Fragment-type 4-azolylcoumarin derivatives with anticancer properties

Article abstract of DOI:10.1002/ardp.202100238

Several coumarin derivatives with a directly attached azole substituent at C-4 were synthesized and biologically studied for their anticancer properties. The cell lines used for this investigation (HeLa, K-562, MDA-MB-53, and MCF-7) demonstrated different sensitivities. The best response in the MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) assay was shown by K-562 cells, with compounds displaying activity (3c, IC₅₀ 3.06 μM; 4a, IC₅₀ 5.24 μM; 4c, IC₅₀ 4.7 μM) similar to that of cisplatin (IC₅₀ ~6 μM), which was used as the standard. The studied azole-substituted coumarins demonstrated weaker activity toward other cell lines, except for compound 4c, which was equally potent in the case of MCF-7 cells. Additional biological evaluations supported interference with the cell cycle as a potential mechanism of action and confirmed the absence of toxicity in zebrafish embryos. On the basis of these initial results, 4-azole coumarins should be explored further. Although their activity would need additional optimization, the fact that these compounds are fragment-like structures with MW <300 and clog P <3 offers enough flexibility to fine-tune their drug-like properties.

Full text of DOI:10.1002/ardp.202100238

Received: 2 July 2021
Revised: 22 July 2021
Accepted: 23 July 2021
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DOI: 10.1002/ardp.202100238
F U L L P A P E R
Fragment‐type 4‐azolylcoumarin derivatives with anticancer
properties
Milena Simic1
| Milos Petkovic1 | Predrag Jovanovic1 | Milos Jovanovic1
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Gordana Tasic1 | Irina Besu2 | Zeljko Zizak2 | Ivana Aleksic3
Jasmina Nikodinovic‐Runic3 | Vladimir Savic1
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1Department of Organic Chemistry, Faculty of
Pharmacy, University of Belgrade, Belgrade,
Serbia
Abstract
Several coumarin derivatives with a directly attached azole substituent at C‐4 were
synthesized and biologically studied for their anticancer properties. The cell lines
used for this investigation (HeLa, K‐562, MDA‐MB‐53, and MCF‐7) demonstrated
different sensitivities. The best response in the MTT (3‐(4,5‐dimethyl‐2‐thiazolyl)‐
2,5‐diphenyl‐2H‐tetrazolium bromide) assay was shown by K‐562 cells, with com-
pounds displaying activity (3c, IC50 3.06 μM; 4a, IC50 5.24 μM; 4c, IC50 4.7 μM)
similar to that of cisplatin (IC50 ~6 μM), which was used as the standard. The studied
azole‐substituted coumarins demonstrated weaker activity toward other cell lines,
except for compound 4c, which was equally potent in the case of MCF‐7 cells.
Additional biological evaluations supported interference with the cell cycle as a
potential mechanism of action and confirmed the absence of toxicity in zebrafish
embryos. On the basis of these initial results, 4‐azole coumarins should be explored
further. Although their activity would need additional optimization, the fact that
these compounds are fragment‐like structures with MW <300 and clog P <3 offers
enough flexibility to fine‐tune their drug‐like properties.
2Department of Experimental Oncology,
Institute of Oncology and Radiology of Serbia,
Belgrade, Serbia
3Laboratory for Eco‐Biotechnology and Drug
Development, Institute for Molecular Genetics
and Genetic Engineering, University of
Belgrade, Belgrade, Serbia
Correspondence
Milena Simic and Vladimir Savic, Department
of Organic Chemistry, Faculty of Pharmacy,
University of Belgrade, Vojvode Stepe 450,
11221 Belgrade, Serbia.
Email: milena@pharmacy.bg.ac.rs and
vladimir.savic@pharmacy.bg.ac.rs
Funding information
Ministry of Education, Science and
Technological Development, Republic of
Serbia through Grant Agreement with
University of Belgrade‐Faculty of Pharmacy
No, Grant/Award Number:
K E Y W O R D S
451‐03‐9/2021‐14/200161
anticancer properties, azoles, cell lines, coumarins, embryotoxicity
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1
INTRODUCTION
important medicinal leads, coumarins have been widely investigated
as fluorescence probes as well.[11–14]
Among the secondary metabolites produced by plants or other or-
ganisms, coumarins are arguably one of the most important classes of
heterocyclic compounds.[1,2] The coumarin core can be considered a
useful structure for both natural products and medicinally important
biologically active compounds.[3] As such, coumarins have been in-
tensively studied in recent decades and numerous classes of these
compounds have been synthesized and biologically explored, yielding
a range of activities such as anti‐inflammatory, anticoagulant, anti-
bacterial, antifungal, antiviral, and anticancer.[4–10] Apart from being
Our recent studies of azole‐substituted isocoumarins identified in-
teresting antimicrobial properties of these compounds.[15,16] We also
showed that some of these derivatives also possessed potent cytotoxic
properties. This prompted our work on easily accessible isosteric 4‐azole
coumarins with the aim of exploring the effect of azole substituents on
their anticancer potential. Surprisingly, 4‐azole‐substituted coumarins
were scarcely studied as medicinal compounds.[17]
Anticancer properties of various coumarin derivatives
were comprehensively examined and it is clear that they can
© 2021 Deutsche Pharmazeutische Gesellschaft
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Arch. Pharm. 2021;e2100238.
wileyonlinelibrary.com/journal/ardp
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https://doi.org/10.1002/ardp.202100238

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inhibit the growth, proliferation, and metastasis of cancer cells
(Figure 1).[18–30]
K‐562, and breast cancer cells MDA‐MB‐453 and MCF‐7. The effect
of synthesized derivatives on healthy fibroblast MRC‐5 cells was also
investigated. For this study, the 3‐(4,5‐dimethyl‐2‐thiazolyl)‐2,5‐
diphenyl‐2H‐tetrazolium bromide (MTT) assay was used, which is
based on the spectrophotometric determination of formazan
produced in the reductive transformation of MTT by a cellular
oxidoreductase.[36,37] The result is in direct correlation to the number
of metabolically active cells.
These effects are exerted via various mechanisms including in-
hibition of microtubule polymerization, angiogenesis, carbonic anhy-
drase, the PI3K/AKT/mTOR signaling pathway, acting on apoptosis
proteins, or inhibiting tumor multidrug resistance. However, the rich
medicinal chemistry of coumarins is yet to create a marketed anticancer
drug, and the search for biologically efficient compounds of this type is
still a challenging task for medicinal chemists.[31]
In addition to biological profiling, we also determined selected
basic molecular properties for the synthesized compounds such as
MW and log P with the aim of retaining these within the fragment‐
like properties (Ro3), providing enough flexibility for potential further
structural modification.[38] The majority of drugs are weak acids or
bases, and some of their properties are related to the ionization
state.[39] Although we did not assume that there would be a sig-
nificant effect of ionization of our compounds on their performance
at this stage, pKa values were calculated and used as a measure of
electron‐donating ability. Under near‐neutral conditions, our com-
pounds were not expected to ionize, but pKa would be important in
any broader structure–activity relationship optimization.
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2
RESULTS AND DISCUSSION
Chemistry
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2.1
In recent years, the anticancer properties of coumarin derivatives have
been intensively investigated. Numerous compounds with different
substitution patterns were synthesized and biologically investigated, and
these efforts resulted in the accumulation of a large amount of data in
this area. The 4‐substituted derivatives seem to be particularly inter-
esting as they act via various mechanisms.[32] Our interest in these
derivatives, particularly 4‐azole‐substituted coumarins, was indirectly
generated by results obtained on the isocoumarin series.[15,16] Inter-
estingly, coumarins of this type are rare in the literature, but very few
reports suggested that they might provide interesting leads in the search
for novel anticancer agents (Figure 2).[33,34]
In our experiments, HeLa cells showed low sensitivity toward the
azole‐derived coumarins (Table 1). The most active compounds 3c
(IC50 20.3 µM) demonstrated significantly lower activity than cispla-
tin (IC50 ~7 µM) used in these experiments as the standard.
The myelogenous leukemia cells K‐562 showed different results.
Several compounds showed anticancer activity against this cell line,
with IC50 values slightly below (Table 1, 3c, IC50 3.06 µM; 4a, IC50
5.24 µM; 4c, IC50 4.7 µM) or slightly above (4b, IC50 10 µM) that of
cisplatin (IC50 ~6 µM). The most active compound was the chlor-
opyrazole derivative 3c, which also showed 12‐fold K‐562/MRC‐5
selectivity. Interestingly, small changes in the structure of the chlor-
opyrazole moiety had a strong impact on the activity against K‐562.
Replacement of chlorine with bromine (Table 1, 3e, IC50 167 µM) or
iodine (Table 1, 3j, IC50 >200 µM) resulted in almost complete loss of
activity, while removal of chlorine (Table 1, 3b, IC50 112.5 µM) also
caused a decrease in potency, but to a lesser extent. In the imidazole
series, the most active compound is the ester derivative (Table 1, 3f,
IC50 22 µM). Removal of this substituent creates the parent com-
pound (Table 1, 3d, IC50 44 µM), which showed lower activity, a trend
observed for the pyrazole series, but with a less drastic effect. In-
troduction of the larger benzimidazole substituent (Table 1, 3h, IC50
66 µM) decreased potency, while the presence of two chlorines on
We synthesized a series of 4‐azolyl derivatives and briefly in-
vestigated their biological properties.[35] These compounds are easily
accessible from coumarin 1 in several straightforward steps as out-
lined in Scheme 1. Nucleophilic displacement of bromine in derivative
2 was performed in refluxing MeCN and in the presence of a weak
base affording product 3 in acceptable yields, while thio‐derivative 4
was prepared using Lawesson's reagent. All synthesized derivatives
are outlined in Figure 3.
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2.2
Biological studies
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2.2.1
Cytotoxicity
The anticancer properties of these compounds were studied on four
cell lines: cervical cancer cells HeLa, myelogenous leukemia cells
FIGURE 1 Examples of anticancer coumarins

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FIGURE 2 Some azole‐derived coumarins
SCHEME 1 Synthesis of 4‐azolylcoumarins
FIGURE 3 Structures of the synthesized
4‐azolylcoumarins
the imidazole ring (Table 1, 3i, IC50 >200 µM) proved to be detri-
mental to the biological properties. Based on compounds from the
pyrazole and the imidazole series, it is evident that the substituent at
the heterocyclic ring is very important, but further study is necessary
to establish its precise role. The comparison of compounds with the
parent azoles as coumarin substituents at the C(4), exemplified by 3a,
3b, and 3d, is very interesting. All these compounds in K562 cell line
experiments were less active than the most potent derivative 3c.
However, within the parent compounds, the activity trend was
somehow unexpected. Namely, the pyrazole derivative was the least
active (Table 1, 3b, IC50 112.5 µM), while triazole (Table 1, 3a, IC50
73 µM) and, in particular, imidazole (Table 1, 3d, IC50 44 µM) showed
better potency. The observed results do not seem to correlate to the
calculated log P or pKa and may suggest the involvement of the ring
nitrogens in binding as an electron donor (e.g., H‐bond acceptor). At
least in the parent series, position 3 for nitrogen in the heterocyclic
substituent and its stronger electron‐donor ability seemed to pro-
mote better potency and this is perhaps further supported by the
activity observed for 3g (Table 1, IC50 59 µM) and 3h (Table 1, IC50
66 µM) in comparison with 3b (Table 1, IC50 112.5 µM). Replacement
of the carbonyl with the thiocarbonyl moiety was found to have a
strong impact on the activity. Thus, thio‐derivative 4a (Table 1, IC50
5.24 µM) showed better potency than the corresponding O‐analogue
3a (Table 1, IC50 73 µM). The same trend was observed for

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TABLE 1 IC50 values of 4‐azolylcoumarins against cancer cell lines and healthy cells
IC50 (µM)
Compound
HeLa
82.8
154.1
20.3
95
K562
73
MDAMB453
100
MCF‐7
129.2
197.2
22.1
>200
194
MRC5
119.7
95.6
36.6
192
146
157
84
log P
0.73
1.42
2.02
1.1
pKa
0.41
1.6
MW
213
212
247
212
290
284
263
262
280
338
229
278
228
–
3a
3b
3c
112.5
3.06
44
156.7
30.2
>200
>200
126
0.64
4.04
0.84
1.23
0.21
2.89
0.41
0.88
0.4
3d
3e
>200
50
167
22
2.19
1.64
2.44
2.5
3f
121
3g
103
68
59
170
111
3h
3i
66
143
198
156
>200
>200
43.2
18
60
>200
>200
5.24
10
32
>200
>200
39.2
20
2.24
2.35
1.62
3.39
2.31
–
3j
>200
30.4
23
>200
49.4
99
4a
4b
4c
2.9
35
4.7
80
6.2
5
1.61
–
Cisplatin
7
6
6.7
5.7
–
FIGURE 4 Cell cycle distribution of HeLa cells
after 24 h of continuous action of the investigated
agents
compounds 4b (Table 1, IC50 10 µM) and 4c (Table 1, IC50 4.7 µM)
compared to compounds 3h (Table 1, IC50 66 µM) and 3b (Table 1,
IC50 112.5 µM). Unfortunately, potency toward healthy MRC‐5 cell
lines also increased, hence downgrading the K‐562/MRC‐5
selectivity.
Replacement of carbonyl with thiocarbonyl produced the most active
compound for this cell line 4c (IC50 6.2 µM) but, unfortunately, with
complete loss of MCF‐7/MRC‐5 selectivity.
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Regarding the MDA‐MB‐453 cell line, compared to cisplatin as
the standard, a weak activity is generally observed, with most active
compounds being chloro derivative 3c (IC50 30.2 µM) and dichloro 3i
(IC50 32 µM). The dichloro derivative showed better MDA‐MB‐453/
MRC‐5 selectivity.
2.2.2
Cell cycle analysis
To further examine the mechanisms of action of our compounds,
determination of the cell cycle distribution was performed in HeLa
cells using compounds 3c, 3f, 3i, and 4a–c. After exposure to the
investigated compounds at concentrations corresponding to IC50
values, cells were harvested, stained with propidium iodide (PI), and
subjected to flow cytometry. As shown in Figure 4 and Table S1, after
24‐h exposure to the investigated compounds, the numbers of HeLa
Finally, profiling all compounds against the MCF‐7 cell line de-
monstrated some of the above‐mentioned trends but also some
specificity. Among the derivatives with a carbonyl group, once again,
chloro compound 3c (IC50 22.1 µM) was the most active.

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FIGURE 5 Effects of 4a, 3c, and 4b on the development of zebrafish embryos: (a) Percent of normal, teratogenic, and normal embryos for
each tested compound at 100, 50, 25, and 1 µM. (b) Images of zebrafish embryos treated with 4a, 3c, and 4b at 1, 100, and 100 µM
concentrations, respectively, and 1% (v/v) dimethyl sulfoxide as a control
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cells in the subG1 phase increased in all the experiments. This ac-
cumulation indicated induction of cell death and this was further
confirmed by the decrease in viable cells in the G2/M phase, parti-
cularly in the case of compounds 4b, 4c, and 3c, implying that the
active compounds interfere with the highly regulated cell cycle pro-
cess. At this stage, it is difficult to predict the origin of the activity of
the studied compounds as the cell cycle is a very complex and finely
regulated process with many potential targets from initially im-
portant CDK4/6 to proteins involved in spindle assembly.[40] The
increase in cell number in the subG1 phase may suggest that the
cells are incapable of crossing the G1 checkpoint, which could be a
result of DNA damage.[41] This effect might be correlated to the
functions of p53/p21, CDK2/4/6, and related cyclines, but for
unambiguous rationalization, this needs to be further investigated.
The studied compounds possess structural properties of kinase
inhibitors as they can adopt a near‐planar conformation (mimicking
the adenine part of ATP) with heteroatoms that can create at least
2.2.3
Embryotoxicity of selected compounds
Biological exploration of the azolylcoumarins revealed a certain level
of toxicity in in vitro experiments on the MRC‐5 cell line. Therefore,
we also explored in vivo toxicity of selected compounds in the zeb-
rafish model. Zebrafish is a vertebrate model system often used to
assess the toxicology of biologically active compounds. It has genetic
similarity to and good correlation with humans, while zebrafish body
transparency allows easy and practical visual inspection for any
malformation during embryonic development.[43,44] The effects of
selected coumarins were examined at four concentrations (100, 50,
25, and 1 µM), close to the IC50 values determined in the cytotoxicity
assessment (Table 1). Embryos treated with 4a died after the first
24 h at the three highest concentrations. When treated with 1 µM of
4a, 80% of the embryos developed normally and 20% showed car-
diovascular abnormalities, which was marked as a teratogenic effect
(Figure 5a). None of the remaining tested substances, at four con-
centrations, showed lethal or teratogenic effects on embryos within 5
days (Figure 5b). The developed embryos did not show any mal-
formations on the skeleton or internal organs, suggesting the suit-
ability of 3c and 4b for further development as anticancer drug leads.
one
H bond with the hinge region (the kinase binding
modes I and II).[42] Interactions with several targets are also con-
ceivable as a small size of the compounds implies more flexibility
toward various biomolecules.

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3
CONCLUSION
heated in a nitrogen atmosphere at 82°C for 16 h. After completion
of the reaction, as indicated by TLC, the mixture was cooled to room
temperature, and the solvent was removed under reduced pressure.
The crude mixture was purified by flash chromatography to afford
the product.
Several azolylcoumarin derivatives with fragment‐type properties were
synthesized and their anticancer potential was studied. Biological profiling
was carried out on selected cell lines, among which the myelogenous
leukemia cells K‐562 proved to be the most sensitive. The most active
derivative 3c showed slightly better potency than cisplatin, which is used
as a standard. In addition, when tested on the healthy fibroblast MRC‐5
cells, this compound demonstrated lower activity with 12‐fold K‐562/
MRC‐5 selectivity. Further biological experiments suggested interference
of 3c with the cell cycle and they also demonstrated the absence of
toxicity in the zebrafish model. The biological profile of 3c, together with
the fact that this is a fragment‐like compound with low molecular weight
and low clog P, makes it a good candidate for further development as an
anticancer agent in this class of compounds.
4‐[1,2,4]Triazol‐1‐yl‐chromen‐2‐one (3a)
Compound 3a was synthesized from 4‐bromocoumarin and
1,2,4‐triazole following the general procedure. Flash chromatography
(SiO2, 8:2 v/v diethyl ether/petroleum ether) afforded the product 3a
(46%) as white needles, mp: 234–236°C. IR (attenuated total re-
flection [ATR]) cm−1: 1771, 1721, 1438, 1282, 1217, 1003, 947, 767;
1H NMR (400 MHz, CDCl3) δ 8.59 (s, 1H), 8.29 (s, 1H), 7.95 (d, 1H,
J = 8.0 Hz), 7.68 (t, 1H, J = 8.0 Hz), 7.47 (d, 1H, J = 8.4 Hz), 7.37 (t, 1H,
J = 7.6 Hz), 6.56 (s, 1H); 13C NMR (101 MHz, CDCl3) δ 159.7, 154.4,
153.9, 144.2, 133.6, 125.4, 124.9, 114.4, 109.4; mass spectrometry
(electrospray ionization) [MS (EI)]: m/z 213.0 [M]+, 185.0, 143.9,
130.9, 115.9, 103.0, 88.0, 76.0, 63.0; high‐resolution mass spectro-
metry (HRMS) (ESI/quadrupole time‐of‐flight [Q‐TOF]) m/z calcd. for
[C11H7N3O2 + H+]: 214.0616; found, 214.0616.
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4
EXPERIMENTAL
Chemistry
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4.1
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4.1.1
General
4‐Pyrazol‐1‐yl‐chromen‐2‐one (3b)
Compound 3b was synthesized from 4‐bromocoumarin and pyrazole
following the general procedure. Flash chromatography (SiO2, 1:1 v/v
diethyl ether/petroleum ether) afforded the product 3b (73%) as a
colorless solid, mp: 125–127°C. IR (ATR) cm−1: 1722, 1619, 1437,
1395, 1188, 969, 942, 752, 653; 1H NMR (400 MHz, CDCl3) δ 8.25
(d, 1H, J = 8.0 Hz), 7.93 (s, 1H), 7.92 (s, 1H), 7.62 (t, 1H, J = 7.6 Hz),
7.43 (d, 1H, J = 8.4 Hz), 7.34 (t, 1H, J = 7.6 Hz), 8.81 (s, 1H), 6.45 (s,
1H); 13C NMR (101 MHz, CDCl3) δ 160.7, 154.5, 149.5, 143.5, 132.8,
130.6, 126.7, 124.5, 117.4, 115.0, 108.9, 107.1; MS (EI): m/z 212.0
[M]+, 184.0, 171.9, 155.0, 144.0, 129.0, 116.0, 103.0, 88.0, 78.0,
Infrared (IR) spectra were recorded on an IR Thermo Scientific
NICOLET iS10 (4950) spectrometer. Melting points were determined
using a Boetius PHMK 05 apparatus without correction. 1H and 13C
nuclear magnetic resonance (NMR) spectra were recorded on a
Bruker Ascend 400 (400 MHz) spectrometer (see the Supporting
Information). Deuterochloroform was used as a solvent, and chemical
shifts are given in parts per million (δ) downfield from tetra-
methylsilane as the internal standard. Mass spectral data were re-
corded using an Agilent Technologies 6520 Q‐TOF spectrometer
coupled with an Agilent 1200 HPLC, LTQ Orbitrap XL and an Agilent
Technologies 5975C MS coupled with an Agilent Technologies
6890N GC. Flash chromatography used a silica gel 60 (230–400
mesh), while thin‐layer chromatography (TLC) was carried out using
alumina plates with a 0.25 mm silica layer (Kieselgel 60 F254; Merck).
Compounds were visualized by staining with potassium permanga-
nate solution and Dragendorff reagent. The starting compound,
4‐bromocoumarin (4‐bromo‐chromen‐2‐one), was synthesized from
4‐hydroxycoumarin, TBAB, and P2O5 following the literature proce-
dure.[45] The physicochemical properties of synthesized compounds
were determined using MarvinSketch version 21.3.0‐12862, 2021,
ChemAxon (http://www.chemaxon.com).
63.0, 51.0, 39.0; HRMS (ESI/Q‐TOF) m/z calcd. for [C12H9N2O2
+
H+]: 213.0664; found, 213.0661.
4‐(4‐Chloro‐pyrazol‐1‐yl)‐chromen‐2‐one (3c)
Compound 3c was synthesized from 4‐bromocoumarin and
4‐chloropyrazole following the general procedure. Flash chromato-
graphy (SiO2, 7:3 v/v petroleum ether/diethyl ether) afforded the
product 3c (50%) as a colorless solid, mp: 187–190°C. IR (ATR) cm−1
:
1722, 1621, 1436, 1246, 986, 947, 871, 758; 1H NMR (400 MHz,
CDCl3) δ 8.16 (dd, 1H, J = 8.0 and 1.2 Hz), 7.92 (s, 1H), 7.84 (s, 1H),
7.64 (t, 1H, J = 7.7 Hz), 7.44 (d,1H, J = 8.0 Hz), 7.36 (t, 1H, J = 8.0 Hz),
6.44 (s, 1H); 13C NMR (101 MHz, CDCl3) δ 160.3, 154.5, 148.8,
142.1, 133.1, 128.2, 126.2, 124.7, 117.6, 114.57, 114.4, 107.6; MS
(EI): m/z 246.0 [M]+, 218.0, 189.0, 163.0, 144.0, 116.0, 103.0, 89.0,
The InChI codes of the investigated compound, together with some
biological activity data, are provided as the Supporting Information.
75.0, 63.0, 39.0; HRMS (ESI/Q‐TOF) m/z calcd. for [C12H7ClN2O2
+
H+]: 247.0274; found, 247.0271.
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4.1.2
General procedure for the synthesis of
4‐azolylcoumarins 3[46]
4‐Imidazol‐1‐yl‐chromen‐2‐one (3d)
Compound 3d was synthesized from 4‐bromocoumarin and imidazole
following the general procedure. Flash chromatography (SiO2, 8:2 v/v
diethyl ether/ethyl acetate) afforded the product 3d (88%) as
The mixture of 4‐bromocoumarin (23 mg, 0.10 mmol), azole
(0.12 mmol), and K2CO3 (21 mg, 0.15 mmol) in acetonitrile (2 ml) was

SIMIC ET AL.
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colorless needles, mp: 181–183.5°C. IR (ATR) cm−1: 1732, 1620,
1608, 1482, 1405, 1243, 1086, 948, 872, 759, 658; 1H NMR
(400 MHz, CDCl3) δ 7.87 (s, 1H), 7.70 (t, 1H, J = 8.0 Hz), 7.58 (d, 1H,
179.0, 145.0, 117.0, 101.0, 89.0, 76.0, 63.0, 50.0, 39.1; HRMS (HESI/
orbitrap) m/z calcd. for [C15H9N3O2 + H+]: 264.07730; found,
264.07658.
J = 8.0 Hz), 7.47 (d, 1H, J = 8.0 Hz), 7.38–7.35 (m, 2H) 7.31 (s, 1H); 13
C
NMR (101 MHz, CDCl3) δ 159.9, 154.3, 147.9, 136.8, 133.5, 131.3,
125.0, 124.2, 119.7, 117.9, 115.6, 110.3; MS (EI): m/z 212.0 [M]+,
185.0, 171.0, 156.0, 145.0, 130.0, 117.0, 101.0, 89.0, 76.0, 63.0,
51.0, 39.1; HRMS (HESI/orbitrap) m/z calcd. for [C12H8N2O2 + H+]:
213.06640; found, 213.06586.
4‐Benzoimidazol‐1‐yl‐chromen‐2‐one (3h)
Compound 3h was synthesized from 4‐bromocoumarin and benzi-
midazole following the general procedure. Flash chromatography
(SiO2, diethyl ether) afforded the product 3h (85%) as a colorless
solid, mp: 184–187°C. IR (ATR) cm−1: 1723, 1623, 1605, 1144, 763,
740; 1H NMR (400 MHz, CDCl3) δ 8.17 (s, 1H), 7.94 (d, 1H,
J = 7.6 Hz), 7.75–7.63 (m, 1H), 7.52 (d, 1H, J = 8.0 Hz), 7.46–7.39 (m,
4H), 7.32 (dd, 1H, J = 11.3, 4.0 Hz), 6.59 (s, 1H); 13C NMR (101 MHz,
CDCl3) δ 196.1, 157.7, 144.0, 141.7, 139.4, 133.7, 133.4, 125.8,
124.8, 124.6, 124.4, 124.1, 121.2, 117.7, 117.4, 111.2; MS (EI): m/z
[M]+ 262.1, 245.0, 233.1, 221.1, 206.1, 179.1, 145.0, 116.0, 89.0,
76.0, 63.0, 39.1; HRMS (HESI/orbitrap) m/z calcd. for [C16H10N2O2 +
H+]: 263.08205; found, 263.08234.
4‐(4‐Bromo‐pyrazol‐1‐yl)‐chromen‐2‐one (3e)
Compound 3e was synthesized from 4‐bromocoumarin and 4‐bromo‐
pyrazole following the general procedure. Flash chromatography
(SiO2, 6:4 v/v petroleum ether/diethyl ether) afforded the product,
which was then washed with petroleum ether. Pure compound 3e
was isolated as white needles (47%), mp: 170–173°C. IR (ATR) cm−1
:
1723, 1452, 1185, 984, 945, 757; 1H NMR (400 MHz, CDCl3) δ 8.15
(dd, 1H, J = 8.0 and 0.8 Hz), 7.95 (s, 1H), 7.87 (s, 1H), 7.65 (dd, 1H,
J = 11.4, 4.2 Hz). 7.44 (d, 1H, J = 8.4 Hz), 7.35 (t, 1H, J = 8.4 Hz), 6.44
(s, 1H); 13C NMR (101 MHz, CDCl3) δ 160.3, 154.5, 148.7, 144.0,
133.1, 130.5, 126.1, 124.7, 117.6, 114.5, 107.7, 97.7; MS (EI): m/z
290.0 [M]+, 262.0, 235.0, 206.9, 183.1, 172.0, 155.0, 144.0, 128.0,
103.0, 89.0, 77.1, 63.1, 51.0, 39.0; HRMS (HESI/orbitrap) m/z calcd.
for [C12H7BrN2O2 + H+]: 290.97692; found, 290.97621.
4‐(4,5‐Dichloro‐imidazol‐1‐yl)‐chromen‐2‐one (3i)
Compound 3i was synthesized from 4‐bromocoumarin and 4,5‐
dichloroimidazole following the general procedure. Flash chromato-
graphy (SiO2, 6:4 v/v petroleum ether/ethyl acetate) afforded the
product 3i (62%) as a colorless solid, mp: 190–192°C. IR (ATR) cm−1
:
1727, 1622, 1606, 1397, 1278, 1249, 945, 882, 765; 1H NMR
(400 MHz, CDCl3) δ 7.69 (d, 1H, J = 7.6 Hz), 7.62 (s, 1H), 7.48 (d, 1H,
J = 8.4 Hz), 7.36 (t, 1H, J = 7.6 Hz), 7.20 (d, 1H, J = 8.0 Hz), 6.49 (s,
1H); 13C NMR (101 MHz, CDCl3) δ 159.1, 154.0, 145.4, 134.6, 133.9,
129.0, 125.3, 124.1, 117.7, 115.9, 115.2, 114.3; MS (EI): m/z 280.0
[M]+, 252.9, 245.0, 217.0, 190.0, 178.0, 156.0, 101.0, 89.0, 75.0,
63.0, 51.0, 39.1; HRMS (HESI/orbitrap) m/z calcd. for [C12H6Cl2N2O2
+ H+]: 280.98846; found, 280.98806.
1‐(2‐Oxo‐2H‐chromen‐4‐yl)‐1H‐imidazole‐4‐carboxylic acid ethyl
ester (3f)
Compound 3f was synthesized from 4‐bromocoumarin and ethyl
imidazole‐4‐carboxylate following the general procedure. Flash
chromatography (SiO2, 9:1 v/v diethyl ether/ethyl acetate) afforded
the product 3f (37%) as a colorless solid, mp: 153–155°C. IR (ATR)
cm−1: 1745, 1720, 1622, 1484, 1217, 945, 763; 1H NMR (400 MHz,
CDCl3) δ 7.96 (d, 1H, J = 1.6 Hz), 7.86 (d, 1H, J = 0.8 Hz), 7.77–7.65
(m, 1H), 7.48 (dd, 2H, J = 14.1, 5.1 Hz), 7.39 (t, 1H, J = 7.6 Hz). 6.48 (s,
1H), 4.44 (q, 2H, J = 7.2 Hz), 1.43 (t, 3H, J = 7.2 Hz); 13C NMR
(101 MHz, CDCl3) δ 162.03, 159.36, 154.23, 146.96, 137.2, 136.1,
133.9, 125.3, 124.9, 123.8, 118.0, 115.1, 111.4, 61.2, 14.4; MS (EI):
m/z 284.1 [M]+, 269.0, 256.0, 239.0, 212.0, 199.0, 185.0, 172.0,
155.1, 145.0, 129.0, 118.0, 89.0, 63.0, 51.0, 39.0; HRMS (HESI/or-
4‐(4‐Iodo‐pyrazol‐1‐yl)‐chromen‐2‐one (3j)
Compound 3j was synthesized from 4‐bromocoumarin and 4‐
iodopyrazole following the general procedure. Flash chromatography
(SiO2, 7:3 v/v petroleum ether/diethyl ether) afforded the product 3j
(93%) as a colorless solid, mp: 173–176°C. IR (ATR) cm−1: 1720,
1621, 1429, 1185, 984, 937, 765, 742; 1H NMR (400 MHz, CDCl3) δ
8.13 (d, 1H, J = 8.4 Hz), 7.98 (s, 1H), 7.89 (s, 1H), 7.63 (t, 1H,
J = 8.0 Hz), 7.42 (d, 1H, J = 8.0 Hz), 7.34 (t, 1H, J = 8.0 Hz), 6.44 (s,
1H); 13C NMR (101 MHz, CDCl3) δ 160.4, 154.4, 148.6, 148.3, 134.9,
133.1, 126.1, 124.6, 117.5, 114.5, 107.6; MS (EI): m/z 338.0 [M]+,
310.0, 255.0, 156.0, 144.0, 127.0, 116.0, 101.0, 89.0, 63.0, 51.1,
39.1; HRMS (HESI/orbitrap) m/z calcd. for [C12H7IN2O2 + H+]:
338.96305, found 338.96321.
bitrap) m/z calcd. for [C15H12N2O4
+
H+]: 285.08753; found,
285.08685.
4‐Benzotriazol‐1‐yl‐chromen‐2‐one (3g)
Compound 3g was synthesized from 4‐bromocoumarin and benzo-
triazole following the general procedure. Flash chromatography
(SiO2, 6:3:1 v/v/v petroleum ether/diethyl ether/dichloromethane)
afforded the product 3g (80%) as a colorless solid, mp: 190–193°C. IR
(ATR) cm−1: 1728, 1623, 1606, 1494, 1426, 1185, 1027, 937, 744;
1H NMR (400 MHz, CDCl3) δ 8.24 (d, 1H, J = 8.4 Hz), 7.85 (d, 1H,
J = 8.0 Hz), 7.72–7.68 (m, 3H), 7.57–7.51 (m, 2H), 7.36 (t, 1H,
J = 7.2 Hz), 6.70 (s, 1H); 13C NMR (101 MHz, CDCl3) δ 160.1, 154.6,
146.8, 146.2, 133.5, 132.8, 129.6, 126.1, 125.6, 124.9, 120.9, 117.6,
115.0, 110.5, 109.9; MS (EI): m/z 263.0 [M]+, 235.0, 207.0, 190.0,
|
4.1.3
General procedure for the synthesis of
4‐azolylthiocoumarins 4[47]
A mixture of 4‐azolylcoumarin (0.10 mmol) and Lawesson's reagent
(40.4 mg, 0.1 mmol) in anhydrous toluene (10 ml) was stirred at
110°C for several hours. After completion of the reaction, as

8 of 10
SIMIC ET AL.
|
indicated by TLC, the mixture was cooled to room temperature and
the solvent was removed under reduced pressure. The crude mixture
was purified by flash chromatography to afford the product.
MB‐453 and MCF7), and the noncancerous cell line MRC‐5 (human
embryonic lung fibroblasts) were grown in complete RPMI‐1640
medium.
4‐[1,2,4]Triazol‐1‐yl‐chromene‐2‐thione (4a)
|
Compound 4a was synthesized from 4‐[1,2,4]triazol‐1‐yl‐chromen‐2‐
one and Lawesson's reagent following the general procedure. Flash
chromatography (SiO2, 8:2 v/v diethyl ether/petroleum ether) af-
forded the product 4a (52%) as a yellow solid, mp: 166–169°C. IR
(ATR) cm−1: 1606, 1554, 1511, 1438, 1366, 1179, 1004, 758, 667;
1H NMR (400 MHz, CDCl3) δ 8.62 (s, 1H), 8.29 (s, 1H), 8.06 (d, 1H,
J = 7.2 Hz), 7.71 (t, 1H, J = 7.2 Hz), 7.58 (d, 1H, J = 8.4 Hz), 7.42 (t, 1H,
J = 8.0 Hz), 7.33 (s, 1H); 13C NMR (101 MHz, CDCl3) δ 195.6, 157.7,
154.1, 144.1, 138.8, 133.6, 125.9, 125.4, 121.9, 117.4, 115.8; MS
(EI): m/z 229.0 [M]+, 212.9, 202.0, 185.0, 175.0, 159.9, 145.9, 131.9,
119.0, 103.0, 89.0, 63.1, 39.1; HRMS (ESI/Q‐TOF) m/z calcd. for
[C11H7N3OS + H+]: 230.0388; found, 230.0385.
4.2.2
Determination of cell survival
Stock solutions of the investigated compounds (10 mM) were pre-
pared in dimethyl sulfoxide (DMSO) and then dissolved in complete
medium to achieve adequate working concentrations. The final
concentration of the DMSO solvent never exceeded 0.5%, a con-
centration considered nontoxic to the cells. Target adherent cells
HeLa (2500 cells/well), MDA‐MB‐453 (3000 cells/well), MCF7
(7000 cells/well), and MRC‐5 (5000 cells/well) were seeded into the
wells of a 96‐well flat‐bottom microtiter plate. Twenty‐four hours
later, after the cell adherence, different concentrations of in-
vestigated compounds were added to the wells, except for the
controls, where only the complete medium was added. For non‐
adherent K562 cells (6000 cells/well), the compounds were applied
2 h after cell seeding. Culture medium with the corresponding con-
centrations of the investigated compounds, but without cells, was
used as a blank. The cultures were incubated for 72 h, and the effects
of the investigated compounds on cancer and normal cell survival
were determined using the MTT variant of the microculture tetra-
zolium assay (MTA), according to Mosmann,[36] with modification by
Ohno and Abe,[37] 72 h after the addition of the investigated com-
pounds. Briefly, 20 μl of MTT dye solution (5 mg/ml of MTT in
phosphate‐buffered saline) was added to each well. Samples were
incubated for an additional 4 h at 37°C in a humidified atmosphere of
5% CO2 (v/v). Afterward, 100 µl of sodium dodecyl sulfate was added
to extract the insoluble formazan, which represents the product of
the conversion of the MTT dye by viable cells. The number of viable
cells in each well is proportional to the intensity of the absorbance (A)
of light, which was measured in a microtiter plate reader at 570 nm
after 24 h. To determine cell survival (S%), the A of a sample with cells
grown in the presence of various concentrations of the investigated
compounds was divided by the control optical density (the A of
control cells grown only in nutrient medium) and multiplied by 100.
The A of the blank was always subtracted from the A of the corre-
sponding sample incubated with the target cells. All experiments
were performed in triplicate.
4‐Benzoimidazol‐1‐yl‐chromene‐2‐thione (4b)
Compound 4b was synthesized from 4‐benzoimidazol‐1‐yl‐chromen‐
2‐one and Lawesson's reagent following the general procedure. Flash
chromatography (SiO2, 8:2 v/v diethyl ether/petroleum ether) afforded
the product 4b (70%) as a yellow solid, mp: 197–200°C. IR (ATR) cm−1
:
1602, 1533, 1506, 1399, 1179, 1113, 727; 1H NMR (400 MHz, CDCl3)
δ 8.18 (s, 1H), 7.93 (d, 1H, J = 7.6 Hz), 7.72 (t, 1H, J = 7.8 Hz). 7.63 (d, 1H,
J = 8.4 Hz), 7.48 (d, 1H, J = 8.0 Hz), 7.45–7.40 (m, 3H), 7.36–7.35 (m,
2H); 13C NMR (101 MHz, CDCl3) δ 196.1, 157.7, 144.0, 141.7, 139.4,
133.7, 133.4, 125.8, 124.8, 124.6, 124.4, 124.1, 121.2, 117.7, 117.4,
111.2; MS (EI): m/z 278.0 [M]+, 262.0, 245.0, 234.0, 221.0, 206.0,
160.9, 101.0, 89.0, 63.0; HRMS (HESI/orbitrap) m/z calcd. for
[C16H10N2OS + H+]: 279.05921; found, 279.05880.
4‐Pyrazol‐1‐yl‐chromene‐2‐thione (4c)
Compound 4c was synthesized from 4‐pyrazol‐1‐yl‐chromen‐2‐one
and Lawesson's reagent following the general procedure. Flash
chromatography (SiO2, 8:2 v/v petroleum ether/diethyl ether) af-
forded the product 4c (30%) as yellow needles, mp: 163–165.5°C. IR
(ATR) cm−1: 1600, 1548, 1441, 1393, 1354, 1139, 975, 773, 751; 1
H
NMR (400 MHz, CDCl3) δ 8.41 (d, 1H, J = 8.0 Hz), 7.96–7.93 (m, 2H),
7.66 (t, 1H, J = 8.0 Hz), 7.55 (d, 1H, J = 8.4 Hz), 7.39 (t, 1H, J = 8.0 Hz),
7.29 (s, 1H), 6.62 (s, 1H); 13C NMR (101 MHz, CDCl3) δ 196.5, 157.9,
144.0, 141.8, 133.0, 130.5, 126.9, 125.5, 120.1, 117.3, 116.3, 109.3;
MS (EI): m/z 228.0 [M]+, 212.0, 201.0, 184.0, 169.0, 132.0, 114.0,
89.0, 63.0, 51.1, 39.1; HRMS (HESI/orbitrap) m/z calcd. for
[C12H8N2OS + H+]: 229.04356; found, 229.04307.
|
4.2.3
Cell cycle determination
Aliquots of 5 × 105 control cells or cells treated with investigated
compounds for 24 h (concentrations corresponded to IC50 values for
72 h determined in the MTT test) were fixed in 70% ethanol on ice
for 1 week and centrifuged. The pellet was treated with RNase A
(100 μg/ml) at 37°C for 30 min and then incubated with 40 μg/ml PI
for at least 30 min. Cells were analyzed using a FACSCalibur flow
cytometer (BD Biosciences Franklin Lakes) equipped with a 15 mW,
air‐cooled 488 nm argon‐ion laser for excitation of PI. PI fluorescence
|
4.2
Biology
|
4.2.1
Cell lines
Human cervix adenocarcinoma cell line (HeLa), human chronic mye-
logenous leukemia cells (K562), human breast cancer cell lines (MDA‐

SIMIC ET AL.
9 of 10
|
was collected with FL2 detector (yellow orange channel) after pas-
sing a 585/42‐nm band pass filter. A FACSCalibur flow cytometer
is equipped with an FL2 upgraded doublet discrimination module,
which enables screening and then exclusion of the possible oc-
currence of cell doublets, clumps, and debris by plotting FL2‐area
versus FL2‐width signals.[48] PI fluorescence data were collected
using linear amplification. A minimum of 10,000 events was col-
lected on each sample. Finally, data were analyzed using CELL-
Quest 3.2.1.f1 software (BD Biosciences).
ORCID
Milena Simic
http://orcid.org/0000-0002-0865-5509
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