Synthesis, in?vitro anticancer and antibacterial activities and in silico studies of new 4-substituted 1,2,3-triazole–coumarin hybrids

Article abstract of DOI:10.1016/j.ejmech.2016.08.062

The 4-substituted 1,2,3-triazole core in designed coumarin hybrids (4–35) with diverse physicochemical properties was introduced by eco-friendly copper(I)-catalyzed Huisgen 1,3-dipolar cycloaddition under microwave irradiation. Coumarin–1,2,3-triazole–ben

Full text of DOI:10.1016/j.ejmech.2016.08.062

Accepted Manuscript
Synthesis, in vitro anticancer and antibacterial activities and in silico studies of new 4-
substituted 1,2,3-triazole–coumarin hybrids
Tatjana Gazivoda Kraljević, Anja Harej, Mirela Sedić, Sandra Kraljević Pavelić, Višnja
Stepanić, Domagoj Drenjančević, Jasminka Talapko, Silvana Raić-Malić
PII:
S0223-5234(16)30719-X
10.1016/j.ejmech.2016.08.062
EJMECH 8858
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 9 June 2016
Revised Date: 25 August 2016
Accepted Date: 26 August 2016
Please cite this article as: T.G. Kraljević, A. Harej, M. Sedić, S.K. Pavelić, V. Stepanić, D. Drenjančević,
J. Talapko, S. Raić-Malić, Synthesis, in vitro anticancer and antibacterial activities and in silico studies of
new 4-substituted 1,2,3-triazole–coumarin hybrids, European Journal of Medicinal Chemistry (2016), doi:
10.1016/j.ejmech.2016.08.062.
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ACCEPTED MANUSCRIPT
R7
O
O
N
N
N
R4
R4
R7
IC50
R4
(
R7
MIC
I
1
7
8 µg/mL (E. faecium)
4 µg/mL (E. faecium VRE)
CH2)4CH3 OH
3
0
OH
8.57 uM (HepG2)
6
N
O
S
O
H
2
3
N
F
16 µg/mL (E. faecium)
64 µg/mL (E. faecium VRE)
OH
N
N
33
CH3
0.9 uM (HepG2)
Cl
N
S
2
7
32 µg/mL (E. faecium)
4 µg/mL (E. faecium VRE)
NH
OH
6
S
5
-LO
ASAH
inhibition
apoptosis
induction
inhibition

ACCEPTED MANUSCRIPT
Synthesis, in vitro anticancer and antibacterial activities and in silico studies of
new 4-substituted 1,2,3-triazole–coumarin hybrids
a
b,c
b,c
b,c
Tatjana Gazivoda Kraljević, Anja Harej, Mirela Sedić, Sandra Kraljević Pavelić,
d
e,f
f
a*
Višnja Stepanić, Domagoj Drenjančević, Jasminka Talapko and Silvana Raić-Malić
a
University of Zagreb, Faculty of Chemical Engineering and Technology,
Department of Organic Chemistry, Marulićev trg 20, HR-10000 Zagreb, Croatia
b
University of Rijeka, Department of Biotechnology, Radmile Matejčić 2,
HR-51000 Rijeka, Croatia
c
University of Rijeka, Centre for high-throughput technologies, Radmile Matejčić 2,
HR-51000 Rijeka, Croatia
d
Division of Molecular Medicine, Ruđer Bošković Institute, Bijenička 54,
HR-10002 Zagreb, Croatia
e
Osijek University Hospital, Department of Transfusion Medicine, Josipa Huttlera 4,
HR-31000 Osijek, Croatia
f
University of Osijek, Faculty of Medicine, Department of Microbiology and Parasitology,
Cara Hadrijana 10/E, HR-31000 Osijek, Croatia
1

ACCEPTED MANUSCRIPT
Abstract. The 4-substituted 1,2,3-triazole core in designed coumarin hybrids (4–35) with
diverse physicochemical properties was introduced by eco-friendly copper(I)-catalyzed
Huisgen 1,3-dipolar cycloaddition under microwave irradiation. Coumarin–1,2,3-triazole–
benzofused heterocycle hybrids emerged as the class of compounds exhibiting the highest
antiproliferative activity. The strong relationship between lipophilicity and antiproliferative
activities was observed indicating that lipophilic 1,2,3-triazole–coumarin hybrids containing
phenylethyl (13), 3,5-difluorophenyl (14), 5-iodoindole (30) and benzimidazole (33 and 35)
subunits showed the most potent cytostatic effects. The 7-methylcoumarin–1,2,3-triazole–2-
methylbenzimidazole hybrid 33 can be highlighted as a lead that exerted the highest
cytotoxicity against hepatocellular carcinoma HepG2 cells with IC value of 0.9 µM and high
5
0
selectivity (SI = 50). This compound induced cell death, mainly due to early apoptosis. Strong
antiproliferative effect of 33 could be associated with its inhibition of 5-lipoxygenase (5-LO)
activity and perturbation of sphingolipid signaling by interfering with intracellular acid
ceramidase (ASAH) activity. Outlined considerable effect of lipophilicity on antiproliferative
activity was not observed for antibacterial activity. The compounds with p-pentylphenyl (17),
2
-chloro-4-fluorobenzenesulfonamide (23) and dithiocarbamate (27) moiety were endowed
with high selectivity against Enterococcus species. Moreover, these compounds found
superior in inhibiting the growth of clinically isolated vancomycin resistant Enterococcus
faecium, while the reference antibiotics exhibited the lack of activity. Our findings indicate
that coumarin–1,2,3-triazole could be used as a model skeleton for structural optimization to
develop more potent and selective anticancer agents and encourage further development of
novel structurally related analogs of 33 as more effective 5-LO inhibitors.
Keywords: 1,2,3-triazole–coumarin hybrids, click chemistry, cytostatic activity, antibacterial
activity, 5-lipoxygenase (5-LO), acid ceramidase (ASAH)
2

ACCEPTED MANUSCRIPT
1
.
Introduction
The exploration of new heterocycles that can accommodate potency to multiple biological
targets remains an intriguing scientific endeavour. Among the oxygen heterocycles,
coumarins are privileged structural motifs widely found in many natural products that have
attracted intense interest in recent years because of their diverse biological and
pharmacological properties [1,2]. Beside clinically proven anticoagulant and antithrombotic
actions, various coumarins have exhibited anticancer [3–6] and antimicrobial [7–11] activities
that have been the prime reason for their inclusion in the hybrids framework. Thus,
hybridizing the coumarin nucleus with other moieties has afforded new molecules with
improved anticancer and antimicrobial activity profiles [2–4]. It was found that coumarin
derivatives target a number of pathways in cancer such as kinase inhibition, cell cycle arrest,
angiogenesis inhibition, heat shock protein (HSP90) inhibition, telomerase inhibition,
antimitotic activity, carbonic anhydrase inhibition, monocarboxylate transporters inhibition,
aromatase inhibition and sulfatase inhibition [12–14]. Some plant-derived or synthetic
coumarins have been also reported to interfere with 5-lipoxygenase (5-LO) metabolic
pathways that are identified to be involved in cancer progression [15,16]. It is demonstrated
that the inhibition of the 5-LO pathway may be useful for cancer therapy through
antiproliferative, pro-apoptotic, pro-differentiation and antiangiogenic effects in cell lines and
animal models [17–19]. Although the 5-LO is recognized as a potential target in prevention
and/or treatment of cancer, to the best of our knowledge reports describing potent and
selective 5-LO inhibitors based on the coumarin core have been limited [20–22].
On the other hand, life-threatening infections caused by multi-drug resistant (MDR)
pathogens continue to present serious public health issues worldwide. A few antibiotics with
coumarin skeleton have been found that are active against MDR bacteria [23]. Moreover, for
many years Enterococcus species were believed to be harmless to humans and, therefore,
considered unimportant medically. During the past few decades, enterococci have emerged as
the only Gram-positive pathogens associated with a high risk of death [24]. In-hospital
mortality associated with enterococcal blood stream infections (BSI) has been estimated
between 25-50%, leading to the recognition of the Enterococcus species as formidable
pathogens [25]. Bacteremia and endocarditis [26,27] are the more common manifestations of
infections due to enterococci. The treatment of enterococcal BSI is complicated by high rates
of antimicrobial resistance. The rapid spread of enterococci with resistance to vancomycin
3

ACCEPTED MANUSCRIPT
(VRE) has been of particular concern. To address this problem, the development of new
antimicrobials remains a high priority for the continued effective treatment of infections
caused by resistant strains.
1
,2,3-Triazoles occupy a prominent place in drug discovery due to their facile synthesis
through click chemistry [28] and wide biological profiles, which include antibacterial [29–34]
and anticancer activities [35–39]. Potential structural features of bioactive triazoles include
stability to metabolic degradation, high selectivity and capability of hydrogen bonding that
could be favorable in the binding of biomolecular targets [40]. It was demonstrated that
different types of chemical bridges at C-4 of the 1,2,3-triazole core eliminated the planarity,
thereby could ameliorate the druggability and facilitate the compounds binding to their
possible receptor through induced fit [36]. In recent years, a library of coumarin derivatives
conjugated with 1,2,3-triazole were synthesized and proved to possess significant anticancer
and antimicrobial activities. Thus, novobiocin analogue I with 1,2,3-triazole at the C-3
position of coumarin displayed potent cytotoxic activity against breast cancer cell lines (Fig.
1
) [41].
O
O
O
O
O
O
N
N
NH
N
O
N N
N
N
O
O
O
O
R
N
N
O
O
R = NO2, CN
HN
O
I
II
III
F
O
HO
HO
O
N
N
N
N
N
O
O
O
N
S
S
N
N
O
N
O
O
N
N
O
O
O
O
O
IV
V
VI
Fig. 1. Representatives of 1,2,3-triazoles–coumarin hybrids as potent anticancer agents.
1
H-1,2,3-triazole linked (arylamidomethyl)-dihydrofurocoumarin hybrid II with the highest
cytotoxicity was studied as inhibitors of phosphodiesterase [42]. Triazoles linked 6-
hydroxycoumarin III with ortho substituted phenyl moiety showed to be selectively cytotoxic
against lung cancer cell line [43]. Chalcone–coumarin hybrid IV with a triazole linker was of
interest as potential anticancer and antimycobacterial agent [44]. Coumarin–1,2,3-triazole–
4

ACCEPTED MANUSCRIPT
dithiocarbamate hybrid V [45,46] and 4-(1,2,3-triazole-1-yl)coumarin VI with p-
fluorophenoxy)methyl substituent exhibited anticancer activity through inducing apoptosis of
the cell lines. Furthermore, among the triazolyl cumarin derivatives, it was observed that
substituent, length and position of alkyl spacer had profound effect on the antimicrobial
potency. Thus, triazolyl 4-methylcumarins VII containing morpholine and piperazine moiety
showed noticeable activity against various bacterial pathogens, particularly against
Pseudomonas aeruginosa (Fig. 2) [29]. Bis-1,2,4-triazole coumarin derivative and its
hydrochloride VIII containing a butylene linker displayed much stronger antibacterial and
antifungal efficacy than corresponding mono-triazole derivative [11]. Moreover, bis-triazole
coumarin alcohol VIII gave better anti-MRSA (methicillin-resistant Staphylococcus aureus)
activity than its mono-triazole and standard drug (Fig. 2) [47].
N
N
N
N
R
O
O
O
N
R
O
O
O
N
n
n = 4
N
O
N
R
O
R =
N
N
N
,
N
O
R = -(CH ) -, CH(OH)
2
2
VII
VIII
Fig. 2. Representatives of triazoles–coumarin hybrids VII and VIII as potent antimicrobial
agents.
Inspired by multifarious bioactivity of coumarin and 1,2,3-triazole heterocycles, and dramatic
increase in multidrug resistance to clinically available drugs, we conceptualized the
hybridization of 7-hydroxycoumarin (umbelliferone) or its 7-methyl analog and 1,2,3-triazole
entity with the aim to develop a new lead possessing efficient and selective pharmacological
activities. Taking into consideration the aforementioned and as a continuation of our project
on searching for new biologically active molecules possessing 1,2,3-triazole moiety [48–50],
we envisaged that coumarin and 1,2,3-triazole pharmacophores if linked together through
rotatable bond would generate novel hybrid molecules with promising anticancer and/or
antibacterial activities (Fig. 3).
5

ACCEPTED MANUSCRIPT
Fig. 3. Conformationally unrestricted 4-substituted 1,2,3-triazole–coumarin hybrids (4–35).
Moreover, varied alkyl, phenyl and heterocycle moieties introduced to C-4 of 1,2,3-triazole
were taken into account in order to evaluate their contribution to the antiproliferative activity.
Therefore, herein we have reported the synthesis of a diverse conformationally unrestricted
hybrids (4–35) of 7-substituted coumarin and 4-substituted 1,2,3-triazole linked through
single methylene unit in order to assess the influence of obtained structural modifications on
their physicochemical properties and consequently on antiproliferative and/or antibacterial
activities. The effect of selected 1,2,3-triazole–coumarin hybrids on the regulation of specific
lipid metabolic pathways mediated by enzymes 5-LO, sphingosine kinase 1 and acid
ceramidase that control major aspects of cancer cell behavior has been also investigated for
the first time.
2
.
Results and discussion
2
.1. Chemistry
Adopting the hybridization approach [1], a series of 1,2,3-triazole–coumarin hybrids (4–35)
was synthesized using copper(I)-catalyzed Huisgen 1,3-dipolar cycloaddition reaction of a
terminal alkyne with an azide (Scheme 1). 4-Azidomethyl coumarin derivatives 1 and 2 were
obtained in the reaction of sodium azide with 4-chloromethyl-7-hydroxycoumarin and 4-
chloromethyl-7-methylcoumarin, which were synthesized in the reaction of m-hydroxyphenol
and m-cresol with 4-chloroacetoacetate ethyl ester under Pechmann cyclization condition
using sulphuric acid to give 7-hydroxy and 7-methylcoumarin derivatives, respectively
[
46,51]. 1,2,3-Triazole–coumarin hybrids 4–19 comprising various 4-alkyl- and substituted 4-
aryl-1,2,3-triazole subunits were subsequently prepared by click chemistry of 4-azidomethyl
coumarin derivative and corresponding alkyne in the presence of Cu(I), as the catalyst, [52]
6

ACCEPTED MANUSCRIPT
(Scheme 1) which was generated in situ by reaction of copper(II) sulfate and metallic copper,
as reducing agent.
OH
O
S
Cl
R'
NH
X = NR', O
O
X
R3
R
R = OH, CH3
3
R' =
O
(i)
(ii)
(
iii)
O
S
O
S
O
R7
O
R'
N
N
S
H
O
X
R
2
H
a 2b 2c 2d 2e
3a 3b
1
a 1b
N3
R
R'
H
H
Cl
H
X
O
NCO(CH3)3
R7 OH CH3
CH
Cl
F
F
NO
2
3
O
(
iv)
O
R7
O
N
N
N
4
-35
R4
Compd
4
R7
R4
Yield Compd
R7
R4
Yield
O
S
H
OH
OH
81
92
20
21
OH
N
CH3
Cl
60
O
O
S
H
5
(CH )
2 3
Cl
OH
N
84
O
O
S
O
O
S
O
H
N
6
7
OH
OH
(CH
2
)
2
OH
39
83
22
23
OH
OH
F
F
14
6
OH
H
N
Cl
O
S
O
H
8
9
OH
OH
(CH
2
2
)
)
3
4
OH
OH
79
43
24
25
OH
OH
N
NO
2
77
S
(CH
N
O
76
S
S
N
N
O
10
11
12
OH
OH
OH
51
51
45
26
27
28
OH
OH
OH
51
S
O
S
41
(CH
(CH
)
CH
CH
N
NH
2
5
7
3
S
S
HN
)
68
2
3
N
7

ACCEPTED MANUSCRIPT
S
N
(
CH
2
)
2
HN
1
3
4
OH
OH
77
29
30
CH
3
71
F
F
I
I
88
1
OH
N
N
85
1
5
OH
Br
54
31
CH
3
78
N
1
1
1
1
6
7
8
9
OH
OH
OH
OH
(
CH
2
)
3
CH
3
95
52
56
81
32
33
34
35
OH
83
81
76
67
N
N
(CH
2
)
4
CH
3
CH
3
N
N
2 5 3
(CH ) CH
OH
N
N
(
2 2 3
CH ) CH
CH
3
N
Scheme 1. Reagents and conditions: (i) a) ethyl 4-chloroacetoacetate, conc. H SO , –5 °C; b) NaN ,
2
4
3
CH CN, reflux; (ii) propargyl amine, pyridine, r.t., 24 h; (iii) CS , Na PO x 12 H O, propargyl
3
2
3
4
2
bromide, acetone, Ar, r.t.; (iv) corresponding azide, Cu, 1M CuSO solution, tert-butanol : H O = 1 : 1,
4
2
DMF, 80 °C, 1 h.
The reactions were performed under microwave irradiation in a mixture of water and organic
solvent to give compounds 4–19 with a good yields ranging from 43–95%. The versatility of
target hybrids was further extended to the synthesis of coumarin–triazole–arylsulfonamide
hybrids 20–24 from coumarin azide derivative 1a and corresponding N-propargylated aryl
sulfonamides 2a–2e prepared by reaction of propargyl amine and arylsulfonyl chloride in the
presence of pyridine, as a base (Scheme 1). Inspired by potent anticancer activity of 1,2,3-
triazole–dithiocarbamate hybrids [46], we prepared 1,2,3-triazole–dithiocarbamate hybrids 25
and 27 containing unprotected morpholine and piperazine subunit. Cu(I)-catalyzed click
reaction of 20–26 were performed in a very good yield, except for 4-(2-fluorosulfonamide)-
(22) and 4-(2-chloro-4-fluorophenylsulfonamide) (23) substituted 1,2,3-triazole with a yield
of 14% and 6%, respectively. The structural diversity was further explored with the synthesis
of triazole-linked nitrogen/sulfur heterocycles to coumarin [48]. Firstly, propargylated
derivatives of benzothiazole, 5-iodoindole, benzimidazole and 2-methylbenzimidazole were
prepared that were subsequently submitted to Cu(I)-catalyzed 1,3-dipolar cycloaddition
reaction with azidomethyl coumarin derivatives 1a and 1b under microwave irradiation to
give 28–35 [48].
8

ACCEPTED MANUSCRIPT
2
.2. In vitro biological evaluations and in silico physicochemical profile of a coumarin–
1,2,3-triazole library
2
.2.1. Antiproliferative evaluations
Results of antiproliferative evaluations of compounds 4–35 on human tumor cell lines: lung
adenocarcinoma (A549), hepatocellular carcinoma (HepG2), ductal pancreatic
adenocarcinoma (CFPAC-1), cervical carcinoma (HeLa) and colorectal adenocarcinoma,
metastatic (SW620), as well as normal human lung fibroblast (WI38) and mouse embryonic
fibroblast (3T3) are presented in Table 1. It can be noted that among the compounds
exhibiting the highest activity (IC50 ≤ 30 ꢀM) most of the compounds showed inhibitory
effect against A549 and HeLa cell lines (Table S1, Supplementary data). However, none of
the compounds exhibited inhibitory effect with IC ≤ 30 ꢀM against SW620 cells. From the
5
0
alkyl and phenyl C-4 substituted coumarin–1,2,3-triazole hybrids 4–19, the compounds 4–12
bearing the 4-alkyl and 4-(hydroxyalkyl) chains showed poor, if any antiproliferative
activities. Introducing of the phenyl- and 4-(p-alkylphenyl)-substituted 1,2,3-triazoles (13–18)
increased the activity, particularly against lung adenocarcinoma (A549; IC = 8.87–32.34
5
0
µM) with 13 and 14 showing the selectivity index (SI) above 4. Whereas the compounds with
benzenesulfonamide (20–24) and dithiocarbamate (25–27) units showed no appreciable
inhibitory effects, the incorporation of benzofused heterocycles, such as benzothiazole (28
and 29), 5-iodoindole (30 and 31) and benzimidazole (32–35) led to improvements in
activities. Thus, 7-hydroxycoumarin–1,2,3-triazole–2-arylbenzothiazole 28 demonstrated
excellent activity against cervical carcinoma (HeLa, IC50 = 7.26 µM). Additionally, 7-
hydroxycoumarin–1,2,3-triazole–5-iodoindole hybrid 30 showed high potency against
hepatocellular carcinoma (HepG2, IC50 = 8.57 µM) with selectivity index of 3. The
substitution at C-7 of the coumarin ring seemed to have considerable impact on the
antiproliferative activity. Thus, inhibitory activities of 7-hydroxycoumarins 28 and 30 were to
7
-fold higher than those of their 7-methylcoumarin congeners 29 and 31. In contrast, among
the coumarin–1,2,3-triazole–benzimidazole conjugates, the activities of 7-hydroxycoumarin
derivatives 32 and 34 were 2–4-fold lower than those of corresponding 7-methylcoumarin
analogs 33 and 35. The 7-methylcoumarin–1,2,3-triazole–2-methylbenzimidazole hybrid 33
exerted the cytotoxicity against HepG2 cells in nM range with the highest selectivity index of
5
0. The hybrids 13, 14, 30 and 35 bearing phenylethyl, 3,5-difluorophenyl, 5-iodoindole and
9

ACCEPTED MANUSCRIPT
benzimidazole subunits, respectively, showed also selective inhibitory activity, particularly
against A549 and HepG2 cell lines. Unfortunately, most of the coumarin–1,2,3-triazole
hybrids have been also cytotoxic against normal fibroblasts WI38 and 3T3.
Table 1.
The growth-inhibition effects in vitro of compounds 4–35 on selected tumor cell lines and
normal fibroblasts.
a
IC (µM)
5
0
Cell lines
HeLa
Compd
A549
HepG2
CFPAC-1
SW620
3T3/b
WI38
4
5
6
7
8
9
91.71
30.07
90.33
>100
96.22
>100
67.59
66.15
46.03
24.78
21.06
32.34
13.83
13.65
8.87
>100
>100
>100
>100
>100
>100
>100
61.56
34.22
50.46
38.91
61.11
48.40
27.34
9.16
>100
75.81
>100
>100
>100
>100
>100
>100
62.65
>100
>100
52.92
69.77
50.40
>100
52.79
68.86
81.91
>100
>100
> 100
>100
49.59
>100
>100
90.20
>100
81.08
59.01
35.14
35.05
27.61
40.17
38.99
24.63
8.73
>100
59.76
>100
>100
>100
>100
>100
>100
50.87
84.38
66.73
97.66
49.24
39.64
36.79
37.52
92.19
>100
>100
>100
> 100
37.98
65.56
>100
57.70
40.83
>100
38.48
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
b
58.50
b
43.39
>100
>100
0.05
0.35
0.71
b
13.96
b
41.03
46.40
47.93
88.46
91.86
> 100
30.41
29.84
31.40
76.70
74.82
> 100
34.26
55.71
58.46
>100
74.88
> 100
7.91
b
0.03
b
0.82
b
24.95
b
1.45
b
<0.01
1
0

ACCEPTED MANUSCRIPT
b
25
26
27
28
29
30
31
32
33
34
35
70.17
35.41
96.04
16.16
46.24
28.63
34.09
>100
16.85
>100
40.13
99.35
56.94
>100
52.82
94.74
8.57
56.30
88.52
>100
10.54
34.89
29.89
41.71
76.51
59.41
>100
80.55
40.80
47.95
>100
7.26
≥100
>100
>100
89.95
≥100
34.34
70.02
>100
>100
>100
>100
59.15
b
2.82
b
>100
5.01
51.42
14.16
34.43
49.15
17.48
>100
23.91
18.24
25.84
8.31
38.06
70.69
0.90
97.46
45.33
>100
96.44
78.06
22.52
a
b
Compound concentration required to inhibit tumor cell proliferation by 50%; Compounds 11, 12,
8–27 were tested in WI38 cell line.
1
2
.2.2. Principle component analysis in terms of the measured IC50 values and calculated
VolSurf+ descriptors
The set of 32 synthetized coumarin–1,2,3-triazole hybrids has been characterized in terms of
molecular interaction field descriptors calculated by VolSurf+ [53] and principle component
analysis (PCA). According to the PCA model, the nine descriptors account for 83.3% of the
variance within the set of compounds differing primarily by substituent at the C-4 of the
1
,2,3-triazole ring (Fig. 4). Following descriptors have been used: molecular volume (V),
hydrophilic volume accounting for polarizability and dispersion forces (W1), the ratio of the
hydrophilic volume over the total molecular surface (CW2), hydrogen bond acceptor volume
(
WN1), the ratio between the polar and the hydrophobic surface areas (PHSAR), a vector
integy moment pointing from the centre of mass to the centre of the hydrophobic regions
ID4) indicating concentration of nonpolar regions in only one part of the molecular surface,
(
intrinsic aqueous solubility (SOLY), membrane permeability (CACO2) and the logarithm of
the partition coefficient between 1-octanol and water (logP). According to the PCA and our
main rationale in selection of C-4 substituents, the synthetized compounds were divided into
subgroups depending on their physicochemical properties (Fig. 4). Substituents at position C-
4
of triazole in described hybrids had considerable impact on their physicochemical
1
1

ACCEPTED MANUSCRIPT
characteristics and hence on the observed antiproliferative activities. The subgroup containing
derivatives with saturated alkyl side-chains (4–12) was the most soluble (SOLY) and the least
lipophilic within the set. A subgroup of soluble derivatives with arylsulfonamide and
dithiocarbamate fragments (20–27) were characterized by a relatively large hydrophilic
surface (W1), concentration of hydrophobic regions in a part of the molecular surface (ID4),
hydrogen bond acceptor volume (WN1) and the least potential within the compound set to
cross membranes. The two benzothiazole derivatives (28 and 29), forming a separate
subgroup, were the most lipophilic and the least soluble compounds with the largest volumes
in the set (Fig. S1, Supplementary data). The fourth group comprises compounds with phenyl-
containing side-chains (13–19), indole (30 and 31) and benzimidazole (32–35) subunits,
which were all lipophilic molecules with a capacity for passive transport across membranes.
2
Fig. 4. The A) score and B) loadings plots for the two-component PCA model (R (cum) =
7
1.0 %, Q2(cum) = 54%, Multibase 2015) built for 32 C-4-substituted coumarin–1,2,3-
triazole hybrids (Table 1) in terms of nine preselected VolSurf+ descriptors (Table S2,
Supplementary data). C) The significant effect of substituents on lipophilicity and cytotoxicity
for the tested set of novel hybrids.
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2

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Considering the antiproliferative activities, the compounds from the most lipophilic group
were the most active ones having the lowest IC values (Fig. 4, Table 1). Furthermore, simple
5
0
two-class decision tree models (active/inactive) based on logP values confirmed that
lipophilicity was a quite good parameter for distinguishing active from inactive compounds,
particularly in A549, HepG2 and HeLa cell lines with the highest number of active
compounds (IC < 90 ꢀM) (Table 2). Thus, 93.7% of the compound set, in which inactive
5
0
compounds were defined to have IC ≥ 90.0 ꢀM, was predicted correctly. Moreover, the
5
0
simple two-class model correctly classified 84.4% and 87.5% of compounds based on their
activities in A549 and HepG2 cancer cell lines, respectively. The prediction with the lowest
accuracy of 75% in CPFAC-1 cell line (with seven misclassified compounds as false
positives) was obtained demonstrating satisfactory accuracy of the applied logP model.
Table 2.
a
The two-class model for cytotoxicity in terms of lipophilicity (logP) against cancer cell lines.
Cancer cell
line
Minimum logP for
activity
# of predicted inactives/
# false negatives
# of predicted actives/
# false positives
A549
HepG2
HeLa
1.65
0.71
0.93
2.88
2.18
15/5
7/0
17/0
25/4
23/1
23/7
14/5
9/1
CPFAC-1
SW620
9/1
18/1
a
IC < 90 ꢀM for active compounds. The values of 1-octanol/water partition coefficient logP were
5
0
calculated by VolSurf+.
2
.2.3. The prediction of activity spectra for substances (PASS) analysis
The Prediction of Activity Spectra for Substances (PASS) has been applied in the present
study to explore the biological potentials of selected compounds and to prioritize them for
further in vitro studies. The PASS is an in silico tool used for predicting biological activity
spectra for natural and synthetic substances, which is based on the structure-activity
relationships knowledgebase for more than 260,000 compounds with known biological
activities including drugs, drug candidates, pharmaceutical leads and toxic compounds [54].
The PASS predicts the tentative biological potential of the compound based on its structure
and reveals the predicted activities as the probability of activity (Pa) and inacitivity (Pi). The
higher Pa value, the lower is the predicted probability of obtaining false positives in biological
1
3

ACCEPTED MANUSCRIPT
testing. The PASS analysis revealed that compounds 30 and 33 have high activity score for
leukotriene synthesis inhibitory effect with Pa values of 0.47 and 0.49, respectively (Table S3,
Supplementary data). The obtained in silico findings were further validated in vitro by
Western blot method as described below.
2
.2.4. Western blot analysis of predicted protein target
The results of PASS analysis indicated that compounds 30 and 33 could be potential
inhibitors of 5-lipoxygenase (5-LO), an enzyme that catalyzes two steps in biosynthesis of
leukotrienes, a group of lipid mediators of inflammation derived from arachidonic acid.
Mounting evidence suggested that 5-LO and 5-LO-catalyzed products were implicated in the
development and progression of different cancer types including lung, colon, pancreatic and
liver cancer [55–57]. Therefore, drugs that directly interfere with the production of 5-LO
metabolites or antagonize the signaling functions of 5-LO products may be effective in
preventing cancer [58]. It was also demonstrated that leukotrienes worked in concert with
other bioactive lipids such as sphingolipids in complex network of cellular signaling [59].
Simple sphingolipids such as ceramide and sphingosine are antiproliferative or pro-apoptotic,
whereas their phosphorylated derivatives ceramide-1-phosphate and sphingosine-1-phosphate
(S1P) have growth-promoting and anti-apoptotic properties. Overexpression of sphingosine
kinase 1 (SK1) and acid ceramidase (ASAH) resulting in an increased production of S1P and
degradation of ceramide, respectively, tips the balance towards survival, growth, and
proliferation, which makes these two enzymes attractive drug targets for cancer therapy.
Results of our study revealed that compound 30 neither inhibits 5-LO nor suppresses the
activities of the two major regulators of sphingolipid metabolism, namely SK1 and ASAH in
hepatocellular carcinoma cells (Fig. 5), in which this compound exerted the most potent
cytostatic effects. Thus, one can conclude that induced growth inhibition of HepG2 cells by
compound 30 is not associated with down-regulation of the key enzymes regulating
leukotriene and sphingolipid biosynthesis. On the contrary, compound 33 proved to be
putative 5-LO inhibitor and negative regulator of sphingolipid metabolic conversion with
selective inhibitory effect towards the ASAH activity (Fig. 5). Interestingly, this compound
did not impose inhibitory effect on the SK1 enzyme activity. Previous study demonstrated
that combination of drugs targeting the key enzymes involved in arachidonic acid metabolism,
1
4

ACCEPTED MANUSCRIPT
namely cyclooxygenase-2 and 5-LO, determined a significant increase in ceramide levels in
colon cancer cells [60]. Additional study in human leukemia-derived HL-60 cells showed that
TNF-α-stimulated release of arachidonic acid stimulated hydrolysis of sphingomyelin by
sphingomyelinase and concomitant ceramide generation [61]. Altogether, the growth-
suppressive properties of compound 33 could be associated with its ability to inhibit
arachidonic acid metabolism resulting in the accumulation of arachidonic acid that could, in
turn, regulate cellular ceramide levels probably through the negative regulation of ASAH
activity.
Fig. 5. Western blot analysis of potential protein targets of compounds 30 and 33 in HepG2
cells. Cells were treated with tested compounds at their 2 x IC values for 48 h. Samples were
5
0
normalized for protein loading (50 ꢀg) by reblotting the membrane-bound protein with an
anti-actin antibody. 5-LO, 5-lipoxygenase; p-SK1, phospho-sphingosine kinase 1; ASAH,
acid ceramidase.
1
5

ACCEPTED MANUSCRIPT
2
.2.5. Apoptosis detection
In order to determine whether antiproliferative effects of compounds 30 and 33 that similarly
showed strong and selective cytostatic activity in HepG2 cells could be ascribed to induction
of apoptosis, Annexin V assay was carried out as previously described [62]. We found that
compound 30 resulted in a slight decline in the viable cell population by 9.2% with
concomitant increase in early apoptotic and late apoptotic/primary necrotic cells by 5.2% and
9
.2%, respectively (Table 3, Fig. 6B). Compound 33 induced marked reductions in the viable
cell population by 32.9% accompanied by dramatic increase in early apoptotic cells by 35.3%
in comparison with untreated cells (Table 3, Fig. 6C). Different extent of cell death response
triggered by tested compounds could be associated with their 5-iodoindole or 2-
methylbenzimidazole heterocycle and hydroxyl or methyl substituent at C-7 of the coumarin
moiety.
Table 3.
a
Results of Annexin V assay for apoptosis detection in HepG2 cells.
HepG2 cells (%)
Control
0
30
9.2
Control
9.6
33
5.6
Late apoptotic/primary
necrotic cells
Viable cells
94.2
0.6
5.2
85
5.8
0
38.46
51.9
9.6
5.6
87.2
7.2
Early apoptotic cells
Secondary necrotic
cells
a
Shown are the percentages of viable cells (PI-/Ann V-), early apoptotic cells (PI-/Ann V+), late
apoptotic/primary necrotic cells (PI+/Ann V+), and secondary necrotic cells (PI+) after 48 h treatment
with compounds 30 and 33 at their 2 x IC values.
50
1
6

ACCEPTED MANUSCRIPT
A)
B)
C)
Fig. 6. Visualization of apoptotic cells stained with annexin V-FITC using fluorescence
microscopy at 40x magnification. PI staining was used as nuclear marker. Shown here are
bright-field images (left) and late apoptotic/primary necrotic cells (PI+/Ann V+, right). A)
Untreated HepG2 cells; B) HepG2 cells treated with 2 x IC of compound 30 for 48 h; and C)
5
0
HepG2 cells treated with 2 x IC of compound 33 for 48 h.
5
0
1
7

ACCEPTED MANUSCRIPT
2
.2.6. Antibacterial evaluations
The in vitro antibacterial activity of novel 1,2,3-triazole–coumarin conjugates 4–35 was tested
against Gram-positive bacteria including Staphylococcus (ATCC 25923), Enterococcus
faecalis, vancomycin-resistant Enterococcus faecium (VRE) and Gram-negative bacteria
including Pseudomonas aeurigonsa (ATCC 27853), Escherichia coli (ATCC 25925),
Acinetobacter baumannii (ATCC 19606), extended-spectrum β-lactamase (ESBL)-producing
Klebisiella pneumoniae. The obtained results were compared with known antibiotics
ceftazidime (CAZ) and ciprofloxacin (CIP). To evaluate the activity of synthesized
compounds against bacteria minimum inhibitory concentrations (MICs) were determined and
given in Table 5 for selected compounds. Compounds having MIC values higher than 256
ꢀ
g/mL against all tested bacterial strains were considered inactive and were not included in
the Table 5. Generally, results of antibacterial evaluations revealed that compounds 4–35 did
not exhibit considerable antibacterial activities against tested Gram-positive and Gram-
negative bacteria, with the exception of some selective activities against Enterococcus
species. Compounds showing antibacterial activity exhibited a wide range of logP values (-
0
.20–5.61) suggesting that the effect of lipophilic property of 1,2,3-triazole–coumarin
conjugates on their inhibitory activity against selected seven Gram-positive and Gram-
negative bacteria was not found (Table 5). It can be observed that from the series of
coumarin–1,2,3-triazole hybrids containing substituted phenyl unit in 13–19, compounds with
p-alkylphenyl (16–18) and (p-alkylcyclohexyl)phenyl (19) 4-substituted 1,2,3-triazole moiety
displayed promising activity against Enterococcus faecalis at MICs ranging from 8 to 64
ꢀ
g/mL. On the contrary to this, variously hydroxyalkyl and alkyl substituted 1,2,3-triazole
moiety in 4–12 were deprived of anti-Enterococcus faecalis activities (MICs were > 256
µg/mL). Among the 4-(benzenesulfonamide)methyl-1,2,3-triazole–coumarin conjugates 20–
2
4, compounds with p-methyl- (20) and p-fluorobenzenesulfonamide (22 and 23) subunits
demonstrated inhibitory effect against E. faecalis at 64 and 32 µg/mL of MICs. In addition,
coumarin–1,2,3-triazole hybrids with 4-(methylthiocarbonothioyl)morpholine (25) and -
piperazine (27) units were found to exhibit good potency at 32 ꢀg/mL and 16 ꢀg/mL of MICs,
respectively. It is interesting to note that representatives of afore-mentioned classes, namely
p-pentylphenyl
methylthiocarbonothioyl)piperazine (27) 4-substituted 1,2,3-triazole–coumarin hybrids
revealed the inhibitory activities against clinically isolated vancomycin resistant Enterococcus
(17),
2-chloro-4-fluorobenzenesulfonamide
(23)
and
(
1
8

ACCEPTED MANUSCRIPT
faecium, while the reference antibiotics ceftazidime and ciprofloxacin exhibited the lack of
activity (MICs were > 256 µg/mL).
Table 5.
Antimicrobial activities of selected compounds 10, 12, 15–20, 22–25 and 27 against Gram-
a
positive bacteria S aureus, E. faecalis, vancomycin-resistant E. faecium (VRE) and Gram-
b
negative bacteria including P. aeurigonsa, E. coli, A. baumannii and ESBL -producing K.
pneumoniae.
MIC (µg/mL)
S.
E.
E. faecium
P.
A.
K.
Compd
E. coli
aureus faecalis
VRE
aeurigonsa
baumannii pneumoniae
ESBL
10
12
15
16
17
18
19
20
22
23
> 256
> 256
> 256
> 256
> 256
> 256
> 256
256
128
256
256
64
> 256
> 256
> 256
256
> 256
> 256
> 256
> 256
> 256
> 256
> 256
> 256
> 256
> 256
> 256
> 256
> 256
> 256
> 256
> 256
> 256
> 256
> 256
> 256
> 256
> 256
> 256
> 256
> 256
> 256
> 256
> 256
> 256
> 256
> 256
> 256
> 256
> 256
> 256
> 256
> 256
> 256
> 256
> 256
8
64
16
> 256
> 256
128
64
64
> 256
> 256
64
128
32
64
2
2
2
4
5
7
> 256
> 256
> 256
64
256
32
> 256
> 256
64
> 256
> 256
> 256
4
> 256
> 256
> 256
0.5
> 256
> 256
> 256
16
> 256
> 256
> 256
> 256
> 256
16
CAZ
CIP
256
0.5
> 256
> 256
b
0.25
< 0.125
< 0.125
< 0.125
a
Vancomycin-resistant E. faecium; Extended-spectrum
β
-lactamases (resistant to most
β
-
lactam antibiotics).
1
9

ACCEPTED MANUSCRIPT
3
.
Conclusions
This work focused on the development of new potentially active cytostatic and antibacterial
agents based on coumarin–1,2,3-triazole pharmacophoric system. A variety of 1,2,3-triazole
tethered coumarins (4–35) containing 4-substituted alkyl, phenyl, alkylphenyl,
dithiocarbamate, benzenesulfonamide and benzofused heterocycle subunit were designed and
synthesized by regioselective copper(I)-catalyzed Huisgen 1,3-dipolar cycloaddition. A
physicochemical rationale in selection of C-4 substituents has been applied for designed
coumarin–1,2,3-triazole hybrid library. While the strong relationship between lipophilicity of
the coumarin–1,2,3-triazole library and antiproliferative activities was indicated, such effect
of lipophilicity was not observed on antibacterial activities. The two-class models based on
logP values confirmed that lipophilicity was a quite good parameter for distinguishing active
from inactive compounds for all tested cancer cells lines. In vitro antiproliferative activity of
compounds evaluated against five human cancer cell lines (A549, HepG2, CFPAC-1, HeLa
and SW620) revealed that five of the compounds comprising phenylethyl (13), 3,5-
difluorophenyl (14), 5-iodoindole (30), 2-methylbenzimidazole (33) and benzimidazole (35)
exhibited high antiproliferative activities. We may conclude that benzofused heterocycle unit
proved to be effective in increasing the cytostatic activity with exceptional profile of
compound 33 in HepG2 cells displaying an IC value of 0.9 µM and selectivity index of 50.
5
0
Similarly, 5-iodoindole heterocycle proved to confer selectivity of compound 30 for HepG2
cells with IC of 8.57 µM and selectivity index of 3. Due to their highly selective cytostatic
5
0
activity in HepG2 cells, compounds 30 and 33 were selected for further biological studies.
The underlying mechanism of cytostatic effects of compound 30 included induction of
apoptosis and primary necrosis, whereas compound 33 triggered mainly early apoptosis.
Importantly, the growth-suppressive properties of compound 33 in HepG2 cells could be
associated with its ability to inhibit 5-lipoxygenase (5-LO) and acid ceramidase activities that
may, in turn, lead to accumulation of pro-apoptotic lipids arachidonic acid and ceramide,
respectively. On the contrary, growth inhibition of HepG2 cells by compound 30 was not
associated with down-regulation of the key enzymes regulating leukotriene synthesis from
arachidonic acid and ceramide turnover, which clearly demonstrated that heterocycle subunit
and 5-substituent at coumarin ring in 30 and 33 were crucial for their impact on the regulation
of lipid metabolism in HepG2 cells. Results of antibacterial evaluations revealed a coumarin–
1
,2,3-triazole hybrids comprising p-pentylphenyl (17), 2-chloro-4-fluorobenzenesulfonamide
2
0

ACCEPTED MANUSCRIPT
(23) and dithiocarbamate (27) subunit with selective anti-Enterococcus species activities.
More importantly, whereas the reference antibiotics ceftazidime and ciprofloxacin exhibited
the lack of activity against clinically isolated vancomycin resistant Enterococcus faecium
(MICs were > 256 µg/mL), compounds 17, 23 and 27 demonstrated the inhibitory effect
against E. faecium (VRE).
4
.
Experimental part
4
.1. Materials and methods
Thin layer chromatography (TLC) was performed on pre-coated Merck silica gel 60F-254
plates using an appropriate solvent system and the spots were detected under UV light (254
nm). For column chromatography silica gel (Fluka, 0.063–0.2 mm) was employed, glass
column was slurry-packed under gravity. Melting points (uncorrected) were determined with
Kofler micro hot-stage (Reichert, Wien). Microwave-assisted syntheses were performed in a
o
Milestone start S microwave oven using glass cuvettes at 80 C and 300 W under the pressure
of 1 bar. Elemental analyses were performed in the Central Analytic Service, Ruđer Bošković
Institute, Zagreb. All elemental compositions were within the 0.4% of the calculated values.
1
13
H and C NMR spectra were acquired on a Bruker 300 and 600 MHz NMR spectrometer.
All data were recorded in DMSO-d at 298 K. Chemical shifts were referenced to the residual
6
1
13
solvent signal of DMSO at δ 2.50 ppm for H and δ 39.50 ppm for C. Individual resonances
were assigned on the basis of their chemical shifts, signal intensities, multiplicity of
resonances and H–H coupling constants. Mass spectra were recorded on an Agilent 6410
instrument equipped with electrospray interface and triple quadrupole analyzer (LC/MS/MS).
High performance liquid chromatography was performed on an Agilent 1100 series system
with UV detection (photodiode array detector) using Zorbax C18 reverse-phase analytical
column (2.1 x 30 mm, 3.5 µm).
4
.2. Procedures for the preparation of compounds
4
-(Azidomethyl)-7-hydroxy-2H-chromen-2-one (1a) [46], 4-(azidomethyl)-7-methyl-2H-
chromen-2-one (1b) [51], tert-butyl 4-[(prop-2-ynylthio)carbonothioyl]piperazine-1-
carboxylate (3b) [46], tert-butyl 4-{[(1-(4-(7-hydroxy-2H-chromen-2-on-4-yl)methyl)-1H-
1
,2,3-triazol-4-yl)methylthio]carbonothioyl}piperazine-1-carboxylate
compounds 28–35 [48] were synthesized in accord with procedures given in the literature.
.2.1.General procedure for the synthesis of 2a-e
(26)
[46]
and
4
2
1

ACCEPTED MANUSCRIPT
Pyridine (6.5 eq) was added dropwise in the propargyl amine (1.9 eq) and the solution was
stirred at 0 °C under an argon atmosphere. Corresponding sulfonyl chloride (1 eq) was added
in small portions, and the reaction mixture was stirred overnight at room temperature. The
solvent was evaporated and ethyl acetate (100 mL) was added, followed by 1M HCl (50 ml).
Organic layer was separated, dried over MgSO and evaporated to dryness to give crude
4
product which was then purified by column chromatography.
4
.2.1.1. 4-Methyl-N-(prop-2-ynyl)benzenesulfonamide (2a). Compound 2a was synthesized
according to the general procedure using pyridine (12.3 mL), propargyl amine (2.43 g, 44
mmol) and p-toluenesulfonyl chloride (2.5 g, 22.8 mmol). After purification by column
chromatography (CH Cl ) compound 2a was isolated as white powder (2.2 g, 47%, m.p. =
2
2
1
8
7
2
8
4
1−83 °C). H-NMR (300 MHz, DMSO) δ 8.03 (1H, s, NH), 7.69 (2H, d, J = 8.3 Hz, H-7),
.39 (2H, d, J = 8.0 Hz, H-8), 3.65 (2H, d, J = 2.3 Hz, H-3), 3.05 (1H, t, J = 2.5 Hz, H-1),
13
.38 (3H, s, CH ) ppm. C-NMR (75 MHz, DMSO) δ 142.77 (C-9), 137.56 (C-6), 129.50 (C-
3
), 128.71 (C-7), 79.39 (C-2), 74.60 (C-1), 31.88 (C-3), 20.99 (CH ) ppm.
3
.2.1.2. 4-Chloro-N-(prop-2-ynyl)benzenesulfonamide (2b). Compound 2b was synthesized
according to the general procedure using pyridine (5.1 mL), propargyl amine (1.01 g, 18
mmol) and 4-chlorobenzenesulfonyl chloride (2 g, 9.5 mmol). After purification by column
chromatography (CH Cl ) compound 2b was isolated as white powder (1.6 g, 73%, m.p. =
2
2
1
1
7
06−108 °C). H-NMR (300 MHz, DMSO) δ 8.25 (1H, s, NH), 7.85–7.75 (2H, m, H-7),
13
.73–7.56 (2H, m, H-8), 3.73 (2H, d, J = 2.5 Hz, H-3), 3.05 (1H, t, J = 2.5 Hz, H-1) ppm. C-
NMR (75 MHz, DMSO) δ 139.93 (C-6), 137.84 (C-9), 129.65 (C-8), 129.17 (C-7), 79.58 (C-
), 75.32 (C-1), 32.32 (C-3) ppm.
-Fluoro-N-(prop-2-ynyl)benzenesulfonamide (2c). Compound 2c was synthesized according
2
4
to the general procedure using pyridine (6.9 mL), propargyl amine (1.37 g, 24 mmol) and 4-
fluorobenzenesulfonyl chloride (2.5 g, 12.8 mmol). After purification by column
chromatography (CH Cl ) compound 2c was isolated as white powder (2 g, 73%, m.p. =
2
2
1
8
8−90 °C). H-NMR (600 MHz, DMSO) δ 8.16 (1H, s, NH), 7.89–7.85 (2H, m, H-7), 7.43
1
3
(
2H, t, J = 6.0 Hz, H-8), 3.72 (2H, d, J = 2.4 Hz, H-3), 3.02 (1H, t, J = 2.4 Hz, H-1) ppm. C-
NMR (151 MHz, DMSO) δ 164.19 (d, JC-F = 250.7 Hz, C-9), 136.93 (d, JC-F = 3.0 Hz, C-6),
29.74 (d, JC-F = 9.1 Hz, C-7), 116.1 (d, JC-F = 22.7 Hz, C-8), 79.14 (C-2), 74.40 (C-1), 31.83
C-3) ppm.
1
(
2
2

ACCEPTED MANUSCRIPT
4
.2.1.3. 2-Chloro-4-fluoro-N-(prop-2-ynyl)benzenesulfonamide (2d). Compound 2d was
synthesized according to the general procedure using pyridine (1.4 mL), propargyl amine
0.26 g, 4.6 mmol) and 2-chloro-4-fluorobenzenesulfonyl chloride (0.6 g, 2.5 mmol). After
purification by column chromatography (CH Cl : CH OH = 100 : 1), compound 2d was
(
2
2
3
1
isolated as white powder (406.7 mg, 66%, m.p. = 97−100 °C). H-NMR (300 MHz, DMSO) δ
8
7
1
.38 (1H, s, NH), 8.04 (1H, dd, J = 8.9, 6.0 Hz, H-8), 7.70 (1H, dd, J = 8.8, 2.6 Hz, H-7’),
.41 (1H, td, J = 8.6, 2.6 Hz, H-8’), 3.78 (2H, d, J = 2.4 Hz, H-3), 2.99 (1H, t, J = 2.4 Hz, H-
1
3
) ppm. C-NMR (75 MHz, DMSO) δ 163.96 (d, J = 252.8 Hz, C-9), 134.79 (d, JC-F = 5.2
C-F
Hz, C-6), 133.06 (d, JC-F = 9.8 Hz, C-7’), 132.97 (d, JC-F = 11.6 Hz, C-7), 119.14 (d, JC-F
=
2
5.5 Hz, C-8), 114.75 (d, JC-F = 21.8 Hz, C-8’), 78.96 (C-2), 74.41 (C-1), 31.75 (C-3) ppm.
4
.2.1.4. 4-Nitro-N-(prop-2-ynyl)benzenesulfonamide (2e). Compound 2e was synthesized
according to the general procedure using pyridine (12.3 mL), propargyl amine (0.2 g, 3 mmol)
and 4-nitrobenzenesulfonyl chloride (0.4 g, 1.9 mmol). After purification by column
chromatography (CH Cl ) compound 2e was isolated as white powder (2 g, 77%, m.p. =
2
2
1
1
8
65−167 °C). H-NMR (300 MHz, DMSO) δ 8.52 (1H, s, NH), 8.45–8.39 (2H, m, H-8),
13
.10–8.04 (2H, m, H-7), 3.79 (2H, d, J = 2.5 Hz, H-3), 3.01 (1H, t, J = 2.5 Hz, H-1) ppm. C-
NMR (75 MHz, DMSO) δ 150.09 (C-9), 146.73 (C-6), 128.89 (C-7), 124.86 (C-8), 79.27 (C-
2
), 75.66 (C-1), 32.35 (C-3) ppm.
4
.2.2. Prop-2-ynyl morpholine-4-carbodithioate (3a). CS (0.62 mL, 10.3 mmol) was added
2
dropwise to the solution of morpholine (300 mg, 3.4 mmol) and Na PO x 12 H O (785 mg,
3
4
2
2
.1 mmol) in acetone (20 mL). The reaction mixture was stirred at room temperature for 0.5
h. Then propargyl bromide (0.32 mL, 3.8 mmol) was added and the reaction mixture was
stirred at room temperature for 30 min. Upon completion, the reaction mixture was filtered
and the filtrate was concentrated under reduced pressure. The residue was dissolved in EtOAc
(
350 mL), washed with water, brine, dried over anhydrous Na SO and concentrated under
2
4
1
vacuum to afford compound 3a as a yellow oil (213 mg, 31%). H-NMR (300 MHz, DMSO)
δ 4.14 (4H, d, J = 2.6 Hz, H-7), 3.69–3.66 (6H, m, H-3, H-8), 3.18 (1H, t, J = 2.6 Hz, H-1).
1
3
C-NMR (75 MHz, DMSO) δ 194.03 (C=S), 79.53 (C-2), 74.69 (C-8), 66.02 (C-1), 40.04
C-7), 25.42 (C-3).
.2.3. General procedure for the synthesis of 4
(
4
−25
A mixture of 4-(azidomethyl)-7-hydroxycoumarin, Cu(0) (1.2 eq) and terminal alkyne (1.2
eq), 1M CuSO (0.06 mL) in t-BuOH/H O (1 : 1) (3 mL) and DMF (0.5 mL) was added into
4
2
2
3

ACCEPTED MANUSCRIPT
glass tube with a magnetic stirring bar and sealed with a plastic cap. The synthesis was carried
out at 80 °C for 1h under microwave irradiation (300 W). Completion of the reaction was
monitored by TLC. After the reaction was completed, the solvent was evaporated under
reduced pressure and the residue was chromatographed on silica column.
4
.2.3.1.
Compound 4 was synthesized according to the general procedure using compound 1a (60 mg,
.28 mmol) and ethynyl cyclopropane (0.019 mL, 0.23 mmol). After purification by column
chromatography (CH Cl : CH OH = 20 : 1), compound 4 (21 mg, 81%, m.p. = 154–156 °C)
[4-(4-Cyclopropyl-1H-1,2,3-triazol-1-yl)methyl]-7-hydroxy-2H-chromen-2-one (4).
0
2
2
3
1
was isolated as light yellow powder. H-NMR (600 MHz, DMSO) δ: 10.62 (1H, s, OH), 7.95
1H, s, H-3'), 7.66 (1H, d, J = 8.8 Hz, H-5), 6.82 (1H, dd, J = 8.7, 2.3 Hz, H-6), 6.76 (1H, d, J
2.3 Hz, H-8), 5.80 (2H, s, H-1'), 5.58 (1H, s, H-3), 1.98–1.95 (1H, m, H-5'), 0.96–0.87 (2H,
(
=
1
3
m, H-6'), 0.76 – 0.68 (2H, m, H-7'). C-NMR (151 MHz, DMSO): 161.59 (C-2), 159.93 (C-
7
1
), 155.08 (C-4), 150.47 (C-8a), 149.45 (C-4'), 126.01 (C-5), 121.76 (C-3'), 113.14 (C-6),
09.40 (C-4a), 109.39 (C-3), 102.49 (C-8), 49.06 (C-1'), 7.65 (C-6', C-7'), 6.45 (C-5'). MS
+
(
ESI): m/z = 284.3 ([M + H] ). Anal. calcd. for C H N O : C, 63.60; H, 4.63; N, 14.83.
15
13
3
3
Found: C, 63.38; H, 4.64; N, 14.80.
4
.2.3.2. {4-[4-(3-Chloropropyl)-1H-1,2,3-triazol-1-yl]methyl}-7-hydroxy-2H-chromen-2-one
(5). Compound 5 was synthesized according to the general procedure using compound 1a (60
mg, 0.28 mmol) and 5-chloropentyne (0.024 mL, 0.23 mmol). After purification by column
chromatography (CH Cl : CH OH = 20 : 1), compound 5 (68 mg, 92%, m.p. = 201–203 °C)
2
2
3
1
was isolated as light yellow powder. H-NMR (600 MHz, DMSO) δ: 10.67 (1H, s, OH), 8.04
1H, s, H-3'), 7.67 (1H, d, J = 8.8 Hz, H-5), 6.83 (1H, dd, J = 8.7, 2.3 Hz, H-6), 6.76 (1H, d, J
2.3 Hz, H-8), 5.85 (2H, s, H-1'), 5.58 (1H, s, H-3), 3.68 (2H, t, J = 6.5 Hz, H-7'), 2.79 (2H,
(
=
1
3
t, J = 7.5 Hz, H-5'), 2.16–1.98 (2H, m, H-6'). C-NMR (75 MHz, DMSO): 163.07 (C-2),
60.42 (C-7), 155.57 (C-4), 151.17 (C-8a), 146.59 (C-4'), 126.52 (C-5), 123.74 (C-3'), 113.62
1
(C-6), 109.89 (C-4a), 109.80 (C-3), 102.99 (C-8), 49.56 (C-1'), 45.13 (C-7'), 32.13 (C-5'),
+
2
4
4
2.76 (C-6'). MS (ESI): m/z = 320.7 ([M + H] ). Anal. calcd. for C H ClN O : C, 56.35; H,
1
5
14
3
3
.41; N, 13.14. Found: C, 56.51; H, 4.42; N, 13.15.
.2.3.3. 7-Hydroxy-{4- 4-(2-hydroxyethyl)-1H-1,2,3-triazol-1-yl]methyl}-2H-chromen-2-one
[
(6). Compound 6 was synthesized according to the general procedure using compound 1a (60
mg, 0.28 mmol) and 3-butyn-1-ol (0.017 mL, 0.23 mmol). After purification by column
chromatography (CH Cl : CH OH = 10 : 1) compound 6 (25 mg, 39%, m.p. = 219–221°C)
2
2
3
2
4

ACCEPTED MANUSCRIPT
1
was isolated as light yellow powder. H-NMR (600 MHz, DMSO) δ: 10.66 (1H, s, OH), 7.98
(1H, s, H-3'), 7.68 (1H, d, J = 8.8 Hz, H-5), 6.81 (1H, dd, J = 8.7, 2.3 Hz, H-6), 6.74 (1H, d, J
=
2.3 Hz, H-8), 5.83 (2H, s, H-1'), 5.55 (1H, s, H-3), 4.67 (1H, s, OH), 3.63 (2H, dd, J = 11.1,
1
3
6
.5 Hz, H-6'), 2.78 (2H, t, J = 6.8 Hz, H-5'). C-NMR (75 MHz, DMSO): 162.06 (C-2),
1
60.45 (C-7), 155.57 (C-4), 151.17 (C-8a), 145.04 (C-4'), 126.55 (C-5), 124.07 (C-3'), 113.63
(C-6), 109.92 (C-4a), 109.71 (C-3), 102.99 (C-8), 60.69 (C-1'), 49.47 (C-6'), 29.56 (C-5'). MS
+
(
ESI): m/z = 288.3 ([M + H] ). Anal. calcd. for C H N O : C, 58.53; H, 4.56; N, 14.63.
14
13
3
4
Found: C, 58.41; H, 4.57; N, 14.59.
4
.2.3.4. 7-Hydroxy-{4-[4-(1-hydroxyethyl)-1H-1,2,3-triazol-1-yl]methyl}-2H-chromen-2-one
(7). Compound 7 was synthesized according to the general procedure using compound 1a (60
mg, 0.28 mmol) and 3-butyn-2-ol (0.018 mL, 0.23 mmol). After purification by column
chromatography (CH Cl : CH OH = 30 : 1) compound 7 (75 mg, 81%, m.p. > 250 °C) was
2
2
3
1
isolated as light brown powder. H-NMR (600 MHz, DMSO) δ: 10.64 (1H, s, OH), 8.05 (1H,
s, H-3'), 7.72 (1H, d, J = 8.7 Hz, H-5), 6.84 (1H, dd, J = 8.7, 2.3 Hz, H-6), 6.76 (1H, d, J = 2.4
Hz, H-8), 5.86 (2H, s, H-1'), 5.57 (1H, s, H-3), 5.25 (1H, d, J = 4.9 Hz, OH), 4.04 (1H, q, J =
1
3
5
1
1
.3 Hz, H-5'), 1.42 (3H, d, J = 6.5 Hz, CH ). C-NMR (151 MHz, DMSO): 161.58 (C-2),
3
59.94 (C-7), 156.07 (C-4), 153.17 (C-8a), 150.69 (C-4'), 126.06 (C-5), 122.42 (C-3'),
13.13 (C-6), 109.40 (C-4a), 109.16 (C-3), 102.49 (C-8), 61.54 (C-5'), 49.03 (C-1'), 23.65
+
(
CH ). MS (ESI): m/z = 288.3 ([M + H] ). Anal. calcd. for C H N O : C, 58.53; H, 4.56; N,
3
14 13
3
4
1
4
4.63. Found: C, 58.72; H, 4.55; N, 14.60.
.2.3.5. 7-Hydroxy-{4-[4-(3-hydroxypropyl)-1H-1,2,3-triazol-1-yl]methyl}-2H-chromen-2
one (8). Compound 8 was synthesized according to the general procedure using compound 1a
60 mg, 0.28 mmol) and 4-pentyn-1-ol (0.021 mL, 0.23 mmol). After purification by column
chromatography (CH Cl : CH OH = 10 : 1), compound 8 (55 mg, 79%, m.p. = 98–100 °C)
(
2
2
3
1
was isolated as light yellow powder. H-NMR (300 MHz, DMSO) δ: 10.69 (1H, s, OH), 7.99
1H, s, H-3'), 7.68 (1H, d, J = 8.7 Hz, H-5), 6.93–6.68 (2H, m, H-6, H-8), 5.84 (2H, s, H-1'),
.54 (1H, s, H-3), 4.49 (1H, t, J = 5.1 Hz, OH), 3.43 (2H, dd, J = 11.5, 6.2 Hz, H-7'), 2.68
(
5
1
3
(
2H, t, J = 7.6 Hz, H-5'), 1.76 (2H, dd, J = 14.6, 6.8 Hz, H-6'). C-NMR (75 MHz, DMSO):
62.07 (C-2), 160.43 (C-7), 155.57 (C-4), 151.17 (C-8a), 147.99 (C-4'), 126.51 (C-5), 123.43
1
(C-3'), 113.63 (C-6), 109.91 (C-4a), 109.71 (C-3), 102.99 (C-8), 60.44 (C-1'), 49.52 (C-7'),
+
3
2.64 (C-5'), 22.07 (C-6'). MS (ESI): m/z = 302.3 ([M + H] ). Anal. calcd. for C H N O : C,
1
5
15
3
4
5
9.79; H, 5.02; N, 13.95. Found: C, 59.60; H, 5.01; N, 13.92.
2
5

ACCEPTED MANUSCRIPT
4
.2.3.6. 7-Hydroxy-{4-
[
4-(4-hydroxybutyl)-1H-1,2,3-triazol-1-yl]methyl}-2H-chromen-2-one
(9). Compound 9 was synthesized according to the general procedure using compound 1a (60
mg, 0.28 mmol) and 5-hexyn-1-ol (0.025 mL, 0.23 mmol). After purification by column
chromatography (CH Cl : CH OH = 10 : 1) compound 9 (31 mg, 43%, m.p. = 152–154 °C)
2
2
3
1
was isolated as light yellow powder. H-NMR (300 MHz, DMSO) δ: 10.69 (1H, s, OH), 7.99
1H, s, H-3'), 7.68 (1H, d, J = 8.8 Hz, H-5), 6.83 (1H, dd, J = 8.7, 2.3 Hz, H-6), 6.76 (1H, d, J
2.3 Hz, H-8), 5.84 (2H, s, H-1'), 5.53 (1H, s, H-3), 4.38 (1H, t, J = 5.1 Hz, OH), 3.41 (2H,
dd, J = 11.5, 6.3 Hz, H-8'), 2.64 (2H, t, J = 7.5 Hz, H-5'), 1.63–1.60 (2H, m, H-7'), 1.45 (2H,
(
=
1
3
dt, J = 10.3, 6.7 Hz, H-6'). C-NMR (75 MHz, DMSO): 162.07 (C-2), 160.43 (C-7), 155.57
C-4), 151.18 (C-8a), 148.04 (C-4'), 126.51 (C-5), 123.38 (C-3'), 113.63 (C-6), 109.91 (C-4a),
(
1
6
1
4
09.71 (C-3), 102.99 (C-8), 60.85 (C-1'), 49.52 (C-8'), 32.43 (C-5'), 25.96 (C-7'), 25.28 (C-
+
'). MS (ESI): m/z = 316.3 ([M + H] ). Anal. calcd. for C H N O : C, 60.94; H, 5.43; N,
1
6
17
3
4
3.33. Found: C, 61.05; H, 5.44; N, 13.30.
.2.3.7. 4- (4-Tert-butyl-1H-1,2,3-triazol-1-yl)methyl
[
]
-7-hydroxy-2H-chromen-2-one
(10).
Compound 10 was synthesized according to the general procedure using compound 1a (60
mg, 0.28 mmol) and 3,3-dimethylbutyne (0.028 mL, 0.23 mmol). After purification by
column chromatography (CH Cl : CH OH = 30 : 1), compound 10 (35 mg, 51%, m.p. > 250
2
2
3
1
°
C) was isolated as light yellow powder. H-NMR (300 MHz, DMSO) δ: 10.68 (1H, s, OH),
8
.00 (1H, s, H-3'), 7.70 (1H, d, J = 8.7 Hz, H-5), 6.88–6.72 (2H, m, H-6, H-8), 5.82 (2H, s, H-
1
3
1
'), 5.56 (1H, s, H-3), 1.29 (9H, s, CH ). C-NMR (151 MHz, DMSO): 161.62 (C-2), 159.96
3
(C-7), 156.78 (C-4), 155.08 (C-8a), 150.62 (C-4'), 126.03 (C-5), 120.86 (C-3'), 113.15 (C-6),
1
09.41 (C-4a), 109.27 (C-3), 102.50 (C-8), 48.98 (C-1'), 39.51 (C-5'), 30.19 (CH ). MS (ESI):
3
+
m/z = 300.3 ([M + H] ). Anal. calcd. for C H N O : C, 64.20; H, 5.72; N, 14.04. Found: C,
1
6
17
3
3
6
3.95; H, 5.73; N, 14.01.
.2.3.8. 4- (4-Hexyl-1H-1,2,3-triazol-1-yl)methyl
4
[
]
-7-hydroxy-2H-chromen-2-one
(11).
Compound 11 was synthesized according to the general procedure using compound 1a (30
mg, 0.14 mmol) and octyne (0.03 mL, 0.17 mmol). After purification by column
chromatography (CH Cl : CH OH = 100 : 1), compound 11 (22.5 mg, 51%, m.p. = 200–203
2
2
3
1
°
C) was isolated as yellow powder. H-NMR (DMSO) δ: 10.74 (1H, s, OH), 8.63 (1H, s, H-
'), 7.64 ( 1H, d, J = 8.8 Hz, H-5), 6.80 (1H, dd, J = 8.8, 2.2 Hz, H-6), 6.73 (1H, d, J = 2.1 Hz,
H-8), 5.82 (2H, s, H-1'), 5.50 (1H, s, H-3), 2.61 (2H, t, J = 7.5 Hz, H-5'), 1.57 (2H, dt, J = 7.7
3
13
Hz, H-6'), 1.25–1.22 (6H, m, H-7', H-8', H-9'), 0.83 (3H, t, J = 6.4 Hz, CH ). C-NMR (75
3
2
6

ACCEPTED MANUSCRIPT
MHz, DMSO): 161.81 (C-2), 160.12 (C-7), 154.67 (C-4), 150.84 (C-8a), 147.63 (C-4'),
1
26.05 (C-5), 123.05 (C-3'), 113.32 (C-6), 109.44 (C-4a), 109.21 (C-3), 102.60 (C-8), 49.15
(
C-1'), 30.99 (C-5'), 28.84 (C-6'), 28.16 (C-7'), 24.95 (C-9'), 22.05 (C-8'), 13.94 (CH ). MS
3
+
(
ESI): m/z = 328.4 ([M + H] ). Anal. calcd. for C H N O : C, 66.04; H, 6.47; N, 12.84.
18
21
3
3
Found: C, 65.89; H, 6.45; N, 12.80.
4
.2.3.9. 7-Hydroxy-
[
4-(4-octyl-1H-1,2,3-triazol-1-yl)methyl
]
-2H-chromen-2-one
(12).
Compound 12 was synthesized according to the general procedure using compound 1a (30
mg, 0.14 mmol) and decyne (0.03 mL, 0.16 mmol). After purification by column
chromatography (CH Cl : CH OH = 100 : 1) compound 12 (22.5 mg, 45%, m.p. = 109–111
2
2
3
1
°
C) was isolated as yellow powder. H-NMR (DMSO) δ: 10.86 (1H, s, OH), 7.97 (1H, s, H-
'), 7.65 (1H, d, J = 8.7 Hz, H-5), 6.81 (1H, dd, J = 8.8, 1.1 Hz, H-6), 6.75 (1H, d, J = 2.1 Hz,
H-8), 5.82 (2H, s, H-1'), 5.50 (1H, s, H-3), 2.61 (2H, t, J = 7.5 Hz, H-5'), 1.59–1.55 (2H, m,
3
1
3
H-6'), 1.26–1.19 (10H, H-7'–11'), 0.83 (3H, t, J = 7.0 Hz, CH ). C-NMR (75 MHz, DMSO):
3
1
61.72 (C-2), 160.16 (C-7), 155.02 (C-4), 150.87 (C-8a), 147.68 (C-4'), 126.15 (C-5), 123.10
(C-3'), 113.32 (C-6), 109.53 (C-4a), 109.31 (C-3), 102.63 (C-8), 49.18 (C-1'), 31.30 (C-5'),
2
8.90 (C-6'), 28.75 (C-7'), 28.53 (C-9'), 28.67 (C-8'), 24.96 (C-10'), 22.15 (C-11'), 14.02
+
(
CH ). MS (ESI): m/z = 356.4 ([M + H] ). Anal. calcd. for C H N O : C, 67.58; H, 7.09; N,
3
20 25
3
3
1
4
1.82. Found: C, 67.47; H, 7.11; N, 11.79.
.2.3.10. 7-Hydroxy-
[
4-(4-phenylethyl-1H-1,2,3-triazol-1-yl)methyl
]
-2H-chromen-2-one
(13). Compound 13 was synthesized according to the general procedure using compound 1a
(60 mg, 0.28 mmol) and 4-phenyl-1-butyne (0.032 mL, 0.23 mmol). After purification by
column chromatography (CH Cl : CH OH = 30 : 1) compound 13 (61 mg, 77%, m.p. = 150–
2
2
3
1
1
7
7
5
52°C) was isolated as white powder. H-NMR (600 MHz, DMSO) δ: 10.66 (1H, s, OH),
.88 (1H, s, H-3'), 7.64 (1H, d, J = 8.8 Hz, H-5), 7.22 (2H, dd, J = 10.3, 4.6 Hz, H-9'), 7.18–
.12 (3H, m, H-8', H-10'), 6.81 (1H, dd, J = 8.7, 2.4 Hz, H-6), 6.75 (1H, d, J = 2.3 Hz, H-8),
1
3
.81 (2H, d, J = 0.9 Hz, H-1'), 5.48 (1H, s, H-3), 2.99–2.81 (4H, m, H-5', H-6'). C-NMR
(151 MHz, DMSO): 161.57 (C-2), 159.93 (C-7), 155.05 (C-4), 150.72 (C-8a), 146.65 (C-4'),
1
1
5
1
4
40.98 (C-7'), 128.33 (C-9'), 128.18 (C-8'), 126.00 (C-5), 125.87 (C-10'), 123.20 (C-3'),
13.14 (C-6), 109.36 (C-4a), 109.05 (C-3), 102.47 (C-8), 49.02 (C-1'), 34.70 (C-6'), 26.87 (C-
+
'). MS (ESI): m/z = 348.4 ([M + H] ). Anal. calcd. for C H N O : C, 69.15; H, 4.93; N,
2
0
17
3
3
2.10. Found: C, 69.28; H, 4.92; N, 12.06.
.2.3.11.{4- 4-(3,5-difluorophenyl)-1H-1,2,3-triazol-1-yl]methyl}-7-hydroxy-2H-chromen-2-
[
2
7

ACCEPTED MANUSCRIPT
one (14). Compound 14 was synthesized according to the general procedure using compound
1
a (50 mg, 0.23 mmol) and 1-ethynyl-3,5-difluorobenzene (0.022 mL, 0.19 mmol). After
purification by column chromatography (CH Cl : CH OH = 50 : 1) compound 14 (59 mg,
2
2
3
1
8
8%, m.p. > 250 °C) was isolated as yellow powder. H-NMR (600 MHz, DMSO) δ: 10.82
(1H, s, OH), 8.80 (1H, s, H-3'), 7.63 (1H, d, J = 8.7 Hz, H-5), 7.57 (2H, d, J = 6.0 Hz, H-6'),
7
5
1
1
.20 (1H, ddd, J = 9.3, 5.8, 2.3 Hz, H-8'), 6.80 (1H, d, J = 8.7 Hz, H-6), 6.75 (1H, s, H-8),
1
3
.95 (2H, s, H-1'), 5.81 (1H, s, H-3). C-NMR (75 MHz, DMSO): 163.45 (d, J = 243.8,
C-F
3.5 Hz, C-7'), 162.29 (C-2), 160.41 (C-7), 155.72 (C-4), 150.06 (C-8a), 145.33 (C-4'),
34.35 (C-5'), 126.48 (C-3'), 124.13 (C-5), 113.78 (C-6), 110.76 (C-3), 109.78 (C-4a), 108.55
(d, JC-F = 26.25 Hz, C-6'), 103.75 (t, JC-F = 26.25 Hz, C-8'), 103.08 (C-8), 50.03 (C-1'). MS
+
(
ESI): m/z = 356.1 ([M + H] ). Anal. calcd. for C H F N O : C, 60.85; H, 3.12; F, 10.69; N,
1
8
11
2
3
3
1
4
1.83. Found: C, 61.02; H, 3.13; N, 10.66.
.2.3.12.{4- 4-(4-Bromophenyl)-1H-1,2,3-triazol-1-yl]methyl}-7-hydroxy-2H-chromen-2-one
[
(15). Compound 15 was synthesized according to the general procedure using compound 1a
(60 mg, 0.28 mmol) and 1-bromo-4-ethynylbenzene (41.6 mg, 0.23 mmol). After purification
by column chromatography (CH Cl : CH OH = 30 : 1) compound 15 (25 mg, 54%, m.p. >
2
2
3
1
2
50 °C) was isolated as light brown powder. H-NMR (300 MHz, DMSO) δ: 10.68 (1H, s,
OH), 8.76 (1H, s, H-3'), 7.82 (2H, d, J = 8.5 Hz, H-6'), 7.72‒7.65 (3H, m, H-5, H-7'), 6.85
1
3
(
1H, d, J = 8.7 Hz, H-6), 6.81 (1H, s, H-8), 5.95 (2H, s, H-1'), 5.77 (1H, s, H-3'). C-NMR
(75 MHz, DMSO): 162.29 (C-2), 160.43 (C-7), 155.65 (C-4), 150.38 (C-8a), 146.21 (C-4'),
1
32.35 (C-7'), 130.13 (C-5'), 127.74 (C-6'), 126.46 (C-5), 123.22 (C-3'), 121.51 (C-8'), 113.76
(C-6), 110.46 (C-3), 109.81 (C-4a), 103.07 (C-8), 49.93 (C-1'). MS (ESI): m/z = 398.0 ([M +
+
H] ). Anal. calcd. for C H BrN O : C, 54.29; H, 3.04; N, 10.55. Found: C, 54.44; H, 3.03;
18
12
3
3
N, 10.57.
.2.3.13.{4-[4-(4-Butylphenyl)-1H-1,2,3-triazol-1-yl]methyl}-7-hydroxy-2H-chromen-2-one
4
(16). Compound 16 was synthesized according to the general procedure using compound 1a
(60 mg, 0.28 mmol) and 1-butyl-4-ethynylbenzene (0.04 mL, 0.23 mmol). After purification
by column chromatography (CH Cl : CH OH = 30 : 1) compound 16 (82 mg, 95%, m.p. =
2
2
3
1
2
21–223 °C) was isolated as white powder. H-NMR (600 MHz, DMSO) δ: 10.64 (1H, s,
OH), 8.62 (1H, s, H-3'), 7.76 (2H, d, J=8.2 Hz, H-6'), 7.69 (1H, d, J=8.8 Hz, H-5), 7.27 (2H,
d, J = 8.1 Hz, H-7'), 6.83 (1H, dd, J = 8.7, 2.4 Hz, H-6), 6.77 (1H, d, J = 2.3 Hz, H-8), 5.93
(2H, s, H-1'), 5.77 (1H, s, H-3), 2.60 (2H, t, J = 7.6 Hz, H-9'), 1.57 (2H, p, J = 7.6 Hz, H-10'),
2
8