Novel 6- and 7-substituted coumarins with inhibitory action against lipoxygenase and tumor-associated carbonic anhydrase IX

Article abstract of DOI:10.3390/molecules23010153

A series of carboxamide derivatives of 6- and 7-substituted coumarins have been prepared by an original procedure starting from the corresponding 6- or 7-hydroxycoumarins which were alkylated with ethyl iodoacetate, and the obtained ester was converted to the corresponding carboxylic acids which were thereafter reacted with a series of aromatic/aliphatic/heterocyclic amines leading to the desired amides. The new derivatives were investigated as inhibitors of two enzymes, human carbonic anhydrases (hCAs) and soy bean lipoxygenase (LOX). Compounds 4a and 4b were potent LOX inhibitors, whereas many effective hCA IX inhibitors (K_Is in the range of 30.2–30.5 nM) were detected in this study. Two compounds, 4b and 5b, showed the phenomenon of dual inhibition. Furthermore, these coumarins did not significantly inhibit the widespread cytosolic isoforms hCA I and II, whereas they were weak hCA IV inhibitors, making them hCA IX-selective inhibitors. As hCA IX and LOX are validated antitumor targets, these results are promising for the investigation of novel drug targets involved in tumorigenesis.

Full text of DOI:10.3390/molecules23010153

molecules
Article
Novel 6- and 7-Substituted Coumarins with
Inhibitory Action against Lipoxygenase and
Tumor-Associated Carbonic Anhydrase IX
Aikaterini Peperidou 1,2, Silvia Bua 1, Murat Bozdag 3, Dimitra Hadjipavlou-Litina 2
and
ID
Claudiu T. Supuran 1,
*
ID
1
Dipartimento Neurofarba, Sezione di Scienze Farmaceutiche e Nutraceutiche, Università degli Studi di
Firenze, Via Ugo Schiff 6, 50019 Sesto Fiorentino (Firenze), Italy; peperidou@pharm.auth.gr (A.P.);
silvia.bua@unifi.it (S.B.)
Department of Pharmaceutical Chemistry, School of Pharmacy, Faculty of Health Sciences,
54124 Thessaloniki, Greece; hadjipav@pharm.auth.gr
Dipartimento di Chimica, Laboratorio di Chimica Bioinorganica, Università degli Studi di Firenze,
Via della Lastruccia 3, 50019 Sesto Fiorentino (Firenze), Italy; bozdag.murat@unifi.it
Correspondence: claudiu.supuran@unifi.it; Tel./Fax: +39-055-4573729
2
3
*
Received: 11 December 2017; Accepted: 11 January 2018; Published: 12 January 2018
Abstract: A series of carboxamide derivatives of 6- and 7-substituted coumarins have been prepared
by an original procedure starting from the corresponding 6- or 7-hydroxycoumarins which were
alkylated with ethyl iodoacetate, and the obtained ester was converted to the corresponding carboxylic
acids which were thereafter reacted with a series of aromatic/aliphatic/heterocyclic amines leading
to the desired amides. The new derivatives were investigated as inhibitors of two enzymes,
human carbonic anhydrases (hCAs) and soy bean lipoxygenase (LOX). Compounds 4a and 4b were
potent LOX inhibitors, whereas many effective hCA IX inhibitors (KIs in the range of 30.2–30.5 nM)
were detected in this study. Two compounds, 4b and 5b, showed the phenomenon of dual inhibition.
Furthermore, these coumarins did not significantly inhibit the widespread cytosolic isoforms hCA I
and II, whereas they were weak hCA IV inhibitors, making them hCA IX-selective inhibitors. As hCA
IX and LOX are validated antitumor targets, these results are promising for the investigation of novel
drug targets involved in tumorigenesis.
Keywords: coumarins; carboxamides; carbonic anhydrase; lipoxygenase; enzyme inhibitor
1. Introduction
Vertebrates, including humans, encode for a multitude of metalloenzymes belonging to the
carbonic anhydrase (CA, EC 4.2.1.1) family of proteins [
are known to date ( -, -, -, -, -, - and -CAs) [ ], only
15 different isoforms, 12 of which are catalytically active and involved in a multitude of physiologic
functions [ ]. By catalyzing the reversible hydration of CO2 to bicarbonate, with the release of a
hydronium ion, in humans CAs are involved in pH regulation, biosynthetic reactions, electrolyte
secretion, excretion, tumorigenesis, etc. [ ]. CA inhibitors (CAIs) are in pharmacological/clinical
use for decades for the treatment of glaucoma [ ], for the imaging and treatment of hypoxic
1–
4]. Although seven CA genetic families
α
β
γ
δ
ζ
η
θ
2,5
α
-CAs are present in humans, but as
3–9
3,4,6–9
6,7
tumors [3,4,8,9], as anti-obesity agents [10], or as diuretics [11]. Recently these pharmacological agents
were validated for the management of neuropathic pain [12], but the sulfonamides, which are the main
class of CAIs [11
catalytically active CA isoforms, and not only the ones targeted for a specific application [
Thus, alternative classes of CAIs to the sulfonamides and their isosteres were explored in the last
–13] possess a rather large number of side effects, as they indiscriminately inhibit all
1–3,13–17].
Molecules 2018, 23, 153; doi:10.3390/molecules23010153
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Molecules 2018, 23, 153
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period [14], which led to the discovery of several totally different inhibition mechanisms and families of
inhibitors [14]. Among them, the coumarins are among the most relevant ones for several reasons [18].
Discovered initially in a natural product library isolated from an Australian biota [18], the coumarins
were demonstrated to possess a very particular inhibition mechanism [18
prodrug, suicide inhibitors which undergo a hydrolytic process within the enzyme active site with
generation of 2-hydroxycinnamic acid derivatives [18 19]. These relatively bulky compounds cannot
bind to the catalytic metal ion, which is a Zn(II) ion in -CAs, and is situated deep within the active
site [ ]. Instead, the hydrolyzed coumarins were observed (by means of X-ray crystallography) to
be bound at the entrance of the active site cavity, which is rather large for the hCAs [ 18 19].
,19]. Indeed, they act as
,
α
1–3
1–3, ,
Furthermore, that is the only region of the active site which is the most variable between the
12 catalytically active isoforms, which may explain why the coumarins and their derivatives are among
the most isoform-selective CAIs known to date [19–25]. Indeed, extensive drug design campaigns in
which various parts of the coumarin moiety were changed, showed the useful as well as the detrimental
substitution patterns as well as the tolerated or less tolerated substituents that can be appended to the
ring system in order to obtain effective and isoform-selective CAIs [18–25]. Among the most effective
coumarin CAIs detected in this way it has been observed that 6- and 7- or 6,7-disubstituted derivatives
possess an effective inhibition of the tumor-associated isoforms CA IX and XII, whereas they are
poor inhibitors or do not significantly inhibit the widespread “house-keeping” isoforms hCA I and II
(the inhibition of which is responsible for the side effects of the sulfonamide CAIs [1–4]). Thus, here we
continue our research in developing non-sulfonamide CAIs and report a new series of coumarins
possessing 6- and 7 moieties which have not been explored earlier, of the ether-carboxamide type.
Lipoxygenase (LOX) plays a major role in many inflammatory diseases including chronic
obstructive pulmonary disease (COPD), asthma, chronic bronchitis, cancer including pancreatic,
gastric and brain tumors. Similarly to different isozymes of CA, such as CA II, LOX is expressed
in pancreatic, gastric as well as brain tumors [26]. It should to be mentioned that morphological
cells changes and CA activity are used to determine the effect of LOX inhibitors on cancer cell
differentiation [26]. LOX is upregulated in cancer cells and arachidonic acid as well as its metabolites,
5-HETE and 12-HETE, stimulate mitogenesis of human pancreatic cancer cells. Furthermore, blockade
of LOX pathways abolishes cancer cell proliferation in vitro and induces cancer cell apoptosis [27].
The development of coumarins as antioxidant agents, anticancer and LOX inhibitors has attracted
much attention recently [27
the knowledge in this field [27
new polyfunctional drugs [30
–
29]. Several reviews and research papers have updated and expanded
29]. In recent years, intensive research has been conducted on creating
31]. For the treatment of complex diseases e.g., neurological disorders,
–
,
cancer and inflammation, in which more than one target is implicated, a combination of drugs is
frequently used. Therefore, novel potent inhibitors of both LOX and CA II are required to explore the
role of these enzymes further and to enable the drug discovery efforts. Thus, we considered of interest
to prepare and test new compounds as dual CA and LOX inhibitors.
2. Results and Discussion
A large number of variously substituted coumarins were reported to act as CAIs [18
–25] and to
also possess diverse other biological/pharmacological actions [30]. For example, the development of
coumarins as antioxidant agents, anticancer and LOX inhibitors has attracted much attention [27,29,31–33]
It has been also reported that antioxidant polyphenols structurally related to coumarins effectively
inhibited CAs [34]. In recent years, intensive research has been conducted for creating new polyfunctional
drugs for the treatment of complex diseases, in which more than one target is implicated [30–35].
No carboxamide derivatives of coumarins at the 6- or 7-position of the ring were explored
so far. Thus, the drug design strategy was to obtain the carboxymethyl-oxy derivatives 3a
and 3b, which possess a reactive COOH moiety, easy to derivatize with aromatic, aliphatic or
heterocyclic amines, in order to generate chemical diversity. Thus, commercially available 6- or
.

Molecules 2018, 23, 153
3 of 17
7-hydroxy-coumarins 1a
–b
were reacted with ethyl iodoacetate leading to esters 2a and 2b, which were
then hydrolysed in alkaline medium to the corresponding acids 3a and 3b (Scheme 1).
Scheme 1. Preparation of the key intermediate carboxylic acids 3a and 3b.
Scheme 2. Preparation of amides 4a,b–13a,b reported in the paper.
The two carboxylic acids 3a and 3b were converted to the corresponding amides by reaction with
aromatic, aliphatic and heterocyclic primary amines, as shown in Scheme 2, by using carbodiimide
chemistry. The nature of the various amines was chosen in such a way as to generate the widest
possible chemical diversity (Scheme 2). All compounds were extensively characterized by spectral and
other physico-chemical procedures which proved their structure (see Experimental part for details).
The new coumarins (5a
8a ) were investigated here for the inhibition of four physiologically relevant CA isoforms,
hCA I and II (cytosolic, widespread isoforms, involved in glaucoma and other eye diseases [ ],
,b–6a,b, 9a,b–13a,b) and previously reported coumarins (2a–b, 3a–b,
7a
–
b,
–b
1,3,6
hCA IV (membrane-bound isoform highly abundant in the kidney and lungs and involved in
diuresis, respiration and retinitis [11] as well as hCA IX (tumor-associated, transmembrane isoforme,
a newly validated antitumor target [3,4,9]. A stopped-flow CO2 hydrase assay has been used
for monitoring the inhibition of these CAs with the new coumarins and acetazolamide (AAZ
,
5-acetamido-1,3,4-thiadiazole-2-sulfonamide, a clinically used CAI) as standard inhibitor [1,3].
As seen from the data in Table 1, like other coumarins investigated by our group these
derivatives also do not inhibit the cytosolic isoforms hCA I and II up to 10 M concentration of
inhibitor in the assay system. hCA IV was also poorly inhibited, with most compounds being
inactive whereas few of them showed activity in the high nanomolar range (e.g., 8a 8b and 11a
with KIs in the range of 350.4–848.3 nM). Several other coumarins, incuding 2a 3a and 7b, were
micromolar hCA IV inhibitors, with KIs in the range of 2.65–8.48 M. Thus, the 4-fluoroanilides of
µ
,
,
,
µ

Molecules 2018, 23, 153
4 of 17
both 2-((2-oxo-2H-chromen-7-yl)oxy)acetic acid as well as its 6-isomer led to the best inhibitors of
this isoform.
hCA IX on the other hand was effectively inhibited by most new coumarins reported here,
except for 12a and 12b which were not hCA IX inhibitors up to 10 µM (Table 1). These compounds
incorporate the morpholine-ethylamide moiety which is obviously inappropriate for obtaining effective
CAIs in that position of the coumarin ring and with this type of substitution pattern. The remaining
compounds showed an interesting hCA IX inhibitory patterns, with several compounds being quite
effective inhibitors, with KIs in the range of 30.2–30.5 nM, similar to AAZ (KI of 25 nM).
Table 1. Inhibition data of CA I, II, IV and IX with compounds reported here and the standard
sulfonamide inhibitor acetazolamide (AAZ) by a stopped flow CO2 hydrase assay [36].
KI (nM) *
Compound
hCA I
hCA II
hCA IV
hCA IX
2a
2b
3a
3b
4a
4b
5a
5b
6a
6b
7a
7b
8a
8b
9a
9b
10a
10b
11a
11b
12a
12b
13a
13b
AAZ
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
250
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
12
5572
>10,000
8480
247.1
2044
290.2
194.9
165.7
30.5
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
2649
83.7
30.2
2536
2785
200.6
201.9
136.1
145.6
2732
2041
2377
2147
122.3
1962
>10,000
>10,000
1969
273.7
25
350.4
848.3
>10,000
>10,000
>10,000
>10,000
766.4
>10,000
>10,000
>10,000
>10,000
>10,000
74
* Mean from 3 different assays, by a stopped flow technique (errors were in the range of
values, data not shown).
±
5−10% of the reported
These compounds, 4b and 5b, are the phenethylamide and benzylamide derivatives of
2-((2-oxo-2H-chromen-6-yl)oxy)acetic acid 3b and they are much more effective CA IX inhibitors
compared to the corresponding 6-isomers 4a and 5a (Table 1). However, this was not always the
case, as for other pairs of isoforms, the 7-isomer was a better hCA IX inhibitor compared to the
corresponding 6-isomer (e.g., 2a, which is a better inhibitor than 2b
IX inhibitor compared to its isomer 11b, etc.). Many other coumarins were slightly less effective hCA
IX inhibitors, with KIs in the range of 83.7–290.2 nM. They include derivatives 2a 3a 3b 4a 5a 7a
7b 8a 8b 11a 13b (Table 1). It is thus obvious that apart from the position in the coumarin ring
; 11a, a much more effective CA
,
,
,
,
,
,
,
,
,
,
where the substituent is appended, the most important factor influencing hCA IX inhibition is the
nature of the moiety present on the amide part of the functionality. Indeed, the effective hCA IX
inhibitors incorporate amides obtained from phenethylamine, benzylamine, aniline and substituted
anilines. The only heterocyclic derivative leading to effective inhibitors was 4-pyridylmethylamine

Molecules 2018, 23, 153
5 of 17
and piperidin-1-yl-ethylamine. The remaining amides (2b
,
6a
,
6b
,
9a
,
9b
,
10a
,
10b
,
11b
,
13a) were
micromolar hCA IX inhibitors, with KIs in the range of 1.96–2.73 µM (Table 1).
An important feature of many coumarins reported here is that they are highly selective hCA IX
versus hCA I/II/IV inhibitors, and in many cases also very effective in inhibiting the tumor-associated
isoform hCA IX without inhibition of the widespread cytosolic/membrane—bound isoforms I; II and
IV. For example 4b and 5b are equipotent to acetazolamide as hCA IX inhibitors but do not inhibit at
all hCA I, II and IV, whereas AAZ inhibits these three isoforms significantly (Table 1).
In vitro inhibition of soybean lipoxygenase (LOX) has also been investigated with the new
coumarins reported here (Table 2). Eicosanoids are oxygenated metabolites of arachidonic acid
with a broad implication in a diversity of diseases among which are included the pathogenesis of
neutrophil-mediated inflammatory diseases with a marked relation to the severity of cardiovascular
diseases, asthma and cancer [36].
Table 2. In vitro inhibition of soybean LOX (IC50 µM or % LOX inhibition) [34] and lipophilicity values
of 6- or 7- substituted coumarin derivatives 1–13 and their clogP values.
% LOX Inhibition at 100 Mµ b
IC50 (µM) b
Compounds
ClogP a
1a (7-HC)
1b (6-HC)
2a d
2b d
3a d
3b d
4a
1.62
1.62
1.89
1.89
1.03
1.03
2.60
2.60
2.39
2.39
2.53
2.53
2.36
2.36
2.76
2.76
2.80
2.80
1.11
1.11
0.89
0.89
0.91
0.91
2.32
2.32
Reference compound
43 µM c
40%
16%
41%
50%
50%
96%
85%
45%
42%
63%
26%
50%
45%
43%
11%
33%
50%
79%
40%
77%
37%
56%
64%
50%
37.6%
93%
a
nt e
nt e
nt e
100 µM
100 µM
10 µM
10 µM
nt e
4b
5a
5b
6a
6b
7a
7b
8a
nt e
47 µM
nt e
100 µM
nt e
nt e
8b
9a
9b
nt e
nt e
100 µM
15 µM
nt e
10a
10b
11a
11b
12a
12b
13a
13b
NDGA
27 µM
nt e
42.5 µM
16.5 µM
100 µM
nt e
0.45 µM
Means within each column differ significantly (p < 0.05). clogP values were measured by using the Biobyte
b
c
C-QSAR [37]. Values are means (
±
SD < 10%) of three or four different determinations. From reference [38].
d
e
Values are referred to 2a
,
3a
,
2b and 3b coumarin acetic acid derivatives [38]. nt, not tested (IC50 values not
found due to the fact that it may be >100 µM).
In this context, we evaluated the synthesized compounds of Table 2 for their ability to
inhibit soybean LOX by the UV absorbance based enzyme assay [34] using compounds samples
with concentrations from 0.1–100 µM. Most of the LOX inhibitors are antioxidants or free radical
scavengers. LOXs contain a non-heme iron per molecule in the enzyme active site as high-spin
Fe2+ in the native state and the high spin Fe3+ in the activated state [35]. Some studies suggest
a relationship between LOX inhibition and the ability of the inhibitors to reduce Fe3+ at the
active site to the catalytically inactive Fe2+, whereas several LOX inhibitors are excellent ligands

Molecules 2018, 23, 153
6 of 17
for Fe3+
used as a reference compound (IC50 0.45
experiments [35]. We determined the IC50 inhibition values for compounds 1a
10a 11a 12a 13a. We did not succeed to evaluate the IC50 values for the rest of the compounds,
since they were not active LOX inhibitors at 100 M (11–46%). The most potent % inhibition at 100
is shown by compound 4a (4a > 4b > 10a~11a > 12b~6a).
Perusal of the IC50’s inhibition values (Table 2) shows that the most potent, and equipotent,
inhibitors are compounds 4a and 4b (10 M) followed by 10a (15 M) and 12b (16.5 M). It is interesting
to note that attachment on the coumarin ring, e.g., in the 6-/7- for compounds 4a and 4b, does not seem
to play any role. Replacement of phenyl (4a) by a 2-pyridyl group (10a) or by a morpholinyl group (12a
leads to a reduction of the inhibitory activity, which is highly significant for 12a (42.5 M). The presence
[
35]. Nordihydroguaiaretic acid (NDGA), a known inhibitor of soybean LOX, has been
M/93% at 100 M) and as a positive control in our
3a 4a 6a 7a 9b
µ
µ
,
–
b,
–b
,
,
,
,
,
,
–b,
µ
µM
µ
µ
µ
)
µ
of a 2-pyridyl group in compound 10b significantly decreased activity (by 40%) compared to 4b. In a
similar manner, the presence of a 4-pyridyl group (11a) resulted in significant loss of inhibitory activity
(27 µM) compared to compound 10a. The replacement by a morpholinyl group (12b) does not induce
a considerable loss in activity.
The length of the chain between the aromatic ring and the NHCO-group [(CH2)n], influenced the
biological response, since compound 4a (10 µM) with n = 2, is more potent compared to 7a (100 µM) in
which n = 0 and 5a (45%) in which n = 1. The same is seen for 7b and 5b. The F-substitution allows
an improved inhibitory activity compared to the unsubstituted compound: for example 6a has an
IC50 of 47 µM, whereas 5a only presents 45% at a concentration of 100 µM (Table 2). As concerns the
acids 3a and 3b they appear to present some inhibitory activities (Table 2). Although lipophilicity is
referred to as an important physicochemical property for LOX inhibitors [35], herein the theoretically
calculated log P values did not always support this observation. The most potent compounds 4a and
4b showed the third higher lipophilicity values (2.60) in this series (Table 2). Furthermore, compounds
with comparable lipophilicities showed in many cases striking different LOX inhibitory activities
(Table 2).
3. Experimental Section
3.1. General Information
All biochemical reagents were of analytical grade and purchased from commercial sources.
Soybean lipoxygenase, sodium linoleate, and NDGA were obtained from Sigma Chemical, Co.
(St. Louis, MO, USA).
3.2. Chemistry
3.2.1. General Procedure for the Synthesis of Compounds 3a–b [39]
A mixture of 7-hydroxycoumarin (1a) or 6-hydroxycoumarin (1b) (1 eq.) and potassium carbonate
(3 eq.) was dissolved in dry DMF (5 mL) and the mixture was stirred at room temperature for
15 min. Then, ethyl 2-iodoacetate (a, 1.5 eq.) was added dropwise to the mixture under nitrogen
atmosphere and heated to 100 ◦C for 30 min. After completion of the reaction (TLC monitoring) the
mixture was cooled to room temperature and quenched with water and 1M aqueous HCl solution.
The precipitated products 2a–b were collected by filtration and washed with water, and used without
further purification.

Molecules 2018, 23, 153
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Ethyl 2-((2-oxo-2H-chromen-7-yl)oxy)acetate (2a). Using 7-hydroxycoumarin and, ethyl 2-iodoacetate as
starting materials and the general procedure described above compound 2a was obtained in 94% yield;
m.p. 112.9–113.0 ◦C; δH (400 MHz, CDCl3) 1.31 (3H, t, J = 7.2 Hz), 4.28 (2H, q, J = 7.2 Hz), 4.68 (2H, s),
6.27 (1H, d, J = 9.5 Hz), 6.78 (1H, d, J = 2.4 Hz), 6.88 (1H, dd, J = 2.4, 8.6 Hz), 7.39 (1H, d, J = 8.6 Hz,),
7.63 (1H, d, J = 9.5 Hz); δC (100 MHz, CDCl3) 14.3, 61.9, 65.5, 101.9, 113.0, 113.5, 113.9, 129.1, 143.3,
155.8, 161.0, 161.0, 168.0; m/z (ESI positive) (C13H12O5) 249.2 [M + H]+. Experimental data are in
agreement with the data reported in [40].
Ethyl 2-((2-oxo-2H-chromen-6-yl)oxy)acetate (2b). Using 6-hydroxycoumarin and, ethyl 2-iodoacetate as
starting materials and the general procedure described above compound 2a was obtained in 86% yield;
m.p. 120–122 ◦C; δH (400 MHz, DMSO-d6) 1.26 (3H, t, J = 7.2 Hz), 4.21 (2H, q, J = 7.2 Hz), 4.88 (2H, s),
6.55(1H, d, J = 9.6 Hz), 7.27 (1H, dd, J = 3.0, 9.0 Hz), 7.33 (1H, d, J = 3.0 Hz), 7.39 (1H, d, J = 9.0 Hz)
,
8.03 (1H, d, J = 9.6 Hz); δC (100 MHz, DMSO-d6) 15.0, 61.6, 66.1, 112.9, 117.6, 118.3, 120.1, 120.6, 144.8,
149.2, 154.9, 161.0, 169.4; m/z (ESI positive) (C13H12O5) 249.2 [M + H]+. Experimental data are in
agreement with those reported in [41].
The crude products 2a or 2b (2.7 mmol) were dissolved in an aqueous solution of 5% NaOH
(5 mL) in ethanol (15 mL) and the mixture was stirred at room temperature for 5 min. The residue
quenched with water and acidified with aqueous 6 M solution HCl. The precipitated white solid was
filtered off and subsequently washed with cool water and DCM to give compounds 3a–b, respectively.
2-((2-Oxo-2H-chromen-7-yl)oxy)acetic acid (3a). Compound 3a was obtained in 98% yield; m.p. 180–182 ◦C;
δH (400 MHz, DMSO-d6) 4.86 (2H, s), 6.34 (1H, d, J = 9.2 Hz), 7.98–7.00 (2H, m), 7.68 (1H, d, J = 9.6 Hz,),
8.03 (1H, d, J = 9.2 Hz); δC (100 MHz, DMSO-d6) 65.8, 102.4, 113.5, 113.7, 113.7, 130.4, 145.2, 156.1, 161.2,
161.8, 170.5; m/z (ESI positive) (C11H8O5) 221.0 [M + H]+. Experimental data are in agreement with those
reported in [40].
◦
2-((2-Oxo-2H-chromen-6-yl)oxy)acetic acid (3b). Compound 3b was obtained in 87% yield; m.p. 163.5–163.7 C;
δH (400 MHz, DMSO-d6) 4.78 (2H, s,), 6.53(1H, d, J = 9.5 Hz), 7.25 (1H, dd, J = 3.0, 9.0 Hz), 7.30 (1H, d,
J = 3.0 Hz,), 7.38 (1H, d, J = 9.0 Hz), 8.03 (1H, d, J = 9.5 Hz); δC (100 MHz, DMSO-d6) 65.9, 112.7, 117.6,
118.2, 120.1, 120.6, 144.9, 149.0, 155.0, 160.1, 170.8; m/z (ESI positive) 221. [M + H]+, m/z (ESI negative)
219.0 [M − H]−. Experimental data are in agreement with those reported in [41].
3.2.2. General Procedure for the Synthesis of 4a,b–13a,b
The appropriate coumarin acetic acid derivative 2-((2-oxo-2H-chromen-7-yl)oxy)acetic acid
(
3a) or 2-((2-oxo-2H-chromen-6-yl)oxy)acetic acid (3b) (1.0 eq.), 1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide hydrochloride salt (EDCI HCl, 1.5 eq.) and 1-hydroxy-7-azabenzotriazole (HOAT,
1.5 eq.), were dissolved in dry DMA (3.0 mL) and stirred for 10 min at r.t., followed by addition of the
corresponding amine 13 (1.0 eq.) and N,N-diisopropylethylamine (DIPEA, 4.0 eq.) or triethylamine
·
4
–
(Et3N, 5.0 eq.) in the same solvent (2.0 mL). The reaction mixture was stirred until the consumption
of starting materials (TLC monitoring) and quenched with water and 6.0 M aqueous HCl solution at
◦
0–5 C. The crude products were collected by filtration and washed with cool water, DCM and diethyl
ether to obtain desired products 4a,b–13a,b.

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2-((2-Oxo-2H-chromen-7-yl)oxy)-N-phenethylacetamide (4a). Using 3a and
4
as starting materials and the
general procedure described above compound 4a was obtained as a white solid in 80% yield; m.p.
◦
178–179 C; silica gel TLC Rf = 0.73 (MeOH/DCM 10% v/v); IR (KBr, cm−1) 1710.1, 1604.1, 1558.2;
δH (400 MHz, DMSO-d6) 2.79 (2H, t, J = 7.2 Hz, 20-H2), 3.39 (2H, q, J = 7.2 Hz, 10-H2), 4.63 (2H, s,
OCH2CO), 6.35 (1H, d, J = 9.5 Hz, 3-H), 7.00 (2H, m, 6, 8-H), 7.20–7.24 (3H, m, 40, 60-H), 7.29–7.33 (2H,
m, 50-H), 7.68 (1H, d, J = 8.6 Hz, 5-H), 8.04 (1H, d, J = 9.5 Hz, 4-H), 8.27 (1H, t, J = 7.2 Hz, exchangeable
with D2O, NH); δC (100 MHz, DMSO-d6) 35.9, 68.1, 102.6, 113.6, 113.7, 113.8, 127.0, 129.2, 129.5, 130.4,
140.1, 145.1, 156.0, 161.0, 161.7, 167.7; m/z (ESI positive) (C19H17NO4) 324.3 [M + H]+.
2-((2-Oxo-2H-chromen-6-yl)oxy)-N-phenethylacetamide (4b). Using 3b and
4 as starting materials and
the general procedure described above compound 4b was obtained as a white solid 64% yield; m.p.
150–151◦C; silica gel TLC Rf = 0.5 (MeOH/DCM 10% v/v); IR (KBr, cm−1) 1699.2, 1670.5, 1610.1,
1560.2; δH (400 MHz, DMSO-d6) 2.74 (2H, t, J = 7.2 Hz, 20-H2), 3.39 (2H, q, J = 7.2 Hz, 10-H2), 4.50 (2H,
s, OCH2CO), 6.52 (1H, d, J = 9.6 Hz, 3-H), 7.20–7.32 (7H, m, 5, 7, 40, 50, 60-H), 7.40 (1H, d, J = 8.8,
8-H), 8.03 (1H, d, J = 9.6 Hz, 4-H), 8.21 (1H, t, J = 7.2 Hz, exchangeable with D2O, NH); δC (100 MHz,
DMSO-d6) 35.9, 40.8, 68.4, 112.9, 117.6, 118.2, 120.0, 120.9, 127.0, 129.2, 129.5, 140.2, 144.9, 149.1, 154.9,
160.9, 168.1; m/z (ESI positive) (C19H17NO4) 324.3 [M + H]+.
N-Benzyl-2-((2-oxo-2H-chromen-7-yl)oxy)acetamide (5a). Using 3a and
5 as starting materials and the
general procedure described above compound 5a was obtained as a white solid 67% yield; m.p.
167–168 ◦C; silica gel TLC Rf = 0.52 (MeOH/DCM 10% v/v); IR (KBr, cm−1) 1699.5, 1672.7, 1612.8,
1550.4; δH (400 MHz, DMSO-d6) 4.40 (2H, d, J = 6.1 Hz, 10-H)0, 4.74 (2H, s, OCH2CO), 6.35 (1H,
0
0
d, J = 9.5 Hz, 3-H), 7.03–7.06 (2H, m, 6, 8-H), 7.25–7.36 (5H, m, 3 , 4 , 5 -H), 7.69 (1H, d, J = 8.4 Hz, 5-H),
8.03 (1H, d, J = 9.6 Hz, 4-H), 8.78 (1H, t, J = 6.1 Hz, exchange with D2O, NH); δC (100 MHz, DMSO-d6)
42.8, 68.2, 102.7, 113.7, 113.8, 113.9, 127.8, 128.2, 129.2, 130.5, 140.2, 145.2, 156.1, 161.2, 161.7, 168.1;
m/z (ESI positive) (C18H15NO4) 310.2 [M + H]+.

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N-Benzyl-2-((2-oxo-2H-chromen-6-yl)oxy)acetamide (5b). Using 3b and
5
as starting materials and the
general procedure described above compound 5b was obtained as a white solid in 67% yield; m.p.
161–162 ◦C; silica gel TLC Rf = 0.63 (MeOH/DCM 10% v/v); IR (KBr, cm−1) 1705.2, 1680.5, 1605.7,
1555.2; δH (400 MHz, DMSO-d6) 4.38 (2H, d, J = 6.2 Hz, 10-H), 4.65 (2H, s, OCH2CO), 6.52 (1H,
d, J = 9.6 Hz, 3-H), 7.25–7.36 (7H, m, 30, 40, 50-H), 7.40 (1H, d, J = 8.6 Hz, 8-H), 8.02 (1H, d, J = 9.6 Hz,
4-H), 8.07 (1H, t, J = 6.2 Hz, exchange with D2O, NH); δC (100 MHz, DMSO-d6) 42.9, 68.5, 113.0,
117.7, 118.4, 120.2, 121.1, 127.8, 128.2, 129.2, 140.3, 145.2, 149.2, 155.1, 161.1, 168.6; m/z (ESI positive)
(C18H15NO4) 310.2 [M + H]+.
N-(4-Fluorobenzyl)-2-((2-oxo-2H-chromen-7-yl)oxy)acetamide (6a). Using 3a and 6 as starting materials
and the genera◦l procedure described above compound 6a was obtained as a white solid in 54% yield;
m.p. 160–161 C; silica gel TLC Rf = 0.62 (MeOH/DCM 10% v/v); IR (KBr, cm−1) 1711.6, 1673.1,
1554.8, 1549.6; δH (400 MHz, DMSO-d6) 4.37 (2H, d, J = 6 Hz, 10-H), 4.73 (2H, s, OCH2CO), 6.36 (1H, d,
J = 9.6 Hz, 3-H), 7.06–7.10 (2H, m, 6, 8-H), 7.18–7.23 (2H, m, 40-H), 7.67–7.71 (3H, m, 5, 30-H), 7.69 (1H,
d, J = 8.4 Hz), 8.04 (1H, d, J = 9.6 Hz, 4-H), 8.77 (1H, t, J = 6 Hz, exchange with D2O, NH); δC (100 MHz,
2
3
DMSO-d6) 42.1, 68.1, 102.6, 113.7, 113.8, 113.8, 115.8 (d, JC-F 22), 130.2 (d, JC-F 8), 130.4, 136.3 (d,
4JC-F 3), 145.2, 156.0, 161.7, 162.1 (d, 1JC-F 238.8), 161.61, 168.0; δF (376 MHz, DMSO-d6)
m/z (ESI positive) (C18H14FNO4) 328.2 [M + H]+.
−
115.9 (1F, s);
N-(4-Fluorobenzyl)-2-((2-oxo-2H-chromen-6-yl)oxy)acetamide (6b). Using 3b and 6 as starting materials
and the genera◦l procedure described above compound 6b was obtained as a white solid in 45% yield;
m.p. 158–159 C; silica gel TLC Rf = 0.42 (MeOH/DCM 10% v/v); IR (KBr, cm−1) 1698.6, 1672.3,
1615.0, 1554.9; δH (400 MHz, DMSO-d6) 4.36 (2H, d, J = 6 Hz, 10-H), 4.65 (2H, s, OCH2CO), 6.54 (1H, d,
J = 9.6 Hz, 3-H), 7.17–7.21 (2H, m, 40-H), 7.33–7.42 (3H, m, 5, 7, 8-H), 7.67–7.70 (2H, m, 30-H), 8.04 (1H,
d, J = 9.6 Hz, 4-H), 8.76 (1H, t, J = 6 Hz, exchange with D2O, NH); δC (100 MHz, DMSO-d6) 42.0,
68.4, 112.9, 115.8 (d, 2JC-F 21), 117.6, 118.3, 120.0, 121.0, 130.2 (d, 3JC-F 8), 136.4 (d, 4JC-F 3), 144.9, 149.1,
1
154.9, 161.0, 162.06 (d, JC-F 240), 168.4. δF (376 MHz, DMSO-d6)
−
116.1 (1F, s); m/z (ESI positive)
(C18H14FNO4) 328.2 [M + H]+.
2-((2-Oxo-2H-chromen-7-yl)oxy)-N-phenylacetamide (7a). Using 3a and
7 as starting materials and the
general procedure described above compound 7a was obtained as a white solid in 70% yield; m.p.

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◦
182–183 C; silica gel TLC Rf = 0.57 (MeOH/DCM 10% v/v); δH (400 MHz, DMSO-d6) 4.96 (2H, s,
COCH2O), 6.35 (1H, d, J = 8 Hz, 3-H), 7.10 (3H, m), 7.36 (2H, m), 7.69 (3H, m), 8.04 (1H, d, J = 8 Hz),
10.22 (1H, s, exchange with D2O, NH); δC (100 MHz, DMSO-d6) 68.2, 102.6, 113.6, 113.8, 113.9, 120.6,
124.7, 129.6, 130.4, 139.2, 145.1, 156.0, 161.1, 161.9, 166.7; m/z (ESI positive) (C17H13NO4) 296.2 [M + H]+.
Experimental data are in agreement with those reported in [39].
2-((2-Oxo-2H-chromen-6-yl)oxy)-N-phenylacetamide (7b) [39]. Using 3b and 7 as starting materials and
the general procedure described above compound 7b was obtained as a white solid in 80% yield;
m.p. 182–183 ◦C; silica gel TLC Rf 0.56 (MeOH/DCM 10% v/v); δH (400 MHz, DMSO-d6) 4.80 (2H, s),
6.54 (1H, d, J = 9.6 Hz), 7.12 (1H, t, J = 7.4 Hz), 7.36 (4H, m), 7.43 (1H, d, J = 8.7), 7.68 (2H, d, J = 7.4 Hz),
8.07 (1H, d, J = 9.6 Hz), 10.14 (1H, s, exchange with D2O, NH); δC (100 MHz, DMSO-d6) 68.6, 113.0,
117.6, 118.3, 120.1, 120.6, 120.8, 124.6, 129.7, 139.2, 144.9, 149.1, 155.1, 161.0, 167.1. m/z (ESI positive)
(C17H13NO4) 296.2 [M + H]+. Experimental data are in agreement with those reported in [39].
N-(4-Fluorophenyl)-2-((2-oxo-2H-chromen-7-yl)oxy)acetamide (8a) [39]. Using 3a and 8 as starting materials
and the general procedure described above compound 8a was obtained as a white solid in 80% yield;
m.p. 208–209 ◦C; silica gel TLC Rf 0.75 (MeOH/DCM 10% v/v); δH (400 MHz, DMSO-d6) 4.87 (2H, s),
6.35 (1H, d, J = 9.5 Hz), 7.08 (2H, m), 7.21 (2H, m), 7.69 (3H, m), 8.03 (1H, d, J = 9.5 Hz), 10.25 (1H, s,
exchange with D2O, NH); δC (100 MHz, DMSO-d6) 68.2, 102.6, 113.7, 113.9, 113.9, 116.4 (d, 2JC-F = 22),
3
4
1
122.6 (d, JC-F = 8), 130.5, 135.6 (d, JC-F = 3), 145.2, 156.1, 159.3 (d, JC-F = 239), 161.2, 161.9, 166.8;
δF (376 MHz, DMSO-d6)
data are in agreement with those reported in [39].
−
118.7 (1F, s); m/z (ESI positive) (C17H12FNO4) 314.2 [M + H]+. Experimental
N-(4-Fluorophenyl)-2-((2-oxo-2H-chromen-6-yl)oxy)acetamide (8b) [39]. Using 3b and
8 as starting
materials and the general procedure described above compound 8b was obtained as a white solid in
80% yield; m.p. 206–207 ◦C; silica gel TLC Rf 0.86 (MeOH/DCM 10% v/v); δH (400 MHz, DMSO-d6)
4.79 (2H, s), 6.52 (1H, d, J = 9.6 Hz), 7.18 (2H, t, J = 8.8), 7.38 (3H, m), 7.68 (2H, m), 8.03 (1H, d,
J = 9.6 Hz), 10.22 (1H, s, exchange with D2O, NH); δC (100 MHz, DMSO-d6) 68.6, 113.1, 116.3 (d,
3
4
2JC-F 22), 117.7, 118.5, 120.2, 121.0, 122.8 (d, JC-F 8), 135.6 (d, JC-F 3), 145.1, 149.3, 155.2, 159.3 (d,
1JC-F 238.8), 161.2, 167.3. δF (376 MHz, DMSO-d6)
118.78 (1F, s); m/z (ESI positive) (C17H12FNO4)
314.2 [M + H]+. Experimental data are in agreement with those reported in [39].
−

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Methyl 2-(2-((2-oxo-2H-chromen-7-yl)oxy)acetamido)benzoate (9a). Using 3a and
9
as starting materials
and the general procedure described above compound 9a was obtained as a white solid in 45% yield;
◦
m.p. 213–214 C; silica gel TLC Rf = 0.69 (MeOH/DCM 10% v/v); IR (KBr, cm−1) 1710.3, 1668.7, 1612.3,
1562.0; δH (400 MHz, DMSO-d6) 3.96 (3H, s, COOCH3), 4.93 (2H, s, OCH2CO), 6.39 (1H, d, J = 9.6 Hz,
3-H), 7.15-7.20 (2H, m, 6, 8-H), 7.28 (1H, t, J = 7.5, 40-H), 7.70 (1H, t, J = 7.5, 50-H), 7.76 (1H, d, J = 9.2,
5-H), 8.03-8.09 (2H, m, 4, 60-H), 8.62 (1H, d, J = 7.5 Hz, 30-H), 11.71 (1H, s, exchange with D2O, NH);
δC (100 MHz, DMSO-d6) 53.5, 68.5, 102.8, 113.7, 114.1, 114.2, 117.2, 121.1, 124.4, 130.7, 131.7, 135.4, 140.3,
145.1, 156.1, 160.9, 161.0, 167.3, 168.4. m/z (ESI positive) (C19H15NO6) 354 [M + H]+.
Methyl 2-(2-((2-oxo-2H-chromen-6-yl)oxy)acetamido)benzoate (9b). Using 3b and 9 as starting materials
and the gener◦al procedure described above compound 9b was obtained as a white solid in 50% yield;
m.p. 213–214 C; silica gel TLC Rf = 0.67 (MeOH/DCM 10% v/v); IR (KBr, cm−1) 1712.9, 1670.4, 1611.9,
1563.8; δH (400 MHz, DMSO-d6) 3.94 (3H, s, COOCH3), 4.85 (2H, s, OCH3CO), 6.56 (1H, d, J = 9.6 Hz,
3-H), 7.27 (1H, t, J = 7.8, 40-H), 7.43 (1H, dd, J 2.8, 9.1 Hz, 7-H), 7.47-7.50 (2H, m, 5, 8-H), 7.71 (1H, t,
J = 7.8, 50-H), 8.05 (1H, d, J = 7.8, 60-H), 8.08 (1H, d, J = 9.6 Hz, 4-H), 8.65 (1H, d, J = 7.8 Hz, 30-H),
11.74 (1H, s, exchange with D2O, NH); δC (100 MHz, DMSO-d6) 51.6, 66.2, 110.4, 113.2, 113.9, 115.6,
118.3, 119.4, 119.5, 124.2, 130.3, 133.6, 141.3, 143.5, 147.8, 157.6, 163.2, 167.7, 168.3; m/z (ESI negative)
(C19H15NO6) 352 [M − H]−.
2-((2-Oxo-2H-chromen-7-yl)oxy)-N-(2-(pyridin-2-yl)ethyl)acetamide (10a). Using 3a and 10 as starting
materials and the general procedure described above compound 10a was obtained as a white solid in
68% yield; m.p. 138–139 ◦C; silica gel TLC Rf = 0.60 (MeOH/DCM 10% v/v); IR (KBr, cm−1) 1710.5,
1672.3, 1612.8, 1554.7; δH (400 MHz, DMSO-d6) 2.95 (2H, t, J = 7.2 Hz, 20-H), 3.54 (2H, q, J = 7.2 Hz,
10-H), 4.63 (2H, s, OCH2CO), 6.36 (1H, d, J = 9.6 Hz, 3-H), 6.97 (1H, d, J = 2.4 Hz, 8-H), 7.00 (1H, dd,
J = 2.4, 8.6 Hz, 6-H), 7.22–7.24 (1H, m, 60-H), 7.26 (1H, d, J = 8.6 Hz, 5-H), 7.67–7.73 (2H, m, 50,80-H),
8.03 (1H, d, J = 9.6 Hz, 4-H), 8.27 (1H, t, J = 7.2 Hz, exchange with D2O, NH), 8.50-8.52 (1H, m, 70-H);
δC (100 MHz, DMSO-d6) 38.2, 39.8, 68.5, 103.2, 114.4, 114.5, 114.6, 123.5, 125.1, 131.3, 138.7, 146.2, 150.4,
156.6, 160.2, 162.3, 162.6, 169.4; m/z (ESI positive) (C18H16N2O4) 325.3 [M + H]+.

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2-((2-Oxo-2H-chromen-6-yl)oxy)-N-(2-(pyridin-2-yl)ethyl)acetamide (10b). Using 3b and 10 as starting
materials and the general procedure described above compound 10b was obtained as a white solid in
60% yield; m.p. 141–142 ◦C; silica gel TLC Rf = 0.60 (MeOH/DCM 10% v/v); IR (KBr, cm−1) 1700.8,
1671.5, 1609.1, 1556.8; δH (400 MHz, DMSO-d6) 2.95 (2H, t, J = 6.4 Hz, 20-H), 3.55 (2H, q, J = 6.4 Hz,
10-H), 4.56 (2H, s, OCH2CO), 6.54 (1H, d, J = 9.6 Hz, 3-H), 7.21-7.30 (4H, m, 7, 5, 60, 80-H), 7.39 (1H,
d, J = 8.8 Hz, 8-H), 7.70 (1H, t, J = 7.8 Hz, 70-H), 8.04 (1H, d, J = 9.6 Hz, 4-H), 8.27 (1H, t, J = 6.4 Hz,
exchange with D2O, NH), 8.51 (1H, m, 50-H); δC (100 MHz, DMSO-d6) 36.4, 54.1, 67.0, 112.9, 113.4,
113.9, 117.6, 118.3, 120.1, 120.9, 136.9, 143.7, 144.9, 149.1, 154.9, 158.7, 160.9, 168.1; m/z (ESI positive)
(C18H16N2O4) 325.3 [M + H]+.
2-((2-Oxo-2H-chromen-7-yl)oxy)-N-(pyridin-4-ylmethyl)acetamide (11a). Using 3a and 11 as starting
materials and the general procedure described above compound 11a was obtained as a white solid in
62% yield; m.p. 158–159 ◦C; silica gel TLC Rf = 0.43 (MeOH/DCM 10% v/v); IR (KBr, cm−1) 1699.7,
1670.3, 1605.2, 1555.1; δH (400 MHz, DMSO-d6) 4.42 (2H, d, J = 6.1 Hz, 10-H), 4.70 (2H, s, OCH2CO),
0
6.55 (1H, d, J = 9.6 Hz, 3-H), 7.27–7.29 (2H, 6, 8-H), 7.33–7.36 (2H, m, 3 -H), 7.42 (1H, d, J = 8.9 Hz, 5-H),
8.05 (1H, d, J = 9.6 Hz, 4-H), 8.50–8.52 (2H, m, 40-H), 8.85 (1H, t, J = 6.1 Hz, exchange with D2O, NH);
δC (100 MHz, DMSO-d6) 41.8, 68.3, 112.9, 117.6, 118.3, 120.0, 121.0, 123.0, 144.8, 149.1, 149.2, 150.3, 154.9,
160.9, 168.8; m/z (ESI positive) (C17H14N2O4) 311.2 [M + H]+.
2-((2-Oxo-2H-chromen-6-yl)oxy)-N-(pyridin-4-ylmethyl)acetamide (11b). Using 3b and 11 as starting
materials and the general procedure described above compound 11b was obtained as a white solid in
62% yield; m.p. 160–161 ◦C; silica gel TLC Rf = 0.40 (MeOH/DCM 10% v/v); IR (KBr, cm−1) 1709.3,
1671.6, 1606.5, 1555.0; δH (400 MHz, DMSO-d6) 4.41 (2H, d, J = 6.1 Hz, 10-H), 4.78 (2H, s, OCH2CO),
6.36 (1H, d, J = 9.6 Hz, 3-H), 7.05–7.07 (2H, m, 6, 8-H), 7.27–7.30 (2H, m, 30-H), 7.70 (1H, d, J = 8.6 Hz,
8-H), 8.06 (1H, d, J = 9.6 Hz, 4-H), 8.51-8.53 (2H, m, 40-H), 8.85 (1H, t, J = 6.1 Hz, exchange with D2O,
NH); δC (100 MHz, DMSO-d6) 41.8, 68.1, 102.7, 113.7, 113.8, 113.9, 123.0, 130.4, 145.1, 149.1, 150.4, 156.1,
157.3, 161.6, 168.4; m/z (ESI positive) (C17H14N2O4) 311.2 [M + H]+.

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N-(2-Morpholinoethyl)-2-((2-oxo-2H-chromen-7-yl)oxy)acetamide (12a). Using 3a and 12 as starting
materials and the general procedure described above compound 12a was obtained as a white solid in
30% yield; m.p. 148–149 ◦C; silica gel TLC Rf = 0.50 (MeOH/DCM 010% v/v); IR (KBr, cm−1) 1712.7,
0
0
1671.9, 1607.3, 1553.8; δH (400 MHz, DMSO-d6) 2.38–2.43 (6H, m, 2 , 4 -H), 3.30 (2H, q, J = 6.3 Hz, 1 -H),
3.57 (4H, t, J = 4.7 Hz, 50-H), 4.66 (2H, s, OCH2CO), 6.36 (1H, d, J = 9.6 Hz, 3-H), 7.00–7.05 (2H, m, 6,
8-H), 7.70 (1H, d, J = 8.5 Hz, 5-H), 8.05 (1H, d, J = 9.6 Hz, 4-H), 8.09 (1H, t, J = 6.3 Hz, exchange with
D2O, NH); δC (100 MHz, DMSO-d6) 39.8, 54.1, 58.0, 67.0, 68.1, 102.6, 113.7, 113.8, 113.9, 130.4, 145.1,
156.0, 161.0, 161.6, 167.7; m/z (ESI positive) (C17H20N2O5) 333.3 [M + H]+.
N-(2-Morpholinoethyl)-2-((2-oxo-2H-chromen-6-yl)oxy)acetamide (12b). Using 3b and 12 as starting
materials and the general procedure described above compound 12b was obtained as a white solid in
30% yield; m.p. 149–150 ◦C; silica gel TLC Rf = 0.57 (MeOH/DCM 10% v/v); IR (KBr, cm−1) 1700.2,
1678.4, 1617.2, 1557.0; δH (400 MHz, DMSO-d6) 2.37–2.43 (6H, m, 20, 40-H), 3.30 (2H, q, J = 6.6 Hz,
10-H), 3.56 (4H, t, J = 4.6 Hz, 50-H), 4.58 (2H, s, OCH2CO), 6.54 (1H, d, J = 9.6 Hz, 3-H), 7.30 (1H, dd,
J = 2.8, 8.9 = Hz, 7-H), 7.33 (1H, d, J = 2.8, 5-H), 7.42 (1H, d, J = 8.9 Hz, 8-H), 8.01 (1H, t, J = 6.6 Hz,
exchange with D2O, NH), 8.05 (1H, d, J = 9.6 Hz, 4-H); δC (100 MHz, DMSO-d6) 39.6, 55.8, 58.9, 66.7,
67.0, 112.9, 113.4, 113.9, 118.1, 120.0, 143.5, 145.3, 156.3, 160.8, 168.6; m/z (ESI positive) (C17H20N2O5)
333.3 [M + H]+.
2-((2-Oxo-2H-chromen-7-yl)oxy)-N-(2-(piperidin-1-yl)ethyl)acetamide (13a). Using 3a and 13 as starting
materials and the general procedure described above compound 13a was obtained as a white solid
◦
in 45% yield; m.p. 116–117 C; silica gel TLC Rf = 0.24 (MeOH/DCM 10% v/v); IR (KBr, cm−1
)
0
0
1669.8, 1670.7, 1605.8, 1558.4; δH (400 MHz, DMSO-d6) 1.37–1.41 (2H, m, 6 -H), 1.45–1.50 (4H, m, 5 -H),
2.33–2.38 (6H, m, 20, 40-H), 3.27 (2H, q, J = 6.4 Hz, 10-H), 4.65 (2H, s, OCH2CO), 6.34 (1H, d, J = 9.6 Hz,
3-H), 7.00–7.04 (2H, m, 6, 8-H), 7.69 (1H, d, J = 8.4 Hz, 5-H), 8.03 (1H, d, J = 9.6 Hz, 4-H); δC (100 MHz,
DMSO-d6) 24.9, 26.4, 36.9, 54.9, 58.3, 68.2, 102.7, 113.7, 113.9, 130.4, 145.1, 115.1, 161.1, 161.6, 167.7;
m/z (ESI positive) (C18H22N2O4) 331.3 [M + H]+.

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2-((2-Oxo-2H-chromen-6-yl)oxy)-N-(2-(piperidin-1-yl)ethyl)acetamide (13b). Using 3b and 13 as starting
materials and the general procedure described above compound 13b was obtained as a white solid
◦
in 45% yield; m.p. 115–116 C; silica gel TLC Rf = 0.30 (MeOH/DCM 10% v/v); IR (KBr, cm−1
)
0
0
1701.6, 1670.4, 1610.0, 1554.8; δH (400 MHz, DMSO-d6) 1.38–1.39 (2H, m, 6 -H), 1.46–1.50 (4H, m, 5 -H),
2.35–2.42 (6H, m, 20, 40-H), 3.27 (2H, q, J= 6.6 Hz, 10-H), 4.57 (2H, s, OCH2CO), 6.52 (1H, d, J = 9.6 Hz,
3-H), 7.29 (1H, dd, J = 2.9, 8.9 Hz, 7-H), 7.33 (1H, d, J = 2.9 Hz, 5-H), 7.41 (1H, d, J = 8.9 Hz), 7.96 (1H, t,
J = 6.6 Hz, exchange with D2O, NH), 8.04 (1H, d, J = 9.6 Hz, 4-H); δC (100 MHz, DMSO-d6) 24.9, 26.4,
36.8, 54.8, 58.3, 68.4, 112.9, 117.6, 118.3, 120.1, 120.9, 144.9, 149.1, 154.9, 160.9, 168.0; m/z (ESI positive)
(C18H22N2O4) 331.3 [M + H]+.
3.3. Soybean Lipoxygenase Inhibition Studies
A DMSO solution of the tested compound was incubated with sodium linoleate (0.1 mM) and
0.2 mL of soybean LOX solution (1/9
The conversion of sodium linoleate to 13-hydroperoxylinoleic acid was recorded at 234 nm and
×
10−4 w/v in saline) in buffer pH 9 (tris) and at room temperature.
compared with the standard inhibitor NDGA (IC50 = 0.45 µM). The results are given in Table 2
expressed as IC50 values or % inhibition at 100 µM [34].
3.4. CA Inhibition Assay
An Applied Photophysics (Oxford, UK) stopped-flow instrument has been used for assaying the
CA catalysed CO2 hydration activity [36]. Phenol red (at a concentration of 0.2 mM) has been used
as indicator, working at the absorbance maximum of 557 nm, with 20 mM HEPES (pH 7.5) as buffer,
and 20 mM Na2SO4 (for maintaining constant the ionic strength), following the initial rates of the
CA-catalyzed CO2 hydration reaction for a period of 10–100 s. The CO2 concentrations ranged from 1.7
to 17 mM for the determination of the kinetic parameters and inhibition constants. For each inhibitor
at least six traces of the initial 5–10% of the reaction have been used for determining the initial velocity.
The uncatalyzed rates were determined in the same manner and subtracted from the total observed
rates. Stock solutions of inhibitor (0.1 mM) were prepared in distilled-deionized water and dilutions
up to 0.01 nM were done thereafter with distilled-deionized water. Inhibitor and enzyme solutions
were preincubated together for 6 h at 4 ◦C prior to assay, in order to allow for the formation of the E-I
complex and for the active site mediated hydrolysis of the inhibitor [18
show the inhibition after 6 h incubation, which led to the completion of the in situ hydrolysis of the
coumarin and formation of the hydroxycinnamic acid [18 19]. The inhibition constants were obtained
by non-linear least-squares methods using PRISM 3, as reported earlier [42 50] and represent the mean
,19]. Data reported in Table 1
,
–
from at least three different determinations. The four CA isoforms were recombinant proteins obtained
as reported earlier in our laboratory [42–50].
4. Conclusions
We report here a series of carboxamide derivatives of 6- and 7-substituted coumarins. They have
been prepared by an original procedure starting from the corresponding 6- or 7-hydroxycoumarins
which were alkylated with ethyl iodoacetate, then the obtained ester was converted to the corresponding
carboxylic acid which was thereafter reacted with a series of aromatic/aliphatic/heterocyclic amines
leading to the desired amides. The present study shows that these compounds represent a promising
class of multi-targeting derivatives which can interact with several biological targets, in this case,

Molecules 2018, 23, 153
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lipoxygenase and carbonic anhydrases. Compounds 4a and 4b were potent LOX inhibitors, whereas
many effective hCA IX inhibitors (KIs in the range of 30.2–30.5 nM) were detected in this study.
Two compounds 4b and 5b showed the phenomenon of dual inhibition. Furthermore, these coumarins
did not significantly inhibit the widespread cytosolic isoforms hCA I and II, whereas they were weak
hCA IV inhibitors, making them hCA IX-selective inhibitors. As hCA IX and LOX are validated
antitumor targets, these results are promising for the investigation of novel drug targets involved
in tumorigenesis.
Acknowledgments: Hadjipavlou-Litina and Peperidou (PhD student) are thankful to Biobyte and A. Leo for the
free use of C-QSAR software. Peperidou had EU financial support as an Erasmus student.
Author Contributions: A.P. and M.B. prepared the compounds, S.B. tested them, D.H.-L. and C.T.S. supervised
all the project, designed the experiments and wrote the manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 2–13b are available from the authors.
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2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
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