes
π–
π
, CH– and
π
π
amino acid residues in different classes of enzymes and receptors. Additionally, the double bond in the
lactone helps to make the planar system, allows charge delocalization between the carbonyl group of
the lactone and the aromatic ring and confers the characteristic fluorescence of this class of compounds.
On the other hand, 1,2,3-triazoles are nitrogen heterocycles capable of forming hydrogen bonds,
which improves their solubility and ability to interact with biomolecular targets [29]. The 1,2,3-triazoles
are highly stable to metabolic degradation, compared to other compounds containing three adjacent
nitrogen (N) atoms [29]. The triazoles have been used for broad therapeutic applications due to
their diverse biological activities [30], i.e. antimicrobial [31–33], antiviral [34], anti-inflammatory [35],
analgesic [35], anticancer [36–38], antifungal [39] and anticonvulsant [40] activities.
Taking into consideration the antimicrobial activity shown by some coumarins and 1,2,3-triazols
mentioned above and as a continuation of our project on searching for new antibacterial molecules [41–45],
we envisaged that the linkage of coumarin and 1,2,3-triazole pharmacophores through –OCH2– or
–NCH2– linkers would generate novel hybrid molecules with promising antibacterial activities.
Therefore, we herein report the synthesis and antibacterial activity of coumarin-1,2,3-triazole
conjugates with varied alkyl, phenyl and heterocycle moieties at C-4 of the triazole nucleus in order to
evaluate their contribution to the antimicrobial activity.
2. Results and Discussion
2.1. Chemistry
The required acetylenic dipolarophiles
the treatment of 4-hydroxy-coumarin ( ) with 3-bromoprop-1-yne (
in anhydrous acetone yielded the O-propargylated coumarin ( ) in 55% yield. The N-propargylated
coumarin ( ) was obtained in 63% yield from 4-bromo-coumarin ( ) through nucleophilic substitution
with prop-2-yn-1-amine (5) in dimethylformamide (DMF).
3
and
6
were obtained as shown in Scheme 1. Thus,
1
2
) employing potassium carbonate
3
6
4
Scheme 1. Formation of O-propargylated coumarin (3) and N-propargylated coumarin (6).

Molecules 2018, 23, 199
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The 4-substituted 1,2,3-triazole-coumarin derivatives were synthesized using a copper(I)-catalysed
Huisgen 1,3-dipolar cycloaddition reaction [46] of the corresponding O-propargylated coumarin (
N-propargylated coumarin (6) with alkyl or aryl azides (Scheme 2).
3) or
Scheme 2. Formation of 4-substituted 1,2,3-triazole-coumarin derivatives 8 and 9.
Azides 7a–7j were prepared from the corresponding boronic acids and sodium azide, in the
presence of CuSO4 in ethanol (EtOH), at room temperature (Table 1 and Scheme 3) [47,48].
Scheme 3. Preparation of azides 7a–7j.
Table 1. Synthesized azides 7a–7j.
Entry
Azide
Ar
Yield a
1
2
3
4
5
6
7
8
9
7a
7b
7c
7d
7e
7f
7g
7h
7i
Ph
58
61
97
96
96
41
31
82
35
2-OMe–Ph
3-OMe–Ph
4-OMe–Ph
3-F, 4-OMe–Ph
4-F–Ph
3-CF3–Ph
3-NO2–Ph
5-1H-indol
3-furyl
b
10
7j
-
a
Isolated yield; b It was not isolated but was reacted in situ.
Azides 7k
–7m were obtained from the corresponding alkyl bromide or aryl bromide and sodium
azide in DMF (Table 2 and Scheme 4) [49,50].
Table 2. Synthesized azides 7k–7m.
Entry
Azide
R
Yield a
1
2
3
7k
7l
7m
(CH2)10CH3
(CH2)13CH3
CH2Ph
86
73
82
a
Isolated yield; b MW irradiation at 120 ◦C for 35 min was used.
Scheme 4. Preparation of azides 7k–7m.
Following the reaction shown in Scheme 2 and using azides 7a
9a–9m were obtained as illustrated in Figure 1.
–7m, compounds 8a–8m and

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Figure 1. Structures of 4-substituted 1,2,3-triazole-coumarin derivatives (8a–8n) and (9a–9n).
As can be seen, two isosteric series of coumarin derivatives were obtained (X=O, X=NH).
Each series presents different substituents at the triazole moiety in order to evaluate their influence on
the antimicrobial activity. Thus, coumarin derivatives with an aromatic ring having electron-donating
groups or electron-withdrawing groups were prepared (8a
with alkyl moieties (8k 9k 8l 9l) and coumarin-indole hybrids (8i
Moderate yields were obtained with aromatic azides while the use of the more stable aliphatic azides
7k 7l and 7m) led to high yields, in agreement with the more favourable HOMO of the dipole in the
–
8h
,
9a
–9h). Coumarin-triazole derivatives
,
,
,
,
9i) were synthesized as well.
(
,
1,3-dipolar cycloaddition. The structures of all adducts were determined by spectroscopic studies.
All of them showed the characteristic proton of the triazol ring in the 1H-NMR spectral region between

Molecules 2018, 23, 199
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δ
7.63 and 9.31. The hydrogen of the coumarin nucleus was detected as a singlet at
δ
5.18–6.22 and the
methylene hydrogens in the oxygenated series appeared as a singlet at
δ
5.35–5.59 and as a doublet at
δ 4.66–4.46 (J = 5.6 Hz) in the nitrogenated series.
The best yields were obtained from the N-propargylated coumarin (
6
) and from the aliphatic
azides (7k, 7l and 7m).
2.2. Biology
Since some coumarins and 1,2,3-triazoles have shown potential as antibacterial drugs [41–45,51],
these combined pharmacophores could offer some advantages e.g. in overcoming drug resistance as
well as improving their biological potency.
The in vitro antimicrobial activity of the novel coumarin-1,2,3-triazole conjugates was tested
against the yeast Candida albicans, Gram-positive bacteria Staphylococcus aureus and Enterococcus
faecalis and Gram-negative bacteria Escherichia coli, Klebsiella pneumonia and Pseudomonas aeruginosa.
The minimum inhibitory concentrations (MICs) were determined and given in Table 3. As can be
seen, most of the coumarin-triazole hybrids did not exhibit considerable activity against the tested
microorganisms. The best results were obtained with conjugates 8a
promising activity against Enterococcus faecalis at MICs ranging from 12.5 to 50.0
8b having a 2-OMe–Ph group attached at the triazol nucleus and an –OCH2– linker was the best of
,
8b
,
8f
,
9h and 9k, which displayed
µg/mL. Compound
the series, while the corresponding isoster 9b (–NHCH2–) turned out to be 64-fold less active than 8b
The position of the OMe group in the phenyl ring also plays an important role in the activity, since
compounds 8c (3-OMe–Ph) and 8d (4-OMe–Ph) showed an 8- and 16-fold lower antibacterial activity,
respectively, than 8b. In the nitrogenated series, compounds 9h (3-NO2–Ph) and 9k having an undecyl
chain showed the best activities.
.
Table 3. Antimicrobial activity (MIC µg/mL) of the synthesized compounds.
Candida
albicans
Staphylococcus
aureus
Enterococcus
faecalis
Escherichia
coli
Klebsiella
pneumoniae
Pseudomonas
aeruginosa
Compound
8a
8b
8c
8d
8e
1600
800
1600
-
400
1600
800
800
1600
1600
-
400
200
800
-
400
1600
1600
400
1600
800
-
50
12.5
100
200
100
50
100
400
200
800
400
400
400
800
400
100
100
1600
-
1600
1600
1600
1600
200
-
1600
1600
1600
1600
800
800
800
1600
1600
1600
1600
800
-
1600
800
800
800
400
400
800
1600
1600
1600
1600
1600
800
1600
800
800
1600
-
8f
1600
800
8g
8h
8i
8j
8k
8l
9a
9b
9c
9d
9e
9f
9g
9h
9i
9j
9k
9l
1600
1600
1600
1600
1600
1600
1600
1600
800
-
-
1600
-
1600
1600
800
400
200
-
200
100
1600
800
200
-
1600
800
800
800
-
200
200
200
200
-
1600
1600
-
1600
1600
n.d.
8
800
1600
-
-
50
800
800
800
1600
-
1600
5
800
1600
-
100
800
50
800
5
1600
1600
-
1600
1.2
-
1600
5
n.d.
CAM
KET
5
n.d.
n.d.
n.d.
n.d.
no activity; n.d.—not determined, CAM—chloramphenicol; KET—ketoconazole.
In other studies, menthyl 1,4-disubstituted 1,2,3-triazole derivatives of hydroxybenzaldehydes,
phenols and bile acids showed a strong inhibitory effect against E. faecium with the minimum
inhibitory concentration (MIC) values in the range of 1–3 µM [52]. Kant and co-workers reported

Molecules 2018, 23, 199
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that 1,2,3-triazole linked chalcone and flavone hybrids showed activity against Gram-positive
bacteria (Staphylococcus aureus, Enterococcus faecalis) and Gram-negative bacteria (Escherichia coli,
Pseudomonas aeruginosa, Shigella boydii, Klebsiella pneumoniae) with MIC values in the range of
6.25–100 µg/mL [53]. In turn, 1,2,4-triazolo[3,4a]phthalazine derivatives showed inhibitory activity
against Staphylococcus aureus (MIC 16–128 µg/mL) [54].
In order to verify if the newly synthesized triazoles could be considered as potential antimicrobial
therapeutics, the most active compounds, namely 8a
, 8b, 8f, 9h and 9k were examined in terms of
their haemolytic activity against human erythrocytes. The results are shown in Figure 2.
Figure 2. Haemolytic activity of compounds 8a
,
8b
,
8f,
9h and 9k
.
Tetracycline (TET) and
chloramphenicol (CAM) in MIC concentrations were used as a control.
Compounds 8b, 8f and 9h exhibit minimal toxicity towards human blood cells (1.6–3.1% of lysed
cells) in MIC. Although compound 8b appears to be the most active antimicrobial agent, simultaneously
it moderately affects the erythrocytes (6.9% of lysed cells) in MIC. The presence of an undecyl chain in
the triazole ring (9k) results in a drastic increase in the haemolytic activity (94% of lysed cells) in MIC.
3. Materials and Methods
3.1. Compounds Synthesis
3.1.1. General Experimental Procedures
IR spectra were obtained using a Fourier Transform Infrared spectrometer. NMR spectra were
1
recorded in CDCl3 or DMSO at 500 or 600 MHz for H NMR and 125 or 150 MHz for 13C-NMR.
1
Chemical shifts are given in (
δ
) parts per million and coupling constants (J) in hertz (Hz). H- and
13C-spectra were referenced using the solvent signal as an internal standard. Melting points were taken
on a capillary melting point apparatus and are uncorrected. Microwave reactions were conducted
in sealed glass vessels (capacity 5 mL) using a CEM Discover microwave reactor. HREIMS were
recorded using a high-resolution magnetic trisector (EBE) mass analyser. The analytical thin-layer
chromatography plates used were Polygram-Sil G/UV254. Preparative thin-layer chromatography
was carried out with Analtech (Newark, NJ, USA) silica gel GF plates (20
using appropriate mixtures of ethyl acetate and hexanes. All solvents and reagents were purified by
standard techniques reported in [55] or used as supplied from commercial sources. All compounds
×
20 cm, 1000 Microns)

Molecules 2018, 23, 199
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were named using the ACD40 Name-Pro program, which is based on IUPAC rules. Azides 7a–7m
were synthesized according to procedures previously described in the literature [47–50,56].
4-(Prop-2-yn-1-yloxy)-2H-chromen-2-one (3). 259 µL (2.4 mmol) of propargyl bromide were slowly added
to a mixture of 330.9 mg (2.0 mmol) of 4-hydroxycoumarin and 552.8 mg (4.0 mmol) of K2CO3 in 15 mL
of acetone. The reaction mixture was refluxed for 8 h until disappearance of the starting coumarin.
Then, the solvent was eliminated under reduced pressure, 30 mL of H2O were added and the mixture
was extracted with AcOEt (3
and brine (20 mL) and dried over anhydrous MgSO4. After filtration and elimination of the solvent,
the crude extract was purified by silica gel column chromatography using DCM as an eluent and
×
30 mL). The organic phases were collected, washed with H2O (20 mL)
224.7mg (55%) of compound
3 were obtained as an amorphous white solid. Compound 3 showed
identical spectroscopic data to those described in the literature [57].
4-(Prop-2-yn-1-ylamino)-2H-chromen-2-one ( ). 47 L (0.72 mmol) of prop-2-yn-1-amine were slowly
6
µ
added to 200 mg (0.89 mmol) of 4-bromocoumarin in 2 mL of dimethylformamide (DMF) under argon
atmosphere. The reaction mixture was stirred at room temperature for 18 h. Then water was added
and the N-propargylated coumarin precipitated. After filtration, 111.8 mg (63%) of compound (
obtained as an amorphous white solid. m.p. 223–224 ◦C; 1H-NMR (600 MHz, (CDCl3)
7.57 (1H, t,
J = 8.1 Hz), 7.46 (1H, d, J = 8.1 Hz), 7.37 (1H, d, J = 8.1 Hz), 7.30 (1H, t, J = 8.1 Hz), 5.46 (1H, s), 5.28
(1H, bs), 4.11 (2H, dd, J = 5.5, 2.5 Hz), 2.41 (1H, t, J = 2.5 Hz); 13C-NMR (150 MHz, (CDCl3)
162.5
6) was
δ
δ
(C=O), 153.6 (C), 151.8 (C), 132.0 (CH), 123.6 (CH), 119.9 (CH), 118.1 (CH), 113.9 (C), 85.9 (CH), 77.6
(C), 73.6 (CH2), 32.9(CH) ppm; EIMS m/z 199 ([M+], 71); 198 (61); 197 (13); 171 (100); 170 (53); 144 (13);
143 (22); 142 (27); 119 (12); 118 (16); 115 (15); 90 (11); 77 (18); 63 (14); 51 (14); HREIMS 199.0638 (calcd.
for C12H9NO2 [M+] 199.0633); FT-IR (ATR)
1552, 1484, 1445, 1389, 1354, 1328, 1271, 1192, 1147, 1045, 983, 937, 865, 811 cm−1
νmax 3325, 3259, 3093, 3074, 2934, 2122, 1807, 1668, 1612,
.
3.1.2. General Procedures for the Preparation of 4-Substituted 1,2,3-Triazole-Coumarin Derivatives
Method A. Corresponding boronic acid (0.24 mmol) and 78.5 mg (1.2 mmol) of sodium azide in 1.5 mL
of H2O were added to a vigorously stirred mixture of 3.4 mg (0.0241 mmol) of Cu2O in 0.06 mL of
20% of NH3 and 0.12 mL of H2O. The reaction mixture was stirred for 16 h at room temperature under
an oxygen atmosphere. Then, 0.14 mmol of propargylated coumarin (3 or 6), 8.11 mg (0.041 mmol)
of sodium ascorbate, 1.5 mL of H2O and 3 mL of acetone were added. The reaction was left at room
temperature for 48h. Then, the reaction mixture was extracted with EtOAc. The aqueous phase was
acidified with 5% HCl until pH = 2 and extracted with EtOAc (3
collected, dried over anhydrous MgSO4 and after elimination of the solvent, the corresponding residue
was purified by silica gel CC or TLC-preparative with DCM or 5% DCM/MeOH.
×
15 mL). The organic phases were
Method B. To a solution of 0.28 mmol of the corresponding azide in 3 mL of DCM, 0.14 mmol of
propargylated coumarin (
CuSO4 5H2O and 3 mL of H2O, were added. The reaction mixture was stirred for 48 h at room
temperature. The reaction mixture was extracted with EtOAc (3 15 mL). The organic phases were
3 or 6), 3.6 mg (0.02 mmol) of sodium ascorbate, 1.2 mg (0.004 mmol) of
·
×
collected, dried over anhydrous MgSO4 and after elimination of the solvent, the corresponding residue
was purified by silica gel CC or TLC-preparative with DCM or 5% DCM/MeOH.
4-((1-Phenyl-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one (8a). Following the experimental procedure
described in method A, from 31.2 mg (0.24 mmol) of phenyl boronic acid and 28.0 mg (0.14 mmol) of
O-propargylated coumarin (
solid [58]. m.p. 193–194 ◦C; 1H-NMR (500 MHz, (CD3)2SO)
(1H, dd, J = 7.9, 1.5 Hz), 7.65–7.63 (3H, m), 7.52 (1H, t, J = 7.4 Hz), 7.41 (1H, dd, J = 8.3, 0.7 Hz), 7.34
(1H, t, J = 7.6 Hz), 6.21 (1H, s), 5.53 (2H, s) ppm; 13C-NMR (125 MHz, (CD3)2SO)
164.4 (C=O), 161.6
3
), 10.9 mg (24%) of compound 8a were obtained as an amorphous orange
δ
9.09 (1H,s), 7.95 (2H, d, J = 7.5 Hz), 7.83
δ
(C), 152.8 (C), 142.3 (C), 136.5 (C), 132.9 (CH), 129.9 (2CH), 128.9 (CH), 124.2 (CH), 123.4 (CH), 123.1
(CH), 120.3 (2CH), 116.5 (CH), 115.1 (C), 91.5 (CH), 62.8 (CH2) ppm; EIMS m/z 319 ([M+], 26); 131 (11);

Molecules 2018, 23, 199
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130 (100); 103 (11); 77 (47); 51 (12); HREIMS319.0967 (calcd. for C18H13N3O3 [M+] 319.0957); FT-IR
(ATR) 3386, 3146, 3097, 1716, 1621, 1563, 1494, 1460, 1397, 1367, 1337, 1243, 1208, 1186, 1136, 1100,
ν
max
1029, 925, 835 cm−1
.
4-((1-(2-Methoxyphenyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one (8b). Following the experimental
procedure described in method B, from 42.3 mg (0.28 mmol) of 1-azido-2-methoxybenzene and 28 mg
(0.14 mmol) of O-propargylated coumarin (
3
), 14.2 mg (27%) of compound 8b were obtained as an
8.33 (1H, s), 7.84 (2H, dd,
amorphous white solid. m.p. 200–201 ◦C; 1H-NMR (500 MHz, CDCl3)
δ
J = 7.9, 1.3 Hz), 7.56 (1H, t, J = 8.5 Hz), 7.47 (1H, t, J = 8.5 Hz), 7.32 (1H, dd, J = 8.3, 0.6 Hz), 7.25 (1H,
td, J = 7.8, 1.1 Hz), 7.16–7.11 (2H, m), 5.93 (1H, s), 5.43 (2H, s), 3.93 (3H, s) ppm; 13C-NMR (125 MHz,
CDCl3)
δ 165.2 (C=O), 162.8 (C), 153.4 (C), 151.1 (C), 140.8 (C), 132.6 (CH), 130.6 (CH), 126.0 (C), 125.9
(CH), 125.5 (CH), 124.0 (CH), 123.4 (CH), 121.4 (CH), 116.8 (CH), 115.6 (C), 112.4 (CH), 91.2 (CH), 62.8
(CH2), 56.2 (CH3) ppm; EIMS m/z 349 ([M+], 37); 161 (12); 160 (100); 145 (28); 120 (14); 92 (11); 77 (16);
HREIMS 349.1050 (calcd. for C19H15N3O4 [M+] 349.1063); FT-IR (ATR)
ν
3400, 3129, 3093, 3009,
max
2942, 2841, 2287, 1707, 1617, 1563, 1507, 1493, 1477, 1460, 1410, 1368, 1233, 1186, 1136, 1103, 1052, 1018,
970, 930, 883, 868, 811 cm−1
.
4-((1-(3-Methoxyphenyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one (8c). Following the experimental
procedure described in method B, from 42.3 mg (0.28 mmol) of 1-azido-2-methoxybenzene and 28 mg
(0.14 mmol) of O-propargylated coumarin (3), 26.8 mg (51%) of compound 8c were obtained as an
◦
1
amorphous white solid. m.p. 188–189 C; H-NMR (500 MHz, CDCl3)
δ 8.16 (1H, s), 7.82 (1H, d,
J = 7.6 Hz), 7.56 (1H, t, J = 7.4 Hz), 7.45 (1H, t, J = 8.1 Hz), 7.37 (1H, s), 7.33 (1H, d, J = 8.1 Hz), 7.30–7.21
(2H, m), 7.00 (1H, d, J = 7.8 Hz), 5.91 (1H, s), 5.43 (2H, s), 3.90 (3H, s) ppm;13C-NMR (125 MHz, CDCl3)
δ
165.1 (C=O), 162.7 (C), 160.8 (C), 153.5 (C), 142.2 (C), 137.9 (C), 132.7 (CH), 130.8 (CH), 124.1 (CH),
123.3 (CH), 121.9 (CH), 116.9 (CH), 115.6 (C), 115.2 (CH), 112.7 (CH), 106.7 (CH), 91.4 (CH), 62.7 (CH2),
55.8 (CH3) ppm; EIMS m/z 349 ([M+], 49); 160 (100); 145 (18); 130 (16); 117 (12); 107 (14); 92 (18); 77
(21); HREIMS 349.1078 (calcd. for C19H15N3O4 [M+] 349.1063); FT-IR (ATR)
ν
3401, 3148, 3093,
max
2933, 2838, 1719, 1620, 1610, 1565, 1493, 1456, 1400, 1368, 1337, 1237, 1189, 1158, 1103, 1044, 1004, 931,
833 cm−1
.
4-((1-(4-Methoxyphenyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one (8d). Following the experimental
procedure described in method B, from 42.3 mg (0.28 mmol) of 1-azido-4-methoxybenzene and 28.0 mg
(0.14 mmol) of O-propargylated coumarin (3), 27.7 mg (53%) of compound 8d were obtained as an
◦
1
amorphous white solid. m.p. 195–196 C; H-NMR (500 MHz, CDCl3)
J = 8.0 Hz), 7.66 (2H, d, J = 8.9 Hz), 7.56 (1H, t, J = 7.8 Hz), 7.33 (1H, d, J = 8.3 Hz), 7.4 (1H, t, J = 7.9 Hz),
7.05 (2H, d, J = 8.9 Hz), 5.91 (1H, s), 5.42 (2H, s), 3.89 (3H, s) ppm; 13C-NMR (125 MHz, CDCl3)
165.1
δ 8.07 (1H, s), 7.83 (1H, d,
δ
(C=O), 162.7 (C), 160.3 (C), 153.5 (C), 142.1 (C), 132.7 (CH), 130.3 (C), 124.1 (CH), 123.3 (CH), 122.5
(2CH), 121.9 (CH), 116.9 (CH), 115.6 (C), 115.0 (2CH), 91.4 (CH), 62.8 (CH2), 55.8 (CH3) ppm; EIMS m/z
349 ([M+], 29); 161 (12); 160 (100); 145 (13); 92 (12); 77 (13); HREIMS 349.1061 (calcd. for C19H15N3O4
[M+] 349.1063); FT-IR (ATR)
ν
3140, 3095, 3006, 2942, 2840, 2381, 2055, 1719, 1619, 1564, 1519, 1457,
max
1400, 1369, 1257, 1240, 1185, 1137, 1102, 1030, 927, 824 cm−1
.
4-((1-(3-Fluoro-4-methoxyphenyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one (8e). Following the
experimental procedure described in method B, from 47.4 mg (0.28 mmol) of 1-azido-3-fluoro-
4-methoxybenzene and 28 mg (0.14 mmol) of O-propargylated coumarin (
3
), 25.3 mg (46%) of
compound 8e were obtained as an amorphous white solid. m.p. 190–191 C; 1H-NMR (600 MHz,
CDCl3) 8.07 (1H, s), 7.82 (1H, dd, J = 8.0, 1.0 Hz), 7.60–7.51 (2H, m), 7.48 (1H, d, J = 8.8 Hz), 7.33
(1H, d, J = 8.3 Hz), 7.24 (1H, t, J = 7.9 Hz), 7.11 (1H, t, J = 8.7 Hz), 5.91 (1H, s), 5.42 (2H, s), 3.97 (3H, s)
ppm; 13C-NMR (150 MHz, CDCl3) 165.1 (C=O), 162.7 (C), 153.4 (C), 152.3 (J1C–F = 249.1 Hz), 148.6
(C, J2C–F = 28.7 Hz), 142.4 (C), 132.8 (CH), 124.1 (CH), 123.3 (CH), 121.8 (CH), 117.0 (CH), 116.8 (CH,
J3 = 3.6 Hz), 115.6 (C), 114.0 (CH, J3 = 1.9 Hz), 110.0 (CH, J2
= 22.6 Hz), 91.5 (CH), 62.7 (CH2),
◦
δ
δ
C–F
C–F
C–F
56.7 (CH3) ppm; EIMS m/z 367 ([M+], 27); 179 (12); 178 (100); 163 (21); HREIMS367.0898 (calcd. for

Molecules 2018, 23, 199
9 of 18
C19H14N3O4F [M+] 367.0968); FT-IR (ATR)
ν
3488, 3143, 3089, 2976, 2944, 2844, 2361, 1715, 1620,
max
1565, 1527, 1493, 1451, 1401, 1368, 1328, 1287, 1243, 1233, 1186, 1159, 1133, 1104, 1017, 952, 933, 880, 833,
808 cm−1
.
4-((1-(4-Fluoro-phenyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one (8f). Following the experimental
procedure described in method A, from 34.8 mg (0.24 mmol) of 4-fluorophenyl boronic acid and 28 mg
(0.14 mmol) of O-propargylated coumarin (
3
), 22.1 mg (43%) of compound 8f were obtained as an
amorphous white solid. m.p. 233–235 C; 1H-NMR (500 MHz, (CD3)2SO)
9.07 (1H, s), 8.02–7.98 (2H,
m), 7.83 (1H, dd, J = 7.9, 1.5 Hz), 7.67 (1H, ddd, J = 8.7, 7.4, 1.6 Hz), 7.49 (2H, t, J = 8.8 Hz), 7.42 (1H, dd,
◦
δ
J = 8.3, 0.6 Hz), 7.35 (1H, t, J = 7.6 Hz), 6.20 (1H, s), 5.52 (2H, s) ppm; 13C-NMR (125 MHz, (CD3)2SO)
δ
164.3 (C=O), 161.5 (C), 161.4 (C, J1C–F = 245.7 Hz), 152.7 (C), 142.3 (C), 133.0 (C, J4C–F = 3.4 Hz), 132.9
(CH), 124.2 (CH), 123.6 (CH), 123.0 (CH), 122.7 (2 CH, J3C–F = 8.6 Hz), 116.8 (2 CH, J2C–F = 24.1 Hz),
116.5 (CH), 115.1 (C), 91.5 (CH), 62.8 (CH2); EIMS m/z 337 ([M+], 23); 149 (11); 148 (100); 95 (25);
HREIMS 337.0859 (calcd. for C18H12N3O3F [M+] 337.0863). FT-IR (ATR)
ν
max 3148, 3098, 2384, 2294,
−1
2050, 1718, 1624, 1567, 1516, 1496, 1465, 1454, 1401, 1370, 1233, 1184, 1105, 1051, 1022, 951, 931, 833 cm
.
4-((1-(3-Trifluoromethyl-phenyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one (8g). Following the
experimental procedure described in method B, from 53.2 mg (0.28 mmol) of 1-azido-3-
trifluoromethylbenzene and 28 mg (0.14 mmol) of O-propargylated coumarin (
of compound 8g were obtained as an amorphous white solid. m.p. 192–193 ◦C; 1H-NMR (500 MHz,
(CD3)2SO) 9.27 (1H, s), 8.34 (1H, s), 8.32 (1H, d, J = 8.2 Hz), 7.93–7.86 (2H, m), 7.85 (1H, dd, J = 8.0,
1.5 Hz), 7.68 (1H, ddd, J = 8.8, 7.4, 1.6 Hz), 7.43 (1H, d, J = 7.7 Hz), 7.37 (1H, t, J = 7.6 Hz), 6.22 (1H,
s), 5.56 (2H, s) ppm; 13C-NMR (125 MHz, (CD3)2SO)
164.3 (C=O), 165.1 (C), 152.7 (C), 142.5 (C),
136.9 (C), 132.8 (CH), 131.3 (CH), 130.5 (C, J2 = 29.6 Hz), 125.4 (CH, J3
= 4.1 Hz), 124.2 (CH),
3), 24.0 mg (41%)
δ
δ
C–F
C–F
124.1 (CH), 123.5 (C, J1C–F = 276.4 Hz), 123.7 (CH), 123.0 (CH), 116.9 (CH, J3C–F = 4.5 Hz), 116.4 (CH),
115.0 (C), 91.5 (CH), 62.7 (CH2);EIMS m/z 387 ([M+], 12); 386 (42); 358 (23); 357 (45); 329 (10); 199 (12);
198 (100); 159 (26); 145 (50); HREIMS 387.0846 (calcd. for C19H12N3O3F3 [M+] 387.0831); FT-IR (ATR)
ν
max 3531, 3409, 1685, 1618, 1559, 1069, 972 cm–1.
4-((1-(3-Nitro-phenyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one (8h). Following the experimental
procedure described in method B, from 46.6 mg (0.28 mmol) of 1-azido-3-nitrobenzene and 28 mg
(0.14 mmol) of O-propargylated coumarin (
3
), 23.1 mg (43%) of compound 8g were obtained as an
amorphous white solid. m.p. 215–217 ◦C; 1H-NMR (600 MHz, (CD3)2SO)
δ
9.31 (1H, s), 8.78 (1H, s),
8.45 (1H, d, J = 7.8 Hz), 8.36 (1H, d, J = 7.9 Hz), 7.93 (1H, t, J = 8.1 Hz), 7.85 (1H, d, J = 7.8 Hz), 7.67 (1H,
t, J = 7.6 Hz), 7.42 (1H, d, J = 8.3 Hz), 7.36 (1H, t, J = 7.5 Hz), 6.21 (1H, s), 5.56 (2H, s) ppm; 13C-NMR
(150 MHz, (CD3)2SO)
δ 164.4 (C=O), 161.6 (C), 152.8 (C), 148.6 (C), 142.8 (C), 137.1 (C), 133.0 (CH),
131.7 (CH), 126.4 (CH), 124.3 (CH), 123.9 (CH), 123.4 (CH), 123.1 (CH), 116.5 (CH), 115.2 (CH), 115.1
(C), 91.6 (CH), 62.8 (CH2) ppm; EIMS m/z 364 ([M+], 59); 176 (11); 175 (100); 162 (23); 145 (14); 129
(92); 128 (37); 121 (14); 120 (39); 92 (18); 77 (11); 76 (37); HREIMS 364.0820 (calcd. for C18H12N4O5 [M+]
364.0808); FT-IR (ATR)
ν
3431, 3146, 3091, 2925, 1713, 1617, 1565, 1531, 1493, 1464, 1406, 1352, 1245,
max
1185, 1138, 1104, 1045, 1009, 980, 952, 929, 830 cm–1.
4-((1-(1H-Indole-5-yl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one (8i). Following the experimental
procedure described in method B, from 44.9 mg (0.28 mmol) of 5-azido-1H-indol and 28 mg (0.14 mmol)
of O-propargylated coumarin (
3
), 18.1 mg (33%) of compound 8i were obtained as an amorphous
11.46 (1H, s), 8.99 (1H, s), 8.05 (1H,
white solid. m.p. 191–193 ◦C; 1H-NMR (500 MHz, (CD3)2SO)
δ
s), 7.84 (1H, d, J = 7.1 Hz), 7.67 (1H, t, J = 7.2 Hz), 7.63–7.57 (2H, m), 7.53–7.51 (1H, m), 7.42 (1H, d,
J = 8.3 Hz), 7.35 (1H, t, J = 7.5 Hz), 6.59 (1H, s), 6.22 (1H, s), 5.52 (2H, s); 13C NMR (125 MHz, (CD3)2SO)
164.4 (C=O), 161.6 (C), 152.8 (C), 141.8 (C), 135.6 (C), 132.9 (CH), 129.3 (C), 127.8 (CH), 127.6 (C), 124.3
(CH), 123.7 (CH), 123.1 (CH), 116.5 (CH), 115.1 (C), 114.4 (CH), 112.4 (CH), 112.3 (CH), 102.0 (CH), 91.4
(CH), 62.9 (CH2) ppm; EIMS m/z 358 ([M+], 28); 169 (100); 168 (19); 162 (30); 121 (13); 120 (39); 116 (28);

Molecules 2018, 23, 199
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92 (14); HREIMS 358.1068 (calcd. for C20H14N4O3 [M+] 358.1066); FT-IR (ATR)
1685, 1616, 1563, 1494, 1403, 1350, 1327, 1228, 1097, 1048, 1025, 993 cm–1.
ν
max 3399, 3299, 1699,
4-((1-(Furan-3-yl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one (8j). Following the experimental
procedure described in method A, from 34.8 mg (0.24mmol) of 3-furylboronic acid and 28 mg
(0.14 mmol) of O-propargylated coumarin (
3
), 8.1 mg (18%) of compound 8j were obtained as an
8.88 (1H,s), 8.48 (1H, s),
amorphous white solid. m.p. 189–190 ◦C; 1H-NMR (500 MHz, (CD3)2SO)
δ
7.91 (1H, t, J = 1.9 Hz), 7.80 (1H, dd, J = 7.9, 1.5 Hz), 7.67 (1H, t, J = 7.6Hz), 7.42 (1H, dd, J = 8.3, 0.7 Hz),
7.35 (1H, t, J = 7.8 Hz), 7.16 (1H, dd, J = 2.0, 0.9 Hz), 6.19 (1H, s), 5.51 (2H, s) ppm; 13C-NMR (125 MHz,
(CD3)2SO) δ 164.4 (C=O), 161.6 (C), 152.8 (C), 144.9 (CH), 141.9 (C), 134.3 (CH), 132.9 (CH), 125.9 (C),
124.3 (CH), 124.2 (CH), 123.0 (CH), 116.5 (CH), 115.1 (C), 105.3 (CH), 91.5 (CH), 62.7 (CH2) ppm; EIMS
m/z 309 ([M+], 59); 279 (25); 198 (21); 159 (23); 120 (100); 94 (18); 65 (13); HREIMS 309.0662 (calcd. for
C16H11N3O4 [M+] 309.0671). FT-IR (ATR)
ν
3514, 3399, 3084, 2924, 2387, 2094, 2064, 1992, 1696,
max
1619, 1606, 1563, 1492, 1450, 1410, 1365, 1270, 1248, 1193, 1105, 1053, 1023, 942, 911, 871, 839 cm−1
.
4-((1-Undecyl-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one (8k). Following the experimental
procedure described in method B, from 56.1 mg (0.28mmol) of 1-azido-undecane and 28 mg (0.14 mmol)
of O-propargylated coumarin (
3
), 51.4 mg (86%) of compound 8k were obtained as an amorphous
◦
white solid. m.p. 144–145 C; 1H-NMR (500 MHz, CDCl3)
δ
7.78 (1H, dd, J = 13.2, 6.5 Hz), 7.75 (1H, s),
7.54 (1H, t, J = 7.5 Hz), 7.30 (1H, d, J = 7.9 Hz), 7.24 (1H, t, J = 7.6 Hz), 5.87 (1H, s), 5.34 (1H, s), 4.41 (2H, t,
J = 7.3 Hz), 1.96 (2H, t, J = 7.2 Hz), 1.38–1.23 (16H, m), 0.87 (3H, t, J = 6.9 Hz) ppm; 13C-NMR (125 MHz,
CDCl3) δ 165.1 (C=O), 162.7 (C), 153.4 (C), 141.4 (C), 132.6 (CH), 124.0 (CH), 123.4 (CH), 123.2 (CH),
116.8 (CH), 115.5 (C), 91.2 (CH), 62.8 (CH2), 50.7 (CH2), 31.9 (CH2), 30.3 (CH2), 29.6 (CH2), 29.5 (CH2),
29.4 (CH2), 29.3 (CH2), 29.0 (CH2), 26.6 (CH2), 22.7 (CH2), 14.2 (CH3) ppm; EIMS m/z 397 ([M+], 18);
236 (17); 209 (15); 208 (100); 68 (21); 57 (16); 55 (15); HREIMS 397.2351 (calcd. for C23H31N3O3 [M+]
397.2365); FT-IR (ATR)
ν
3134, 3075, 2918, 2849, 1725, 1623, 1610, 1566, 1492, 1458, 1424, 1381, 1328,
max
1272, 1248, 1187, 1154, 1138, 1106, 1059, 1031, 979, 931, 882, 851, 814 cm−1
.
4-((1-Benzyl-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one (8l). Following the experimental procedure
described in method B, from 40.8 mg (0.28 mmol) of benzylazide and 28 mg (0.14 mmol) of
O-propargylated coumarin (
3
), 47.0 mg (94%) of compound 8l were obtained as an amorphous white
solid. m.p. 210–211 C; H-NMR (500 MHz, CDCl3) 7.76 (1H, dd, J = 7.9, 1.5 Hz), 7.63 (1H, s),
7.56–7.52 (1H, t, J = 7.6 Hz), 7.47–7.36 (3H, m), 7.35–7.28 (3H, m), 7.23 (1H, t, J = 7.6 Hz), 5.84 (s, 1H),
5.59 (s, 2H), 5.31 (2H, d, J = 3.8 Hz) ppm; 13C-NMR (125 MHz, CDCl3)
165.1 (C=O), 162.5 (C), 153.7
◦
1
δ
δ
(C), 142.1 (C), 134.4 (C), 132.6 (CH), 129.5 (2CH), 129.2 (CH), 128.4 (2CH), 124.0 (CH), 123.3 (CH), 123.2
(CH), 117.0 (CH), 115.8 (C), 91.5 (CH), 62.9 (CH2), 54.6 (CH2) ppm; EIMS m/z 333 ([M+], 39); 172 (13);
144 (62); 104 (11); 92 (14); 91 (100); HREIMS 333.1116 (calcd. for C19H15N3O3[M+] 333.1113); FT-IR
(ATR)
ν
3075, 1722, 1625, 1569, 1498, 1459, 1421, 1377, 1331, 1277, 1250, 1187, 1141, 1111, 1063, 1035,
max
983, 933, 885, 849, 814 cm−1
.
4-((1-Tetradecyl-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one (8m). Following the experimental
procedure described in method B, from 68.0 mg (0.28 mmol) of 1-azido-tetradecane and 28 mg
(0.14 mmol) of O-propargylated coumarin (
3
), 61.3 mg (93%) of compound 8m were obtained as an
7.79 (1H, dt, J = 7.9, 1.5 Hz),
amorphous white solid. m.p. 146–147 ◦C; 1H-NMR (500 MHz, CDCl3)
δ
7.72 (1H, s), 7.54 (1H, tt, J = 9.1, 1.7 Hz), 7.31 (1H, dd, J = 8.4, 1.5 Hz), 7.24 (1H, t, J = 7.6 Hz), 5.87
(1H, s), 5.35 (2H, s), 4.41 (2H, t, J = 7.3 Hz), 1.96 (2H, t, J = 7.1 Hz), 1.38–1.23 (22H, m), 0.88 (3H, t,
J = 6.9 Hz) ppm; 13C-NMR (125 MHz, CDCl3)
δ 165.1 (C=O), 162.8 (C), 153.4 (C), 141.4 (C), 132.6
(CH), 124.0 (CH), 123.4 (CH), 123.3 (CH), 116.8 (CH), 115.6 (C), 91.2 (CH), 62.8 (CH2), 50.8 (CH2),
32.0 (CH2), 31.0 (CH), 30.4 (CH2), 29.9 (CH2), 29.8 (CH2), 29.7 (2CH2), 29.6 (CH2), 29.5 (CH2), 29.4
(CH2), 29.1 (CH2), 26.6 (CH2), 22.8 (CH2), 14.2 (CH3) ppm; EIMS m/z 439 ([M+], 11); 278 (26); 251 (19);
250 (100); 215 (20); 120 (10); 71 (14); 70 (12); 68 (23); 57 (22); 56 (10); 55 (19); HREIMS 439.2852 (calcd.

Molecules 2018, 23, 199
11 of 18
for C26H37N3O3 [M+] 439.2835); FT-IR (ATR)
ν
max 3135, 3075, 2918, 2849, 1817, 1726, 1624, 1567, 1468,
1423, 1381, 1328, 1273, 1248, 1185, 1154, 1139, 1107, 1058, 979, 931, 882, 851, 814 cm−1
.
4-(((1-Phenyl-1H-1,2,3-triazol-4-yl)methyl)amino)-2H-chromen-2-one (9a). Following the experimental
procedure described in method A, from 31.2 mg (0.24 mmol) of phenyl boronic acid and 28 mg
(0.14 mmol) of N-propargylated coumarin (
amorphous white solid. m.p. 194–195 ◦C; 1H-NMR (500 MHz, (CD3)2SO)
J = 5.6 Hz), 8.10 (1H, dd, J = 8.0, 1.0 Hz), 7.90 (2H, d, J = 7.6 Hz), 7.61–7.56 (3H, m), 7.48 (1H, t, J = 7.4 Hz),
7.36–7.30 (2H, m), 5.30 (1H, s), 4.64 (2H, d, J = 5.6 Hz) ppm; 13C-NMR (125 MHz,(CD3)2SO)
161.5
6), 13.8 mg (28%) of compound 9a were obtained as an
δ
8.82 (1H, s), 8.31 (1H, t,
δ
(C=O), 153.1 (C),153.0 (C), 144.6 (C), 136.6 (C), 132.0 (CH), 130.0 (CH), 128.7 (CH), 123.4 (CH), 122.6
(CH), 121.5 (CH), 120.0 (CH), 117.0 (CH), 114.5 (C), 82.5 (CH), 37.8 (CH2) ppm; EIMS m/z 318([M+],
64); 290 (21); 289 (44); 261 (16); 198 (15); 159 (22); 130 (100); 77 (56); HREIMS 318.1117 (calcd. for
C18H14N4O2 [M+] 318.1117); FT-IR (ATR)
1480, 1146, 1377, 1340, 1321, 1188, 1054, 954, 922, 861, 823 cm−1
ν
3500, 3297, 3138, 3082, 2940, 1707, 1609, 1557, 1503,
max
.
4-(((1-(2-methoxyphenyl)-1H-1,2,3-triazol-4-yl)methyl)amino)-2H-chromen-2-one (9b). Following the
experimental procedure described in method B, from 68.0 mg (0.28 mmol) of 1-azido-2-methoxybenzene
and 28 mg (0.14 mmol) of N-propargylated coumarin (
6
), 16.9 mg (32%) of compound 9b were obtained
8.36 (1H, s), 8.09
as an amorphous white solid. m.p. 187–189 ◦C; 1H-NMR (500 MHz, (CD3)2SO)
δ
(1H, t, J = 5.5 Hz), 8.01 (1H, dd, J = 8.1, 1.2 Hz), 7.54 (1H, dd, J = 7.9, 1.6 Hz), 7.51 (1H, dd, J = 11.3,
4.2 Hz), 7.44 (1H, t, J = 7.9 Hz), 7.27–7.20 (m, 3H), 7.06 (1H, td, J = 7.7, 1.0 Hz, ), 5.30 (1H, s), 4.56 (2H,
d, J = 5.7 Hz), 3.77 (3H, s) ppm; 13C-NMR (125 MHz, (CD3)2SO)
δ 161.2 (C=O), 153.0 (C), 152.8 (C),
151.4 (C), 142.9 (C), 131.7 (CH), 130.4 (CH), 125.7 (C), 125.4 (CH), 125.1 (CH), 123.1 (CH), 122.4 (CH),
120.8 (CH), 116.7 (CH), 114.4 (C), 113.0 (CH), 82.5 (CH), 56.0 (CH3), 37.5 (CH2) ppm; EIMS m/z 348
([M+], 20); 320 (17); 319 (20); 160 (100); 159 (20); 145 (11); 120 (11); 77 (19); HREIMS 348.1228 (calcd.
for C19H16N4O3 [M+] 348.1222); FT-IR (ATR)
ν
max 3524, 3318, 3173, 3085, 2944, 2847, 2376, 1649, 1607,
1555, 1505, 1476, 1446, 1384, 1327, 1290, 1260, 1244, 1197, 1120, 1044, 1017, 957, 937, 865 cm−1
.
4-(((1-(3-Methoxyphenyl)-1H-1,2,3-triazol-4-yl)methyl)amino)-2H-chromen-2-one (9c). Following the
experimental procedure described in method B, from 42.3 mg (0.28 mmol) of 1-azido-3-methoxybenzene
and 28 mg (0.14 mmol) of N-propargylated coumarin (
6
), 26.9 mg (51%) of compound 9c were obtained
as an amorphous white solid. m.p. 246–248 ◦C; 1H-NMR (500 MHz, (CD3)2SO)
δ
8.71 (1H, s), 8.08
(1H, t, J = 5.5 Hz), 8.02 (1H, dd, J = 8.1, 1.2 Hz), 7.52 (1H, t, J = 7.8 Hz), 7.44–7.36 (3H, m), 7.27 (1H, d,
J = 0.8 Hz), 7.24 (1H, t, J = 7.7 Hz), 6.97 (1H, dt, J = 6.8, 2.5 Hz), 5.25 (1H, s), 4.57 (2H, d, J = 5.6 Hz), 3.78
(3H, s) ppm; 13C-NMR (125 MHz, (CD3)2SO)
δ 161.2 (C=O), 160.1 (C), 153.0 (C), 152.7 (C), 144.3 (C),
137.5 (C), 131.7 (CH), 130.6 (CH), 123.1 (CH), 123.0 (CH), 121.5 (CH), 116.7 (CH), 114.4 (C), 114.2 (CH),
111.9 (CH), 105.7 (CH), 82.5 (CH), 55.5 (CH3), 37.7 (CH2) ppm; EIMS m/z 348 ([M+], 40); 319 (25); 291
(10); 198 (11); 161 (13); 160 (100); 159 (30); 123 (11); 107 (24); 92 (19); 77 (26); HREIMS 348.1221 (calcd.
for C19H16N4O3 [M+] 348.1222); FT-IR (ATR)
1483, 1446, 1373, 1322, 1246, 1192, 1158, 1142, 1118, 1048, 954, 922, 859, 846, 819 cm−1
νmax 3297, 3143, 3085, 3000, 2936, 2830, 1708, 1609, 1558,
.
4-(((1-(4-Methoxyphenyl)-1H-1,2,3-triazol-4-yl)methyl)amino)-2H-chromen-2-one (9d). Following the
experimental procedure described in method B, from 42.3 mg (0.28 mmol) of 1-azido-4-methoxybenzene
and 28 mg (0.14 mmol) of N-propargylated coumarin (
6
), 29.6 mg (56%) of compound 9d were obtained
8.58 (1H, s), 8.07
as an amorphous white solid. m.p. 246–248 ◦C; 1H-NMR (500 MHz, (CD3)2SO)
δ
(1H, t, J = 5.4 Hz), 8.01 (1H, dd, J = 8.1, 1.1 Hz), 7.74–7.69 (2H, m), 7.52 (1H, t, J = 7.8 Hz), 7.24 (2H, dd,
J = 14.4, 7.6 Hz), 7.07–7.01 (2H, m), 5.25 (1H, s), 4.55 (2H, d, J = 5.6 Hz), 3.75 (3H, s) ppm; 13C-NMR
(125 MHz, (CD3)2SO)
δ 161.2 (C=O), 159.2 (C), 153.0 (C), 152.8 (C), 144.1 (C), 131.7 (CH), 130.0 (C),
123.1 (CH), 122.4 (CH), 121.6 (2CH), 121.3 (CH), 116.7 (CH), 114.7 (2CH), 114.4 (C), 82.5 (CH), 55.4
(CH3), 37.7 (CH2) ppm; EIMS m/z 348 ([M+], 21); 320 (14); 319 (22); 161 (12); 160 (100); 159 (26); 123
(10); 77 (15); HREIMS 348.1225 (calcd. for C19H16N4O3 [M+] 348.1222); FT-IR (ATR)
νmax 3528, 3285,

Molecules 2018, 23, 199
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3137, 3081, 3067, 3003, 2945, 2924, 2829, 2310, 2051, 1700, 1609, 1556, 1517, 1446, 1378, 1307, 1247, 1187,
1140, 1055, 1041, 989, 953, 920, 860, 822 cm−1
.
4-(((1-(3-Fluoro-4-methoxyphenyl)-1H-1,2,3-triazol-4-yl)methyl)amino)-2H-chromen-2-one (9e). Following
the experimental procedure described in method B, from 47.4 mg (0.28 mmol) of 1-azido-3-fluoro-
4-methoxybenzene and 28 mg (0.14 mmol) of N-propargylated coumarin (
6
), 27.3 mg (49 %) of
compound 9e were obtained as an amorphous white solid. m.p. 204–205 C; 1H-NMR (500 MHz,
(CD3)2SO) 8.76 (1H, s), 8.29 (1H, t, J = 5.7 Hz), 8.09 (1H, dd, J = 8.1, 1.2 Hz), 7.87 (1H, dd, J = 12.1,
2.6 Hz), 7.72 (1H, ddd, J = 8.9, 2.5, 1.4 Hz), 7.60 (1H, t, J = 7.8 Hz), 7.39–7.30 (3H, m), 5.28 (1H, s), 4.62
(2H, d, J = 5.6 Hz), 3.90 (3H, s); 13C-NMR (125 MHz, (CD3)2SO)
162.4 (C=O), 153.7 (C), 153.4 (C),
◦
δ
δ
151.6 (C, J1C–F = 247.8 Hz), 147.7 (C, J2C–F = 20.3 Hz), 144.9 (C), 132.7 (CH), 129.9 (C, J3C–F = 9.0 Hz),
124.1 (CH), 122.8 (CH), 122.1 (CH), 117.4 (CH), 117.0 (CH,J3C–F = 2.8 Hz), 114.9 (C), 114.7 (CH), 109.2
(CH, J2C–F = 22.5 Hz), 82.8 (CH), 56.7 (CH3), 38.0 (CH2) ppm; EIMS m/z 366 ([M+], 30); 338 (16); 337
(22); 198 (15); 179 (13); 178 (100); 159 (22); HREIMS 366.1138 (calcd. for C19H15N4O3F [M+] 366.1128);
FT-IR (ATR)
ν
3301, 3133, 3078, 3009, 2936, 2849, 1699, 1609, 1557, 1518, 1477, 1446, 1377, 1317, 1184,
max
1123, 1082, 1053, 953, 921, 884, 859, 817 cm−1
.
4-(((1-(4-Fluoro-phenyl)-1H-1,2,3-triazol-4-yl)methyl)amino)-2H-chromen-2-one (9f). Following the experimental
procedure described in method A, from 34.8 mg (0.24mmol) of 4-fluoro-phenyl boronic acid and
28 mg (0.14 mmol) of N-propargylated coumarin (
6
), 15.2 mg (30 %) of compound 9f were obtained
as an amorphous white solid. m.p. 212–213 ◦C; 1H-NMR (500 MHz, (CD3)2SO)
δ
8.79 (1H, s), 8.32
(1H, t, J = 6.0 Hz), 8.16 (1H, d, J = 7.8 Hz), 7.97–7.91 (2H, m), 7.61 (1H, t, J = 8.4 Hz), 7.45 (2H, t,
J = 8.8 Hz), 7.35–7.30 (2H, m), 5.30 (1H, s), 4.6 (2H, d, J = 5.5 Hz) ppm; 13C-NMR (125 MHz, (CD3)2SO)
δ
J3
161.4 (C=O), 153.1 (C), 153.0 (C), 144.7 (C), 133.1 (C), 132.0 (CH), 123.4 (CH), 122.6 (CH), 122.3 (2CH,
= 9.1 Hz), 121.7 (C), 116.9 (CH), 116.7 (2CH, J2
= 23.9 Hz), 114.5 (C), 82.6 (CH), 37.4 (CH2)
C–F
C–F
ppm; EIMS m/z 336 ([M+], 54); 308 (19); 307 (36); 198 (15); 159 (27); 148 (100); 95 (38); HREIMS 336.1030
(calcd. for C18H13N4O2F [M+] 336.1023); FT-IR (ATR)
ν
3537, 3417, 3307, 3143, 3084, 2454, 2288,
max
−1
2167, 2051, 1985, 1707, 1610, 1558, 1541, 1516, 1481, 1447, 1377, 1230, 1183, 1051, 993, 957, 920, 825 cm
.
4-(((1-(3-Trifluoromethylphenyl)-1H-1,2,3-triazol-4-yl)methyl)amino)-2H-chromen-2-one (9g). Following
the experimental procedure described in method B, from 53.2 mg (0.28 mmol) of 1-azido-3-
trifluoromethylbenzene and 28 mg (0.14 mmol) of N-propargylated coumarin (
of compound 9g were obtained as an amorphous white solid. m.p. 231–232 ◦C; 1H-NMR (500 MHz,
(CD3)2SO) 9.00 (1H, s), 8.32 (1H, t, J = 5.7 Hz), 8.29–8.24 (2H, m), 8.09 (1H, dd, J = 8.1, 1.2 Hz),
7.88–7.80 (2H, m), 7.60 (1H, dd, J = 15.6, 1.4 Hz), 7.34 (2H, ddd, J = 9.2, 8.2, 1.0 Hz), 5.29 (1H, s), 4.66
6), 26.2 mg (45 %)
δ
(2H, d, J = 5.7 Hz) ppm; 13C-NMR (125MHz, (CD3)2SO)
δ 161.5 (C=O), 153.1 (C), 153.0 (C), 145.0 (C),
137.1 (C), 132.0 (CH), 131.4 (CH), 130.5 (C, J2C–F = 32.5 Hz), 125.2(CH, J3C–F = 3.1 Hz), 123.9 (CH), 123.6
(C, J1C–F = 272.5 Hz), 123.4 (CH), 122.6 (CH), 121.8 (CH), 117.0 (CH), 116.7 (CH, J3C-F= 3.8 Hz), 114.5
(C), 82.6 (CH), 37.8 (CH2); EM-IE m/z 386 ([M+], 72); 358 (26); 357 (50); 198 (100); 159 (29); 145 (49);
HREIMS 386.0977 (calcd. for C19H13N4O2F3 [M+] 386.0991); FT-IR (ATR)
νmax 3425, 3318, 3142, 3085,
2942, 1701, 1610, 1557, 1540, 1482, 1448, 1377, 1342, 1321, 1298, 1266, 1248, 1172, 1142, 1110, 1070, 1046,
955, 923, 896, 861, 819 cm–1.
4-(((1-(3-Nitrophenyl)-1H-1,2,3-triazol-4-yl)methyl)amino)-2H-chromen-2-one (9h). Following the experimental
procedure described in method B, from 46.6 mg (0.28 mmol) of 1-azido-3-nitrobenzene and 28 mg
(0.14 mmol) of N-propargylated coumarin (
6
), 21.2 mg (39 %) of compound 9h were obtained as an
amorphous white solid. m.p. 240–241 ◦C; 1H-NMR (600 MHz, (CD3)2SO)
δ
9.06 (1H, s), 8.73 (1H, t,
J = 1.9 Hz), 8.42 (1H, d, J = 7.8 Hz), 8.32 (2H, dd, J = 8.2, 2.1 Hz), 8.10 (1H, d, J = 7.9 Hz), 7.93–7.83
(1H, m), 7.61 (1H, t, J = 7.4 Hz), 7.37–7.27 (2H, m), 5.29 (1H, s), 4.67 (2H, d, J = 5.4 Hz) ppm; 13C-NMR
(150 MHz, (CD3)2SO)
δ 161.4 (C=O), 153.1 (C), 153.0 (C), 148.6 (C), 145.2 (C), 137.1 (C), 132.0 (CH),
131.5 (CH), 126.0 (CH), 123.4 (CH), 123.1 (CH), 122.5 (CH), 122.0 (CH), 116.9 (CH), 114.7 (CH), 114.5
(C), 82.61 (CH), 37.7 (CH2) ppm; EIMS m/z 363 ([M+], 48); 335 (31); 334 (69); 333 (100); 286 (23); 242

Molecules 2018, 23, 199
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(30); 198 (28); 197 (31); 175 (40); 161 (38); 159 (31); 129 (40); 92 (27); 76 (30); 65 (27); HREIMS 363.0983
(calcd. for C18H13N5O4 [M+] 363.0968); FT-IR (ATR)
3426, 3137, 3106, 3082, 2927, 1707, 1614, 1561,
ν
max
1531, 1481, 1448, 1352, 1315, 1269, 1189, 1142, 1119, 1043, 1001, 960, 925, 863, 818 cm−1
.
4-(((1-(1H-Indole-5-yl)-1H-1,2,3-triazol-4-yl)methyl)amino)-2H-chromen-2-one (9i). Following the experimental
procedure described in method B, from 44.9 mg (0.28 mmol) of 5-azido-1H-indole and 28.0 mg
(0.14 mmol) of N-propargylated coumarin (
6
), 20.7 mg (37 %) of compound 9i were obtained as an
amorphous white solid. m.p. 236–237 ◦C;1H-NMR (500 MHz, (CD3)2SO)
δ
11.42 (1H, bs), 8.72 (1H, s),
8.29 (1H, t, J = 5.6 Hz), 8.11 (1H, d, J = 7.2 Hz), 8.00 (1H, s), 7.63–7.53 (3H, m), 7.50 (1H, s), 7.33 (2H, dd,
J = 12.4, 7.8 Hz), 6.55 (1H, s, J = 2.1 Hz), 5.34 (1H, s), 4.63 (2H, d, J = 5.6 Hz) ppm; 13C-NMR (125 MHz,
(CD3)2SO)δ162.4 (C=O), 161.5 (C), 153.1 (C), 153.0(C), 144.1 (C), 135.5 (C), 132.0 (CH), 129.5 (C), 127.7
(CH), 127.6 (C), 123.4 (CH), 122.6 (CH), 121.9 (CH), 116.9 (CH), 114.5 (C), 114.2 (CH), 112.3 (CH), 111.9
(CH), 101.9 (CH), 82.5 (CH), 37.9 (CH2); EIMS m/z 357 ([M+], 27); 329 (14); 328 (23); 170 (16); 169 (100);
168 (30); 156 (23); 132 (21); 116 (57); 115 (16); 89 (19); HREIMS 357.1217 (calcd. for C20H15N5O2 [M+]
357.1226); FT-IR (ATR) νmax 3522, 3397, 3324, 1682, 1616, 1562, 1486, 1354, 1092, 1053, 886 cm−1
.
4-(((1-(Furan-3-yl)-1H-1,2,3-triazol-4-yl)methyl)amino)-2H-chromen-2-one (9j). Following the experimental
procedure described in method A, from 21.3 mg (0.24 mmol) of 3-furyl boronic acid and 28 mg
(0.14 mmol) of N-propargylated coumarin (
6
), 5.4 mg (12%) of compound 9j were obtained as an
8.58 (1H, s), 8.42 (1H, d,
amorphous white solid. m.p. 198–199 ◦C; 1H-NMR (500 MHz, (CD3)2SO)
δ
J = 0.8 Hz), 8.30 (1H, t, J = 5.6 Hz), 8.08 (1H, d, J = 6.9 Hz), 7.86 (1H, t, J = 1.8 Hz), 7.60 (1H, t, J = 7.8 Hz),
7.32 (2H, dd, J = 8.1, 4.6 Hz), 7.12 (1H, dd, J = 2.0, 0.8 Hz), 5.27 (1H, s), 4.62 (2H, d, J = 5.6 Hz) ppm;
13C-NMR (125 MHz, (CD3)2SO)
δ 161.4 (C=O), 153.1 (C), 153.0 (C), 144.8 (CH), 144.3 (C), 133.9 (CH),
132.0 (CH), 126.0 (C), 123.4 (CH), 122.6 (CH), 122.2 (CH), 117.0 (CH), 114.5 (C), 105.1 (CH), 82.5 (CH),
37.7 (CH2) ppm; EIMS m/z 308 ([M+], 99); 120 (67); 93 (28); 91 (12); 66 (23); 65 (100); 58 (17); HREIMS
308.1000 (calcd. for C16H12N4O3 [M+] 308.0988); FT-IR (ATR)
ν
max 3293, 3134, 3083, 2924, 2853, 2285,
1706, 1610, 1557, 1480, 1446, 1378, 1323, 1262, 1230, 1191, 1143, 1118, 1089, 1039, 1017, 955, 921, 867,
821 cm−1
.
4-(((1-Undecyl-1H-1,2,3-triazol-4-yl)methyl)amino)-2H-chromen-2-one (9k). Following the experimental
procedure described in method B, from 56.1 mg (0.28 mmol) of 1-azido-undecane and 28 mg (0.14 mmol)
of N-propargylated coumarin (
6
), 50.3 mg (84%) of compound 9k were obtained as an amorphous
white solid. m.p. 154–156 C; 1H-NMR (500 MHz, (CD3)2SO)
8.08–7.93 (3H, m), 7.51 (1H, t, J = 7.7 Hz),
7.25 (1H, s), 7.22 (1H, t, J = 7.9 Hz), 5.18 (1H, s), 4.46 (2H, d, J = 5.6 Hz), 4.24 (2H, t, J = 7.0 Hz), 1.80–1.59
(2H, m), 1.14 (16H, s), 0.78 (3H, t, J = 6.9 Hz) ppm; 13C-NMR (125 MHz, (CD3)2SO)
161.1 (C=O),
◦
δ
δ
153.0 (C), 152.7 (C), 143.1 (C), 131.6 (CH), 123.1 (CH), 122.8 (CH), 122.3 (CH), 116.7 (CH), 114.4 (C), 82.3
(CH), 49.2 (CH2), 37.7 (CH2), 31.0 (CH2), 29.4 (CH2), 28.7 (CH2), 28.6 (CH2), 28.6 (CH2), 28.4 (CH2),
28.1 (CH2), 25.6 (CH2), 21.8 (CH2), 13.6 (CH3) ppm; EIMS m/z 396 ([M+], 88); 395 (16); 368 (40); 367
(100); 339 (15); 297 (18); 283 (17); 235 (20); 227 (37); 199 (23); 198 (21); 162 (19); 57 (19); 55 (23); HREIMS
396.2504 (calcd. for C23H32N4O2 [M+] 396.2525); FT-IR (ATR)
2920, 2849, 1679, 1607, 1553, 1481, 1465, 1446, 1376, 1096, 1055 cm−1
νmax 3523, 3399, 3325, 3127, 3069, 2955,
.
4-(((1-Benzyl-1H-1,2,3-triazol-4-yl)methyl)amino)-2H-chromen-2-one (9l). Following the experimental
procedure described in method B, from 56.1 mg (0.28 mmol) of azidomethyl-benzene and 28 mg
(0.14 mmol) of N-propargylated coumarin (
amorphous white solid. m.p. 217–219 ◦C; 1H-NMR (500 MHz, (CD3)2SO)
7.96 (1H, dd, J = 8.0, 1.2 Hz), 7.50 (1H, t, J = 7.8 Hz), 7.31–7.19 (7H, m), 5.50 (2H, s), 5.19 (1H, s), 4.47
(2H, d, J = 5.8 Hz) ppm; 13C-NMR (125 MHz, (CD3)2SO)
161.2 (C=O), 153.0 (C), 152.8 (C), 143.5 (C),
6
), 45.5 mg (91%) of compound 9l were obtained as an
δ
8.03 (2H, d, J = 8.7 Hz),
δ
135.9 (C), 131.7 (CH), 128.5 (2CH), 127.9 (CH), 127.7 (2CH), 123.2 (CH), 123.1 (CH), 122.3 (CH), 116.7
(CH), 114.3 (C), 82.4 (CH), 52.7 (CH2), 37.6 (CH2) ppm; EIMS m/z 332 ([M+], 41); 303 (14); 213 (18); 144
(13); 91 (100); 65 (11); HREIMS 332.1279 (calcd. for C19H16N4O2 [M+] 332.1273); FT-IR (ATR)
νmax 3283,

Molecules 2018, 23, 199
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3141, 3075, 2922, 2852, 2366, 2323, 1697, 1608, 1557, 1482, 1446, 1380, 1322, 1262, 1222, 1188, 1124, 1058,
955, 920, 861, 827 cm−1
.
4-(((1-Tetradecyl-1H-1,2,3-triazol-4-yl)methyl)amino)-2H-chromen-2-one (9m). Following the experimental
procedure described in method B, from 68.0 mg (0.28 mmol) of 1-azido-tetradecane and 28 mg
(0.14 mmol) of N-propargylated coumarin (
6
), 63.5 mg (96%) of compound 9m were obtained as an
8.23 (1H, t, J = 5.7 Hz),
amorphous white solid. m.p. 156–157 ◦C; 1H-NMR (500 MHz, (CD3)2SO)
δ
8.07 (1H, s), 8.05 (1H, d, J = 8.1 Hz), 7.58 (1H, d, J = 7.5 Hz), 7.33–7.29 (2H, m), 5.24 (1H, s), 4.53 (2H, d,
J = 5.7 Hz), 4.31 (2H, t, J = 7.0 Hz), 1.75 (2H, t, J = 7.3 Hz ), 1.23–1.13 (22H, m), 0.85 (3H, t, J = 6.9 Hz)
ppm; 13C-NMR (150 MHz, (CD3)2SO)
δ 161.8 (C=O), 153.5 (C), 153.3 (C), 143.7 (C), 132.4 (CH), 123.8
(CH), 123.5 (CH), 122.9 (CH), 117.4 (CH), 114.9 (C), 82.8 (CH), 49.7 (CH2), 31.7 (CH2), 30.1 (CH2), 29.5
(2CH2), 29.4 (2CH2), 29.3 (2CH2), 29.1 (CH2), 28.7 (CH2), 26.2 (CH2), 22.5 (CH2), 14.4 (CH3) ppm; EIMS
m/z 438 ([M+], 92); 410 (55); 409 (100); 367 (37); 227 (51); 199 (44); 198 (60); 162 (39); 57 (42); 55 (47);
HREIMS 438.2978 (calcd. for C26H38N4O2 [M+] 438.2995); FT-IR (ATR)
ν
3320, 3127, 3070, 2918,
max
2848, 2416, 2167, 2051, 1983, 1658, 1606, 1550, 1467, 1377, 1322, 1258, 1202, 1150, 1118, 1055, 966, 938,
867, 835 cm−1
.
3.2. Microbial Strains
Staphylococcus aureus (ATCC 6538), Enterococcus faecalis (PCM 2673), Escherichia coli (ATCC 8739),
Klebsiella pneumoniae (PCM1, Pseudomonas aeruginosa (PCM 2562) and yeast Candida albicans (ATCC
10231) were obtained from the Department of Molecular Biology, The John Paul II Catholic University
of Lublin, Poland.
3.3. MIC Determination
The in vitro antimicrobial studies were carried out with the microbroth dilution method against
test organisms, as described previously [59,60]. The bacterial strains were inoculated in Mueller
Hinton Broth medium (Biocorp, Warsaw, Poland) and the Candida strain was inoculated in Sabouraud
Dextrose liquid medium (Biocorp, Poland) and incubated at 37 ◦C and at 30 ◦C, respectively, with
vigorous shaking (200 rpm) for 24 h. Bacterial cell suspensions at initial inoculums of 5
×
105 in
Mueller-Hinton liquid medium and adequate yeast suspensions at initial inoculums of 3
×
103 cfu/mL
in Sabouraud Dextrose Broth were exposed to the examined compound at relevant concentrations
(range 0.001–2 mg/mL) for 24 h at 37 ◦C for the bacteria and for 48 h at 30 ◦C in the case of the fungi.
Simultaneously, the standard antibiotics, chloramphenicol for antibacterial activity and ketoconazole
for antifungal activity (as a positive control), were tested against the pathogens. The MIC was
the lowest concentration of the compounds that inhibited the visible growth of the microorganism.
The experiments were performed in triplicate.
3.4. Haemolytic Assay
Haemolytic properties of the selected compounds were determined according to the method
described previously [45]. The human blood samples were centrifuged at 500
×
g for 10 min at 4 ◦C
and the supernatant was discarded. Next, the erythrocytes were resuspended with PBS buffer (10 mM
phosphate, pH 7.5; 150 mM NaCl) and centrifuged as previously. The washing procedure was repeated
until a transparent supernatant was obtained. The washed erythrocytes were finally resuspended
in PBS buffer to a final concentration of 2%. Simultaneously, appropriate concentrations (5, 10, 25,
50, 100 and 500 µg/mL for 8a, 8f, 9h and 9k, or 2, 5, 12.5, 50, 125 and 250 µg/mL for 8b) of the
examined compounds were prepared in a final volume of 50 mL DMSO. The compounds prepared
in this way were mixed with 450 mL of 2% erythrocyte suspension and incubated for 1 h at 37 ◦C.
Then, the samples were centrifuged at 5000
was measured.
×
g for 10 min and absorbance at wavelength 415 nm

Molecules 2018, 23, 199
15 of 18
4. Conclusions
In conclusion, twenty-eight coumarin-triazole conjugates were synthesized through a copper(I)-
catalysed Huisgen 1,3-dipolar cycloaddition reaction of the corresponding O-propargylated coumarin
(3
) or N-propargylated coumarin (
6
) with alkyl or aryl azides. Five of them (8a
,
8b
,
8f,
9h and 9k
)
displayed promising activity against Enterococcus faecalis at MICs ranging from 12.5 to 50.0
µg/mL.
Compound 8b having a 2-OMe-Ph group attached at the triazol nucleus and an –OCH2– linker was the
best of the series. The most active compounds showed minimal toxicity towards human blood cells.
1
Supplementary Materials: The following are available online: H-NMR and 13C-NMR spectra of compounds
6,
8a–8m and 9a–9m.
Acknowledgments: We gratefully acknowledge the financial support from the Spanish MINECO SAF
2015-65113-C2-1-R to A.E.B. This project is also co-funded by the European Regional Development Fund (FEDER).
PLR thanks to Spanish MINECO for a pre-doctoral grant (FPU-Program).
Author Contributions: Ana Estévez-Braun, Ángel Amesty, and Maciej Masłyk conceived and designed the
experiments; Priscila López-Rojas, Monika Janeczko, Konrad Kubin´ski, and Ángel Amesty performed the
experiments; Ana Estévez-Braun, Ángel Amesty and Maciej Masłyk wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 8a–l, and 9a–l are available from the authors.
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