Inclusion of a 5-fluorouracil moiety in nitrogenous bases derivatives as human carbonic anhydrase IX and XII inhibitors produced a targeted action against MDA-MB-231 and T47D breast cancer cells

Article abstract of DOI:10.1016/j.ejmech.2020.112112

A new series of pyrimidine derivatives as human carbonic anhydrases (CA, EC 4.2.1.1) inhibitors is here designed by including a 5-fluorouracil (5-FU) moiety, broadly used anticancer medication, in nitrogenous base modulators of the tumor-associated CAs. Most sulfonamide derivatives efficiently inhibit the target CA IX (K_Is in the range 0.47–44.7 nM) and CA XII (K_Is in the range 2.9–83.1 nM), while the 5-FU coumarin derivatives showed a potent and totally selective inhibitory action against the target CA IX/XII over off-target CA I/II. The X-ray solved crystal structure of CA II in adduct with a representative uracil derivative provided insights on the binding mode to the target of such pyrimidine derivatives. On the basis of potency and selectivity inhibition profiles, coumarin 12a, the sulfonamide CAIs showing the greatest II/IX specificity (4e, 6b and 6d) and the unique subnanomolar CA IX inhibitor 10a were tested in vitro for their antiproliferative action against a panel of eight cancer cell lines. The breast cancer cell lines MDA-MB-231 and T47D were the most susceptible with IC₅₀ values in low to medium micromolar ranges (2.45 ± 0.07–18.86 ± 0.72 μM and 6.86 ± 0.31–40.92 ± 1.59 μM, respectively). A cell cycle analysis showed that 4e and 6d arrest T-47D cells mainly in the G2/M phase. Using an annexin V-FITC apoptosis assay, 4e and 6d were shown to induce an approximately 23.6-fold and 34.8-fold total increase in apoptosis compared to the control, corroborating the concrete potential of 5-FU CAIs for the design of new effective anticancer strategies.

Full text of DOI:10.1016/j.ejmech.2020.112112

European Journal of Medicinal Chemistry 190 (2020) 112112
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Inclusion of a 5-fluorouracil moiety in nitrogenous bases derivatives as
human carbonic anhydrase IX and XII inhibitors produced a targeted
action against MDA-MB-231 and T47D breast cancer cells
Andrea Petreni a, Alessandro Bonardi a, Carrie Lomelino b, Sameh M. Osman c,
Zeid A. ALOthman c, Wagdy M. Eldehna d, Radwan El-Haggar e, Robert McKenna b,
,
, **
Alessio Nocentini a *, Claudiu T. Supuran a
a University of Florence, Department of NEUROFARBA, Section of Pharmaceutical and Nutraceutical Sciences, via Ugo Schiff 6, 50019, Sesto Fiorentino,
Florence, Italy
b University of Florida, Department of Biochemistry and Molecular Biology, 1200 Newell Dr, Gainesville, FL, 32610, USA
c Chemistry Department, College of Science, King Saud University, P. O. Box 2455, Riyadh, 11451, Saudi Arabia
d Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Kafrelsheikh University, Kafrelsheikh, 33516, Egypt
e Pharmaceutical Chemistry, Department, Faculty of Pharmacy, Helwan University, 11795, Cairo, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
A new series of pyrimidine derivatives as human carbonic anhydrases (CA, EC 4.2.1.1) inhibitors is here
designed by including a 5-fluorouracil (5-FU) moiety, broadly used anticancer medication, in nitrogenous
base modulators of the tumor-associated CAs. Most sulfonamide derivatives efficiently inhibit the target
CA IX (KIs in the range 0.47e44.7 nM) and CA XII (KIs in the range 2.9e83.1 nM), while the 5-FU
coumarin derivatives showed a potent and totally selective inhibitory action against the target CA IX/
XII over off-target CA I/II. The X-ray solved crystal structure of CA II in adduct with a representative uracil
derivative provided insights on the binding mode to the target of such pyrimidine derivatives. On the
basis of potency and selectivity inhibition profiles, coumarin 12a, the sulfonamide CAIs showing the
greatest II/IX specificity (4e, 6b and 6d) and the unique subnanomolar CA IX inhibitor 10a were tested
in vitro for their antiproliferative action against a panel of eight cancer cell lines. The breast cancer cell
lines MDA-MB-231 and T47D were the most susceptible with IC50 values in low to medium micromolar
Received 15 January 2020
Received in revised form
31 January 2020
Accepted 31 January 2020
Available online 3 February 2020
Keywords:
Anticancer
5-Fluorouracil
Carbonic anhydrase
Selectivity
Annexin
Breast cancer
ranges (2.45 0.07e18.86 0.72 mM and 6.86 0.31e40.92 1.59 mM, respectively). A cell cycle analysis
showed that 4e and 6d arrest T-47D cells mainly in the G2/M phase. Using an annexin V-FITC apoptosis
assay, 4e and 6d were shown to induce an approximately 23.6-fold and 34.8-fold total increase in
apoptosis compared to the control, corroborating the concrete potential of 5-FU CAIs for the design of
new effective anticancer strategies.
© 2020 Elsevier Masson SAS. All rights reserved.
1. Introduction
pH are the carbonic anhydrases (CA, EC 4.2.1.1) [3], that cata-
lyze the reversible hydration of carbon dioxide to bicarbonate
Hypoxic induced cancer cells adapt their metabolism via a
glycolytic shift, which decreases extracellular pH and modifies
their genes expression pattern to survive in an environment
incompatible for normal cell growth [1,2]. Among the mem-
brane proteins overexpressed by hypoxic tumors to regulate
ion and a proton. In this context, the transmembrane isoforms
CA IX and XII participate in the pH regulation of the extra-
cellular environment [3e5]. CA IX is not appreciably expressed
in most normal tissues, while its up-regulation is associated
with hypoxic cancer phenotypes, where it is strongly induced
via the hypoxia inducible factor-1
a (HIF-1a) [6e8]. CA XII is
also implicated in the extracellular acidification in many tumor
types [4,9]. As a result, CA IX and XII are factors driving tumor
growth, invasiveness, proliferation, metastasis, as well as
resistance to radio- and chemotherapy [10]. This makes agents
* Corresponding author.
** Corresponding author.
E-mail addresses: alessio.nocentini@unifi.it (A. Nocentini), claudiu.supuran@
unifi.it (C.T. Supuran).
https://doi.org/10.1016/j.ejmech.2020.112112
0223-5234/© 2020 Elsevier Masson SAS. All rights reserved.

2
A. Petreni et al. / European Journal of Medicinal Chemistry 190 (2020) 112112
inhibiting CA IX and XII, in a selective manner, a valuable
therapeutic for the targeting of the primary tumor growth,
invasion metastasis, and the reduction of cancer stem cell
population [10e12].
2. Results and discussion
2.1. Drug design and chemistry
The issue with sulfonamide-like CA inhibitors (CAIs), the main
inhibitory chemotypes adopted for therapeutic purposes, is a
promiscuous action against almost all isoforms encoded in
humans, that results in non-selective targeting, and therefore
undesired side effects [6]. In fact, the comparison of the 12
catalytically active human CAs demonstrates a high sequence
homology within the active sites that increases the difficulties in
the design of disease-targeted inhibitors [13]. Among the strate-
gies adopted to bypass this issue are the optimization of CAIs (i.e.
the tail approach [12], whose application produced SLC-0111 [14],
the first-in-class CAI entering clinical trials for the treatment of
hypoxic tumors) or the use of alternative chemotypes, such as
coumarin and bioisosters which use different chemotypes for
enzymatic inhibition [4,11].
As an application of the tail approach, we recently reported two
series of uracil/adenine-tailed benzenesulfonamides as potent CA
IX inhibitors (series 1 and 2, Fig. 1) [15]. Purine/pyrimidine-based
scaffolds are considered privileged structures in the drug-design
of bioactive compounds because of synthetic accessibility and
ability to confer drug-like properties [16,17]. In particular, pyrimi-
dines and purines are widely used as antitumor and antiviral
pharmacophores in medicinal chemistry, due their ability to
interfere with the DNA functions in diverse manners [18,19]. A se-
lection of the reported CAIs showed significant anti-proliferative
activity against HT-29 colon cancer cell lines [15].
5-FU is widely used medication in the treatment of cancer [20].
Over the past 20 years, increased understanding of the mechanism
of action of 5-FU, principally based on the inhibition of thymidylate
synthase, has led to the development of strategies that increase its
anticancer activity.
A molecular hybridization approach is here adopted combining
5-FU with CAI scaffolds to synergize the inhibition of the tumor-
associated isozymes with intrinsic anticancer effects resulting by
the pyrimidine pharmacophore [21].
5-FU was incorporated through the N1 atom on a wealth of
benzenesulfonamide structures by amide or triazole linkers to
explore the chemical space within the target and off-target CAs
binding sites. A small subset of ester, amide or triazole linked 5-FU
coumarin derivatives was also produced to yield markedly selective
CA IX/XII 5-FU-based inhibitors. In fact, coumarins act as prodrug
which undergo a CA mediated hydrolysis and the formed species
(E- or Z-cinnamic acid) accomplishes the enzymatic inhibition by
occluding the entrance of the active site [11,12]. The latter is the
most variable region, in terms of amino acid residues, among the
human CAs. The cancer-related CAs were shown to be more prone
to such a kind of inhibition mechanism than ubiquitous CAs.
Intermediate 2 was provided by reacting 5-FU with chloro-
acetic acid in KOH(aq) (Scheme 1). The carboxylic acid 2 was thus
coupled with a variety of primary (3a-e) and secondary (5a-f)
amine benzenesulfonamide derivatives using 1-ethyl-3-(3-
As a continuation of the previous work, the uracil derivatives
series is here developed by swapping the nitrogenous base to a 5-F-
uracil (5-FU), a clinically used chemotherapeutic agent, to produce
series 3 (Fig. 1). A major number of benzenesulfonamide scaffolds
as CAIs was investigated as well as coumarin cores to achieve a
more targeted CA IX/XII inhibition. A subset of 5-FU CAIs was
evaluated in vitro against eight cancer cell lines. The effects of the
two more active compounds on the phases of cell cycle and annexin
V-FITC-positive staining in breast T47D cells were thereafter
assessed.
Scheme 1. Synthesis of intermediate 2.
Fig. 1. Drug-design of 5-fluorouracil derivatives as antitumor CAIs.

A. Petreni et al. / European Journal of Medicinal Chemistry 190 (2020) 112112
3
Scheme 2. Synthesis of amides 4a-4e.
Scheme 3. Synthesis of amides 6a-6f.
to generate in situ the Cu(I) catalyst.
The coumarin derivatives 12a and 12b were prepared by
coupling intermediate 2 with 7-amino-4-methylcoumarin 11a and
umbelliferone 11b, respectively, adopting again EDC as coupling
agent (Scheme 5). The CuAAC between alkyne 7 and coumarin
azide 13 yielded triazole 14 (Scheme 6).
Compounds 4a-e, 6a-f, 10a-d, 12a-b, 14 were obtained in high
yields and thoroughly characterized by 1H-, 13C, 19F NMR and HRMS.
2.2. Carbonic anhydrases inhibition
The CA inhibitory action of 5-FU compounds 4a-e, 6a-f, 10a-d,
12a-b,14, was assessed against the cytosolic CA I and II (ubiquitous)
and CA IX and XII (cancer-related) isoforms by a stopped flow CO2
hydrase assay, in addition to acetazolamide (AAZ) as standard in-
hibitor [22]. The following structureeactivity relationship (SAR)
can be drawn up from the inhibition data reported in Table 1.
The cytosolic off-target CA I was the least inhibited isoform. The
inhibition constants (KIs) of the sulfonamide 5-FU derivatives
spanned between 207.6 and 9679.8 nM. The m-substitution at the
benzenesulfonamide scaffold produced the least CA I inhibition,
Scheme 4. Synthesis of triazoles 10a-d.
such as in compounds 4b (KI of 5.1
mM), 4e (KI of 9.7 mM) and 10b (KI
of 8.9 M). Additionally, the p-chloro substituted secondary amide
m
6b inhibited CA I by a micromolar value (KI of 2.6 mM). The removal
Scheme 5. Synthesis of coumarins 12a-b.
dimethylaminopropyl)carbodiimide (EDC) in presence of 1-
hydroxybenzotriazole (HOBt) and 4-dimethylaminopyridine
(DMAP) to yield amides 4a-e (Scheme 2) and 6a-f (Scheme 3)
[15].
The triazole derivatives (10a-d) were generated by a Cu(I) cat-
alysed azideꢀalkyne cycloaddition (CuAAC) between the N1-
propargyluracil 7 and the freshly prepared azides 9a-d yielded by
a Sand-Meyer reaction (Scheme 4). CuSO4 and vitamin C were used
Scheme 6. Synthesis of coumarin 14.

4
A. Petreni et al. / European Journal of Medicinal Chemistry 190 (2020) 112112
Table 1
Table 3
Inhibition data of human CA isoforms I, II, IX and XII with 5-FU derivatives 4a-e, 6a-f,
10a-d, 12a-b, 14 and the standard acetazolamide (AAZ) by a stopped flow CO2 hy-
drase assay [22]. Compounds selected for in vitro anticancer assays are highlighted in
gray.
KI ratio between 5-FU derivatives and corresponding non-fluorinated compounds
from series A (uracil derivatives) when applicable.
Cmpd
KI uracil derivative (series A)/KI 5-FU derivative (series
C)
Cmpd
KI (nM)a
CA I
CA II
CA IX
CA I
323.5
5158.3
738.6
299.3
9679.8
207.6
2965.0
933.9
CA II
6.8
81.5
101.3
19.6
9083.9
0.59
34.8
13.4
8.0
CA IX
4.8
CA XII
4.7
66.3
40.1
7.0
59.5
2.9
6.2
4a
4b
4c
4d
4e
2.0
1.5
2.1
2.2
1.0
0.4
0.5
2.6
8.6
4.9
2.1
0.5
1.4
4.3
5.3
4a
4b
4c
4d
4e
6a
6b
6c
69.8
13.9
1.8
1.0
10.2
4.4
44.7
29.2
24.8
452.8
3.5
4.2
11.8
1.9
10a
10b
4.8
9.0
6d
727.4
6e
6f
383.6
581.9
651.0
8911.2
656.1
0.71
5.1
0.67
89.2
2.4
0.81
>10,000
>10,000
>10,000
12.5
4.9
28.6
15.5
9.8
83.1
8.4
derivatives inhibited CA II in a subnanomolar range, that are the
unsubstituted and p-CN benzyl amide compounds 6a (KI of
0.59 nM) and 6e (KI of 0.71 nM) and the p-triazole benzenesulfo-
namide derivatives 10a (KI of 0.71 nM) and 10d, that is also
substituted in meta by a bromine atom (KI of 0.81 nM). Swapping
the p-CN group with a chlorine atom (6b), a fluorine atom (6c), a
methoxy group (6d) and a dimethylamino group (6e) reduced the
CA inhibitory action by 7 to 50-fold.
26.8
0.47
105.3
11.2
3.8
16.5
36.8
173.4
25.0
10a
10b
10c
10d
12ab
12bb
14b
AAZ
626.3
3.9
>10,000
>10,000
>10,000
250.0
23.2
14.4
408.4
5.7
The main antitumor target CA IX was also efficiently inhibited by
most 5-FU sulfonamide derivatives with KIs ranging between 0.47
and 44.7 nM. Uniquely, the m-substituted 4e and 10b induced in-
hibition above 100 nM, with KIs of 452.8 and 105.3, respectively. A
single subnanomolar CA inhibitor was identified against CA IX, that
is the triazole 10a, whose KI of 0.47 nM represents the best CA in-
hibition here reported. Among the N-benzyl substituted secondary
amides 6a-f, the p-OCH3 derivative 6d stood out as the most active
one with a KI of 1.9 nM. Notably, a significant subset of 5-FU CAIs
inhibit CA IX more capably than the clinically used AAZ (KI of
25.0 nM).
CA XII, the second tumor-related CA, was inhibited by most
sulfonamide CAIs reported here with an overall comparable range
with CA II and IX, though with diverse SAR. KIs spanned in the range
2.9e83.1 nM and no subnanomolar inhibition was detected. The
worst KIs, detected in the range 59.5e83.1 nM, are consistently
associated with the m-substituted 4b, 4e and 10b. The introduction
of a 1-carbon atom linker between zinc-binding portion and amide
linker (4c) decreased CA IX inhibition below the average as well (KI
of 40.1 nM). The N-benzyl substituted secondary amides showed a
rather flat inhibitory trend with KIs spanning in the range
a
Mean from 3 different assays, by a stopped flow technique (errors were in the
range of 5e10% of the reported values).
b
Incubation time of 6h
of the chlorine atom in the latter compound led instead to best CA I
inhibitor that is 6a (KI of 207.6 nM). The elongation of the spacer
between the benzenesulfonamide and the primary amide moiety
reduced the CA I inhibitory activity passing from 0 (4a) to 1 (4c)
carbon atom, while it is restored to a 300 nM value approximately
when a second carbon atom unit was introduced (4d).
The most physiologically relevant isoform CA II was effectively
inhibited by most 5-FU based sulfonamide derivatives. In fact, KIs
spanned in the range 0.59e34.8 nM, except for 4b (KI of 81.5 nM),
4c (KI of 101.3 nM) and 10b (KI of 89.2 nM), while 4e barely
inhibited CA II below 10 mM (KI of 9.1 mM). Again, m-substitutions at
the benzenesulfonamide core was detrimental for CA inhibition
with respect to p-substitutions, as well as a 1-carbon atom linker
between zinc-binding portion and amide linker (4c). A subset of
Table 2
Selectivity index (SI) calculated for human CA IX and XII over off-targets isoforms as
ratio between KIs.
Table 4
X-ray crystallography statistics for structure of CA II in complex with compound A.
Cmpd
Selectivity Index (SI)
I/IX
II/IX
I/XII
II/XII
CA II - A
4a
4b
4c
4d
4e
6a
67.4
115.4
25.3
12.1
21.4
1.4
1.8
3.5
0.8
20.1
0.2
68.8
77.8
18.4
42.8
162.7
71.6
1.4
1.2
2.5
2.8
152.7
0.2
PDB Accession Code
Space Group
6VJ3
P21
Cell Dimensions (Å;ꢁ)
a ¼ 42.5, b ¼ 41.4, c ¼ 72.2;
b
¼ 104.4
Resolution (Å)
Total Reflections
32.2e1.4 (1.40e1.35)
164,794
59.3
6b
6c
6d
6e
706.0
79.1
382.8
78.3
21.7
1385.1
84.6
8.3
1.1
4.2
0.1
0.2
1.4
0.8
0.2
478.2
194.6
80.8
13.4
37.5
66.4
107.2
78.1
160.6
>431.0
>694.4
>24.5
43.9
5.6
2.8
0.9
<0.1
0.3
0.1
1.1
0.3
0.2
>431.0
>694.4
>24.5
2.2
I/I
s
12.9 (1.5)
3.1 (2.1)
98.0 (88.0)
15.1 (27.2)
15.2 (28.4)
2069
Redundancy
Completeness (%)
Rcryst (%)
6f
Rfree (%)
10a
10b
10c
10d
12a
12b
14
# of Protein Atoms
# of Ligand Atoms
Ramachandran stats (%):
favored, allowed,
generously allowed
Avg. B factors (Å2): Main-chain,
Ligand, Solvent
51
58.6
97.3, 2.7, 0.0
164.8
>606.1
>271.7
>57.7
10.0
0.2
>606.1
>271.7
>57.7
0.5
15.8, 23.8, 25.7
0.007, 1.12
AAZ
rmsd for bonds, angles (Å,ꢁ)

A. Petreni et al. / European Journal of Medicinal Chemistry 190 (2020) 112112
5
Fig. 2. A) Surface representation of CA II in complex with compound A from series 1, the uracil (F-devoid) analog of compound 4a (shown as cyan sticks). Hydrophobic and
hydrophilic residues of the active site are labeled and colored orange and purple, respectively. B) Active site view of compound A binding. Hydrogen bonds are shown as black
dashes with distances labeled in Å. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
As for target/off-target CAs selectivity ratio of action of sulfon-
amide 5-FU derivatives, all compounds exhibited remarkable I/IX
and I/XII inhibitory specificity, as the calculated selectivity index
(SI) spans in the ranges 12.1e1385.1 and 13.4e478.2, respectively
(Table 2). In contrast, only two distinct subsets of sulfonamide
derivatives (approximately half of the compounds) produced a
preferred inhibition of the target CA IX and XII over the main off-
target CA II. In detail, only the 1-carbon atom spacer derivative
4c, the m-substituted 4e and the N-benzyl amides 6b and 6d re-
ported significant II/IX SI spanning between 3.5 and 20, that are
greater than that measured for the standard AAZ. As for II/XII SI
values, oddly the 1- and 2-carbon atoms spacer derivative 4c and
4d, the m-substituted 4e and the N-benzyl amides 6b and 6c solely
had values above 2, with that of 4e even reaching the value of 153.
On the basis of the data of Table 1 and 2, coumarin 12a, the
sulfonamide CAIs showing the greatest II/IX SI, namely 4e, 6b and
6d, and the unique subnanomolar CA IX inhibitor 10a were chosen
for assessing in vitro their antiproliferative action against a panel of
cancer cell lines.
The corresponding nonfluorinated analog (uracil derivative,
from series A) was previously reported and characterized for a
subset of 5-FU derivatives, namely 4a-e, 10a-b, and tested as in-
hibitor of CA I, II and IX [15]. Table 3 shows the KI ratio against CA I,
II and IX between each uracil derivative and its corresponding 5-FU
analog. A significant enhancement in the CA inhibitory action was
detected for most 5-FU compounds against CA II (ratio of 0.5e8.6)
and CA IX (ratio of 1.0e69.8) compared to the uracil correspon-
dents. As in most cases the increase was more intense against the
target CA IX over CA II, it can be speculated that the incorporation of
a fluorine atom in position 5 of the nitrogenous base scaffold in-
creases both potency and selectivity against the target CA IX.
Fig. 3. Overlay of compound A (cyan sticks) and compound B, an adenine derivate
(adenine analog of 4d) from series 2 (green sticks in CA II and yellow sticks in CA IX-
mimic binding) in surface representation of CA II. (For interpretation of the references
to color in this figure legend, the reader is referred to the Web version of this article.)
2.9e28.6 nM. The best value is associated to the unsubstituted 6a
while the worst value is related to the p-CN substitution of 6e.
Among the triazole derivatives 10a-d the introduction of m-Br
substituent on the benzenesulfonamide scaffold produced the best
CA XII inhibition (KI of 3.9 nM).
In contrast, the 5-FU coumarin derivatives 12a-b and 14 showed
a potent and totally selective inhibitory action against the target CA
IX/XII over CA I/II. In detail, the three compounds did not inhibit CA
2.3. X-ray Crystallography
I and II, lower than 10 mM. Whereas, CA IX and XII were inhibited in
X-ray crystallography was utilized to observe the binding to CA
II of a representative compound from the pyrimidine series, com-
pound A from series 1, the uracil (F-devoid) analog of compound 4a
from series 3. This structure was determined to a resolution of 1.4 Å
(Table 4). As expected for a sulfonamide-based compound, the zinc-
binding group (ZBG) displaced the active site zinc-bound water
similar ranges, 16.5e173.4 nM and 14.4e408.4 nM, respectively.
However, while amide 12a was the best CA IX inhibitor (KI of
16.5 nM), CA XII turned out to be slightly more efficiently inhibited
by ester 12b. The triazole coumarin 14 exhibited a decrease of ef-
ficacy against both cancer-related CAs.

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A. Petreni et al. / European Journal of Medicinal Chemistry 190 (2020) 112112
Fig. 4. Anti-proliferative activity (cell growth inhibitory activity at 10 and 100 mM concentrations) of compounds 4e, 6b, 6d, 10a and 12a against Hela, Colo-205, A549 and HL-60
cancer cell lines.
Fig. 5. Anti-proliferative activity (cell growth inhibitory activity at 10 and 100 mM concentrations) of compounds 4e, 6b, 6d, 10a and 12a toward SKOV-3, MDA-MB-231, MCF-7 and
T47D cancer cell lines.
(ZBW) and bound directly to the zinc. The ZBG also formed a
hydrogen bond between the sulfonamide oxygen and backbone
amide of T199 (3.0 Å). The tail component of compound A was
observed to exhibit dual conformations, attributed to the freedom
of rotation about the amide linker, resulting in one conformation
oriented towards the hydrophobic side and another extending to-
wards the hydrophilic side of the active site (Fig. 2A). The minor tail
conformation, oriented towards the hydrophobic pocket, exhibited
a lower occupancy (43%) as indicated by the weaker electron
density (Fig. 2A). In the minor conformer, the carbonyl of the linker
forms a hydrogen bond with a water that bridges to the side chain
of T199 (2.2 and 2.9 Å, respectively; Fig. 2B). In the major tail
conformation, occupancy (55%), a hydrogen bond is formed be-
tween the linker carbonyl and side chain of Q92 (2.8 Å) in addition
to the uracil moiety and side chain of N67 (3.2 Å) (Fig. 2B). The
additional hydrogen bonding is thought to stabilize the compound
tail, resulting in the observed higher occupancy. Both conforma-
tions are further stabilized by interactions with active site residues
F131, V135, L198, P202, L204 and F131, I91, V121 (Fig. 2B).
a shift of the linker and tail portion of compound A by approxi-
mately 2e3 Å due to the steric hindrance of F131, these two binding
modes of compound B are oriented similarly to the two observed
conformations of compound A in CA II (Fig. 3).
2.4. In vitro anti-proliferative activity
Sulfonamides 4e, 6b, 6d, 10a and coumarin 12a displayed effi-
cient and/or selective inhibitory actions against the tumor-related
isoforms CA IX and XII over the cytosolic off-target CA I and II
(Tables 1 and 2). Accordingly, the five compounds were selected to
be evaluated in vitro for their potential anti-proliferative activity,
utilizing Sulforhodamine B colorimetric assay (SRB) as described by
Table 5
IC50 of anti-proliferative activity of compounds 4e, 6b, 6d, 10a and 12a against breast
MDA-MB-231 and T47D cancer cell lines in normoxia.
Cmpd
IC50 (m
M)a
MDA-MB-231
25.46 1.38
40.92 1.59
6.86 0.31
22.85 0.91
32.04 1.84
4.25 0.16
T47D
Interestingly, the two orientations of compound A are reminis-
cent of the dual binding modes of a previously studied purine-
based inhibitor, compound B from series 2, namely the adenine
analog of 4d from series 3. Compound B exhibited unique binding
modes based on the CA isoform with the inhibitor tail orienting in
the hydrophobic side in the active site of CA II and extending to-
ward the hydrophilic side of the active site of CA IX-mimic. Despite
4e
6b
6d
10a
7.56 0.29
18.86 0.72
2.45 0.07
33.51 1.24
13.16 0.71
4.52 0.19
12a
Staurosporine
a
IC50 values are the mean S.D. of three separate experiments.

A. Petreni et al. / European Journal of Medicinal Chemistry 190 (2020) 112112
7
Fig. 6. Effect of sulfonamides 4e and 6d on the phases of cell cycle of T47D cells.
Skehan et al.
(IC50 ¼ 33.51
sulfonamides 4e and 6d emerged as the most potent analogues in
this study with IC50 values of 7.56 0.29 and 2.45 0.07 M,
respectively. Besides, 6d was the most efficient anti-proliferative
agent herein reported, specifically against MDA-MB-231 cells
1.24 mM). As for the activity against T47D cells,
The anti-proliferative activities of 4e, 6b, 6d, 10a and 12a were
first evaluated in a preliminary screening against eight cancer cell
lines, namely cervical (Hela), colon (Colo-205), lung (A-549), leu-
kemia (HL-60), ovarian (SKOV-3), and breast (MDA-MB-231, MCF-7
and T47D) cancer cell lines. This initial assessment of antitumor
m
with IC50 value of 6.86 0.31 mM.
activity tested each derivative in triplicate at 10 and 100 mM con-
centrations, and the data were presented as percentage cell
viability caused by each tested compound (Figs. 4 and 5).
2.5. Cell cycle analysis
The tested derivatives showed different levels of growth inhib-
itory activity and possessed a distinctive pattern of selectivity to-
ward the examined eight cancer cell lines. Close examination of the
cell viability % in Figs. 4 and 5 hinted out that breast cancer MDA-
MB-231 and T47D cells were the most susceptible examined cell
lines to the influence of the tested compounds, with cell viability %
The efficient anti-proliferative impact of sulfonamides 4e and 6d
on breast T-47D cancer cell line (Table 4) prompted a further
investigation on their growth inhibitory action. The impact of both
sulfonamides 4e and 6d on cell cycle distribution, after incubation
with T-47D cells at their IC50 concentration (7.56 and 2.45
mM,
respectively) for 24 h, was analyzed by a DNA flow cytometry assay
(Fig. 6).
range of (49e61 and 36e58, respectively) at 10
mM and (34e41 and
49e61, respectively) at 100 M (Fig. 5). Moreover, lung A-549 and
leukemia HL-60 cells were moderately affected by the tested
compounds, with cell viability % range of (58e91 and 59e99,
m
The obtained results highlighted that, compared to control cells,
breast cancer T-47D cells treated with 4e and 6d were arrested at
the G2/M phase with an increase in cell population from 16.59%
(control) to 26.83% and 35.47%, respectively. Moreover, the number
of cells in the sub-G1 phase was dramatically increased from 2.41%
(control) to 12.72% and 18.04%, respectively (Fig. 6).
respectively) at 10
mM and (53e80 and 41e65, respectively) at
100 M (Fig. 4), whereas, the tested compounds (4e, 6b, 6d, 10a and
m
12a) displayed weak or no growth inhibitory activities towards
cervical Hela, colon Colo-205, ovarian SKOV-3, and breast MCF-7
cancer cell lines.
2.6. Annexin V-FITC apoptosis assay
Therefore, the quantitative IC50 values were determined for
sulfonamides 4e, 6b, 6d and 10a and coumarin 12a against the most
susceptible cancer cell lines in the preliminary screening (MDA-
MB-231 and T47D), testing concentrations of 100, 10, 1, 0.1 and
To determine whether the growth inhibitory action of sulfon-
amides 4e and 6d is consistent with the induction of apoptosis
suggested by the elevated population of sub-G1 in the treated T-
47D cells (Fig. 6), Annexin V-FITC/PI double staining (AV/PI)
apoptosis assay was carried out (Fig. 7).
The results of this AV-FITC/PI assay revealed that incubation of
T-47D cells with sulfonamides 4e and 6d resulted in an induction of
apoptosis in these cells, that was evidenced via the marked eleva-
tion in the apoptotic cells percentage for the early apoptosis (from
0.01
mM for 72h (Table 5).
As indicated in Table 5, the tested compounds were more
effective against T47D (IC50 range: 2.45 0.07e18.86 0.72
than MDA-MB-231 cells (IC50 range: 6.86 0.31e40.92 1.59
m
M)
mM),
except sulfonamide 10a that exhibited enhanced activity against
MDA-MB-231 cells (IC50 ¼ 22.85 0.91 M) than T47D cells
m

8
A. Petreni et al. / European Journal of Medicinal Chemistry 190 (2020) 112112
Fig. 7. Effect of sulfonamides 4e and 6d on the percentage of annexin V-FITC-positive staining in breast T47D cells. The experiments were done in triplicates. The four quadrants
identified as: LL, viable; LR, early apoptotic; UR, late apoptotic; UL, necrotic.
0.39% to 3.59% and 7.31, respectively) and the late apoptosis (from
0.17% to 9.62% and 12.19, respectively) phases which represents
about 23.6-fold and 34.8-fold total apoptosis increase, compared to
the untreated control (Fig. 7).
representative uracil derivative provided insights on the binding
mode to the target of such pyrimidine derivatives.
Coumarin 12a, the sulfonamide CAIs showing the greatest II/IX
SI, namely 4e, 6b and 6d, and the unique subnanomolar CA IX in-
hibitor 10a were chosen for assessing in vitro their antiproliferative
action against a panel of eight cancer cell lines. The IC50 values,
determined against the most susceptible cancer cell lines MDA-MB-
231 and T47D, settle in a low to medium micromolar range
3. Conclusions
The addition of a fluorine atom in the structure of previously
reported uracil benzenesulfonamide inhibitors of tumor-associated
CAs produced a novel series of derivatives of 5-FU, a widely used
medication in the treatment of cancer. A major number of benze-
nesulfonamide scaffolds as CAIs was here investigated as well as
(2.45 0.07e18.86 0.72 mM and 6.86 0.31e40.92 1.59 mM,
respectively). A cell cycle analysis showed that 4e and 6d, the most
active compounds, arrested T-47D cells mainly in the G2/M phase.
Using an annexin V-FITC apoptosis assay, 4e and 6d were shown to
induce an approximately 23.6-fold and 34.8-fold total increase in
apoptosis compared to the control, corroborating the concrete
potential of these 5-FU CAIs for the design of new effective anti-
cancer strategies.
coumarin fragments to achieve
inhibition.
a more targeted CA IX/XII
Most sulfonamide derivatives efficiently inhibit the target CA IX
(KIs in the range 0.47e44.7 nM) and CA XII (KIs in the range
2.9e83.1 nM), whereas the 5-FU coumarin derivatives 12a-b and 14
showed a potent and totally selective inhibitory action against the
target CA IX/XII over the off-target CA I/II. In contrast, while all
sulfonamide compounds exhibited remarkable I/IX and I/XII
inhibitory specificity, only two distinct subsets of sulfonamide de-
rivatives produced a preferred inhibition of the target CA IX and XII
over the main off-target CA II. When applicable, the comparison of
KI against CA I, II and IX between a 5-FU derivative and the corre-
sponding nonfluorinated analog previously reported showed that
the incorporation of a fluorine atom in position 5 of the nitrogenous
base scaffold increases both potency and selectivity against the
target CA IX.
4. Experimental section
4.1. Chemistry
Anhydrous solvents and all reagents were purchased from
Sigma-Aldrich, Fluorochem 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,
19F NMR) 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
The X-ray solved crystal structure of CA II in adduct with a

A. Petreni et al. / European Journal of Medicinal Chemistry 190 (2020) 112112
9
expressed in Hertz (Hz). Splitting patterns are designated as fol-
lows: s, singlet; d, doublet; sept, septet; t, triplet; q, quadruplet; m,
multiplet; bs, broad singlet; dd, double of doublets. The assignment
of exchangeable protons (OH and NH) was confirmed by the
addition of D2O. Two tautomeric forms of the amide bond were
detected for compounds 6a-f which partially double the signals in
the 1H and 13C NMR spectra.
Analytical thin-layer chromatography (TLC) was carried out on
Sigma Aldrich silica gel F-254 plates. Flash chromatography puri-
fications were performed on Sigma Aldrich Silica gel 60 (230e400
mesh ASTM) as the stationary phase and ethyl acetate/n-hexane or
MeOH/DCM were used as eluents. Melting points (mp) were
CH2Cl2 10% v/v); dH (400 MHz, DMSO‑d6): 11.98 (s, 1H, exchange
with D2O, CONHCO), 10.67 (s, 1H, exchange with D2O, CONH), 8.14
(d, J ¼ 6.7 Hz, 1H, AreH), 7.82 (d, J ¼ 8.7 Hz, 2H, AreH), 7.76 (d,
J ¼ 8.8 Hz, 2H, AreH), 7.31 (s, 2H, exchange with D2O, SO2NH2), 4.57
(s, 2H, CH2); dC (100 MHz, DMSO‑d6): 166.89, 158.46 (d, J2
¼ 25.7 Hz), 150.74, 142.26, 140.21 (d, J2 CF ¼ 228.6 Hz). 139.70,
CF
131.97 (d, J2
¼ 34.0 Hz), 127.78, 119.70, 51.22; dF (376 MHz,
CF
DMSO‑d6): 170.40 (d, J ¼ 5.7 Hz); m/z (ESI negative) calcd for
C12H10FN4O5S [M ꢀ H]- 341.0, found 341.1.
4.1.2.2. 2-(5-Fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-N-(3-
sulfamoylphenyl)acetamide 4b. Compound 4b was obtained ac-
cording the general procedure earlier reported using 1-
carboxymethyl 5-fluorouracil 2 (0.2 g, 1.0 eq), HOBt (0.5 eq),
DMAP (0.03 eq) and 3-aminobenzenesulfonamide 3b (1.1 eq) in dry
DMF (2 ml). 54% yield; m.p. >300 ꢁC; silica gel TLC Rf 0.25 (MeOH/
CH2Cl2 20% v/v); dH (400 MHz, DMSO‑d6): 11.97 (s, 1H, exchange
with D2O, CONHCO), 10.62 (s, 1H, exchange with D2O, CONH), 8.18
(d, J ¼ 15.2 Hz, 1H, AreH), 8.14 (d, J ¼ 6.5 Hz, 1H, AreH), 7.74 (s, 1H,
AreH), 7.57 (s, 2H, AreH), 7.42 (s, 2H, exchange with D2O, SO2NH2),
4.56 (s, 2H, CH2); dC (100 MHz, DMSO‑d6): 166.73, 158.47 (d, J2
measured 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 MU,
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/mL and stored at 4 ꢁC.
Working solutions of each analyte were freshly prepared by
diluting stock solutions in a mixture of mQ water:acetonitrile 1:1
¼ 25.8 Hz), 150.74, 145.70, 140.22 (d, J2 CF ¼ 228.5 Hz), 139.72,
CF
131.98 (d, J2 CF ¼ 34.0 Hz), 130.57, 122.92, 121.62, 117.16, 51.19; dF
(376 MHz, DMSO‑d6): 170.44 (d, J ¼ 3.3 Hz); m/z (ESI negative) calcd
for C12H10FN4O5S [M ꢀ H]- 341.0, found 341.1.
(v/v) up to a concentration of 1.0
analyte were acquired by introducing, via syringe pump at
10
L minꢀ1, the working solution. Raw-data were collected and
m
g mLꢀ1 The mass spectra of each
m
processed by Varian Workstation Vers. 6.8 software.
4.1.2.3. 2-(5-Fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-N-(4-
sulfamoylbenzyl)acetamide 4c. Compound 4c was obtained ac-
cording the general procedure earlier reported using 1-
carboxymethyl 5-fluorouracil 2 (0.2 g, 1.0 eq), HOBt (0.5 eq),
DMAP (0.03 eq) and 4-(aminomethyl)benzenesulfonamide hydro-
chloride 3c (1.1 eq) and DIPEA (1.1 eq) in dry DMF (2 ml). 25% yield;
m.p. >300 ꢁC; silica gel TLC Rf 0.30 (MeOH/CH2Cl2 10% v/v); dH
(400 MHz, DMSO‑d6): 11.87 (s, 1H, exchange with D2O, CONHCO),
8.79 (t, J ¼ 5.8 Hz,1H, exchange with D2O, CONH), 8.10 (d, J ¼ 6.8 Hz,
1H, AreH), 7.80 (d, J ¼ 8.2 Hz, 2H, AreH), 7.47 (d, J ¼ 8.2 Hz, 2H,
AreH), 7.35 (s, 2H, exchange with D2O, SO2NH2,), 4.41 (d, J ¼ 5.8 Hz,
2H, CH2), 4.38 (s, 2H, CH2); dC (100 MHz, DMSO‑d6): 167.78, 158.68
4.1.1. Synthesis of 2-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-
1(2H)-yl)acetic acid 2
An aqueous solution of chloroacetic acid (1.1 eq in 4 ml of water)
was added dropwise to a solution of 5-fluorouracil 1 (1.0 g, 1.0 eq)
and KOH (3.0 eq) in water (4 ml) at 100 ꢁC. The reaction mixture
was stirred for 2h at the same temperature, then cooled to r.t. and
acidified to pH 2. The formed precipitate was collected by filtration
and washed with water and Et2O. The crude solid was recrystallized
from the minimum amount of water to obtain the titled compound
as a white powder. 82% yield; m.p. 280e282 ꢁC; silica gel TLC Rf 0.05
(MeOH/CHCl3 10% v/v); dH (400 MHz, DMSO‑d6): 13.29 (s, 1H, ex-
change with D2O, COOH), 11.96 (s, 1H, exchange with D2O, CON-
HCO), 8.12 (d, J ¼ 6.7 Hz, 1H, AreH), 4.40 (s, 2H, CH2); dC (100 MHz,
DMSO‑d6): 170.32, 158.51 (d, J2 CF ¼ 25.8 Hz), 150.72, 140.38 (d, J2
(d, J2
¼
27.2 Hz), 150.74, 144.04, 143.65, 140.40 (d, J2
CF
¼ 238.7 Hz), 131.82 (d, J2 CF ¼ 45.1 Hz), 128.45, 126.58, 50.86,
CF
42.75; dF (376 MHz, DMSO‑d6): 170.46 (d, J ¼ 3.7 Hz); m/z (ESI
negative) calcd for C13H12FN4O5S [M ꢀ H]- 355.1, found 355.0.
¼ 228.9 Hz), 131.56 (d, J2
¼ 33.9 Hz), 49.70; dF (376 MHz,
CF
CF
DMSO‑d6): 170.04 (s); m/z (ESI negative) calcd for C6H4FN2O4
[M ꢀ H]- 187.0, found 186.9.
4.1.2.4. 2-(5-Fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-N-(4-
sulfamoylphenethyl)acetamide 4d. Compound 4d was obtained ac-
cording the general procedure earlier reported using 1-
carboxymethyl 5-fluorouracil 2 (0.2 g, 1.0 eq), HOBt (0.5 eq),
DMAP (0.03 eq) and 4-(aminoethyl)benzenesulfonamide 3d (1.1
eq) in dry DMF (2 ml). 63% yield; m.p. 258e260 ꢁC; silica gel TLC Rf
0.55 (MeOH/CH2Cl2 10% v/v); dH (400 MHz, DMSO‑d6): 11.88 (s, 1H,
exchange with D2O, CONHCO), 8.33 (t, J ¼ 5.3 Hz, 1H, exchange with
D2O, CONH), 8.05 (d, J ¼ 6.7 Hz, 1H, AreH), 7.78 (d, J ¼ 8.2 Hz, 2H,
AreH), 7.45 (d, J ¼ 8.2 Hz, 2H, AreH), 7.34 (s, 2H, exchange with
D2O, SO2NH2), 4.28 (s, 2H, CH2), 3.35 (d, J ¼ 15.0 Hz, 2H, CH2), 2.80
(dd, J ¼ 31.9, 24.9 Hz, 2H, CH2); dC (100 MHz, DMSO‑d6): 167.50,
4.1.2. General synthetic procedure of benzenesulfonamides 4a-4e
EDCI (2.0 eq) was added to a solution of 1-carboxymethyl 5-
fluorouracil 2 (0.2 g, 1.0 eq), HOBt (0.5 eq) and DMAP (0.03 eq) in
dry DMF (2 ml) under a nitrogen atmosphere and the reaction
mixture was stirred at r.t. until the consumption of the starting
material (TLC monitoring). Thereafter the proper amino-
benzenesulfonamide 3a-3e (1.1 eq) was added to the reaction
mixture, that was stirred at r.t. until the disappearance of the
activated ester was observed (TLC monitoring) and then quenched
with ice and HCl(aq) 2M. The formed precipitate was filtered under
vacuo and recrystallized from the minimum amount of water to
afford the titled compounds as white powders.
158.60 (d, J2
¼ 35.1 Hz), 150.65, 144.43, 143.05, 140.23 (d, J2
CF
¼ 234.2 Hz), 131.99 (d, J2
¼ 35.9 Hz), 130.11, 126.63, 50.61,
CF
CF
35.54; dF (376 MHz, DMSO‑d6): 170.66 (s); m/z (ESI negative) calcd
for C14H14FN4O5S [M ꢀ H]- 369.1, found 369.0.
4.1.2.1. 2-(5-Fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-N-(4-
sulfamoylphenyl)acetamide 4a. Compound 4a was obtained ac-
cording the general procedure earlier reported using 1-
carboxymethyl 5-fluorouracil 2 (0.2 g, 1.0 eq), HOBt (0.5 eq),
DMAP (0.03 eq) and 4-aminobenzenesulfonamide 3a (1.1 eq) in dry
DMF (2 ml). 36% yield; m.p. >300 ꢁC; silica gel TLC Rf 0.50 (MeOH/
4.1.2.5. 2-(5-Fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-N-(2-
hydroxy-5-sulfamoylphenyl)acetamide 4e. Compound 4e was ob-
tained according the general procedure earlier reported using 1-
carboxymethyl 5-fluorouracil 2 (0.2 g, 1.0 eq), HOBt (0.5 eq),
DMAP (0.03 eq) and 3-amino-4-hydroxybenzenesulfonamide 3e

10
A. Petreni et al. / European Journal of Medicinal Chemistry 190 (2020) 112112
(1.1 eq) in dry DMF (2 ml). 46% yield; m.p. >300 ꢁC; silica gel TLC Rf
0.20 (MeOH/CH2Cl2 10% v/v); dH (400 MHz, DMSO‑d6): 11.92 (s, 1H,
exchange with D2O, CONHCO), 10.85 (s, 1H, AreH), 9.82 (s, 1H, ex-
change with D2O, OH), 8.49 (s, 1H, exchange with D2O, CONH), 8.13
(d, J ¼ 6.6 Hz, 1H, AreH), 7.45 (d, J ¼ 8.1 Hz, 1H, AreH), 7.16 (s, 2H,
exchange with D2O, SO2NH2), 7.03 (d, J ¼ 8.4 Hz, 1H, AreH), 4.71 (d,
J ¼ 52.0 Hz, 2H, CH2); dC (100 MHz, DMSO‑d6): 166.85, 158.45 (d, J2
4.1.3.3. 2-(5-Fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-N-(4-
fluorobenzyl)-N-(4-sulfamoylphenethyl)acetamide 6c.
Compound 6c was obtained according the general procedure
earlier reported using1-carboxymethyl 5-fluorouracil 2 (0.2 g, 1.0
eq), HOBt (0.5 eq), DMAP (0.03 eq) and 4-(2-((4-fluorobenzyl)
amino)ethyl)benzenesulfonamide 5c (1.1 eq) in dry DMF (2 ml).
65% yield; m.p. 257e259 ꢁC; silica gel TLC Rf 0.83 (MeOH/CH2Cl2
20% v/v); dH (400 MHz, DMSO‑d6): 11.91 (s, 1H, exchange with D2O,
CONHCO), 8.08 (d, J ¼ 6.8 Hz, 0.5H, AreH), 8.02 (d, J ¼ 6.7 Hz, 0.5H,
AreH), 7.80 (d, J ¼ 8.2 Hz, 1H, AreH), 7.77 (d, J ¼ 8.2 Hz, 1H, AreH),
7.52 (d, J ¼ 8.2 Hz, 1H, AreH), 7.32 (m, 7H, partially exchange with
D2O, AreH þ SONH2), 4.70 (s, 1H, CH2), 4.66 (s, 1H, CH2), 4.59 (s, 1H,
CH2), 4.55 (s, 1H, CH2), 3.48 (m, 2H, CH2), 3.01 (m, 1H, CH2), 2.84 (m,
1H, CH2); dC (100 MHz, DMSO‑d6): 167.55, 158.65 (d, J2 CF ¼ 14.0 Hz),
¼ 25.7 Hz), 151.30, 150.74, 140.17 (d, J2 CF ¼ 227.7 Hz), 135.53,
CF
132.07 (d, J2 CF ¼ 33.5 Hz), 126.63, 123.56, 120.60, 115.59, 51.24; dF
(376 MHz, DMSO‑d6): 170.64 (d, J ¼ 14.1 Hz); m/z (ESI negative)
calcd for C12H10FN4O6S [M ꢀ H]- 357.0, found 356.9.
4.1.3. General synthetic procedure of benzenesulfonamides 6a-6f
EDCI (2.0 eq) was added to a solution of 1-carboxymethyl 5-
fluorouracil 2 (0.2 g, 1.0 eq), HOBt (0.5 eq) and DMAP (0.03 eq) in
dry DMF (2 ml) under a nitrogen atmosphere and the reaction
mixture was stirred at r.t. until the consumption of the starting
material (TLC monitoring). Thereafter the proper amino-
benzenesulfonamide 5a-5f (1.5 eq) was added to the reaction
mixture, that was stirred at r.t. until the disappearance of the
activated ester was observed (TLC monitoring) and then quenched
with ice and HCl(aq) 2M. The collected, water washed precipitate
was triturated in MeOH to afford the pure titled compounds as
powders.
150.73, 143.99, 143.26 (d, J2
¼
22.8 Hz), 140.23 (d, J2
CF
¼ 212.0 Hz), 132.00 (d, J2 CF ¼ 40.0 Hz), 130.72 (d, J2 CF ¼ 7.9 Hz),
CF
130.27,129.98,126.79,116.53,116.01, 50.04 (d, J2 CF ¼ 34.5 Hz), 49.12
(d, J2 CF ¼ 77.7 Hz), 48.28, 34.18 (d, J2 CF ¼ 112.8 Hz); dF (376 MHz,
DMSO‑d6): 115.29, ꢀ170.13; m/z (ESI negative) calcd for
C21H19F2N4O5S [M ꢀ H]- 477.1, found 477.2.
4.1.3.4. 2-(5-Fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-N-(4-
methoxybenzyl)-N-(4-sulfamoylphenethyl)acetamide
6d.
Compound 6d was obtained according the general procedure
earlier reported using1-carboxymethyl 5-fluorouracil 2 (0.2 g, 1.0
eq), HOBt (0.5 eq), DMAP (0.03 eq) and 4-(2-((4-methoxybenzyl)
amino)ethyl)benzenesulfonamide 5d (1.1 eq) in dry DMF (2 ml).
69% yield; m.p. 210e212 ꢁC; silica gel TLC Rf 0.63 (MeOH/CH2Cl2
20% v/v); dH (400 MHz, DMSO‑d6): 11.88 (s, 1H, exchange with D2O,
CONHCO), 8.07 (d, J ¼ 6.8 Hz, 0.5H, AreH), 8.00 (d, J ¼ 6.7 Hz, 0.5H,
AreH), 7.77 (d, J ¼ 8.2 Hz, 1H, AreH), 7.74 (d, J ¼ 8.2 Hz, 1H, AreH),
7.48 (d, J ¼ 8.2 Hz, 1H, AreH), 7.35 (d, J ¼ 8.2 Hz, 1H, AreH), 7.29 (s,
2H, exchange with D2O, SO2NH2), 7.28 (d, J ¼ 8.2 Hz, 1H, AreH), 7.21
(d, J ¼ 8.5 Hz, 1H, AreH), 6.96 (d, J ¼ 8.6 Hz, 1H AreH), 6.90 (d,
J ¼ 8.6 Hz, 1H, AreH), 4.65 (s, 2H, CH2), 4.48 (s, 1H, CH2), 4.46 (s, 1H,
CH2), 3.76 (s, 1.5H, CH2), 3.74 (s, 1.5H, CH2), 3.44 (m, 2H, CH2), 2.96
(m, 1H, CH2), 2.79 (m, 1H, CH2); dC (100 MHz, DMSO‑d6): 167.45,
159.75 (d, J2 CF ¼ 8.6 Hz), 150.86, 144.20, 143.53 (d, J2 CF ¼ 9.2 Hz),
143.26, 135.71 (d, J2 CF ¼ 243.0 Hz), 130.39, 130.11, 129.73, 126.92,
115.18, 114.94, 56.13, 49.79 (d, J2 CF ¼ 31.0 Hz), 48.73, 48.17 (d, J2
4.1.3.1. N-benzyl-2-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-
yl)-N-(4-sulfamoylphenethyl)acetamide 6a. Compound 6a was ob-
tained according the general procedure earlier reported using 1-
carboxymethyl 5-fluorouracil 2 (0.2 g, 1.0 eq), HOBt (0.5 eq),
DMAP (0.03 eq) and 4-(2-(benzylamino)ethyl)benzenesulfonamide
5a (1.1 eq) in dry DMF (2 ml). 23% yield; m.p. 234e236 ꢁC; silica gel
TLC Rf 0.75 (MeOH/CH2Cl2 15% v/v); dH (400 MHz, DMSO‑d6): 11.87
(s, 1H, exchange with D2O, CONHCO), 8.09 (d, J ¼ 6.8 Hz, 0.5H,
AreH), 8.04 (d, J ¼ 6.7 Hz, 0.5H, AreH), 7.80 (d, J ¼ 8.1 Hz,1H, AreH),
7.77 (d, J ¼ 8.1 Hz, 1H, AreH), 7.51 (d, J ¼ 8.2 Hz, 2H, AreH), 7.43 (m,
7H, partially exchange with D2O, AreH þ SONH2), 4.72 (s, 1H, CH2),
4.67 (s, 1H, CH2), 4.60 (s, 1H, CH2), 4.57(s, 1H, CH2), 3.48 (m, 2H,
CH2), 3.02 (m, 1H, CH2), 2.84 (m, 1H, CH2); dC (100 MHz, DMSO‑d6):
167.47, 161.16, 158.57 (d, J2 CF ¼ 26.3 Hz), 150.82, 143.36, 140.39 (d, J2
¼ 544.7 Hz), 138.41, 130.27, 129.82 (d, J2 CF ¼ 32.7 Hz), 129.38,
CF
128.62, 128.10, 126.78, 50.94, 49.44 (d, J2 CF ¼ 28.2 Hz), 48.38 (d, J2
¼ 21.2 Hz), 34.31 (d, J2 CF ¼ 104.7 Hz); dF (376 MHz, DMSO‑d6):
CF
¼ 15.2 Hz), 34.22 (d, J2 CF ¼ 95.9 Hz); dF (376 MHz, DMSO‑d6):
CF
170.66; m/z (ESI negative) calcd for C22H22FN4O6S [M ꢀ H]- 489.1,
170.62; m/z (ESI negative) calcd for C21H20FN4O5S [M ꢀ H]- 459.1,
found 489.0.
found 459.1.
4.1.3.2. N-(4-Chlorobenzyl)-2-(5-fluoro-2,4-dioxo-3,4-
dihydropyrimidin-1(2H)-yl)-N-(4-sulfamoylphenethyl)acetamide 6b.
Compound 6b was obtained according the general procedure
earlier reported using1-carboxymethyl 5-fluorouracil 2 (0.2 g, 1.0
eq), HOBt (0.5 eq), DMAP (0.03 eq) and 4-(2-((4-chlorobenzyl)
amino)ethyl)benzenesulfonamide 5b (1.1 eq) in dry DMF (2 ml).
65% yield; m.p. 260e262 ꢁC; silica gel TLC Rf 0.80 (MeOH/CH2Cl2
20% v/v); dH (400 MHz, DMSO‑d6: 11.86 (s, 1H, exchange with D2O,
CONHCO), 8.09 (d, J ¼ 6.7 Hz, 0.5H, AreH), 8.04 (d, J ¼ 6.7 Hz, 0.5H,
AreH), 7.81 (d, J ¼ 8.2 Hz, 1H, AreH), 7.77 (d, J ¼ 8.2 Hz, 1H, AreH),
7.53 (d, J ¼ 8.2 Hz, 1H, AreH), 7.50 (d, J ¼ 8.4 Hz, 1H, AreH), 7.42 (m,
3H, AreH), 7.33 (m, 3H, partially exchange with D2O,
AreH þ SONH2), 4.72 (s, 1H, CH2), 4.64 (s, 1H, CH2), 4.62 (s, 1H, CH2),
4.56 (s, 1H, CH2), 3.53 (m, 1H, CH2), 3.46 (m, 1H, CH2), 3.03 (m, 1H,
CH2), 2.86 (m, 1H, CH2); dC (100 MHz, DMSO‑d6): 167.62, 158.48 (d,
J2 CF ¼ 23.7 Hz), 150.72, 143.32, 140.25 (d, J2 CF ¼ 243.4 Hz), 137.52,
132.86 (d, J2 CF ¼ 39.0 Hz), 130.52, 130.28, 129.97, 129.59, 129.32,
126.78, 50.03 (d, J2 CF ¼ 41.8 Hz), 49.20 (d, J2 CF ¼ 65.6 Hz), 48.45,
34.20 (d, J2 CF ¼ 108.8 Hz); dF (376 MHz, DMSO‑d6): 170.59; m/z (ESI
negative) calcd for C21H19ClFN4O5S [M ꢀ H]- 493.1, found 493.0.
4.1.3.5. N-(4-Cyanobenzyl)-2-(5-fluoro-2,4-dioxo-3,4-
dihydropyrimidin-1(2H)-yl)-N-(4-sulfamoylphenethyl)acetamide 6e.
Compound 6e was obtained according the general procedure
earlier reported using1-carboxymethyl 5-fluorouracil 2 (0.2 g, 1.0
eq), HOBt (0.5 eq), DMAP (0.03 eq) and 4-(2-((4-cyanobenzyl)
amino)ethyl)benzenesulfonamide 5e (1.1 eq) in dry DMF (2 ml).
43% yield; m.p. 230e232 ꢁC; silica gel TLC Rf 0.60 (MeOH/CH2Cl2
20% v/v); dH (400 MHz, DMSO‑d6): 11.92 (s, 1H, exchange with D2O,
CONHCO), 8.02 (d, J ¼ 6.7 Hz, 0.5H, AreH), 7.99 (d, J ¼ 6.7 Hz, 0.5H,
AreH), 7.86 (d, J ¼ 8.2 Hz, 1H, AreH), 7.74 (m, 3H, AreH), 7.49 (m,
3H, AreH), 7.43 (d, J ¼ 8.1 Hz, 1H, AreH), 7.36 (d, J ¼ 8.2 Hz, 1H,
AreH), 7.27 (s, 2H, exchange with D2O, SO2NH2), 4.70 (s, 2H, CH2),
4.62 (s, 1H, CH2), 4.56 (s, 1H, CH2), 3.53(m, 1H, CH2), 3.44 (m, 1H,
CH2), 2.98 (m, 1H, CH2), 2.82 (m, 1H, CH2); dC (100 MHz, DMSO‑d6):
167.99, 158.68 (d, J2
¼ 35.5 Hz), 150.86, 144.59, 143.92 (d, J2
CF
¼ 14.9 Hz), 140.33 (d, J2 CF ¼ 258.6 Hz), 133.70, 133.42, 130.46,
CF
129.37, 126.91, 119.91, 111.00, 50.40 (d, J2 CF ¼ 96.5 Hz), 49.65, 49.01
(d, J2 CF ¼ 8.1 Hz), 34.35 (d, J2 CF ¼ 111.3 Hz); dF (376 MHz, DMSO‑d6):
170.34; m/z (ESI negative) calcd for C22H19FN5O5S [M ꢀ H]- 484.1,
found 484.2.

A. Petreni et al. / European Journal of Medicinal Chemistry 190 (2020) 112112
11
4.1.3.6. N-(4-(Dimethylamino)benzyl)-2-(5-fluoro-2,4-dioxo-3,4-
dihydropyrimidin-1(2H)-yl)-N-(4-sulfamoylphenethyl)acetamide 6f.
Compound 6f was obtained according the general procedure earlier
reported using1-carboxymethyl 5-fluorouracil 2 (0.2 g, 1.0 eq),
HOBt (0.5 eq), DMAP (0.03 eq) and 4-(2-((4-(dimethylamino)
benzyl)amino)ethyl)benzenesulfonamide 5f (1.1 eq) in dry DMF
(2 ml). 28% yield; m.p. 188e190 ꢁC; silica gel TLC Rf 0.78 (MeOH/
CH2Cl2 20% v/v); dH (400 MHz, DMSO‑d6): 11.93 (s, 1H, exchange
with D2O, CONHCO), 9.24 (s, 1H, AreH), 7.82 (d, J ¼ 7.7 Hz, 2H,
AreH), 7.47 (d, J ¼ 8.1 Hz, 2H, AreH), 7.36 (m, 4H, partially exchange
with D2O, AreH þ SONH2), 6.77 (d, J ¼ 7.5 Hz, 2H, AreH), 4.68 (s,1H,
CH2), 4.43 (s, 1H, CH2), 4.06 (s, 2H, CH2), 3.11 (s, 2H, CH2), 2.95 (s, 6H,
2 x CH3), 2.90 (m, 1H, CH2), 2.79 (m, 1H, CH2); dC (100 MHz,
DMSO‑d6): 167.27, 151.79, 150.80 (d, J2 CF ¼ 13.9 Hz), 143.79, 143.37
(d, J2 CF ¼ 20.3 Hz),142.47, 138.05 (d, J2 CF ¼ 215.0 Hz),132.16, 130.21,
127.05, 126.88, 119.52, 113.05, 50.82, 49.86 (d, J2 CF ¼ 36.8 Hz), 48.47
(d, J2 CF ¼ 58.6 Hz), 47.56, 33.01 (d, J2 CF ¼ 160.3 Hz); dF (376 MHz,
DMSO‑d6): 170.70; m/z (ESI negative) calcd for C23H25FN5O5S
[M ꢀ H]- 502.2, found 502.1.
7.38 (d, J ¼ 8.6, 1H, AreH), 7.23 (s, 2H, exchange with D2O, SO2NH2);
dC (100 MHz, DMSO‑d6): 141.9, 134.8, 131.3, 129.5, 126.3, 117.7; m/z
(ESI negative) calcd for C6H4BrN4O2S [M ꢀ H]- 274.9, found 274.8.
4.1.4.5. 7-Azido-4-methyl-chromen-2-one 13. Compound 13 was
obtained according the general procedure earlier reported 7-
amino-4-methyl-chromen-2-one 11a (0.5 g, 1.0 eq), NaNO2 (1.2
eq) and NaN3 (1.5 eq) in a HCl 2M aqueous solution. 88% yield; m.p.
122e124 ꢁC; silica gel TLC Rf0.57 (EtOAc/n-hexane 20% v/v); dH
(400 MHz, DMSO‑d6): 7.83 (d, J ¼ 8.4, 1H, AreH), 7.18 (m, 2H, AreH),
6.38 (d, J ¼ 1.2, 1H, AreH), 2.46 (d, J ¼ 1.2, 3H, CH3); dC(100 MHz,
DMSO‑d6): 160.4, 155.0, 153.8, 144.2, 127.9, 117.7, 116.5, 114.1, 107.7,
19.0; m/z (ESI positive) calcd for C10H8N3O2 [M ꢀ H]- 202.1, found
202.0.
4.1.5. Synthesis of 5-fluoro-1-(prop-2-yn-1-yl)pyrimidine-2,4(1H,3H)-
dione 7
Propargyl bromide (1.0 eq) was added dropwise to a solution of
5-fluorouracil 1 (0.5 g, 1.0 eq) and DBU (1.0 eq) in dry CH3CN (7 ml)
at 0 ꢁC under a nitrogen atmosphere. The reaction mixture was
refluxed for 1.5h, then quenched with HCl(aq) 1M (15 ml) and
extracted with EtOAc (25 ml). The organic layer was dried over
Na2SO4, filtered-off and concentrated under vacuo to give a residue
that was purified by silica gel coloumn chromatography eluting
with 40% EtOAc in n-hexane to afford the title compound 7 as white
solid. 63% yield; m.p. 140e142 ꢁC; silica gel TLC Rf 0.20 (EtOAc/n-
hexane 40% v/v); dH (400 MHz, DMSO‑d6): 11.92 (s, 1H, exchange
with D2O, CONHCO), 8.17 (d, J ¼ 6.6 Hz, 1H, AreH), 4.49 (d,
J ¼ 2.3 Hz, 2H, CH2), 3.48 (t, J ¼ 2.3 Hz, 1H, CH); dC (100 MHz,
4.1.4. General synthetic procedure of azides
The proper amine 3a, 3b, 8a, 8b, 11a (0.5 g, 1.0 eq) was dissolved
in a 2M HCl aqueous solution (5 ml). NaNO2 (1.2 eq) was slowly
added at 0 ꢁC and the reaction mixture was stirred at the same
temperature for 0.5h. Thereafter NaN3 (1.5 eq) was added portion-
wise and the mixture was stirred at r.t. for 0.5h. The obtained
precipitate was filtered-off and washed with water to afford the
corresponding azides 9a-9d and 13.
4.1.4.1. 4-Azido-benzenesulfonamide 9a. Compound 9a was ob-
tained according the general procedure earlier reported 4-
aminobenzenesulfonamide 3a (0.5 g, 1.0 eq), NaNO2 (1.2 eq) and
NaN3 (1.5 eq) in a HCl 2M aqueous solution. 63% yield; m.p.
120e121 ꢁC; silica gel TLC Rf 0.47 (EtOAc/n-hexane 50% v/v); dH
(400 MHz, DMSO‑d6): 7.33 (d, J ¼ 8.8, 2H, Ar), 7.41 (s, 2H, exchange
with D2O, SO2NH2), 7.87 (d, J ¼ 8.8, 2H, Ar); dC (100 MHz, DMSO‑d6):
120.5, 128.6, 141.5, 143.9; m/z (ESI negative) calcd for C6H5N4O2S
[M ꢀ H]- 197.0, found 197.1.
DMSO‑d6): 158.40 (d, J2
¼ 25.9 Hz), 150.12, 140.87 (d, J2
CF
¼ 230.5 Hz), 129.96 (d, J2 CF ¼ 33.9 Hz), 79.18, 77.18, 38.05; dF
CF
(376 MHz, DMSO‑d6): 168.41; m/z (ESI negative) calcd for
C7H4FN2O2 [M ꢀ H]- 167.0, found 167.1.
4.1.6. General synthetic procedure of triazoles 10a-10d and 14
The proper azide 9a-9d and 13 (1.1 eq) was added to a solution
of propargyl derivate 7 (0.1 g, 1.0 eq) in H2O/tBuOH 1/1 (8 ml) at r.t.,
followed by a suspension of CuSO4 (0.1 eq) and Na ascorbate (0.5
eq) in water (0.5 ml). The reaction mixture was stirred at 40 ꢁC
overnight, then quenched with H2O (20 ml) and the formed pre-
cipitate was collected by filtration in vacuo. A solution of the residue
in DMF (4 ml) was filtered-off through Celite521® and then poured
on slush (20 ml). The precipitate was filtered in vacuo and washed
with Et2O to afford the titled compounds 10a-10d and 14 as
powders.
4.1.4.2. 3-Azido-benzenesulfonamide 9b. Compound 9b was ob-
tained according the general procedure earlier reported 3-
aminobenzenesulfonamide 3b (0.5 g, 1.0 eq), NaNO2 (1.2 eq) and
NaN3 (1.5 eq) in a HCl 2M aqueous solution. 60% yield; m.p. 110 ꢁC;
silica gel TLC Rf 0.35 (EtOAc/n-hexane 50% v/v); dH (400 MHz,
DMSO‑d6): 7.37 (m, 2H, AreH), 7.48 (s, 2H, exchange with D2O,
SO2NH2), 7.52 (s, 1H, AreH), 7.63 (m, 1H, AreH); dC (100 MHz,
DMSO‑d6): 117.2, 123.1, 123.5, 131.8, 141.4, 146.9; m/z (ESI negative)
calcd for C6H5N4O2S [M ꢀ H]- 197.0, found 197.1.
4.1.6.1. 4-(4-((5-Fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)
methyl)-1H-1,2,3-triazol-1-yl)benzenesulfonamide 10a.
Compound 10a was obtained according the general procedure earlier
reported using 5-fluoro-1-(prop-2-yn-1-yl)pyrimidine-2,4(1H,3H)-
dione 7 (0.1 g, 1.0 eq), 4-azidobenzenesulfonamide 9a (1.1 eq), CuSO4
(0.1 eq) and Na ascorbate (0.5 eq) in H2O/tBuOH 1/1 (8 ml). 73% yield;
m.p. >300 ꢁC; silica gel TLC Rf 0.20 (MeOH/CH2Cl2 10% v/v); dH
(400 MHz, DMSO‑d6): 11.94 (s, 1H, exchange with D2O, CONHCO),
8.95 (s, 1H, AreH), 8.28 (d, J ¼ 6.7 Hz, 1H, AreH), 8.16 (d, J ¼ 8.7 Hz,
2H, AreH), 8.05 (d, J ¼ 8.6 Hz, 2H, AreH), 7.57 (s, 2H, exchange with
D2O, SO2NH2), 5.06 (s, 2H, CH2); dC (100 MHz, DMSO‑d6): 158.61 (d, J2
4.1.4.3. 4-Azido-3-chlorobenzenesulfonamide 9c. Compound 9c was
obtained according the general procedure earlier reported 4-
amino-3-chlorobenzenesulfonamide 8a (0.5 g, 1.0 eq), NaNO2 (1.2
eq) and NaN3 (1.5 eq) in a HCl 2M aqueous solution. 65% yield; m.p.
140e142 ꢁC; silica gel TLC Rf 0.38 (MeOH/CH2Cl2 10% v/v); dH
(400 MHz, DMSO‑d6): 8.23 (s, 1H, AreH), 7.63 (d, J ¼ 8.8, 1H, AreH),
7.43 (d, J ¼ 8.6, 1H, AreH), 7.23 (s, 2H, exchange with D2O, SO2NH2);
dC (100 MHz, DMSO‑d6): 141.1, 134.6, 130.5, 126.6, 125.4, 105.9; m/z
(ESI negative) calcd for C6H4ClN4O2S [M ꢀ H]- 231.0, found 230.9.
¼ 25.9 Hz), 150.53, 145.09, 144.98, 140.94 (d, J2 CF ¼ 225.6 Hz),
CF
139.56, 130.95 (d, J2 CF ¼ 33.7 Hz), 128.57, 123.00, 121.34, 43.86; dF
(376 MHz, DMSO‑d6): 168.94; m/z (ESI negative) calcd for
C13H10FN6O4S [M ꢀ H]- 365.1, found 365.0.
4.1.4.4. 4-Azido-3-bromobenzenesulfonamide 9d. Compound 9d
was obtained according the general procedure earlier reported 4-
amino-3-bromobenzenesulfonamide 8b (0.5 g, 1.0 eq), NaNO2 (1.2
eq) and NaN3 (1.5 eq) in a HCl 2M aqueous solution. 70% yield; m.p.
154e156 ꢁC; silica gel TLC Rf 0.29 (MeOH/CH2Cl2 10% v/v); dH
(400 MHz, DMSO‑d6): 8.18 (s, 1H, AreH), 7.73 (d, J ¼ 8.8, 1H, AreH),
4.1.6.2. 3-(4-((5-Fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)
methyl)-1H-1,2,3-triazol-1-yl)benzenesulfonamide 10b.
Compound 10b was obtained according the general procedure earlier

12
A. Petreni et al. / European Journal of Medicinal Chemistry 190 (2020) 112112
reported using 5-fluoro-1-(prop-2-yn-1-yl)pyrimidine-2,4(1H,3H)-
dione 7 (0.1 g, 1.0 eq), 3-azidobenzenesulfonamide 9b (1.1 eq), CuSO4
(0.1 eq) and Na ascorbate (0.5 eq) in H2O/tBuOH 1/1 (8 ml). 67% yield;
m.p. 255e257 ꢁC; silica gel TLC Rf 0.24 (MeOH/CH2Cl2 10% v/v); dH
(400 MHz, DMSO‑d6): 11.93 (s, 1H, exchange with D2O, CONHCO),
8.94 (s, 1H, AreH), 8.39 (s, 1H, AreH), 8.27 (d, J ¼ 6.6 Hz, 1H, AreH),
8.16 (d, J ¼ 7.5 Hz, 1H, AreH), 7.96 (d, J ¼ 7.3 Hz, 1H, AreH), 7.84 (t,
J ¼ 7.9 Hz, 1H, AreH), 7.62 (s, 2H, exchange with D2O, SO2NH2), 5.06
4.1.8. Synthesis of 2-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-
1(2H)-yl)-N-(4-methyl-2-oxo-2H-chromen-7-yl)acetamide 12a
EDCI (2.0 eq) was added to a solution of 1-carboxymethyl 5-
fluorouracil 2 (0.2 g, 1.0 eq) and HOBt (0.5 eq) in dry DMF (2 ml)
under a nitrogen atmosphere and the reaction mixture was stirred
at r.t. until the consumption of the starting material (TLC moni-
toring). Thereafter 7-amino-4-methylcoumarin 11a (1.5 eq) was
added to the reaction mixture, that was stirred at r.t. until the
disappearance of the activated ester was observed (TLC moni-
toring) and then quenched with ice and HCl(aq) 2M. The collected,
water washed precipitate was triturated in MeOH to afford the pure
titled compound as a powder. 24% yield; m.p. 274e276 ꢁC; silica gel
TLC Rf 0.33 (MeOH/CH2Cl2 10% v/v); dH (400 MHz, DMSO‑d6): 11.97
(s, 1H, exchange with D2O, CONHCO), 10.76 (s, 1H, exchange with
D2O, CONH), 8.17 (s, 1H, AreH), 7.78 (s, 1H, AreH), 7.72 (d, J ¼ 1.9 Hz,
1H, AreH), 7.53 (dd, J ¼ 8.7, 2.0 Hz,1H, AreH), 6.32 (d, J ¼ 1.0 Hz,1H,
AreH), 4.59 (s, 2H, CH2), 2.44 (s, 3H, CH3).; dC (100 MHz, DMSO‑d6):
(s, 2H, CH2); dC (100 MHz, DMSO‑d6): 158.55 (d, J2
¼ 38.3 Hz),
CF
150.55, 146.85, 145.02, 140.86 (d, J2 CF ¼ 224.8 Hz), 137.70, 131.98,
130.95 (d, J2 CF ¼ 45.9 Hz), 126.65, 124.15, 123.11, 118.23, 43.85; dF
(376 MHz, DMSO‑d6): 168.95; m/z (ESI negative) calcd for
C13H10FN6O4S [M ꢀ H]- 365.1, found 365.0.
4.1.6.3. 3-Chloro-4-(4-((5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-
1(2H)-yl)methyl)-1H-1,2,3-triazol-1-yl)benzenesulfonamide
10c.
Compound 10c was obtained according the general procedure
earlier reported using 5-fluoro-1-(prop-2-yn-1-yl)pyrimidine-
167.21, 160.99, 158.58 (d, J2
¼ 25.8 Hz), 154.73, 154.11, 150.87,
CF
2,4(1H,3H)-dione
7
(0.1
g,
1.0
eq),
4-azido-3-
142.78, 140.38 (d, J2
¼ 228.7 Hz), 132.07 (d, J2
¼ 33.8 Hz),
CF
CF
chlorobenzenesulfonamide 9c (1.1 eq), CuSO4 (0.1 eq) and Na
ascorbate (0.5 eq) in H2O/tBuOH 1/1 (8 ml). 48% yield; m.p.
276e278 ꢁC; silica gel TLC Rf 0.30 (MeOH/CH2Cl2 10% v/v); dH
(400 MHz, DMSO‑d6): 11.91 (s, 1H, exchange with D2O, CONHCO),
8.70 (s, 1H, AreH), 8.29 (s, 1H, AreH), 8.17 (d, J ¼ 1.7 Hz, 1H, AreH),
8.01 (dd, J ¼ 8.3, 1.8 Hz, 1H, AreH), 7.96 (d, J ¼ 8.3 Hz, 1H, AreH),
7.76 (s, 2H, exchange with D2O, SO2NH2), 5.08 (s, 2H, CH2); dC
127.23, 116.43, 116.19, 113.59, 106.76, 51.43, 19.03; dF (376 MHz,
DMSO‑d6): 170.35; m/z (ESI negative) calcd for C16H11FN3O5
[M ꢀ H]- 344.1, found 344.0.
4.1.9. Synthesis of 2-oxo-2H-chromen-7-yl 2-(5-fluoro-2,4-dioxo-
3,4-dihydropyrimidin-1(2H)-yl)acetate 12b
EDCI (2.0 eq) was added to a solution of 1-carboxymethyl 5-
fluorouracil 2 (0.2 g, 1.0 eq) and DMAP (0.03 eq) in dry DMF
(2 ml) under a nitrogen atmosphere and the reaction mixture was
stirred at r.t. until the consumption of the starting material (TLC
monitoring). Thereafter 7-hydroxycoumarin 11b (1.5 eq) was added
to the reaction mixture, that was stirred at r.t. until the disap-
pearance of the 1-carboxymethyl 5-fluorouracil was observed (TLC
monitoring) and then quenched with ice and sodium bicarbonate.
The collected, water-washed precipitate triturated with ethyl-
acetate to afford the pure titled compound 12b as a powder. 15%
yield; m.p. 270e272 ꢁC; silica gel TLC Rf 0.45 (MeOH/CH2Cl2 20% v/
v); dH (400 MHz, DMSO‑d6): 12.08 (s, 1H, exchange with D2O,
CONHCO), 8.17 (d, J ¼ 6.7 Hz, 1H, AreH), 8.09 (d, J ¼ 9.6 Hz, 1H,
AreH), 7.80 (dd, J ¼ 17.8, 8.5 Hz, 1H, AreH), 7.30 (s, 1H, AreH), 7.21
(dd, J ¼ 8.5, 2.2 Hz, 1H, AreH), 6.51 (d, J ¼ 9.6 Hz, 1H, AreH), 4.78 (d,
J ¼ 32.0 Hz, 2H, CH2); dC (100 MHz, DMSO‑d6): 167.62, 160.66,
158.47 (d, J2 CF ¼ 25.8 Hz),155.16,153.26,150.77,144.80,140.57 (d, J2
(100 MHz, DMSO‑d6): 158.52 (d, J2
¼ 37.0 Hz), 150.51, 147.61,
CF
143.75,140.96 (d, J2 CF ¼ 233.5 Hz),137.73,130.99 (d, J2 CF ¼ 33.9 Hz),
130.22, 129.97, 128.76, 126.86, 126.66, 43.66; dF (376 MHz,
DMSO‑d6): 169.03; m/z (ESI negative) calcd for C13H9ClFN6O4S
[M ꢀ H]- 399.0, found 399.1.
4.1.6.4. 3-Bromo-4-(4-((5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-
1(2H)-yl)methyl)-1H-1,2,3-triazol-1-yl)benzenesulfonamide
10d.
Compound 10d was obtained according the general procedure
earlier reported using 5-fluoro-1-(prop-2-yn-1-yl)pyrimidine-
2,4(1H,3H)-dione
7
(0.1
g,
1.0
eq),
4-azido-3-
bromobenzenesulfonamide 9d (1.1 eq), CuSO4 (0.1 eq) and Na
ascorbate (0.5 eq) in H2O/tBuOH 1/1 (8 ml). 45% yield; m.p.
275e277 ꢁC; silica gel TLC Rf 0.28 (MeOH/CH2Cl2 10% v/v); dH
(400 MHz, DMSO‑d6): 11.91 (s, 1H, exchange with D2O, CONHCO),
8.67 (s, 1H, AreH), 8.30 (s, 2H, AreH), 8.03 (d, J ¼ 7.9 Hz, 1H, AreH),
7.90 (d, J ¼ 8.2 Hz, 1H, AreH), 7.75 (s, 2H, exchange with D2O,
SO2NH2), 5.08 (s, 2H, CH2); dC (100 MHz, DMSO‑d6): 158.58 (d, J2
¼ 230.2 Hz), 131.21 (d, J2 CF ¼ 34.7 Hz), 130.69, 119.25, 118.16,
CF
116.95, 110.76, 49.85; dF (376 MHz, DMSO‑d6): 169.21; m/z (ESI
negative) calcd for C15H8FN2O6 [M ꢀ H]- 331.0, found 331.0.
¼ 31.9 Hz), 150.49, 147.79, 143.65, 140.83 (d, J2 CF ¼ 230.1 Hz),
CF
139.47, 131.73, 130.95 (d, J2
¼ 37.0 Hz), 130.48, 127.10, 126.83,
CF
120.16, 43.63; dF (376 MHz, DMSO‑d6): 169.02; m/z (ESI negative)
4.2. Carbonic anhydrases inhibition
calcd for C13H9BrFN6O4S [M ꢀ H]- 443.0, found 442.9.
An Applied Photophysics stopped-flow instrument has been
used for assaying the CA catalysed CO2 hydration activity [22].
Phenol red (at a concentration of 0.2 mM) has been used as indi-
cator, 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 10e100 s. The
CO2 concentrations ranged from 1.7 to 17 mM for the determination
of the kinetic parameters and inhibition constants. For each in-
hibitor at least six traces of the initial 5e10% 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 the assay buffer. Inhibitor and enzyme
solutions were preincubated together for 15 min (sulfonamides) or
6 h (coumarins) at room temperature prior to assay, in order to
4.1.7. 5-Fluoro-1-((1-(4-methyl-2-oxo-2H-chromen-7-yl)-1H-
1,2,3-triazol-4-yl)methyl)pyrimidine-2,4(1H,3H)-dione 14
Compound 14 was obtained according the general procedure
earlier reported using 5-fluoro-1-(prop-2-yn-1-yl)pyrimidine-
2,4(1H,3H)-dione 7 (0.1 g, 1.0 eq), 7-azido-4-methyl-chromen-2-
one 13 (1.1 eq), CuSO4 (0.1 eq) and Na ascorbate (0.5 eq) in H2O/
tBuOH 1/1 (8 ml). 47% yield; m.p. 282e284 ꢁC; silica gel TLC Rf 0.30
(MeOH/CH2Cl2 10% v/v); dH (400 MHz, DMSO‑d6): 11.93 (s, 1H, ex-
change with D2O, CONHCO), 9.00 (s, 1H, AreH), 8.28 (d, J ¼ 6.5 Hz,
1H, AreH), 8.00 (d, J ¼ 9.3 Hz, 3H, AreH), 6.51 (s, 1H, AreH), 5.03 (s,
2H, CH2), 2.51 (s, 3H, CH3); dC (100 MHz, DMSO‑d6): 160.43, 158.71
(d, J2
¼
22.7 Hz), 154.68, 153.74, 150.53, 143.65 (d, J2
CF
¼ 311.3 Hz), 139.80, 139.44, 130.99 (d, J2 CF ¼ 42.8 Hz), 128.27,
CF
122.96, 120.56, 116.45, 115.7, 108.45, 43.87, 19.15; dF (376 MHz,
DMSO‑d6): 168.91; m/z (ESI negative) calcd for C17H11FN5O4
[M ꢀ H]- 368.1 found 368.0.

A. Petreni et al. / European Journal of Medicinal Chemistry 190 (2020) 112112
13
allow for the formation of the E-I complex. The inhibition constants
Acknowledgements
were obtained by non-linear least-squares methods using PRISM 3
and the Cheng-Prusoff equation, as reported earlier [23,24], and
represent the mean from at least three different determinations. All
CA isoforms were recombinant ones obtained in-house as reported
earlier [25].
MIUR is gratefully acknowledged for a grant to A.N (PRIN: rot.
2017XYBP2R). This work was in part funded by the Researchers
Supporting Project No. (RSP-2019/1) King Saud University, Riyadh,
Saudi Arabia.
4.3. X-ray crystallography
Appendix A. Supplementary data
CA II was expressed and purified as previously described [26]. In
brief, CA II was expressed in BL21(DE3) E. coli cells and induced
with IPTG. Protein was purified from lysed cells using affinity
chromatography with p-(aminomethyl)benzenesulfonamide resin.
Final protein purity was confirmed via SDS-PAGE and the purified
CA II diluted to a final concentration of 10 mg/mL for crystallization.
Complex formation was induced by co-crystallization using the
hanging drop vaporization method in which CA II crystals were
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2020.112112.
Associated content
The atomic coordinates of the CA II/A complex have been
deposited in the Protein Data Bank with accession code 6VJ3.
grown in the presence of 350 mM compound A. A precipitant so-
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