Click-tailed coumarins with potent and selective inhibitory action against the tumor-associated carbonic anhydrases IX and XII

Article abstract of DOI:10.1016/j.bmc.2015.09.041

Coumarins behave as inhibitors of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1) with a mechanism of inhibition distinct from other classes of inhibitors. A series of 7-substituted coumarins incorporating aryl-triazole moieties were prepared by click chemistry procedures starting from 7-hydroxycoumarin or 4-methyl-7-aminocoumarin. The panel of new compounds was assayed for the inhibition of the cytosolic, widespread human (h) isoforms hCA I and II, and the transmembrane, tumor-associated ones hCA IX and XII. Most of the coumarins were weak inhibitors or did not inhibit significantly hCA I and II, but showed low nanomolar inhibitory action against the transmembrane isoforms (K_I of 14.3-34.4 nM against hCA IX and of 4.7-37.8 nM against hCA XII). Since many hypoxic tumors overexpress hCA IX/XII, and as these targets were recently validated for obtaining antitumor/antimetastatic agents, with one inhibitor in Phase I clinical trials, the present findings constitute an interesting extension to the knowledge of non-sulfonamide, selective inhibitors of CA isoforms involved in serious pathologies.

Full text of DOI:10.1016/j.bmc.2015.09.041

Bioorganic & Medicinal Chemistry xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Click-tailed coumarins with potent and selective inhibitory action
against the tumor-associated carbonic anhydrases IX and XII
Alessio Nocentini a, Fabrizio Carta a,b, Mariangela Ceruso b, Gianluca Bartolucci a, Claudiu T. Supuran a,b,
⇑
a Università degli Studi di Firenze, Dipartimento Neurofarba, Sezione di Scienze Farmaceutiche, Polo Scientifico, Via U. Schiff 6, 50019 Sesto Fiorentino, Florence, Italy
b Università degli Studi di Firenze, Dipartimento di Chimica, Laboratorio di Chimica Bioinorganica, Rm. 188, Via della Lastruccia 3, 50019 Sesto Fiorentino, Florence, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Coumarins behave as inhibitors of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1) with a
mechanism of inhibition distinct from other classes of inhibitors. A series of 7-substituted coumarins
incorporating aryl-triazole moieties were prepared by click chemistry procedures starting from
7-hydroxycoumarin or 4-methyl-7-aminocoumarin. The panel of new compounds was assayed for the
inhibition of the cytosolic, widespread human (h) isoforms hCA I and II, and the transmembrane,
tumor-associated ones hCA IX and XII. Most of the coumarins were weak inhibitors or did not inhibit sig-
nificantly hCA I and II, but showed low nanomolar inhibitory action against the transmembrane isoforms
(KI of 14.3–34.4 nM against hCA IX and of 4.7–37.8 nM against hCA XII). Since many hypoxic tumors over-
express hCA IX/XII, and as these targets were recently validated for obtaining antitumor/antimetastatic
agents, with one inhibitor in Phase I clinical trials, the present findings constitute an interesting extension
to the knowledge of non-sulfonamide, selective inhibitors of CA isoforms involved in serious pathologies.
Ó 2015 Elsevier Ltd. All rights reserved.
Received 3 September 2015
Revised 24 September 2015
Accepted 25 September 2015
Available online xxxx
Keywords:
Carbonic anhydrase
Tumor-associated isoforms
Coumarin
Click chemistry
1. Introduction
of the active site cavity, thus obstructing it.1b,2 As this is the
most variable part of the active site among the various hCAs,2–4
The coumarins are a relatively new class1 of inhibitors of the
metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1).2–4 Discovered
initially in a library of natural products (the first coumarin CA
inhibitor (CAI) was isolated from the Australian plant Leionema
elipticum),1 a large number of synthetic and natural product
coumarins were investigated as inhibitors5–9 of a large number of
isoforms of the 15 presently described in humans, hCA I–hCA XIV
(there are two V-type isoforms, hCA VA and hCA VB).2,10,11
Coumarins are an important class of CAIs for several reasons: (i)
they were the first CAIs showing a high selectivity for inhibiting CA
isoforms of interest for pharmacologic applications, such as the
tumor-associated ones (hCA IX and XII, which are targets for anti-
tumor/antimetastatic drugs)12,13 or the mitochondrial ones (CA VA
and VB, which are targets for anti-obesity agents).14,15 Further-
more, their selectivity for the target isoforms (e.g., CA IX, XII, VA,
VB, etc.) over the ubiquitous ones (such as CA I and II) was
explained from the structural viewpoint, after the report of several
X-ray crystal structures of adducts of some coumarins bound to
hCA II.1b,2 In fact the coumarins act as prodrug inhibitors, being
hydrolysed by the esterase activity of the enzyme to
2-hydroxycinnamic acid derivatives, which bind at the entrance
this particular binding mode explains the very high selectivity of
many coumarin derivatives for inhibiting isoforms of interest, as
those mentioned above, whereas their inhibitory action against
the house-keeping enzymes hCA I and II is generally low or
insignificant.1,5–11(ii) The chemical simplicity and possibility to
explore various facile synthetic approaches for this ring system,
allows for the possibility to generate a large number of structurally
diverse coumarin derivatives easily, which may lead thereafter to
interesting applications. For example some 6-glycosyl-substituted
coumarins reported earlier by our group7a showed selective
inhibitory action against hCA IX and XII (over hCA I and II), and
excellent in vivo antitumor/antimetastatic activity, in an animal
model of the disease.7a,12a Actually a rather large number of
substitution patterns at the coumarin ring and a variety of
diverse substituents have been investigated in order to obtain
CAIs with desired physico-chemical and pharmacologic
properties.1,5–11(iii) Considering the coumarins as lead compounds,
a large number of structurally diverse classes of CAIs were
obtained, among which the sulfocoumarins, the thio-coumarins,
thioxo-coumarins, the coumarin oximes, as well as mono-cyclic
5- and 6-membered lactones/thiolactones.8–11Similar to the parent
compounds, most of these new classes of CAIs act as prodrug inhi-
bitors, being hydrolysed within the enzyme active site to deriva-
tive which thereafter inhibit the enzyme.8–11
⇑
Corresponding author. Tel.: +39 055 4573005; fax: +39 055 4573385.
E-mail address: claudiu.supuran@unifi.it (C.T. Supuran).
http://dx.doi.org/10.1016/j.bmc.2015.09.041
0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Nocentini, A.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.09.041

2
A. Nocentini et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
Although a large number of synthetic approaches were reported
Starting from 7-hydroxy-coumarin 1, the propargyl ether key
intermediate 2 was obtained as an alkyne for the click chemistry.
Reaction of 2 with aromatic azides A–J afforded a first series of tri-
azoles, 5–14, incorporating the coumarin ring and diverse aryl
groups at the triazole moiety (Scheme 1).4-Methyl-7-amino-cou-
marin 3 was transformed into the corresponding azide 4 by diazo-
tization followed by reaction with sodium azide, and was
subsequently used for cycloaddition reactions with alkyne 2 (lead-
ing to a bis-coumarin derivative, 15) or propargyl derivatives 19–
21 (obtained from phenol, thiophenol or aniline, and propargyl
bromide), leading to derivatives 16–18 (Scheme 1).
to date for derivatizing coumarins in order to obtain CAIs, the click
chemistry has been not investigated in detail for this class of com-
pounds (although many sulfocoumarins incorporating a variety of
substitution patterns were reported by using this cycloaddition
reaction).10,11 Here we report a series of 7-substituted coumarins
obtained by using click chemistry, which showed excellent CA
IX/XII inhibitory activity as well as selectivity for the inhibition
of the tumor-associated isoforms over the cytosolic ones CA I and
II.
The rationale for derivatizing the 7-hydroxy-/amino coumarins
was furnished by our earlier work in which we showed that cou-
marins incorporating various functionalities in this position lead
to effective and isoform-selective CAIs.1,5–7 Thus, we decided to
incorporate substituted-aromatic moieties either directly attached
to the triazole ring (as in 5–14) or via a CH2X linker (as in 16–18, in
which X = O, S or NH). For the first sub-group of derivatives (5–14),
the aryl azides used incorporated various 3- or 4-substituted
groups (halogens, OH, OMe, carboxyl, sulfamoyl, etc.) in order to
delineate the structure–activity relationship (SAR) for the inhibi-
tion of various CA isoforms with the derivatives reported here.
2. Results and discussion
2.1. Chemistry
The rationale of this work was the following one: click chem-
istry is a versatile and facile methodology for generating chemical
space, which was successfully used for obtaining interesting sul-
fonamide and sulfamate CAIs.16,17 Here we apply this methodology
for obtaining 7-substituted coumarins, since this reaction was not
used up until now for obtaining CAIs belonging to the coumarin
class.
O
O
O
R
N
N
= m-OCH ; 9
N
3
= p-F; 10
R= H; 5
= p-CF ;11
3
= m-F; 6
= m-Cl; 7
=m-Br; 8
=
p-OH; 12
= p-CO H; 13
2
= p-SO NH ; 14
2
2
= m-OCH3; E
= p-F; F
R= H; A
= m-F; B
= m-Cl;C
=m-Br; D
N
3
Cu(0) nanosized, TMACl
H2O/tBuOH 1/1
=
=
p
-CF ;
G
H
3
R
-OH;
p
= p-CO2H; I
= p-SO2NH2; J
Br
K CO
2
3
HO
O
O
O
O
O
DMF
1
2
Cu(0) nanosized,
TMACl
O
O
O
N
N
H2O/tBuOH 1/1
N
O
NaNO , NaN
2
O
15
3
O
O
N
3
HCl 2M
O
O
NH
2
3
4
Cu(0) nanosized, TMACl
H2O/tBuOH 1/1
X
X= O; 19
= S; 20
= NH; 21
X
O
O
N
N
N
X= O; 16
= S; 17
= NH; 18
Scheme 1. General synthetic scheme of compounds 5–18.
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A. Nocentini et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
3
2.2. Carbonic anhydrase inhibition
investigated here, which showed highly effective inhibitory action,
with KIs in the range of 4.7–37.8 nM. Only the bis-coumarin 15 was
slightly less inhibitory (KI of 37.8 nM) whereas the remaining
derivatives showed KIs <10 nM (except one compound, 8, which
has a KI of 27.1 nM). Overall, the coumarins reported here show
excellent inhibitory action against both tumor-associated isoforms
hCA IX and XII.(v) As many coumarins and sulfocoumarins
reported earlier,1,5–11 the derivatives investigated here are hCA
IX/XII selective inhibitors over hCA I and II, which constitutes a
very important feature for this class of compounds. Indeed, one
of the main problems with CAIs is constituted by their wide range
of side effects, mainly due to inhibion of the offtarget isoforms such
as hCA I and II.2–4,23
Coumarins 5–18 were screened for the inhibition of four human
(h) CA isoforms involved in important physiologic/pathologic
processes, that is, the cytosolic, hCA I and II (offtargets in this case)
and the transmembrane, tumor-associated hCA IX and XII
(anticancer drug targets).2,3,12,13 Table 1 shows inhibition data of
coumarins 5–18 and the sulfonamide acetazolamide AAZ (as stan-
dard inhibitor) against hCA I, II, IX and XII, after a period of 6 h of
incubation of the enzyme and inhibitor solutions.18–21 It should be
mentioned that assaying the inhibition with the usual 15 min incu-
bation period (as for the sulfonamides)1,5 leads to the measure-
ment of a very weak inhibition (data not shown). For this reason,
a 6 h incubation time has been used for assaying all coumarins
as CAIs.1,5–11The following SAR should be noted regarding the inhi-
bition data of Table 1.(i) Isoform hCA I was moderately or poorly
inhibited by coumarins 5–18 investigated here. Six derivatives
(9, 11–14 and 18) showed KIs in the range of 172.8–246.7 nM
(the same range as the sulfonamide acetazolamide, AAZ) whereas
the remaining ones were weaker, micromolar inhibitors (6, 10,
16 and 17) or did not inhibit significantly the enzyme up until
3. Conclusions
A series of 7-substituted coumarins incorporating aryl-triazole
moieties were prepared by click chemistry procedures starting
from 7-hydroxycoumarin or 4-methyl-7-aminocoumarin. The
panel of new compounds was assayed for the inhibition of the
cytosolic, widespread human hCA I and II isoforms, and the trans-
membrane, tumor-associated ones hCA IX and XII. Most of the cou-
marins were weak inhibitors or did not inhibit significantly hCA I
and II, but showed low nanomolar inhibitory action against the
transmembrane isoforms (KI of 14.3–34.4 nM against hCA IX and
of 4.7–37.8 nM against hCA XII). Since many hypoxic tumors over-
express hCA IX/XII, and as these targets were recently validated for
obtaining antitumor/antimetastatic agents, with one inhibitor in
Phase I clinical trials, the present findings constitute an interesting
extension to the knowledge of non-sulfonamide, selective inhibi-
tors of CA isoforms involved in serious pathologies.
10 lM (Table 1). The compounds showing some inhibitory activity,
had the following substitution patterns at the aryl moiety linked to
the triazole ring: 3-methoxyphenyl, 4-trifluoromethylphenyl,
4-hydroxy-/carboxy-and sulfamoyl-phenyl.(ii) Isoform II, the phys-
iologically dominant one (as it is present in virtually all cells, being
particularly abundant in blood red cells, gastro-intestinal tract,
lungs and kidneys among others) 2–4 was poorly inhibited by
the new coumarins reported here except the carboxylate derivative
13 which showed KI of 99.6 nM. The other derivatives did not inhi-
bit significantly the enzyme up until 10 lM (Table 1). Carboxylates
may have a multitude of inhibition mechanisms towards CAs, as
the COOꢀ moiety may be a zinc-binding group, it may anchor to
the zinc-coordinated water molecule or even promotes binding
outside the active site.19–22(iii) The transmembrane isoform hCA
IX was effectively inhibited by all coumarins reported here, with
KIs in the range of 14.3–34.4 nM. It may be observed that the range
of the inhibitory power is very small, meaning that all the substi-
tution patterns explored in the paper lead to highly effective CAIs
(in fact AAZ shows an inhibition of 25 nM against this isoform and
it has been shown by Williams’ group to show potent antitumor
activity in vivo).12d
4. Experimental protocols
4.1. Chemistry
Anhydrous solvents and all reagents were purchased from
Sigma–Aldrich, Alfa Aesar and TCI. All reactions involving air- or
moisture-sensitive compounds were performed under a nitrogen
atmosphere using dried glassware and syringes techniques to
transfer solutions. Nuclear magnetic resonance (1H NMR, 13C
NMR, DEPT-135, DEPT-90, HSQC, HMBC) spectra were recorded
using a Bruker Advance III 400 MHz spectrometer in DMSO-d6.
Chemical shifts are reported in parts per million (ppm) and the
coupling constants (J) are expressed in Hertz (Hz). Splitting pat-
terns are designated as follows: s, singlet; d, doublet; sept, septet;
t, triplet; q, quartet; m, multiplet; brs, broad singlet; dd, doublet of
doublets, appt, apparent triplet, appq, apparent quartet. The
assignment of exchangeable protons (OH and NH) was confirmed
by the addition of D2O. Analytical thin-layer chromatography
(TLC) was carried out on Merck silica gel F-254 plates. Flash chro-
matography purifications were performed on Merck Silica gel 60
(230–400 mesh ASTM) as the stationary phase and ethyl acetate/
n-hexane were used as eluents. Melting points (mp) were mea-
sured in open capillary tubes with a Gallenkamp MPD350.BM3.5
apparatus and are uncorrected.
The solvents used in MS measures were acetone, acetonitrile
(Chromasolv grade), purchased from Sigma–Aldrich (Milan–Italy),
and mQ water 18 MX, obtained from Millipore’s Simplicity system
(Milan–Italy). The mass spectra were obtained using a Varian
1200L triple quadrupole system (Palo Alto, CA, USA) equipped by
Electrospray Source (ESI) operating in both positive and negative
ions. Stock solutions of analytes were prepared in acetone at
1.0 mg mLꢀ1 and stored at 4 °C. Working solutions of each analyte
were freshly prepared by diluting stock solutions in a mixture of
(iv) A similar situation to what mentioned above for hCA IX has
also been observed for the inhibition of hCA XII with the coumarins
Table 1
Inhibition data of human CA isoforms hCA I, II, IX and XII with coumarins 5–18
reported here and the standard sulfonamide inhibitor acetazolamide (AAZ) by a
stopped flow CO2 hydrase assay18
Compound
R
X
KI (nM)*
hCA I
hCA II
hCA IX
hCA XII
5
6
7
8
H
3-F
—
—
—
—
—
—
—
—
—
—
—
O
>10000
921
>10000
>10000
210.7
1568
239.5
242.5
240.9
172.8
>10000
1243.2
2137.9
246.7
250
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
99.6
>10000
>10000
>10000
>10000
9743
24.5
24.9
31.2
27.5
23.6
24.0
23.0
26.9
28.8
14.3
22.8
33.1
29.6
34.4
25
4.8
5.1
5.5
27.1
5.2
4.7
5.5
5.3
4.9
9.9
37.8
5.0
5.1
4.9
5.7
3-Cl
3-Br
3-OCH3
4-F
4-CF3
4-OH
4-COOH
4-SO2NH2
—
9
10
11
12
13
14
15
16
17
18
AAZ
—
—
—
S
NH
12
*
Mean from 3 different assays, by a stopped flow technique (errors were in the
range of 5–10% of the reported values).
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4
A. Nocentini et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
mQ H2O/ACN 1:1 (v/v) up to a concentration of 1.0
mass spectra of each analyte were acquired by introducing, via syr-
inge pump at 10
L minꢀ1, of the its working solution. Raw-data
were collected and processed by Varian Workstation Vers. 6.8
software.
l
g mLꢀ1 The
(t, J = 7.6, 1H), 7.66 (t, J = 7.6, 2H), 7.70 (d, J = 8.6, 1H), 7.96 (d,
J = 7.6, 2H), 8.05 (d, J = 9.6, 1H), 9.05 (s, 1H); dC (100 MHz,
DMSO-d6): 63.0, 102.6, 113.6, 113.7, 113.8, 121.2, 124.1, 129.8,
130.5, 130.9, 138.0, 144.1, 145.2, 156.2, 161.2, 162.0; m/z (ESI pos-
itive) 320.0 [M+H]+.
l
4.2. Procedure 1
4.2.1.2. Synthesis of 7-[1-(3-fluoro-phenyl)-1H-[1,2,3]triazol-4-
ylmethoxy]-chromen-2-one 6.
4.2.1. General synthetic procedure of compounds 5–1824
N3
Cu(0) nanosized, TMACl
H2O/tBuOH 1/1
O
O
O
O
O
O
F
N
N
N
F
2
B
6
The appropriate alkyne 2, 19–21 (1.0 equiv) was added to a sus-
pension of aryl azide A–J, 4 (1.1 equiv) in H2O/tBuOH 1:1 (3.5 ml)
at rt, followed by copper (0) nanosized (0.1 equiv) and TMACl
(1.0 equiv). The suspension was stirred at 60 °C until starting mate-
rials were consumed (TLC monitoring), then quenched with H2O
(20 ml) and the formed precipitate was filtered-off and washed
with H2O. The solid was dissolved in a minimal amount of acetone,
the obtained solution was filtered through Celite 521Ò and then
concentrated under vacuo to give a residue that was triturated
with Et2O or DCM to afford the titled compounds 5–18.
7-[1-(3-Fluoro-phenyl)-1H-[1,2,3]triazol-4-ylmethoxy]-chromen-
2-one 6 was obtained according the general procedure 1 earlier
reported using 3-fluorophenylazide B (1.1 equiv), 7-(prop-2-yny-
loxy)-2H-chromen-2-one 2 (0.05 g, 1.0 equiv) in tBuOH/H2O (1:1,
3.5 ml), tetramethylamonium chloride (1.0 equiv) and copper
nanosize (0.1 equiv). The reaction mixture was stirred for 1.5 h to
give the titled compound 6 as a white solid.
7-[1-(3-Fluoro-phenyl)-1H-[1,2,3]triazol-4-ylmethoxy]-chromen-
2-one 6: 77% yield; mp 158–160 °C; silica gel TLC Rf 0.45 (Ethyl
acetate/n-hexane 50% v/v); dH (400 MHz, DMSO-d6): 5.42 (s, 2H,
CH2), 6.36 (d, J = 9.6, 1H), 7.11 (dd, J = 2.4, 8.6, 1H), 7.25 (d,
J = 2.4, 1H), 7.42 (m, 1H), 7.71 (m, 2H), 7.86 (m, 1H), 7.91 (m,
1H), 8.05 (d, J = 9.6, 1H), 9.10 (s, 1H); dF (376 MHz, DMSO-d6):
4.2.1.2.
Synthesis
of
7-(1-phenyl-1H-[1,2,3]triazol-4-yl-
methoxy)-chromen-2-one 5.
N
3
Cu(0) nanosized, TMACl
H O/tBuOH 1/1
2
O
O
O
O
O
O
N
N
N
2
A
5
7-(1-Phenyl-1H-[1,2,3]triazol-4-ylmethoxy)-chromen-2-one 5 was
obtained according the general procedure 1 earlier reported using
phenylazide A (1.1 equiv), 7-(prop-2-ynyloxy)-2H-chromen-2-one
2 (0.05 g, 1.0 equiv) in tBuOH/H2O (1:1, 3.5 ml), tetramethylamo-
nium chloride (1.0 equiv) and copper nanosize (0.1 equiv). The
reaction mixture was stirred for 5 h to give the titled compound
5 as a white solid.
ꢀ110.52 (s, 1F); dC (100 MHz, DMSO-d6): 62.5, 102.5, 108.1 (d,
J2CF = 26.3), 113.6, 113.7, 113.8, 116.4 (d, J2CF = 21.0), 117.0, 124.2,
130.5, 132.8 (d, J3CF = 9.0), 138.6 (d, J3CF = 10.4), 144.3, 145.1,
156.2, 161.2, 161.9, 162.8 (d, J1CF = 262.7); m/z (ESI positive)
338.0 [M+H]+.
4.2.1.3. Synthesis of 7-[1-(3-chloro-phenyl)-1H-[1,2,3]triazol-4-
ylmethoxy]-chromen-2-one 7.
N3
Cu(0) nanosized, TMACl
H2O/tBuOH 1/1
O
O
O
O
O
O
Cl
N
N
N
Cl
2
C
7
7-(1-Phenyl-1H-[1,2,3]triazol-4-ylmethoxy)-chromen-2-one
5:
1-Azido-3-chlorobenzene C (1.1 equiv) and 7-(prop-2-ynyloxy)-
2H-chromen-2-one (0.05 g, 1.0 equiv) were dissolved in
tBuOH/H2O 1:1 (3.5 ml) and then tetramethylamonium chloride
74% yield; mp 172–175 °C; silica gel TLC Rf 0.52 (EtOAc/n-hexane
50% v/v); dH (400 MHz, DMSO-d6): 5.41 (s, 2H, CH2), 6.35
(d, J = 9.6, 1H), 7.11 (dd, J = 2.4, 8.6, 1H), 7.25 (d, J = 2.4, 1H), 7.56
2
(1.0 equiv) and copper nanosize (10% mol) were added. The
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A. Nocentini et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
5
mixture was stirred at 60 °C for 3.5 h andthen treated as described
in general procedure 1 to afford 7 as a white solid.
H2O 1:1 (3.5 ml) and then tetramethylamonium chloride
(1.0 equiv) and copper nanosize (10% mol) were added. The mix-
ture was stirred at 60 °C for 2.5 h and then treated as described
in general procedure 1 to afford 9 as a white solid.
7-[1-(3-Methoxy-phenyl)-1H-[1,2,3]triazol-4-ylmethoxy]-chro-
men-2-one 9: 81% yield; mp 131–133 °C; silica gel TLC Rf 0.36
(Ethyl acetate/n-hexane 50% v/v); dH (400 MHz, DMSO-d6): 3.89
(s, 3H, CH3), 5.40 (s, 2H, CH2), 6.36 (d, J = 9.6, 1H), 7.11 (m, 2H),
7.24 (d, J = 2.4, 1H), 7.54 (m, 3H), 7.70 (d, J = 8.6, 1H), 8.05
(d, J = 9.6, 1H), 9.07 (s, 1H); dC (100 MHz, DMSO-d6): 56.6, 62.7,
102.6, 106.6, 113.1, 113.6, 113.7, 113.8, 115.5, 124.2, 130.5,
131.8, 138.5, 144.0, 145.2, 156.2, 131.1, 161.2, 162.0; m/z (ESI pos-
itive) 350.0 [M+H]+.
7-[1-(3-Chloro-phenyl)-1H-[1,2,3]triazol-4-ylmethoxy]-chromen-
2-one 7: 77% yield; mp 182–183 °C; silica gel TLC Rf 0.41
(Ethyl acetate/n-hexane 50% v/v); dH (400 MHz, DMSO-d6): 5.41
(s, 2H, CH2), 6.36 (d, J = 9.6, 1H), 7.10 (dd, J = 2.4, 8.6, 1H), 7.24
(d, J = 2.4, 1H), 7.66 (m, 3H), 7.97 (d, J = 8.1, 1H), 8.05 (d, J = 9.6,
1H), 8.11 (d, J = 2.0, 1H), 9.12 (s, 1H); dC (100 MHz, DMSO-d6):
62.5, 102.3, 113.6, 113.7, 113.8, 119.7, 120.9, 124.3, 129.6, 130.5,
132.6, 135.1, 138.5, 144.3, 145.2, 156.2, 161.1, 161.9; m/z
(ESI positive) 354.0 [M+H]+.
4.2.1.4. Synthesis of 7-[1-(3-bromo-phenyl)-1H-[1,2,3]triazol-4-
ylmethoxy]-chromen-2-one 8.
N
3
Cu(0) nanosized, TMACl
H O/tBuOH 1/1
2
O
O
O
O
O
O
Br
N
N
N
Br
2
D
8
1-Azido-3-bromobenzene D (1.1 equiv) and 7-(prop-2-ynyloxy)-
2H-chromen-2-one 2 (0.05 g, 1.0 equiv) were dissolved in tBuOH/
4.2.1.6. Synthesis of 7-[1-(4-fluoro-phenyl)-1H-[1,2,3]triazol-4-
ylmethoxy]-chromen-2-one 10.
N3
Cu(0) nanosized, TMACl
H2O/tBuOH 1/1
F
O
O
O
O
O
O
N
N
F
N
2
F
10
H2O 1:1 (3.5 ml) and then tetramethylamonium chloride
(1.0 equiv) and copper nanosize (10% mol) were added. The mix-
ture was stirred at 60 °C for 6 h andthen treated as described in
general procedure 1to afford 8 as a white solid.
7-[1-(3-Bromo-phenyl)-1H-[1,2,3]triazol-4-ylmethoxy]-chromen-
2-one 8: 78% yield; mp 170–172 °C; silica gel TLC Rf 0.41 (Ethyl
acetate/n-hexane 50% v/v); dH (400 MHz, DMSO-d6): 5.41 (s, 2H,
CH2), 6.36 (d, J = 9.6, 1H), 7.10 (dd, J = 2.4, 8.6, 1H), 7.24 (d,
J = 2.4, 1H), 7.61 (t, J = 8.2, 1H), 7.71 (d, J = 8.6, 1H), 7.76 (d,
J = 8.2, 1H), 8.02 (d, J = 8.2, 1H), 8.05 (d, J = 9.6, 1H), 8.23 (s, 1H),
9.12 (s, 1H); dC (100 MHz, DMSO-d6): 62.5, 102.6, 113.6, 113.7,
113.8, 120.1, 123.4, 123.7, 124.3, 130.5, 132.5, 132.8, 138.5,
144.3, 145.2, 156.0, 161.1, 161.9; m/z (ESI positive) 398.0 [M+H]+.
7-[1-(4-fluoro-phenyl)-1H-[1,2,3]triazol-4-ylmethoxy]-chromen-
2-one 10 was obtained according the general procedure
earlier reported using 1-azido-4-fluorobenzene (1.1 equiv),
7-(prop-2-ynyloxy)-2H-chromen-2-one 2 (0.05 g, 1.0 equiv) in
tBuOH/H2O 1:1 (3.5 ml), tetramethylamonium chloride
(1.0 equiv) and copper nanosize (0.1 equiv). The reaction
mixture was stirredfor 7.5 h to give the titled compound 10 as
a whitesolid.
7-[1-(4-Fluoro-phenyl)-1H-[1,2,3]triazol-4-ylmethoxy]-chromen-
2-one 10: 68% yield; mp 201–203 °C; silica gel TLC Rf 0.34
(Ethyl acetate/n-hexane 50% v/v); dH (400 MHz, DMSO-d6): 5.40
(s, 2H, CH2), 6.36 (d, J = 9.2, 1H), 7.10 (dd, J = 2.4, 8.6, 1H), 7.24
(d, J = 2.4, 1H), 7.51 (m, 2H), 7.70 (d, J = 8.6, 1H), 8.00 (m, 2H),
8.05 (d, J = 9.2, 1H), 9.02 (s, 1H); dF (376 MHz, DMSO-d6):
ꢀ112.94 (s, 1F); dC (100 MHz, DMSO-d6): 62.4, 102.4, 113.5,
113.6, 113.7, 117.6 (d, J2CF = 23.1), 123.4 (d, J3CF = 8.8), 124.3,
1
F
4.2.1.5. Synthesis of 7-[1-(3-methoxy-phenyl)-1H-[1,2,3]triazol-
4-ylmethoxy]-chromen-2-one 9.
N
3
Cu(0) nanosized, TMACl
H O/tBuOH 1/1
2
O
O
O
O
O
O
O
N
N
N
O
2
E
9
1-Azido-3-methoxybenzene E (1.1 equiv) and 7-(prop-2-ynyloxy)-
2H-chromen-2-one 2 (0.05, 1.0 equiv) were dissolved in tBuOH/
130.7, 133.9 (d, J4CF = 2.9), 144.0, 145.1, 156.1, 161.0, 161.8, 162.5
(d, J1CF = 244.4); m/z (ESI positive) 338.0 [M+H]+.
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6
A. Nocentini et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
4.2.1.7. Synthesis of 7-[1-(4-trifluoromethyl-phenyl)-1H-[1,2,3]-
triazol-4-ylmethoxy]-chromen-2-one 11.
mixture was stirred at 60 °C for 1.5 h and then treated as
described in general procedure 1 to afford 12 as a white solid.
N3
Cu(0) nanosized, TMACl
H2O/tBuOH 1/1
F3C
O
O
O
O
O
O
N
N
CF3
N
2
G
11
7-[1-(4-trifluoromethyl-phenyl)-1H-[1,2,3]triazol-4-ylmethoxy]-
chromen-2-one 11 was obtained according the general procedure 1
7-[1-(4-Hydroxy-phenyl)-1H-[1,2,3]triazol-4-ylmethoxy]-chro-
men-2-one 12: 77% yield; mp 241–242 °C; silica gel TLC Rf 0.21
(Ethyl acetate/n-hexane 50% v/v); dH (400 MHz, DMSO-d6): 5.37
(s, 2H, CH2), 6.35 (d, J = 9.6, 1H), 6.98 (d, J = 8.6, 2H), 7.10 (dd,
J = 2.4, 8.6, 1H), 7.24 (d, J = 2.4, 1H), 7.70 (m, 3H), 8.05 (d, J = 9.6,
1H), 8.85 (s, 1H), 10.00 (s, 1H,, exchange with D2O, OH); dC
(100 MHz, DMSO-d6): 62.6, 102.6, 113.6, 113.7, 113.8, 117.0,
123.0, 124.0, 129.6, 130.5, 143.7, 145.2, 156.2, 158.8, 161.2,
162.0; m/z (ESI positive) 336.0 [M+H]+.
earlier reported using 1-azido-4-trifluoromethylbenzene
G
(1.1 equiv), 7-(prop-2-ynyloxy)-2H-chromen-2-one (0.05 g,
2
1.0 equiv) in tBuOH/H2O 1:1 (3.5 ml), tetramethylamonium
chloride (1.0 equiv) and copper nanosize (0.1 equiv). The reaction
mixture was stirred for 2.5 h to give the titled compound 11 as a
white powder.
7-[1-(4-Trifluoromethyl-phenyl)-1H-[1,2,3]triazol-4-ylmethoxy]-
chromen-2-one 11: 70% yield; mp 185–187 °C; silica gel TLC Rf 0.50
(Ethyl acetate/n-hexane 50% v/v); dH (400 MHz, DMSO-d6): 5.43
(s, 2H, CH2), 6.35 (d, J = 9.6, 1H), 7.10 (dd, J = 2.4, 8.6, 1H), 7.24
4.2.1.9. Synthesis of 7-[1-(4-carboxy-phenyl)-1H-[1,2,3]triazol-
4-ylmethoxy]-chromen-2-one 13.
N3
Cu(0) nanosized, TMACl HO
H2O/tBuOH 1/1
O
O
O
O
O
O
N
N
O
N
HO
O
2
I
13
(d, J = 2.4, 1H), 7.70 (d, J = 8.6, 1H), 8.05 (m, 3H), 8.22 (d, J = 8.4,
2H), 9.19 (s, 1H); dC (100 MHz, DMSO-d6): 62.5, 102.6, 113.7,
113.7, 113.8, 121.6, 123.9 (d, J1CF = 270.6), 124.4, 128.2 (q,
J3CF = 3.4), 129.8 (d, J2CF = 32.7), 130.5, 140.2, 144.5, 145.2, 156.2,
161.2, 161.9; m/z (ESI positive) 388.0 [M+H]+.
1-Azido-4-carboxybenzene I (1.1 equiv) and 7-(prop-2-ynyloxy)-
2H-chromen-2-one 2 (0.05, 1.0 equiv) were dissolved in tBuOH/
H2O 1:1 (3.5 ml) and then tetramethylamonium chloride
(1.0 equiv) and copper nanosize (10% mol) were added. The
mixture was stirred at 60 °C for 12 h and treated as described in
general procedure 1 to afford 13 as a white solid.
4.2.1.8. Synthesis of 7-[1-(4-hydroxy-phenyl)-1H-[1,2,3]triazol-
4-ylmethoxy]-chromen-2-one 12.
7-[1-(4-Carboxy-phenyl)-1H-[1,2,3]triazol-4-ylmethoxy]-chromen-
2-one 13: 55% yield; mp 290–292 °C d; silica gel TLC Rf 0.34 (MeOH/
N
3
Cu(0) nanosized, TMACl
H O/tBuOH 1/1
HO
2
O
O
O
O
O
O
N
N
OH
N
2
H
12
1-Azido-4-hydroxybenzene
H
(1.1 equiv) and 7-(prop-2-yny-
CH2Cl2 10% v/v); dH (400 MHz, DMSO-d6): 5.42 (s, 2H, CH2), 6.36 (d,
J = 9.2, 1H), 7.10 (dd, J = 2.4, 8.6, 1H), 7.25 (d, J = 2.4, 1H), 7.71 (d,
J = 8.6, 1H), 8.05 (d, J = 9.2, 1H), 8.15 (bq, 4H), 9.16 (s, 1H), 13.34 (br
s, 1H, exchange with D2O, COOH); m/z (ESI negative) 362.17 [MꢀH]ꢀ.
loxy)-2H-chromen-2-one 2 (0.05 g, 1.0 equiv) were dissolved in
tBuOH/H2O 1:1 (3.5 ml) and then tetramethylamonium chloride
(1.0 equiv) and copper nanosize (10% mol) were added. The
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A. Nocentini et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
7
4.2.1.10. Synthesis of 4-[4-(2-oxo-2H-chromen-7-yloxymethyl)-
n-hexane from 20% to 50% to afford the title compound 15 as a
[1,2,3]triazol-1-yl]-benzenesulfonamide 14.
yellow solid.
N
3
Cu(0) nanosized, TMACl
H O/tBuOH 1/1
H NO S
2
O
O
O
O
O
O
2
2
N
N
N
SO NH
2
2
2
J
14
4-Azidobenzenesulfonamide J (1.1 equiv) and 7-(prop-2-ynyloxy)-
2H-chromen-2-one 2 (0.05, 1.0 equiv) were dissolved in tBuOH/
H2O 1:1 (3.5 ml) and then tetramethylamonium chloride
(1.0 equiv) and copper nanosize (10% mol) were added. The mix-
ture was stirred at 60 °C for 2.5 h and then treated as described
in general procedure 1 to afford 14 as a light yellow solid.
4-[4-(2-Oxo-2H-chromen-7-yloxymethyl)-[1,2,3]triazol-1-yl]-ben-
zenesulfonamide 14: 80% yield; mp 279–281 °C; silica gel TLC
Rf 0.43 (MeOH/CH2Cl2 10% v/v); dH (400 MHz, DMSO-d6): 5.43
(s, 2H, CH2), 6.36 (d, J = 9.2, 1H), 7.10 (dd, J = 2.4, 8.6, 1H), 7.25
(d, J = 2.4, 1H), 7.59 (s, 2H, exchange with D2O, SO2NH2), 7.71
(d, J = 8.6, 1H), 8.06 (m, 3H), 8.19 (d, J = 8.8, 2H), 9.15 (s, 1H); dC
(100 MHz, DMSO-d6): 62.5, 102.6, 113.7, 113.8, 113.9, 121.4,
4-Methyl-7-(4-((2-oxo-2H-chromen-7-yloxy)methyl)-1H-1,2,3-
triazol-1-yl)-2H-chromen-2-one 15: 88% yield; silica gel TLC Rf 0.32
(Ethyl acetate/n-hexane 50% v/v); dH (400 MHz, DMSO-d6) 2.11
(3H, s, CH3), 5.44 (2H, s, 30-H2), 6.35 (1H, d, J 9.6, 300-H), 6.53 (1H,
d, J 1.2, 3-H), 7.11 (1H, dd, J 8.4, 2.4, 600-H), 7.25 (1H, d, J 2.4,
800-H), 7.71 (1H, d, J 8.4, 500-H), 8.06 (4H, m, 5, 6, 8, 400-H), 9.22
(1H, s, 10-H); dC (100 MHz, DMSO-d6) 161.9, 161.0, 160.3, 156.2,
154.3, 153.7, 145.2, 144.5, 139.3, 130.5, 128.2, 124.4, 120.6,
116.6, 115.8, 113.9, 113.7, 108.6, 108.0, 102.6, 62.5, 32.2; m/z
(ESI positive) 402.0 [M+H]+.
4.2.1.12. Synthesis of 4-methyl-7-(4-phenoxymethyl-[1,2,3]tria-
zol-1-yl)-chromen-2-one 16.
O
O
Cu (0) nanosized, TMACl
H2O/tBuOH 1/1
O
O
N
N
O
O
N3
N
4
16
19
124.4, 128.5, 130.5, 139.4, 144.5, 144.9, 145.2, 156.3, 161.2,
7-Azido-4-methyl-2H-chromen-2-one 4 (1.1 equiv) and prop-2-
ynyloxy-benzene 19 (0.05 g, 1.0 equiv) were dissolved in tBuOH/
H2O 1:1 (3.5 ml) and then tetramethylamonium chloride
(1.0 equiv) and copper nanosize (10% mol) were added. The mix-
ture was stirred at 60 °C for 2 h and then treated as described in
general procedure 1 to afford 16 as a yellow solid.
161.9; m/z (ESI negative) 397.0 [MꢀH]ꢀ.
4.2.1.1. Synthesis of 4-methyl-7-[4-(2-oxo-2H-chromen-7-ylox-
ymethyl)-[1,2,3]triazol-1-yl]-chromen-2-one 15.
5
8
4
3
2
6
Cu(0) nanosized, TMACl
H O/tBuOH 1/1
O
1'
7
5"
6"
7"
2'
3'
4"
2
N
N
O
O
O
O
O
N
3
O
O
3"
2"
N
8"
2
4
O
15
O
4-Methyl-7-[4-(2-oxo-2H-chromen-7-yloxymethyl)-[1,2,3]triazol-
1-yl]-chromen-2-one 15 was obtained according the general
procedure 1 earlier24 reported using 7-azido-4-methyl-2H-chro-
men-2-one 4 (1.1 equiv) and 7-(prop-2-ynyloxy)-2H-chromen-2-
one 2 (0.05 g, 1.0 equiv) in tBuOH/H2O 1:1 (3.5 ml), tetramethyla-
monium chloride (1.0 equiv) and copper nanosize (10% mol). The
reaction mixture was stirred for 6 h to give a solid that was purified
by silica gel column chromatography eluting with ethyl acetate in
4-Methyl-7-(4-phenoxymethyl-[1,2,3]triazol-1-yl)-chromen-2-one
16: 70% yield; mp 208–209 °C; silica gel TLC Rf 0.40 (EtOAc/n-hex-
ane 50% v/v); dH (400 MHz, DMSO-d6): 2.51 (s, 3H, CH3), 5.30 (s,
2H, CH2), 6.53 (d, J = 1.2, 1H), 7.02 (t, J = 7.6, 1H), 7.13 (d, J = 7.6,
2H), 7.37 (t, J = 7.6, 2H), 8.06 (m, 3H), 9.18 (s, 1H); dC (100 MHz,
DMSO-d6): 19.0, 61.8, 108.5, 115.6, 115.7, 116.5, 120.5, 121.9,
124.0, 128.2, 130.5, 139.4, 145.3, 153.7, 154.5, 158.9, 160.3; m/z
(ESI positive) 334.0 [M+H]+.
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8
A. Nocentini et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
4.2.1.13. Synthesis of 4-methyl-7-(4-phenylsulfanylmethyl-
[1,2,3]triazol-1-yl)-chromen-2-one 17.
4.3. Procedure 2
4.3.1. General synthetic procedure of alkynes 2, 19–2125
S
S
O
O
N
N
Cu (0) nanosized, TMACl
H O/tBuOH 1/1
O
O
N
3
N
2
4
17
20
7-azido-4-methyl-2H-chromen-2-one 4 (1.1 equiv) and prop-2-
ynylsulfanyl-benzene 20 (0.05 g, 1.0 equiv) were dissolved in
tBuOH/H2O 1:1 (3.5 ml) and then tetramethylamonium chloride
(1.0 equiv) and copper nanosize (10% mol) were added. The mix-
ture was stirred at 60 °C for 2 h and then treated as described in
general procedure 1 to afford 17 as a yellow solid.
4-Methyl-7-(4-phenylsulfanylmethyl-[1,2,3]triazol-1-yl)-chro-
men-2-one 17: 68% yield; mp 178–179 °C; silica gel TLC Rf 0.44
(EtOAc/n-hexane 50% v/v); dH (400 MHz, DMSO-d6): 2.51 (s,
3H, CH3), 4.44 (s, 2H, CH2), 6.52 (d, J = 1.2, 1H), 7.24 (t, J = 7.6,
1H), 7.37 (t, J = 7.6, 2H), 7.45 (d, J = 7.6, 2H), 8.00 (m, 3H),
8.94 (s, 1H); dC (100 MHz, DMSO-d6): 19.0, 28.1, 108.3,
115.6, 116.3, 120.3, 122.6, 127.0, 128.2, 129.3, 130.2, 136.4.
139.3, 146.2, 153.7, 154.6, 160.4; m/z (ESI positive) 350.0
[M+H]+.
Br
X
X
K CO
2
3
DMF
X= O; 19
= S; 20
X= OH; 19a
= SH; 20a
= NH; 21
= NH2; 21a
Br
K CO
2
3
HO
O
O
DMF
O
O
O
1
2
Propargyl bromide (1.2 equiv) was added to a suspension of com-
pound 1, 19a–21a (0.5 g, 1.0 equiv) and K2CO3 (2.0 equiv) in dry
4.2.1.14. Synthesis of 4-Methyl-7-(4-phenylaminomethyl-[1,2,3]
triazol-1-yl)-chromen-2-one 18.
HN
HN
N
N
O
O
Cu (0) nanosized, TMACl
H2O/tBuOH 1/1
O
O
N3
N
4
18
21
DMF (4 ml) under a nitrogen atmosphere and that was stirred at
rt until starting material was consumed (TLC monitoring). The reac-
tion mixture was quenched with H2O (20 ml) and extracted with
Et2O or EtOAc (25 ml). The organic layer was washed with brine
(4 ꢁ 15 ml), dried over Na2SO4, filtered-off and concentrated under
vacuo to give the titled compounds 2, 19–21.
7-azido-4-methyl-2H-chromen-2-one
4
(1.1 equiv)
and
phenyl-prop-2-ynyl-amine 21 (0.05 g, 1.0 equiv) were dissolved
in tBuOH/H2O 1:1 (3.5 ml) and then tetramethylamonium
chloride (1.0 equiv) and copper nanosize (10% mol) were added.
The mixture was stirred at 60 °C for 2.5 h and then treated
as described in general procedure 1 to afford 18 as a yellow
solid.
4.3.1.1. Synthesis 7-prop-2-ynyloxy-chromen-2-one 2.
4-Methyl-7-(4-phenylaminomethyl-[1,2,3]triazol-1-yl)-chromen-
2-one 18: 85% yield; mp 197–200 °C; silica gel TLC Rf 0.30 (EtOAc/
n-hexane 50% v/v); dH (400 MHz, DMSO-d6): 2.51 (s, 3H, CH3), 4.43
(d, J = 6.0, 2H, CH2), 6.20 (t, J = 6.0, 1H, exchange with D2O, NH),
6.52 (d, J = 1.2, 1H), 6.60 (t, J = 7.8, 1H), 6.71 (d, J = 7.8, 2H), 7.12
(t, J = 7.8, 2H), 8.03 (m, 3H), 8.94 (s, 1H); dC (100 MHz, DMSO-d6):
19.0, 39.5, 108.2, 113.3, 115.5, 116.2, 117.1, 120.2, 122.3, 128.2,
129.8, 139.5, 148.3, 149.2, 153.7, 154.6, 160.4; m/z (ESI positive)
333.0 [M+H]+.
O
O
O
2
7-Prop-2-ynyloxy-chromen-2-one 2 was obtained according the
general procedure 2 reported earlier.25 The reaction mixture was
Please cite this article in press as: Nocentini, A.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.09.041

A. Nocentini et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
9
stirred at rt for 2 h, quenched with H2O and extracted with EtOAc
4.4. Procedure 3
to give the titled compound 2 as a white powder.
7-Prop-2-ynyloxy-chromen-2-one 2: 73% yield; mp 118–119 °C;
silica gel TLC Rf 0.56 (EtOAc/n-hexane 50% v/v); dH (400 MHz,
DMSO-d6): 3.69 (t, J = 2.4, 1H), 4.97 (d, J = 2.4, 2H), 6.36
(d, J = 9.6, 1H), 7.03 (dd, J = 2.4, 8.6, 1H), 7.09 (d, J = 2.4, 1H), 7.70
(d, J = 8.6, 1H), 8.04 (d, J = 9.6, 1H); dC (100 MHz, DMSO-d6): 57.0,
79.4, 79.8, 102.7, 113.7, 113.8, 113.9, 130.4, 145.1, 156.0, 161.0,
161.1. Experimental in agreement with reported data.26
4.4.1. Synthesis of phenylazides 4, A–J6
NH
2
N
3
i) NaNO2, H+
R
R
ii) NaN3
The proper aniline (0.5 g, 1.0 equiv) was dissolved in a 4 M HCl
aqueous solution (5 ml) at 0 °C. NaNO2 (1.2 equiv) was slowly
added and the resulting solution was stirred at the same tempera-
ture for 0.5 h. Then NaN3 (1.5 equiv) was added portion-wise and
the mixture was stirred at rt for 0.5 h. Reaction mixture was fil-
tered-off or extracted with Et2O (2 ꢁ 15 ml) and the combined
organic layers were dried over Na2SO4, filtered-off and the solvent
evaporated in vacuo to afford the corresponding phenylazide
which was used without further purification.
4.3.1.2. Synthesis of prop-2-ynyloxy-benzene 19.
O
19
4.4.2. Synthesis of 7-azido-4-methyl-chromen-2-one 4
Prop-2-ynyloxy-benzene 19 was obtained according the general
procedure 2 earlier reported. The reaction mixture was stirred at
rt for 2 h to give the titled compound 19.
Prop-2-ynyloxy-benzene 19: 74%yield; silica gel TLC Rf 0.68
(EtOAc/n-hexane 20% v/v); dH (400 MHz, CDCl3): 2.58 (t, J = 2.4,
1H), 4.76 (d, J = 2.4, 2H), 7.06 (m, 3H), 7.37 (t, J = 8.8, 2H); dC
(100 MHz, CDCl3): 56.1, 75.8, 79.0, 115.3, 121.9, 129.8, 157.9.
Experimental in agreement with reported data.25
N3
O
O
4
7-Azido-4-methyl-chromen-2-one 4 was obtained according the
general procedure 3reported earlier. 7-Amino-4-methyl-coumar-
in3was treated with NaNO2 and NaN3 in a 2 M HCl aqueous
solution and the formed precipitate was filtered-off to afford the
title compound 4 as a yellow solid.
4.3.1.3. Synthesis of prop-2-ynylsulfanyl-benzene 20.
7-Azido-4-methyl-chromen-2-one 4: 88% yield; mp 122–124 °C;
silica gel TLC Rf 0.57 (EtOAc/n-hexane 20% v/v); dH (400 MHz,
DMSO-d6): 2.46 (d, J = 1.2, 3H, CH3), 6.38 (d, J = 1.2, 1H), 7.18 (m,
2H), 7.83 (d, J = 8.4, 1H); dC (100 MHz, DMSO-d6): 19.0, 107.7,
114.1, 116.5, 117.7, 127.9, 144.2, 153.8, 155.0, 160.4. Experimental
in agreement with reported data.29
S
20
Prop-2-ynylsulfanyl-benzene 20was obtained according the
general procedure 2 earlier reported.25The reaction mixture was
stirred at rt for 2 h to give the titled compound 20.
4.4.3. Synthesis of phenylazide A
Prop-2-ynylsulfanyl-benzene 20: 96% yield; silica gel TLC Rf 0.61
(EtOAc/n-hexane 10% v/v); dH (400 MHz, CDCl3): 2.30 (t, J = 2.4,
1H), 3.67 (d, J = 2.4, 2H), 7.32 (m, 1H), 7.39 (t, J = 7.6, 2H), 7.52
(m, 2H); dC (100 MHz, CDCl3): 22.9, 71.9, 80.2, 127.3, 129.3,
129.4, 130.4. Experimental in agreement with reported data.27
N
3
F
B
Phenyl azide A was obtained according the general procedure 3
reported earlier.
4.3.1.4. Synthesis of phenyl-prop-2-ynyl-amine 21.
Phenyl azide A: 60% yield; silica gel TLC Rf 0.76 (EtOAc/n-hexane
50% v/v); dH (400 MHz, DMSO-d6):7.12 (d, J = 7.6, 2H), 7.20 (t,
J = 7.6, 1H), 7.42 (t, J = 7.6, 2H); dC (100 MHz, DMSO-d6): 120.0,
126.1, 131.0, 140.3. Experimental in agreement with reported
data.29
NH
21
4.4.4. Synthesis of 3-fluorophenylazide B
Phenyl-prop-2-ynyl-amine 21was obtained according the general
procedure 2 earlier reported.25 The reaction mixture was stirred
at rt for 4 h and the obtained oil was purified by silica gel column
chromatography eluting with EtOAc/petroleum ether 1:20 to
afford the title compounds 21.
N
3
F
Phenyl-prop-2-ynyl-amine 21: 35% yield; silica gel TLC Rf 0.76
(EtOAc/n-hexane 20% v/v); dH (400 MHz, DMSO-d6): 2.24
(t, J = 2.4, 1H), 3.96 (d, J = 2.4, 2H), 6.78 (d, J = 7.4, 2H), 6.85
(t, J = 7.4, 1H), 7.31 (t, J = 7.4, 2H); dC (100 MHz, CDCl3): 34.4,
72.1, 80.8, 114.5, 119.7, 129.6, 146.3. Experimental in agreement
with reported data.28
B
3-Fluorophenylazide B was obtained according to the general
procedure3reported earlier.30
3-Fluorophenyl azide B: 62% yield; silica gel TLC Rf 0.81 (EtOAc/
n-hexane 50% v/v); dH (400 MHz, DMSO-d6): 7.06 (m, 3H), 7.46
Please cite this article in press as: Nocentini, A.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.09.041

10
A. Nocentini et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
(q, J = 7.2, 1H); dF (376 MHz, DMSO-d6): ꢀ110.52 (s, 1F); dC
(100 MHz, DMSO-d6): 107.6 (d, J2CF = 25.0), 112.8 (d, J2CF = 21.0),
116.2, 132.5 (d, J3CF = 9.0), 142.5 (d, J3CF = 9.0), 163.7 (d,
J1CF = 245.0). Experimental in agreement with reported data.30
J = 8.8, 2H); dF (376 MHz, DMSO-d6): ꢀ117.77 (s, 1F); dC (100 MHz,
DMSO-d6): 117.7 (d, J2CF = 23), 121.8 (d, J3CF = 9.0), 136.4, 160.3 (d,
J1CF = 241.0). Experimental in agreement with reported data.30
4.4.9. Synthesis of 4-trifluoromethyl-phenyl azide G
4.4.5. Synthesis of 3-chlorophenylazide C
N
3
N3
CF
3
Cl
G
C
4-Trifluoromethylphenylazide G was obtained according to the
general procedure 3 reported earlier.30
3-Chlorophenylazide C was obtained according to the general pro-
cedure 3 reported earlier.30
4-Trifluoromethylphenylazide G: 67% yield; silica gel TLC Rf 0.84
(EtOAc/n-hexane 50% v/v); dH (400 MHz, DMSO-d6): 7.35 (d, J = 8.8,
2H), 7.78 (d, J = 8.8, 2H); dF (376 MHz, DMSO-d6): ꢀ56.18 (s, 3F); dC
3-Chlorophenyl azide C: 86% yield; silica gel TLC Rf 0.84
(EtOAc/n-hexane 50% v/v); dH (400 MHz, DMSO-d6): 7.14 (dd,
J = 2.2, 8.0, 1H), 7.24 (t, J = 2.2, 1H), 7.30 (d, J = 2.2, 1H), 7.46
(t, J = 8.0, 1H); dC (100 MHz, DMSO-d6): 118.9, 120.1, 126.0, 132.4,
135.1, 142.2. Experimental in agreement with reported data.30
(100 MHz, DMSO-d6): 120.8, 125.0 (d, J1CF = 269.6), 126.2 (q, J2
=
CF
32.0), 130.0, 144.7. Experimental in agreement with reported data.33
4.4.10. Synthesis of 4-hydroxy-phenyl azide H
4.4.6. Synthesis of 3-bromophenylazide D
N
3
N3
Br
OH
D
H
3-Bromophenylazide D was obtained according to the general pro-
cedure 3reported earlier.
4-Hydroxyphenylazide H was obtained according the general pro-
cedure 3 reported earlier.31
3-Bromophenylazide D: 88% yield; silica gel TLC Rf 0.83 (EtOAc/
n-hexane 50% v/v); dH (400 MHz, DMSO-d6):7.18 (dt, J = 1.6, 7,2,
1H), 7.38 (m, 3H); dC (100 MHz, DMSO-d6): 119.3, 122.8, 123.5,
128.9, 132.7, 142.3. Experimental in agreement with reported data.30
4-Hydroxyphenylazide H: 46% yield; silica gel TLC Rf 0.84 (EtOAc/
n-hexane 50% v/v); dH (400 MHz, DMSO-d6):6.84 (d, J = 8.8, 2H),
6.97 (d, J = 8.8, 2H), 9.56 (br s, 1H, exchange with D2O, OH); dC
(100 MHz, DMSO-d6): 117.5, 121.1, 130.6, 156.0. Experimental in
agreement with reported data.31
4.4.7. Synthesis of 3-methoxyphenylazide E
4.4.11. Synthesis of 4-azidobenzoic acid I
N3
N
3
O
E
O
OH
3-Methoxyphenyl azide E was obtained according to the general
procedure 3 reported earlier.
I
3-Methoxyphenyl azide E: 74% yield; silica gel TLC Rf 0.78
(EtOAc/n-hexane 50% v/v); dH (400 MHz, DMSO-d6): 3.80 (s, 3H,
CH3), 6.66 (t, J = 2.4, 1H), 6.74 (ddd, J = 0.8, 2.4, 8.2, 1H), 6.81
(ddd, J = 0.8, 2.4, 8.2, 1H), 7.36 (t, J = 8.2, 1H); dC (100 MHz,
DMSO-d6): 56.3, 105.7, 112.0, 112.1, 131.7, 141.5, 161.5. Experi-
mental in agreement with reported data.29
4-Azidobenzoic acid I was obtained according the general procedure
3 reported earlier. 4-Aminobenzoic acid was treated with NaNO2
and NaN3 in a 4 M HCl aqueous solution and the formed precipitate
was filtered-off to afford the title compound I as a yellow solid.
4-Azidobenzoic acid I: 73% yield; mp 188–190 °C d; silica gel TLC
Rf 0.71 (EtOAc/n-hexane 50% v/v); dH (400 MHz, DMSO-d6): 7.25
(d, J = 8.4, 2H), 7.99 (d, J = 8.4, 2H), 13.02 (br s, 1H, exchange with
D2O, COOH); dC (100 MHz, DMSO-d6): 120.2, 128.3, 132.2, 144.9,
167.6. Experimental in agreement with reported data.32
4.4.8. Synthesis of 4-fluorophenylazide F
N3
4.4.12. Synthesis of 4-azido-benzenesulfonamide J
N3
F
F
4-Fluorophenylazide F was obtained according the general proce-
dure 3 reported earlier.29
SO2NH2
4-Fluorophenylazide F: 89% yield; silica gel TLC Rf 0.79 (EtOAc/
n-hexane 50% v/v); dH (400 MHz, DMSO-d6): 67.19 (m, 2H), 7.29 (t,
J
Please cite this article in press as: Nocentini, A.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.09.041

A. Nocentini et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
11
Maresca, A.; Winum, J. Y.; Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2013, 28,
397; (c) Sharma, A.; Tiwari, M.; Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2014,
29, 292.
4-Azidobenzenesulfonamide J was obtained according the general
procedure 3 reported earlier.34 Sulfanilamide was treated with
NaNO2 and NaN3 in a HCl 2 M aqueous solution and the formed
precipitate was filtered-off to afford the title compound J as a yel-
low solid.
8. (a) Carta, F.; Maresca, A.; Scozzafava, A.; Supuran, C. T. Bioorg. Med. Chem. Lett.
2012, 22, 267; (b) Carta, F.; Maresca, A.; Scozzafava, A.; Supuran, C. T. Bioorg.
Med. Chem. 2012, 20, 2266; (c) Carta, F.; Vullo, D.; Maresca, A.; Scozzafava, A.;
Supuran, C. T. Bioorg. Med. Chem. Lett. 2012, 22, 2182.
4-Azidobenzenesulfonamide J: 60% yield; mp 120–121 °C; silica
gel TLC Rf 0.47 (EtOAc/n-hexane 50% v/v); dH (400 MHz, DMSO-
d6): 7.33 (d, J = 8.8, 2H), 7.41 (s, 2H, exchange with D2O, SO2NH2),
7.87 (d, J = 8.8, 2H); dC (100 MHz, DMSO-d6): 120.5, 128.6, 141.5,
143.9. Experimental in agreement with reported data.34
9. (a) Davis, R. A.; Vullo, D.; Maresca, A.; Supuran, C. T.; Poulsen, S. A. Bioorg. Med.
Chem. 2013, 21, 1539; (b) Carreyre, H.; Coustard, J. M.; Carre, G.; Vandebrouck,
C.; Bescond, J.; Ouedraogo, M.; Marrot, J.; Vullo, D.; Supuran, C. T.; Thibaudeau,
S. Bioorg. Med. Chem. 2013, 21, 3790.
10. (a) Tanc, M.; Carta, F.; Bozdag, M.; Scozzafava, A.; Supuran, C. T. Bioorg. Med.
Chem. 2013, 15, 4502; (b) Grandane, A.; Tanc, M.; Zalubovskis, R.; Supuran, C. T.
Bioorg. Med. Chem. Lett. 2014, 5, 1256; (c) Grandane, A.; Tanc, M.; Zalubovskis,
R.; Supuran, C. T. Bioorg. Med. Chem. 2014, 5, 1522; (d) Tars, K.; Vullo, D.;
Kazaks, A.; Leitans, J.; Lends, A.; Grandane, A.; Zalubovskis, R.; Scozzafava, A.;
Supuran, C. T. J. Med. Chem. 2013, 56, 293.
4.5. CA inhibition
11. (a) Tanc, M.; Carta, F.; Scozzafava, A.; Supuran, C. T. Org. Biomol. Chem. 2015, 13,
77; (b) Grandane, A.; Tanc, M.; Di Cesare Mannelli, L.; Carta, F.; Ghelardini, C.;
An Applied Photophysics stopped-flow instrument has been
used for assaying the CA catalysed CO2 hydration activity.18 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-catalysed CO2 hydration reaction for a period of 10–100 s. The
CO2 concentrations ranged from 1.7 to 17 mM for the determina-
tion 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 uncatalysed
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 the assay buffer. Inhibitor and enzyme
solutions were preincubated together for 15 min–6 h at room
temperature (15 min) or 4 °C (6 h) prior to assay, in order to allow
for the formation of the E–I complex. Data from Table 1 were
obtained after 6 h incubation of enzyme and inhibitor, as for the
sulfocoumarins and coumarins reported earlier.1,5–11 The inhibi-
tion constants were obtained by non-linear least-squares methods
using PRISM 3 and the Cheng-Prusoff equation, as reported
earlier,19–21and represent the mean from at least three different
determinations. All CA isofoms were recombinant ones obtained
in-house as reported earlier.19–21
ˇ
Zalubovskis, R.; Supuran, C. T. J. Med. Chem. 2015, 58, 3975.
12. (a) Pacchiano, F.; Carta, F.; McDonald, P. C.; Lou, Y.; Vullo, D.; Scozzafava, A.;
Dedhar, S.; Supuran, C. T. J. Med. Chem. 2011, 54, 1896; (b) Lou, Y.; McDonald, P.
C.; Oloumi, A.; Chia, S. K.; Ostlund, C.; Ahmadi, A.; Kyle, A.; Auf dem Keller, U.;
Leung, S.; Huntsman, D. G.; Clarke, B.; Sutherland, B. W.; Waterhouse, D.; Bally,
M. B.; Roskelley, C. D.; Overall, C. M.; Minchinton, A.; Pacchiano, F.; Carta, F.;
Scozzafava, A.; Touisni, N.; Winum, J. Y.; Supuran, C. T.; Dedhar, S. Cancer Res.
2011, 71, 3364; (c) Krall, N.; Pretto, F.; Decurtins, W.; Bernardes, G. J. L.;
Supuran, C. T.; Neri, D. Angew. Chem., Int. Ed. 2014, 53, 4231; (d) Gieling, R. G.;
Parker, C. A.; De Costa, L. A.; Robertson, N.; Harris, A. L.; Stratford, I. J.; Williams,
K. J. J. Enzyme Inhib. Med. Chem. 2013, 28, 360.
13. (a) Dubois, L.; Lieuwes, N. G.; Maresca, A.; Thiry, A.; Supuran, C. T.; Scozzafava,
A.; Wouters, B. G.; Lambin, P. Radiother. Oncol. 2009, 92, 423; (b) Akurathi, V.;
Dubois, L.; Lieuwes, N. G.; Chitneni, S. K.; Cleynhens, B. J.; Vullo, D.; Supuran, C.
T.; Verbruggen, A. M.; Lambin, P.; Bormans, G. M. Nucl. Med. Biol. 2010, 37, 557;
(c) Dubois, L.; Peeters, S.; Lieuwes, N. G.; Geusens, N.; Thiry, A.; Carta, F.;
Scozzafava, A.; Dogné, J. M.; Supuran, C. T.; Harris, A. L.; Masereel, B.; Lambin, P.
Radiother. Oncol. 2011, 99, 424; (d) Groves, K.; Bao, B.; Zhang, J.; Handy, E.;
Kennedy, P.; Cuneo, G.; Supuran, C. T.; Yared, W.; Peterson, J. D.; Rajopadhye,
M. Bioorg. Med. Chem. Lett. 2012, 22, 653.
14. (a) Singh, S.; Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2012, 27, 666; (b)
Arechederra, R. L.; Waheed, A.; Sly, W. S.; Supuran, C. T.; Minteer, S. D. Bioorg.
Med. Chem. 2013, 21, 1544; (c) Scozzafava, A.; Supuran, C. T.; Carta, F. Expert
Opin. Ther. Pat. 2013, 23, 725; (d) Harju, A. K.; Bootorabi, F.; Kuuslahti, M.;
Supuran, C. T.; Parkkila, S. J. Enzyme Inhib. Med. Chem. 2013, 28, 231.
15. (a) Supuran, C. T. Expert Opin. Ther. Pat. 2003, 13, 1545; (b) De Simone, G.;
Supuran, C. T. Curr. Top. Med. Chem. 2007, 7, 879; (c) Supuran, C. T.; Di Fiore, A.;
De Simone, G. Expert Opin. Emerg. Drugs 2008, 13, 383.
16. (a) Wilkinson, B. L.; Bornaghi, L. F.; Houston, T. A.; Innocenti, A.; Supuran, C. T.;
Poulsen, S. A. J. Med. Chem. 2006, 49, 6539; (b) Wilkinson, B. L.; Bornaghi, L. F.;
Houston, T. A.; Innocenti, A.; Vullo, D.; Supuran, C. T.; Poulsen, S. A. J. Med.
Chem. 2007, 50, 1651; (c) Wilkinson, B. L.; Innocenti, A.; Vullo, D.; Supuran, C.
T.; Poulsen, S. A. J. Med. Chem. 2008, 51, 1945.
Acknowledgment
17. (a) Lopez, M.; Paul, B.; Hofmann, A.; Morizzi, J.; Wu, Q.; Charman, S. A.;
Innocenti, A.; Vullo, D.; Supuran, C. T.; Poulsen, S. A. J. Med. Chem. 2009, 52,
6421; (b) Lopez, M.; Salmon, A. J.; Supuran, C. T.; Poulsen, S. A. Curr. Pharm. Des.
2010, 16, 3277; (c) Lopez, M.; Trajkovic, J.; Bornaghi, L. F.; Innocenti, A.; Vullo,
D.; Supuran, C. T.; Poulsen, S. A. J. Med. Chem. 2011, 54, 1481; (d) Buller, F.;
Steiner, M.; Frey, K.; Mircsof, D.; Scheuermann, J.; Kalisch, M.; Bühlmann, P.;
Supuran, C. T.; Neri, D. ACS Chem. Biol. 2011, 6, 336.
This work was financed in part by a Marie Curie ITN FP7 project
(Dynano).
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