Carbonic anhydrase inhibitors based on sorafenib scaffold: Design, synthesis, crystallographic investigation and effects on primary breast cancer cells

Article abstract of DOI:10.1016/j.ejmech.2019.111600

Carbonic anhydrase inhibitors (CAIs) of the sulfonamide, sulfamate and coumarin classes bearing the phenylureido tail found in the clinically used drug Sorafenib, a multikinase inhibitor actually used for the management of hepatocellular carcinomas, are r

Full text of DOI:10.1016/j.ejmech.2019.111600

Journal Pre-proof
Carbonic anhydrase inhibitors based on sorafenib scaffold: Design, synthesis,
crystallographic investigation and effects on primary breast cancer cells
Murat Bozdag, Marta Ferraroni, Carol Ward, Fabrizio Carta, Silvia Bua, Andrea
Angeli, Simon P. Langdon, Ian H. Kunkler, Abdul-Malek S. Al-Tamimi, Claudiu T.
Supuran
PII:
S0223-5234(19)30734-2
DOI:
https://doi.org/10.1016/j.ejmech.2019.111600
EJMECH 111600
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 15 July 2019
Revised Date: 5 August 2019
Accepted Date: 5 August 2019
Please cite this article as: M. Bozdag, M. Ferraroni, C. Ward, F. Carta, S. Bua, A. Angeli, S.P. Langdon,
I.H. Kunkler, A.-M.S. Al-Tamimi, C.T. Supuran, Carbonic anhydrase inhibitors based on sorafenib
scaffold: Design, synthesis, crystallographic investigation and effects on primary breast cancer cells,
European Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2019.111600.
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition
of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of
record. This version will undergo additional copyediting, typesetting and review before it is published
in its final form, but we are providing this version to give early visibility of the article. Please note that,
during the production process, errors may be discovered which could affect the content, and all legal
disclaimers that apply to the journal pertain.
© 2019 Published by Elsevier Masson SAS.

Graphical abstract

Carbonic Anhydrase Inhibitors based on sorafenib scaffold: Design, Synthesis,
Crystallographic Investigation and Effects on Primary Breast Cancer Cells
Murat Bozdag a,*, Marta Ferraroni b, Carol Ward c, Fabrizio Carta a, Silvia Bua a, Andrea Angeli a,
Simon P. Langdon c, Ian H. Kunkler c, Abdul-Malek S. Al-Tamimi d, Claudiu T. Supuran a,*
a
University of Florence, NEUROFARBA Dept., Sezione di Scienze Farmaceutiche e
Nutraceutiche, Via Ugo Schiff 6 , 50019 Sesto Fiorentino (Florence), Italy.
b
University of Florence, Department of Chemistry “Ugo Schiff”, Via della Lastruccia 3, 50019
Sesto Fiorentino (Florence), Italy.
c
Breakthrough Breast Unit and Division of Pathology, Institute of Genetics and Molecular
Medicine, Edinburgh EH4 2XU, UK
d
Department of Pharmaceutical Chemistry, College of Pharmacy, Prince Sattam Bin Abdulaziz
University, PO Box 173, Alkharj 11942. Saudi Arabia.
Abstract: Carbonic anhydrase inhibitors (CAIs) of the sulfonamide, sulfamate and coumarin
classes bearing the phenylureido tail found in the clinically used drug Sorafenib, a multikinase
inhibitor actually used for the management of hepatocellular carcinomas, are reported. All
compounds were assayed on human (h) CA isoforms I, II, VII and IX, involved in various
pathologies. Among the sulfonamides, several compounds were selective for inhibiting hCA IX,
with KI values in the low nanomolar ranges (i.e. 0.7-30.2 nM). We explored the binding modes of
such compounds by means of X-ray crystallographic studies on isoform hCA I in adduct with one
sulfonamide and a sulfamate inhibitor. Antiproliferative properties of some sulfamates on breast
tumor cell lines were also investigated.
Keywords: Carbonic Anhydrase; Sorafenib; Enzyme inhibitor; sulfonamide; sulfamate; coumarin.
*
Corresponding authors: bozdag.murat@unifi.it (MB) Phone: +39-055-4573666;
claudiu.supuran@unifi.it. (CTS) Phone: +39-055-4573729

1.Introduction
The 15 carbonic anhydrases (CAs, EC 4.2.1.1) expressed humans in are zinc(II) dependent
metalloenzymes, differently distributed at subcellular and tissue levels [1,2]. Human (h) hCAs are
involved in vital physiological processes by means of the reversible conversion of carbon dioxide to
bicarbonate and protons which they catalyze [3–7]. Abnormalities in hCA expressions may lead to
diseases, such as hypertension, glaucoma, epilepsy, obesity, altitude sickness, cancers and pH
disequilibria [7,8]. CA inhibitors (CAIs) of the sulfonamide type or their bioisosteres, are routinely
used as drugs. The most used are acetazolamide (AAZ), methazolamide (MTZ), dichlorophenamide
(DCP), brinzolamide (BRZ), sulthiame (SLT), zonisamide (ZNS), the sulfamate topiramate (TPM)
and various COXIBs with dual CA-cycloxygenase activity such as celecoxib and polmacoxib [7–9].
Besides the sulfonamide moiety, many new types of CAIs acting as zinc binders were discovered
(i.e. dithiocarbamates [10–13] xanthates [14] and monothiocarbamates [15]). In addition, other
classes of CAIs include molecules able to interact with the zinc coordinated water/hydroxide ion
(i.e. phenols [16–20], carboxylic acids [20,21], polyamines [22,23], thioxocoumarins [24,25] and
the sulfonic acid group formed from hydrolysis of sulfocoumarins [26–32]). Compounds occluding
the entrance of the CA active site (cinnamic acid derivatives from in situ hydrolyzed coumarins
[24,33–36]) and out of the site CAIs such as the 2-(benzylsulfonyl)-benzoic acid were also shown to
act as CAIs [37]. Although many CAI chemical classes were reported, the selectivity in targeting
specific enzymatic isoforms was not always granted. Therefore, any medicinal chemistry
application of CAIs resulted hampered. Interestingly, the decoration of the sulfonamide CAI
warhead by means of appropriate tails revealed to be a valid strategy to overcome the lack of
enzymatic selectivity. In particular the arylureido-tails were particularly efficient in solving the
selectivity issue among the many hCA isoforms [38–40], and in particular such a moiety led to
compounds with a preferential inhibition towards the tumor associated isoforms (i.e. hCAs IX and
XII) over the cytosolic off-target ones (i.e. hCAs I and II). Such results were confirmed in in vivo
experiments using an appropriate breast cancer model [38–40], and were rationalized at molecular
level by using X-ray crystallography techniques. The flexibility of the ureido-group has been
claimed to be responsible for allowing the tails to better fit within the CA active site, and thus in
maximizing the interactions occurring between the ligand and the amino acid residues exposed
within
the
enzymatic
cleft
[38–40].
The
compound
(4-(3-(4-
fluorophenyl)ureido)benzenesulfonamide, also known as SLC-0111, was obtained by application of
such a strategy and is currently facing phase II clinical trials in combination with gemcitabine for
the treatment of metastatic pancreatic ductal cancer [41]. SLC-0111 is a selective inhibitor of hCA
IX and XII, which are almost exclusively expressed in hypoxic tumors [42–44], being responsible

of the extracellular pH acidification [39,42–44]. Recent applications of the ureido tail strategy were
reported by our group with the preparation of ureido- substituted sulfamates, coumarins, 4-hydroxy-
3-(3-(phenylureido)benzenesulfonamides, N'-phenyl-N-hydroxyureas and N-aryl-N’-ureido-O-
sulfamates [24,45–49].
Our pursuit to find more efficient ureido-bearing molecules to treat hypoxic tumors directed us to
consider sorafenib (Nexavar®), an orally available multikinase inhibitor approved by FDA to treat
hepatocellular carcinoma (HCC), as an interesting lead. Clinical studies showed sorafenib to exert
promising effects on thyroid cancer, myeloid leukemia, mesothelioma and prostate cancers [50].
HCC is one of the most common cancers associated to high ratio of mortality [50–52]. Sorafenib
was proved to extend the median time of progression in patients with advanced HCC and the life
span of patients by an average of three months [50–53].
2. Results and Discussion
2.1. Drug Design and Chemistry.
The first studies on phenylureido-tailed benzenesulfonamides which revealed selective
inhibition of the hypoxia related hCAs IX and XII were published by some of us [38–40] and
among the series reported a molecule, known as SLC-0111, showed impressive antimetastatic
effects on breast cancer in vivo[39]. Such a molecule currently is under phase II clinical trials in
association with gemcitabine for the management of pancreatic tumors [41]. Such results allowed to
validate the membrane bound hCA IX as pharmaceutical target for the treatment of hypoxic tumors.
Another important study within this field was on sulfamate derivatives of SLC-0111 [54]. In vitro
kinetics on the series reported showed highly potent inhibitors against the tumor associated hCAs.
In particular, three CAIs were further selected for in vitro cellular assays and showed inhibition of
cells migration and spreading in anoxic conditions. Moreover one of such inhibitors was tested on
orthotopic MDA-MB-231 breast cancer model in mice and showed inhibition of metastases in lungs
without relevant toxicity [54]. As expansion the same authors reported a new series of sulfamates
which showed in vitro potent hCA IX inhibitory activity and antiproliferative properties on breast
cancer cell lines (i.e. SKBR3, MCF10A, ZR-75-1, MDA-MB-361 in normoxia and MCF7 in
hypoxia) [45]. The ureido moiety was also inserted in seleno containing compounds as CAIs and
were investigated on prostate (PC3), breast (MDA-MB-231) and colon cancer (HT-29) cells in
comparison with SLC-0111 [55]. Finally it is worth reporting the 2-aminophenol-4-sulfonamide
ureido containing derivatives as CAIs [47]. Again the series showed isoform selective inhibition on
the tumor associated hCAs, and the 4-fluorophenylureido derivative was investigated in vivo and

showed similar results to SLC-0111 [47]. In light of the high potency of the ureido containing CAIs
on the tumor associated isoforms we decided to merge the structures of sorafenib and SLC-0111 to
obtain a novel series of CAIs potentially useful to manage such types of pathologies, as
schematically represented in Figure 1.
Figure 1. Sorafenib, SLC-0111 and 13 obtained by our synthetic strategy.
Our design strategy was further extended to include series of compounds bearing the
sulfonamides, sulfamates and coumarin moieties as warheads.
As reported in Schemes 1-3, compounds 12-17 and 21 were obtained by coupling the amino
containing derivatives 1-6 and 10 with the commercially available 4-chloro-3-
(trifluoromethyl)phenyl isocyanate 11. The same reaction was applied to the synthesis of
intermediates 18-20. In particular 18 and 19 were further decorated with the sulfonamide bioisoster
sulfamate, in order to afford 23 and 24 (scheme 2). On the other hand 20 was activated by means of
its NHS ester 25 which was used to obtain elongated CAIs of the sulfonamide (i.e. 26, 27, 29, 31)
[56] and coumarin type (i.e. 33 and 35) [36].
Scheme 1. General synthesis of ureido derivatives 12-21

Scheme 2. General synthesis of sulfamates 23-24
Scheme 3. General synthesis of carbamides 26-27, 29, 31, 33, 35
1
All compounds were characterized by means of H-, 13C-, 19F-NMR and ESI mass
spectroscopy techniques.
2.2. Carbonic Anhydrase Inhibition
The reported compounds having the sulfonamides (i.e. 12-17, 26, 27, 29, 31), the sulfamates (i.e.
23, 24) and the coumarin (i.e. 21, 33, 35) moieties were tested in vitro for their inhibitory properties
against the physiologically relevant hCA isoforms I, II, VII, IX in comparison with sorafenib and
the clinically used drug AAZ, by means of the stopped-flow carbon dioxide assay [57] (Table 1).
KI (nM)*
Cmp
hCA I
hCA II
hCA VII
hCA IX

12
0.63
0.24
0.38
0.13
3.9
13
84.5
1.8
198.5
916.3
893.0
801.2
911.3
>10000
5.7
14
6422.9
4042.3
513.6
913.4
>10000
48.7
71.5
1.8
15
744.5
51.4
30.2
0.7
16
17
385.6
>10000
3.7
3972
1588.1
1.3
21
23
24
39.3
4.0
3.5
0.8
26
432.1
4545.1
4601.0
6348.3
>10000
>10000
>10000
5080
90.0
210.1
488.1
363.3
584.9
>10000
>10000
8136.4
8550
4.2
27
2660.2
932.9
5761.4
>10000
>10000
>10000
960
3298
188.3
102.4
3412.4
2254.4
>10000
45.1
25.8
29
31
33
35
Sorafenib
SLC-0111**
AAZ
250.0
12.1
6.0
* Mean from 3 different assays, by a stopped flow technique (errors were in the range of ± 5-10 %
of the reported values). ** From reference [38,58].
The inhibition data are showed in Table 1 and the following SARs are below discussed:
(i)
The off target cytosolic isoform hCA I was inhibited by all compounds bearing the zinc
binding functionality of the sulfonamide and sulfamate type 12-17, 23-24, 26-27, 29, 31
with a variety of potencies. The derivative of AAZ 12 acted as sub-nanomolar hCA I
inhibitor with a KI of 0.63 nM. The sulfanilamide derivative 13 and two sulfamates 23-24
were potent hCA I inhibitors with KIs ranging between 39.3 to 84.5 nM. The remaining
sulfonamides 14-17, 26-27, 29 and 31 were medium to weak potency inhibitors of hCA I
with KIs in the range of 432.1-6422.9 nM and they acted as weaker inhibitors compare to
AAZ (KI of 250 nM). The coumarins 21, 33, 35 did not inhibit the slow cytosolic isoform

(hCA I) with inhibition constants > 10 µM. This result has been observed before by many
coumarin derivatives [24,33–35].
(ii)
The second abundant cytosolic isoform hCA II was inhibited by sulfonamides and
sulfamates with KIs in a range of 0.24-5761.4 nM. Compounds 12-13 and 23-24 were potent
hCA II inhibitors with KIs of 0.24-4.0 nM. The AAZ derivative 12 was the most potent
inhibitor of cytosolic isoform hCA II with a KI of 0.24 nM, the second most potent inhibitor
was the sulfanilamide derivative 13 with a KI of 1.8 nM. Two sulfamates 23-24 were also
acted as potent inhibitor of this isoform with KIs of 3.7 and 4.0, respectively. The
sulfonamide derivatives 14-17 were medium to less potent inhibitors of this isoform with a
variety of KIs between 51.4-744.5 nM. We observed a dramatic change of KI (39.7 fold) by
switching sulfonamide group from para- position 13 to meta- position 14 which had an
inhibition constant of 71.5 nM. When a hydroxy group was appended at the para- position
towards the sulfonamide of 14 to afford 15 resulted decrease of the inhibition constant (KI)
to 744.5 nM (10.4 fold). Very recently we investigated inhibition properties of ureido-
derivatives of 4 which are same chemotype of 15 which were also weak inhibitors of hCA II
isoform [47]. The introduction of aliphatic linker between the ring bearing zinc binding
group (ZBG) and urea group (16-17) was showed weaker hCA II inhibitory properties, with
KIs 51.4 for 16 and 385.6 for 17 (28.6, 214.2 fold, respectively compared to 13). When a
benzamide group was inserted between the phenylureido tail and inhibitor heads to
investigate inhibition properties of longer compounds, 26-27, 29, 31 were resulted as weak
inhibitors of hCA II isoform with KIs in range of 90-5761 nM. The coumarins 21, 33, 35 did
not inhibit the fast cytosolic isoform (hCA II) with inhibition constants > 10 µM. This result
has been observed before by many coumarin derivatives [24,33–35].
(iii) Compounds investigated here also showed a variety of different inhibition constants on the
cytosolic isoform hCA VII. The sulfonamides and the sulfamates were inhibited hCA VII
with KIs in the range of 0.38-916.3 nM. Again, the AAZ derivative 12 was the most potent
inhibitor with a KI of 0.38 nM. Followed by two potent sulfamates 23-24 with KIs 5.7, 3.5
nM, respectively. These three compounds were more effective inhibitors compare to AAZ
(KI of 6 nM). Remaining compounds were weak inhibitors of hCA VII isoform KIs in the
range of 198.5-916.3 nM. The coumarins did not inhibit hCA VII as well with KIs > 10 µM.
(iv)
The sulfonamides and the sulfamates reported here showed a variety of inhibitory effects on
the tumor associated extracellular isoform hCA IX (KIs spanning from 0.13 to 3972 nM).
AAZ derivative 12 was a sub-nanomolar inhibitor, and the most potent inhibitor of the
series (with a KI of 0.13 nM) followed by methylbenzenesulfonamide derivative 16 (KI of

0.7 nM). Ethylbenzene sulfonamide derivative 17 was a weak inhibitor with a KI of 3972
nM, that showed a dramatic decrease on inhibition effect by extension of linker between the
ureido and sulfonamide possessing phenyl ring of inhibitor (5674-fold ineffective compare
to 16). Whereas, direct attachment of ureido group to sulfonamide possessing phenyl ring,
13 (the derivative of SLC-0111) showed potent inhibitory effect on hCA IX with a KI of 3.9
nM (5.6-fold weaker inhibitor compare to 16). Manipulation of the sulfonamide group from
4-position to 3-position resulted 14 which enhanced the inhibitory profile with a KI of 1.8
nM (2.16-fold more potent compare to 13). Whereas 15, a derivative of 14 possessing -OH
moiety at the para position of sulfonamide showed dramatic decrease of inhibition with a KI
of 30.2 nM (16.78-fold compare to 14). Two sulfamates 23-24 were excellent inhibitor of
this isoform with KIs of 1.3 and 0.8 nM, respectively. Similar results were reported by
previously investigated
ureido-possessing
sulfamates
[54].
The
methylene
benzenesulfonamide head possessing derivative 26 showed excellent inhibitory effect with a
KI of 4.2 nM whereas ethylbenzenesulfonamide derivative 27 was ineffective inhibitor with
a KI of 3298 nM, again a carbon chain extention shows a dramatic effect on inhibition
capacity between 26 and 27. 29 and 31 were medium potency inhibitors with KIs of 188.3
and 102.4 nM, respectively. 12-14, 16, 23-24, 26 (KIs between 0.13-4.2 nM) were revealed
more potent inhibitory effects on hCA IX compare to clinically used drug AAZ (KI of 25.8).
Coumarins 21, 33, 35 inhibited hCA IX with low efficiency (KIs 1.59-3.41 µM), whereas
did not inhibit any of cytosolic isoforms (CA I, II, VII), (Table 1).
Overall the inhibition data showed a preferential activity of the sulfonamides 14-16 and 26 and the
sulfamates 23-24 on hCA IX, with sub-to low nanomolar KI values for many such derivatives.
2.3. X-Ray crystallography.
We assessed the binding modes of compounds 17 and 23 within the active site of hCA I by
means of X-ray crystallographic experiments. Our intention was to investigate a hCA isoform
usually less investigated, since the majority of CA X-ray crystal adducts reported are related to the
ubiquitous hCA II or to the tumor associated hCA IX mimic [20]. hCA I is abundant in red blood
cells (at 1 µM concentration level), and it might influence the pharmacokinetics of drugs interacting
with it or other CA isoforms of pharmacological interest.
The difference |Fo Fc| electron density maps of adducts of hCA I with inhibitors showed
well ordered structures within the active site of hCA I and clearly assignable to compounds 17 and
23 bound by means of their sulfonamide and sulfamate group to the active site metal ion (Figure 3).

Figure 2. hCA I complexes of compound 17 (A; PDB accession code 6I0L) and 23 (B; PDB
accession code 6I0J) at 1.4 and 1.35 Å resolution respectively.
A further look into the structural features revealed 17 and 23 coordinated to the Zn(II) ion
by means of the deprotonated sulfonamide or sulfamate moieties (SO2NH-, OSO2NH-), which in
turn are also involved in a strong H-bond with the OH of Thr199 residue. One of the
sulfonamide/sulfamate oxygen was also involved in a hydrogen bond interaction with the amide
nitrogen of Thr199. This coordination pattern, which is typical for all primary
sulfonamides/sulfamates investigated so far, is respected also in these cases [20]. The two inhibitors
have a similar orientation inside the cavity, but 23 presents a bended conformation. This could be
the result of the strong H-bond involvement of the ureido-moiety of 23 with Gln92 residue, further
reinforced by a perpendicular π-stacking between its phenyl ring and the imidazole of His94 at the
bottom of the active site cavity site (Figure 3).

Figure 3. Superimposition of hCA I-17 (purple) and 23 (cyan) adducts.
The 3-(trifluoromethyl)-4-chlorophenyl tail of 17 is involved in hydrophobic interactions with
Ala135 and Tyr204 whereas the same tail moiety of 23 is slightly retained within the enzymatic
cleft and makes hydrophobic interactions with Leu131 and Ala135. The additional interactions of
23 when compared to 17, within the catalytic cavity site (i.e. π stacking and the hydrogen bond
interaction with Gln92) are reflected into the inhibition results on the same CA isoform. As reported
in Table 1 and above discussed, compound 17 is 18.8 fold less effective on hCA I when compared
to 23 which is bound tighter.
2.4. Effects of Compounds 23 and 24 on Primary Breast Tumor Growth.
The in vitro obtained results led us to select less investigated sulfamate type inhibitors 23 and 24
(which are excellent inhibitors of hCA IX with KIs between 0.8-1.3 nM, Table 1) for their effects on
primary breast tumor HBL100, MDA-MB-231 and MCF7 cell lines in normoxic and hypoxic
conditions.

HBL100 Normoxia
HBL100 Hypoxia
120
100
80
60
40
20
0
120
100
80
60
40
20
0
21
22
21
22
0 µM 3 µM 10 µM 30 µM 100 µM
Drug Concentration
0 µM 3 µM 10 µM 30 µM 100 µM
Drug concentration
MDA-MB-231 Normoxia
MDA-MB-231 Hypoxia
120
100
80
60
40
20
0
120
100
80
60
40
20
0
21
22
21
22
0 µM 3 µM 10 µM 30 µM 100 µM
Drug Concentration
0 µM 3 µM 10 µM 30 µM 100 µM
Drug concentration
MCF7 Hypoxia
MCF7 Normoxia
120
100
80
60
40
20
0
120
100
80
60
40
20
0
21
22
21
22
0 µM 3 µM 10 µM 30 µM 100 µM
Drug Concentration
0 µM
3 µM
10 µM 30 µM 100 µM
Drug Concentration
Figure 4. The effect of sulfamate type inhibitors 23 and 24 on proliferation of breast cancer cells in
normoxic and hypoxic conditions.
HBL100, MDA-MB-231 and MCF7 breast cancer cells (1 x 103)/well, were allowed to adhere for
24h. Cells were either cultured in normoxia (21% O2), or hypoxia (0.5% O2), for a further 24 h
before the addition of inhibitors at the concentrations indicated above and cultured in these
conditions for a further 5 days. Proliferation was assessed using SRB assays. Results shown mean ±
SEM (n=6; 6 replicates per experiment).

Figure 4 demonstrates that the sulfamate inhibitors 23 and 24 decreased the proliferation of all
three breast cancer cell lines over the time period examined at concentration greater than 10 µM. At
30 µM, both sulfamates reduced proliferation by approximately 50%. This occurred regardless of
the oxygenation status of the cells, with similar concentration responses to both inhibitors, in
normoxic or hypoxic conditions and in all cell lines studied.
3. Conclusion.
Herein we reported a series of CAIs obtained by merging the multikinase inhibitor Sorafenib
with the benzene sulfonamide hCA IX inhibitor SLC-0111. All compounds here share the same tail
moiety of Sorafenib (i.e. 4-chloro-3-(trifluoromethyl)phenyl ureido group) and were assayed in
vitro as inhibitors of four hCA isoforms of pharmacological relevance, the cytosolic isoforms hCA
I, II and VII, and the tumor associated transmembrane isoform hCA IX. Interestingly the in vitro
results showed a preferential inhibition of the tumor associated isoform with KI values spanning
between the low to medium nanomolar concentration. We determined the binding modes of
compounds 17 and 23 on the hCA I isoform by means of X-ray crystallographic studies of their
adducts with hCA I. Moreover, two sulfamates 23 and 24 were investigated for their
antiproliferative activities on HBL100, MDA-MB-231 and MCF7 cell lines at normoxic and
hypoxic conditions. Both sulfamates were found to decrease proliferation of these breast cancer cell
lines at concentrations above 10 µM regardless of oxygenation status.

4. Experimental Section
4.1. Chemistry
All anhydrous solvents and reagents used in this study were purchased from Alfa Aesar, TCI, and
Sigma-Aldrich. The synthetic reactions involving air- or moisture-sensitive chemicals were carried
out under a nitrogen atmosphere using dried glassware and syringe techniques in order to transfer
the solutions. Nuclear magnetic resonance (1 H-, 13C-, and 19F-NMR) spectra were recorded using a
Bruker Avance III 400 MHz spectrometer using DMSO-d6 as solvent. The chemical shifts are
reported in parts per million (ppm), and the coupling constants (J) are expressed in Hertz (Hz). The
splitting patterns are designated as s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; brs, broad
singlet; dd, doublet of doublets. The correct assignment of exchangeable protons (i.e., OH and NH)
was carried out by means of the addition of D2O. Analytical thin-layer chromatography (TLC) was
done on Merck silica gel F-254 plates. The HPLC was performed by using a Waters 2690
separation module coupled with a photodiode array detector (PDA Waters 996) using a Nova-Pak
C18 4 µm 3.9 mm × 150 mm (Waters) silica-based reverse phase column. The sample was
dissolved in 10% acetonitrile/H2O and an injection volume of 45 µL. The mobile phase (flow rate
1.0 mL/min) was a gradient of H2O + trifluoroacetic acid (TFA) 0.1% (A) and acetonitrile + TFA
0.1% (B), with steps as follows: (A%/B%), 0−10 min 90:10, 10−25 min gradient to 60:40, 26:28
min isocratic 20:80, 29−35 min isocratic 90:10. TFA 0.1% in water as well in acetonitrile was used
as counterion. All compounds reported here were ≥95% HPLC pure. The solvents used in MS
measures were acetone, acetonitrile (Chromasolv grade), and mQ water 18 MU. The high resolution
mass spectrometry (HRMS) analysis was performed with a Thermo Finnigan LTQ Orbitrap mass
spectrometer coupled with an electrospray ionization source (ESI). Analysis was carried out in
positive ion mode [M + H]+, and it was used a proper dwell time acquisition to achieve 60,000 units
of resolution at full width at half maximum (fwhm). Elemental composition of compounds was
calculated on the basis of their measured accurate masses, accepting only results with an attribution
error less than 5 ppm and a not integer RDB (double bond/ring equivalents) value. Stock solutions
of analytes were prepared using acetone (1.0 mg mL−1) and stored at 4 °C. Then working solutions
of each analyte were prepared by dilution of the stock solutions using mQ H2O/ACN 1/1 (v/v) up to
a concentration of 1.0 µg mL−1. The HRMS analysis was performed by introducing the analyte
working solution via syringe pump at 10 µL min−1.
4.2. Synthesis of reported compounds
4.2.1. General synthetic procedure a to synthesis of urea derivatives 12-21

A solution of amine 1-10 (0.2g, 1 equiv) was treated with 4-chloro-3-(trifluoromethyl)phenyl
isocyanate 11 (1.0 equv) in dry acetonitrile (3-4 mL). The reaction mixture was stirred at rt until the
consumption of starting materials (TLC monitoring). Reaction was quenched with H2O to give a
precipitate that was filtered-off and washed with diethyl ether (3 x 5 mL) and dried under vacuum
to afford the products 12-21.
4.2.2. General synthetic procedure b to synthesis of sulfamates 23-24
A solution of phenols 18-19 (0.2 g, 1.0 equiv) was treated with freshly prepared sulfamoylchloride
22 (6.0 equiv, portion-wise) in dry DMA at 0 °C. The reaction mixture was warmed to rt and stirred
for 15 min then quenched with slush. The obtained precipitate was filtered-off, washed with DCM
(3 x 5 mL) and dried under vacuum to afford desired products 23-24.
4.2.3 Synthesis of 2,5-dioxopyrrolidin-1-yl4-(3-(4-chloro-3-(trifluoromethyl)phenyl)ureido)
benzoate (25): A solution of 20 (1.0 g, 1.0 equiv) was treated with NHS (1.5 equiv) at 0 ˚C in dry
DMF (5.0 ml), followed by addition of EDCI (1.5 equiv). The reaction mixture was stirred until the
consumption of starting materials (TLC monitoring). The reaction was quenched with H2O to obtain
a precipitate which was filtered-off, washed with H2O (3 x 5 mL) and dried under vacuum to obtain
compound 25.
4.2.4. General synthetic method c for the synthesis of compounds 26-27, 29, 31, 33, 35: A solution
of amine 5-6, 28, 30, 32 and 34 (50-150 mg, 1.0 equiv) in dry DMF (3-4 mL) was treated with
activated ester 25 (1.0 equiv), followed by addition of Et3N (2.5 equiv). The reaction mixture was
stirred until the consumption of the starting materials, then quenched with H2O (2-3 mL) and
acidified by 1M aqueous solution of HCl. The obtained precipitate was filtered-off, washed with
diethyl ether (3 x 5 mL) and dried under vacuum to obtain desired products 26-27, 29, 31, 33 and
35.
4.2.5. 5-(3-(4-chloro-3-(trifluoromethyl)phenyl)ureido)-1,3,4-thiadiazole-2-sulfonamide (12): white
solid; yield 65%; silica gel TLC Rf 0.35 (MeOH/DCM 10% v/v); mp 242-243 °C; δH (400 MHz,
DMSO-d6) 7.72 (1H, d, J 8.8), 7.83 (1H, m), 8.13 (1H, s), 8.35 (2H, exchange with D2O, SO2NH2),
9.72 (1H, s, exchange with D2O, NH), 11.88 (1H, s, exchange with D2O, NH); δC (100 MHz,
3
1
2
DMSO-d6) 118.9 (q, JC-F 6), 123.6 (1, JC-F 271), 125.0, 125.1, 127.7 (q, JC-F 30), 133.1, 138.8,
153.3, 164.1, 164.5; δF (376 MHz, DMSO-d6) -61.5; m/z (ESI negative) 399.9 [M-H]-.
4.2.6. 4-(3-(4-chloro-3-(trifluoromethyl)phenyl)ureido)benzenesulfonamide (13): White solid, yield
60%; silica gel TLC Rf 0.55 (MeOH/DCM 20% v/v); mp 260-261 °C; δH (400 MHz, DMSO-d6)
7.26 (2H, s, exchange with D2O, SO2NH2), 7.66 (4H, m), 7.77 (2H, d, J 8.0), 8.16 (1H, m), 9.28

(1H, s, exchange with D2O, NH), 9.31 (1H, s, exchange with D2O, NH); δC (100 MHz, DMSO-d6)
117.9 (q, 3JC-F6), 118.9, 123.7 (q, 4JC-F 2), 123.8 (q, 1JC-F 271.3), 124.2, 127.7 (q, 2JC-F 30.4), 127.8,
133.0, 138.3, 139.9, 143.3, 153.2; δF (376 MHz, DMSO-d6) -61.4; m/z (ESI negative) 392.0 [M-H]-.
4.2.7. 3-(3-(4-chloro-3-(trifluoromethyl)phenyl)ureido)benzenesulfonamide (14): White solid, yield
76%; silica gel TLC Rf 0.45 (MeOH/DCM 10% v/v); mp 245-246 ºC; δH (400 MHz, DMSO-d6)
7.40 (2H, s, exchange with D2O, SO2NH2), 7.51 (2H, m), 7.60 (1H, dt, J 2.0, 7.6), 7.68 (2H, m),
8.12 (1H, m), 8.17 (1H, d, J 2.0), 9.23-9.25 (2H, m, exchange with D2O, NH); δC (100 MHz,
3
4
1
DMSO-d6) 116.5, 117.9 (q, JC-F 22), 120.3, 122.5, 123.6 (q, JC-F 2), 123.8 (q, JC-F 271), 124.2,
3
127.7 (q, JC-F 30), 130.4, 132.9, 140.1, 140.7, 145.7, 153.3; δF (376 MHz, DMSO-d6) -61.5; m/z
(ESI positive) 392.0 [M-H]-.
4.2.8. 3-(3-(4-chloro-3-(trifluoromethyl)phenyl)ureido)-4-hydroxybenzenesulfonamide (15): White
solid, yield 62%; silica gel TLC Rf 0.12 (MeOH/DCM 10% v/v); mp 254-255 ºC; δH (400 MHz,
DMSO-d6) 6.99 (1H, d, J 8.4), 7.16 (2H, s, exchange with D2O, SO2NH2), 7.36 (1H, dd, J 2.4, 8.4),
7.60 (1H, dd, J 2.0, 8.8), 7.66 (1H, d, J 8.8), 8.20 (1H, d, J 2.0), 8.48 (1H, s, exchange with D2O,
NH), 8.66 (1H, d, J 2.4), 9.89 (1H, s, exchange with D2O, NH), 10.96 (1H, s, exchange with D2O,
OH); δC (100 MHz, DMSO-d6) 114.7, 117.1, 117.3 (q, 3JC-F 6), 121.4, 123.2 (q, 1JC-F 273), 123.4 (q,
2
4JC-F 2), 123.6, 127.8 (q, JC-F 30), 128.3, 133.1, 135.9, 140.2, 149.5, 153.1; δF (376 MHz, DMSO-
d6) -61.5; m/z (ESI negative) 408.0 [M-H]-.
4.2.9. 4-((3-(4-chloro-3-(trifluoromethyl)phenyl)ureido)methyl)benzenesulfonamide (16): White
solid, yield 20%; mp 158-159 °C; δH (400 MHz, DMSO-d6) 4.40 (2H, d, J 5.6), 7.00 (1H, t, J 5.6,
exchange with D2O, NH), 7.34 (2H, s, exchange with D2O, SO2NH2), 7.50 (2H, d, J 8.4), 7.58 (1H,
d, J 8.8), 7.64 (1H, dd, J 2.4, 8.8), 7.81 (2H, d, J 8.4), 8.11 (1H, d, J 2.4), 9.25 (1H, s, exchange
with D2O, NH); δC (100 MHz, DMSO-d6) 43.4, 117.1 (q, 3JC-F 6), 122.4 (q, 4JC-F 2), 123.3, 123.8 (q,
2
1JC-F 271), 126.7, 127.6 (q, JC-F 30), 128.2, 132.7, 141.0, 143.5, 145.2, 155.9; δF (376 MHz,
DMSO-d6) -61.4; m/z (ESI positive) 452.0 [M+HCOO]-.
4.2.10. 4-(2-(3-(4-chloro-3-(trifluoromethyl)phenyl)ureido)ethyl)benzenesulfonamide (17): White
solid, yield 78%; silica gel TLC Rf 0.67 (MeOH/DCM 20% v/v); mp 229-230 °C; δH (400 MHz,
DMSO-d6) 2.88 (2H, t, J 6.8), 3.41 (2H, q, J 6.8), 6.39 (1H, t, J 6.8, exchange with D2O, NH), 7.33
(2H, s, exchange with D2O, SO2NH2), 7.46 (2H, d, J 8.2), 7.58 (2H, m), 7.79 (2H, d, J 8.2), 8.10
(1H, m), 9.03 (1H, s, exchange with D2O, NH); δC (100 MHz, DMSO-d6) 36,3, 41.3, 117.1 (q, 3JC-F
4
1
2
6), 122.4 (q, JC-F 2), 123.2, 123.8 (q, JC-F 271), 126.7, 127.6 (q, JC-F 30), 130.1, 132.7, 141.0,
143.1, 144.7, 155.8; δF (376 MHz, DMSO-d6) -61.4; m/z (ESI positive) 422.0 [M+H]+.

4.2.11. 1-(4-chloro-3-(trifluoromethyl)phenyl)-3-(4-hydroxyphenyl)urea (18): White solid, yield
55%; silica gel TLC Rf 0.33 (MeOH/DCM 10% v/v); δH (400 MHz, DMSO-d6) 6.73 (2H, d, J 8.8),
7.26 (2H, d, J 8.8), 7.64 (2H, m), 8.13 (1H, d, J 2.4), 8.51 (1H, exchange with D2O, NH), 9.05 (1H,
exchange with D2O, NH), 9.15 (1H, s, exchange with D2O, OH); δC (100 MHz, DMSO-d6) 116.2,
3
4
1
2
117.5 (q, JC-F 6), 122.0, 122.9 (q, JC-F 2), 123.7, 123.8 (q, JC-F 271), 127.8 (q, JC-F 30), 131.5,
132.8, 140.6, 153.6, 154.0; δF (376 MHz, DMSO-d6) -61.5.
4.2.12. 1-(4-chloro-3-(trifluoromethyl)phenyl)-3-(3-hydroxyphenyl)urea (19): White solid, yield
60%; silica gel TLC Rf 0.47 (MeOH/DCM 10% v/v); δH (400 MHz, DMSO-d6) 6.44 (1H, m), 6.84
(1H, m), 7.08 (2H, m), 7.64 (1H, d, J 1.2), 8.15 (1H, t, J 1.2), 8.75 (1H, exchange with D2O, NH),
9.10 (1H, exchange with D2O, NH), 9.37 (1H, s, exchange with D2O, OH); δC (100 MHz, DMSO-
3
4
1
d6) 106.6, 110.2, 110.4, 117.6 (q, JC-F 6), 123.1 (q, JC-F 2), 123.7 (q, JC-F 271), 123.9, 127.5 (q,
2JC-F 30), 130.4, 132.9, 140.3, 141.1, 153.2, 158.7; δF (376 MHz, DMSO-d6) -61.5.
4.2.13. 4-(3-(4-chloro-3-(trifluoromethyl)phenyl)ureido)benzoic acid (20): White solid, yield 77%;
silica gel TLC Rf 0.45 (EtOAc/n-hexane 80% v/v); δH (400 MHz, DMSO-d6) 7.62 (2H, d, J 8.8),
7.68 (2H, m), 7.91 (2H, d, J 8.8), 8.15 (1H, m), 9.26 (1H, exchange with D2O, NH), 9.30 (1H, s,
exchange with D2O, NH), 12.68 (1H, s, exchange with D2O, COOH); δC (100 MHz, DMSO-d6)
3
4
1
2
117.9 (q, JC-F 6), 118.6, 123.6 (q, JC-F 2), 123.7 (q, JC-F 271), 124.2, 125.1, 127.7 (q, JC-F 30),
131.5, 133.0, 140.0, 144.5, 153.1, 167.9; δF (376 MHz, DMSO-d6) -61.5.
4.2.14. 1-(4-chloro-3-(trifluoromethyl)phenyl)-3-(4-methyl-2-oxo-2H-chromen-7-yl)urea (21):
White solid; yield 70%; mp>300°C; δH (400 MHz, DMSO-d6) 2.44 (3H, s), 6.27 (1H, s), 7.40 (1H,
d, J 8.6), 7.64-7.74 (4H, m), 8.13 (1H, m), 9.39 (1H, s, exchange with D2O, NH), 9.42 (1H, s,
exchange with D2O, NH); δC (100 MHz, DMSO-d6) 18.8, 105.6, 112.6, 115.0, 115.4, 117.8 (q, 3JC-F
4
1
2
6), 123.5 (q, JC-F 2), 123.6 (q, JC-F 270), 124.2, 126.7, 127.7 (q, JC-F 30), 132.9, 139.8, 143.8,
153.0, 154.0, 154.8, 160.9; δF (376 MHz, DMSO-d6) -61.5; m/z (ESI negative) 395.0 [M-H]-.
4.2.15. 4-(3-(4-chloro-3-(trifluoromethyl)phenyl)ureido)phenyl sulfamate (23): White solid, yield
77%; silica gel TLC Rf 0.70 (MeOH/DCM 10% v/v); mp 175-176°C (dec); δH (400 MHz, DMSO-
d6) 7.24 (2H, d, J 9.0), 7.55 (2H, d, J 9.0), 7.66 (2H, m), 7.95 (2H, s, exchange with D2O, SO2NH2),
8.14 (1H, d, J 2.2), 9.00 (1H, exchange with D2O, NH), 9.21 (1H, exchange with D2O, NH);δC (100
3
4
1
MHz, DMSO-d6) 117.7 (q, JC-F 6), 120.6, 123.3 (q, JC-F 3), 123.6, 123.8 (q, JC-F 271), 124.0,
127.7 (q, 2JC-F 30), 132.9, 138.6, 140.2, 145.9, 153.4; m/z (ESI negative) 408.0 [M-H]-.
4.2.16. 3-(3-(4-chloro-3-(trifluoromethyl)phenyl)ureido)phenyl sulfamate (24): White solid, yield
65%; silica gel TLC Rf 0.55 (MeOH/DCM 10% v/v); mp 190-191°C (dec); δH (400 MHz, DMSO-

d6) 6.95 (1H, m), 7.39 (2H, m), 7.57 (1H, m), 7.67 (2H, m), 8.03 (2H, exchange with D2O,
SO2NH2), 8.15 (1H, d, J 2.0), 9.11 (1H, exchange with D2O, NH), 9.21 (1H, s, exchange with D2O,
3
4
NH);δC (100 MHz, DMSO-d6) 113.2, 116.6, 117.4, 117.8 (q, JC-F 6), 123.4 (q, JC-F 2), 123.7 (q,
2
1JC-F 271), 124.1, 127.7 (q, JC-F 30), 130.7, 132.9, 140.1, 141.4, 151.4, 153.2; δF (376 MHz,
DMSO-d6) -61.5; m/z (ESI negative) 408.0 [M-H]-.
4.2.17. 2,5-dioxopyrrolidin-1-yl 4-(3-(4-chloro-3-(trifluoromethyl)phenyl)ureido)benzoate (25):
White solid; yield 92%; δH (400 MHz, DMSO-d6) 2.92 (4H, s), 7.70 (2H, m), 7.77 (2H, d, J 9.0),
7.99 (1H, s), 8.07 ( 2H, d, J 9.0), 8.17 (1H, d, J 2.2), 9.45 (1H, exchange with D2O, NH), 9.57 (1H,
3
s, exchange with D2O, NH); δC (100 MHz, DMSO-d6) 26.4, 118.1, 118.1 (q, JC-F 6), 119.0, 123.8
(q, 4JC-F 2), 123.7 (q, 1JC-F 271), 124.4, 127.7 (q, 2JC-F 30), 132.4, 132.9, 139.7, 146.8, 152.9, 162.2,
171.3; δF (376 MHz, DMSO-d6) -61.5.
4.2.18. 4-(3-(4-chloro-3-(trifluoromethyl)phenyl)ureido)-N-(4-sulfamoylbenzyl)benzamide (26):
White solid; yield 60%; silica gel TLC Rf 0.26 (MeOH/DCM 10% v/v); mp195-196 °C; δH (400
MHz, DMSO-d6) 4.56 (2H, d, J 6.0), 7.35 (2H, s, exchange with D2O, SO2NH2), 7.52 (2H, d, J 8.3),
7.59 (2H, d, J 8.8), 7.68 (2H, m), 7.81 (2H, d, J 8.3), 7.90 (2H, d, J 8.8), 8.16 (1H, m), 9.04 (1H, t, J
6.0, exchange with D2O, NH), 9.18 (1H, s, exchange with D2O, NH), 9.30 (1H, s, exchange with
D2O, NH); δC (100 MHz, DMSO-d6) 43.2, 117.8 (q, 3JC-F 6), 118.5, 123.5 (q, 4JC-F 2), 123.7 (q, 1JC-F
270), 124.2, 126.6, 127.7 (q, 2JC-F 30), 128.4, 128.6, 129.2, 133.0, 140.1, 143.1, 143.5, 144.9, 153.2,
166.8; δF (376 MHz, DMSO-d6) -61.5; m/z (ESI negative) 525.1 [M-H]-.
4.2.19. 4-(3-(4-chloro-3-(trifluoromethyl)phenyl)ureido)-N-(4-sulfamoylphenethyl)benzamide (27):
White solid; yield 62%; silica gel TLC Rf 0.22 (EtOAc/n-hexane 80% v/v); mp 269-270 °C; δH (400
MHz, DMSO-d6) 2.96 (2H, t, J 7.2), 3.54 (2H, q, J 7.2), 7.32 (2H, s, exchange with D2O, SO2NH2),
7.47 (2H, d, J 8.4), 7.57 (2H, d, J 8.8), 7.67 (2H, m), 7.80 (4H, m), 8.15 (1H, m), 8.49 (1H, t, J 7.2,
exchange with D2O, NH), 9.14 (1H, s, exchange with D2O, NH), 9.29 (1H, s, exchange with D2O,
NH);δC (100 MHz, DMSO-d6) 35.8, 41.3, 117.8 (q, 3JC-F 6), 118.5, 123.5 (q, 4JC-F 2), 123.7 (q, 1JC-F
270), 124.1, 126.6, 127.7 (q, 2JC-F 30), 129.0, 129.0, 130.1, 133.0, 140.1, 142.8, 143.0, 144.8, 153.2,
166.7; δF (376 MHz, DMSO-d6) -61.5; m/z (ESI negative) 539.1 [M-H]-.
4.2.20.
4-(3-(4-chloro-3-(trifluoromethyl)phenyl)ureido)-N-(3-(4-
sulfamoylphenoxy)propyl)benzamide (29): White solid, yield 64%; silica gel TLC Rf 0.25
(MeOH/DCM 10% v/v); mp 231-232 °C; δH (400 MHz, DMSO-d6) 2.03 (2H, pent, J 6.5), 3.45 (2H,
q, J 6.5), 4.15 (2H, t, J 6.5), 7.12 (2H, d, J 8.9), 7.23 (2H, s, exchange with D2O, SO2NH2), 7.57
(2H, d, J 8.8), 7.67 (2H, m), 7.78 (2H, d, J 8.9), 7.84 (2H, d, J 8.8), 8.15 (1H, m), 8.46 (1H, t, J 6.5,
exchange with D2O, NH), 9.14 (1H, s, exchange with D2O, NH), 9.27 (1H, s, exchange with D2O,

3
4
NH); δC (100 MHz, DMSO-d6) 29.8, 37.1, 66.8, 115.4, 117.8 (q, JC-F 6), 118.4, 123.5 (q, JC-F 2),
1
2
123.7 (q, JC-F 270), 124.1, 127.7 (q, JC-F 30), 127.8, 128.6, 129.0, 129.1, 132.9, 137.0, 140.1,
142.8, 153.2, 162.0, 166.8; δF (376 MHz, DMSO-d6) -61.5; m/z (ESI negative) 569.1 [M-H]-.
4.2.21.
4-(3-(4-chloro-3-(trifluoromethyl)phenyl)ureido)-N-(6-(4-
sulfamoylphenoxy)hexyl)benzamide (31): White solid; yield 55%; mp 237-238°C; δH (400 MHz,
DMSO-d6) 1.44 (4H, m), 1.58 (2H, pent, J 7.0), 1.77 (2H, pent, J 7),3.28 (2H, q, J 7.0), 4.08 (2H, t,
J 7.0), 7.10 (2H, d, J 8.8), 7.23 (2H, s, exchange with D2O, SO2NH2), 7.57 (2H, d, J 8.6), 7.66 (2H,
m), 7.78 (2H, d, J 8.8), 7.83 (2H, d, J 8.6), 8.16 (1H, s), 8.36 (1H, t, J 7.0, exchange with D2O,
NH), 9.15 (1H, s, exchange with D2O, NH), 9.30 (1H, s, exchange with D2O, NH); δC (100 MHz,
3
4
DMSO-d6) 26.2, 27.2, 29.4, 30.1, 68.8, 115.3,117.8 (q, JC-F 6), 118.4, 123.5 (q, JC-F 2), 123.7 (q,
2
1JC-F 270), 124.1, 127.7 (q, JC-F 30), 127.8, 128.6, 129.0, 129.2, 133.0, 136.9, 140.1, 142.7, 153.2,
162.0, 166.5; δF (376 MHz, DMSO-d6) -61.5; m/z (ESI negative) 611.2 [M-H]-.
4.2.22. 4-(3-(4-chloro-3-(trifluoromethyl)phenyl)ureido)-N-(2-((2-oxo-2H-chromen-7-yl)oxy)ethyl)
benzamide (33): White solid; yield 35%; mp 217-218 °C; δH (400 MHz, DMSO-d6) 3.67 (2H, q, J
5.6), 4.26 (2H, t, J 5.6), 6.32 (1H, d, J 9.5), 7.00 (1H, dd, J 2.4, 8.6), 7.08 (1H, d, J 2.4), 7.57 (2H,
d, J 8.8), 7.66 (3H, m), 7.86 (2H, d, J 8.8), 8.02 (1H, d, J 9.5), 8.15 (1H, m), 8.65 (1H, t, J 5.6,
exchange with D2O, NH), 9.21 (1H, s, exchange with D2O, NH), 9.36 (1H, s, exchange with D2O,
3
NH); δC (100 MHz, DMSO-d6) 39.5, 67.8, 102.2, 113.4, 113.5, 113.7, 117.7 (q, JC-F 6), 118.4,
123.5 (q, 4JC-F 2), 123.8 (q, 1JC-F 270), 124.1, 127.6 (q, 2JC-F 30), 127.8, 128.6, 128.6, 129.2, 130.5,
133.0, 140.1, 143.1, 145.3, 153.2, 156.4, 161.3, 162.6, 167.0; δF (376 MHz, DMSO-d6) -61.5; m/z
(ESI negative) 544.1 [M-H]-.
4.2.23. 4-(3-(4-chloro-3-(trifluoromethyl)phenyl)ureido)-N-(2-((2-oxo-2H-chromen-6-yl)oxy)ethyl)
benzamide (35): White solid, yield 42%; mp 219-220 °C; δH (400 MHz, DMSO-d6) 3.68 (2H, q, J
5.6), 4.20 (2H, t, J 5.6), 6.53 (1H, d, J 9.6), 7.27 (1H, dd, J 2.4, 8.8), 7.38 (2H, m), 7.58 (2H, d, J
8.8), 7.67 (2H, m), 7.86 (2H, d, J 8.8), 8.04 (1H, d, J 9.6), 8.15 (1H, m), 8.66 (1H, t, J 5.6,exchange
with D2O, NH), 9.29 (1H, s, exchange with D2O, NH), 9.45 (1H, s, exchange with D2O, NH); δC
(100 MHz, DMSO-d6) 39.7, 67.7, 112.5, 117.5, 117.7 (q, 3JC-F 6), 118.3, 118.4, 120.2, 120.8, 123.4
(q, 4JC-F 2), 124.0, 124.2 (q, 1JC-F 270), 127.6 (q, 2JC-F 30), 128.6, 129.2, 133.0, 140.1, 143.0, 145.0,
148.8,153.2, 155.7, 161.1, 167.0 ; δF (376 MHz, DMSO-d6) -61.5; m/z (ESI negative) 544.1 [M-H]-.
4.3. CA inhibition
An Applied Photophysics stopped-flow instrument has been used for assaying the CA catalyzed
CO2 hydration activity [57]. Phenol red (at a concentration of 0.2 mM) has been used as indicator,

working at the absorbance maximum of 557 nm, with 20 mM Hepes (pH 7.5) as buffer, and 20 mM
Na2SO4 (for maintaining constant the ionic strength), following the initial rates of the CA-catalyzed
CO2 hydration reaction for a period of 10-100 s. The CO2 concentrations ranged from 1.7 to 17 mM
for the determination of the kinetic parameters and inhibition constants. For each inhibitor at least
six traces of the initial 5-10% of the reaction have been used for determining the initial velocity.
The uncatalyzed rates were determined in the same manner and subtracted from the total observed
rates. Stock solutions of inhibitor (0.1 mM) were prepared in distilled-deionized water and dilutions
up to 0.01 nM were done thereafter with the assay buffer. Inhibitor and enzyme solutions were
preincubated together at room temperature (15 min) prior to assay, in order to allow for the
formation of the E-I complex. Data from Table 1 were obtained after 15 minutes incubation of
enzyme and inhibitor, as for the sulfonamides reported earlier [9,45,59,60]. The inhibition constants
were obtained by non-linear least-squares methods using PRISM 3 and the Cheng-Prusoff equation,
as reported earlier [9,45,59,60] and represent the mean from at least three different determinations.
All CA isoforms were recombinant ones obtained in-house as reported earlier [9,45,59,60].
4.4. Crystallization and X-ray data collection
Crystals of hCA I complexed with compounds 17 and 23 were obtained using the sitting drop vapor
diffusion method. 2 µl of 10 mg/ml solution of hCA I in Tris-HCl 20 mM pH 9.0 were mixed with 2
µl of a solution of 28-31% PEG4000, 0.2 M Sodium acetate, 0.1 M Tris pH 8.5-9.0 and were
equilibrated against the same solution at 296 K. Crystals of the protein grew in fifteen days.
Afterwards hCAI crystals were soaked in 5mM inhibitor solutions for 3 days.
The crystals were flash-frozen at 100 K using a solution obtained by adding 15% (v/v) glycerol to
the mother liquor solution as cryoprotectant. Data on crystals of the complexes were collected using
synchrotron radiation at the ID30A-1 beamline at ESRF (Grenoble, France) with a wavelength of
0.966 Å and a PILATUS3 2M Dectris CCD detector. Data were integrated and scaled using the
program XDS [61]. Data processing statistics are shown in Supporting Information Table S1.
4.4.1. Structure determination
The crystal structure of hCA I (PDB accession code 1JV0) without solvent molecules and other
heteroatoms was used to obtain initial phases of the structures using Refmac5 [62]. 5% of the
unique reflections were selected randomly and excluded from the refinement data set for the
purpose of Rfree calculations. The initial |Fo - Fc| difference electron density map unambiguously
showed the inhibitor molecules. Atomic models for inhibitors were calculated and energy

minimized using the program JLigand 1.0.40 [63]. Refinements proceeded using normal protocols
of positional, isotropic atomic displacement parameters alternating with manual building of the
models using COOT [64]. Solvent molecules were introduced automatically using the program ARP
[65]. The quality of the final models were assessed with COOT and RAMPAGE [66]. Crystal
parameters and refinement data are summarized in Table S1 in the Supporting information. Atomic
coordinates were deposited in the Protein Data Bank (PDB accession code: 6I0L and 6I0J).
Graphical representations were generated with Chimera [67].
4.5. Cell Lines and Culture
The breast cancer cell lines, HBL100, MCF7 and MDA.MB.231 were cultured in Dulbecco’s
modified Eagle medium (DMEM) supplemented with 10% foetal bovine serum, 100ꢀU/ml penicillin
and 100ꢀµg/ml streptomycin in 21% O2. Hypoxic cells were grown at 0.5% O2 in a Don Whitley
H35 Hypoxystation. All cell lines used were tested and authenticated using STR profiling by Public
Health England, Porton Down, Salisbury, UK.
4.5.1. Sulforhodamine B assay
Cells (1 x 103/well) were seeded into 96-well flat-bottomed plates and incubated for 48ꢀh before the
sulfamates were added using concentrations between 0 – 300 µM. After 5ꢀdays treatment, plates
were fixed by the addition of 50ꢀµl 25% trichloroacetic acid solution per well for 1ꢀh at 4ꢀ°C. Plates
were washed with H2O and air dried before the addition of 50ꢀµl 0.4% sulforhodamine B solution in
1% acetic acid for 30ꢀmin. Plates were washed in 1% acetic acid and air dried before the addition of
150ꢀµl 10ꢀmM Tris solution (pHꢀ10.5). After 30ꢀmin, the absorbance of each well was measured at
540ꢀnm on a BP900 biohit plate reader.
Disclosure Statement
The authors declare no competing financial interest.
References
[1] C.T. Supuran, Carbonic anhydrases, Bioorg. Med. Chem. 21 (2013) 1377–1378.
doi:10.1016/j.bmc.2013.02.026.
[2] C.T. Supuran, Structure and function of carbonic anhydrases, Biochem. J. 473 (2016) 2023–
2032. doi:10.1042/BCJ20160115.

[3] M. Sentürk, I. Gülçin, A. Daştan, O.I. Küfrevioğlu, C.T. Supuran, Carbonic anhydrase
inhibitors. Inhibition of human erythrocyte isozymes I and II with a series of antioxidant
phenols, Bioorg. Med. Chem. 17 (2009) 3207–3211. doi:10.1016/j.bmc.2009.01.067.
[4] T. Abdülkadir Coban, Ş. Beydemir, İ. Gücin, D. Ekinci, A. Innocenti, D. Vullo, C.T. Supuran,
Sildenafil is a strong activator of mammalian carbonic anhydrase isoforms I–XIV, Bioorganic
& Medicinal Chemistry. 17 (2009) 5791–5795. doi:10.1016/j.bmc.2009.07.019.
[5] A. Innocenti, S. Beyza Öztürk Sarıkaya, İ. Gülçin, C.T. Supuran, Carbonic anhydrase inhibitors.
Inhibition of mammalian isoforms I-XIV with a series of natural product polyphenols and
phenolic acids, Bioorg. Med. Chem. 18 (2010) 2159–2164. doi:10.1016/j.bmc.2010.01.076.
[6] S.B. Oztürk Sarikaya, F. Topal, M. Sentürk, I. Gülçin, C.T. Supuran, In vitro inhibition of α-
carbonic anhydrase isozymes by some phenolic compounds, Bioorg. Med. Chem. Lett. 21
(2011) 4259–4262. doi:10.1016/j.bmcl.2011.05.071.
[7] C.T. Supuran, Carbonic anhydrases: novel therapeutic applications for inhibitors and activators,
Nat Rev Drug Discov. 7 (2008) 168–181. doi:10.1038/nrd2467.
[8] D. Neri, C.T. Supuran, Interfering with pH regulation in tumours as a therapeutic strategy, Nat
Rev Drug Discov. 10 (2011) 767–777. doi:10.1038/nrd3554.
[9] M. Abdoli, A. Angeli, M. Bozdag, F. Carta, A. Kakanejadifard, H. Saeidian, C.T. Supuran,
Synthesis and carbonic anhydrase I, II, VII, and IX inhibition studies with a series of
benzo[d]thiazole-5- and 6-sulfonamides, J Enzyme Inhib Med Chem. 32 (2017) 1071–1078.
doi:10.1080/14756366.2017.1356295.
[10] M. Bozdag, F. Carta, D. Vullo, A. Akdemir, S. Isik, C. Lanzi, A. Scozzafava, E. Masini,
C.T. Supuran, Synthesis of a new series of dithiocarbamates with effective human carbonic
anhydrase inhibitory activity and antiglaucoma action, Bioorg. Med. Chem. 23 (2015) 2368–
2376. doi:10.1016/j.bmc.2015.03.068.
[11] F. Carta, M. Aggarwal, A. Maresca, A. Scozzafava, R. McKenna, C.T. Supuran,
Dithiocarbamates: a new class of carbonic anhydrase inhibitors. Crystallographic and kinetic
investigations, Chem. Commun. (Camb.). 48 (2012) 1868–1870. doi:10.1039/c2cc16395k.
[12] F. Carta, M. Aggarwal, A. Maresca, A. Scozzafava, R. McKenna, E. Masini, C.T. Supuran,
Dithiocarbamates strongly inhibit carbonic anhydrases and show antiglaucoma action in vivo, J.
Med. Chem. 55 (2012) 1721–1730. doi:10.1021/jm300031j.
[13] S. Avram, A.L. Milac, F. Carta, C.T. Supuran, More effective dithiocarbamate derivatives
inhibiting carbonic anhydrases, generated by QSAR and computational design, J Enzyme Inhib
Med Chem. 28 (2013) 350–359. doi:10.3109/14756366.2012.727410.

[14] F. Carta, A. Akdemir, A. Scozzafava, E. Masini, C.T. Supuran, Xanthates and
trithiocarbonates strongly inhibit carbonic anhydrases and show antiglaucoma effects in vivo, J.
Med. Chem. 56 (2013) 4691–4700. doi:10.1021/jm400414j.
[15] D. Vullo, M. Durante, F.S. Di Leva, S. Cosconati, E. Masini, A. Scozzafava, E. Novellino,
C.T. Supuran, F. Carta, Monothiocarbamates Strongly Inhibit Carbonic Anhydrases in Vitro
and Possess Intraocular Pressure Lowering Activity in an Animal Model of Glaucoma, J. Med.
Chem. 59 (2016) 5857–5867. doi:10.1021/acs.jmedchem.6b00462.
[16] A. Innocenti, D. Vullo, A. Scozzafava, C.T. Supuran, Carbonic anhydrase inhibitors:
interactions of phenols with the 12 catalytically active mammalian isoforms (CA I-XIV),
Bioorg. Med. Chem. Lett. 18 (2008) 1583–1587. doi:10.1016/j.bmcl.2008.01.077.
[17] R.A. Davis, A. Hofmann, A. Osman, R.A. Hall, F.A. Mühlschlegel, D. Vullo, A. Innocenti,
C.T. Supuran, S.-A. Poulsen, Natural product-based phenols as novel probes for mycobacterial
and fungal carbonic anhydrases, J. Med. Chem. 54 (2011) 1682–1692. doi:10.1021/jm1013242.
[18] A. Innocenti, I. Gülçin, A. Scozzafava, C.T. Supuran, Carbonic anhydrase inhibitors.
Antioxidant polyphenols effectively inhibit mammalian isoforms I-XV, Bioorg. Med. Chem.
Lett. 20 (2010) 5050–5053. doi:10.1016/j.bmcl.2010.07.038.
[19] A. Innocenti, D. Vullo, A. Scozzafava, C.T. Supuran, Carbonic anhydrase inhibitors:
inhibition of mammalian isoforms I-XIV with a series of substituted phenols including
paracetamol and salicylic acid, Bioorg. Med. Chem. 16 (2008) 7424–7428.
doi:10.1016/j.bmc.2008.06.013.
[20] V. Alterio, A. Di Fiore, K. D’Ambrosio, C.T. Supuran, G. De Simone, Multiple binding
modes of inhibitors to carbonic anhydrases: how to design specific drugs targeting 15 different
isoforms?, Chem. Rev. 112 (2012) 4421–4468. doi:10.1021/cr200176r.
[21] E. Langella, K. D’Ambrosio, M. D’Ascenzio, S. Carradori, S.M. Monti, C.T. Supuran, G.
De Simone, A Combined Crystallographic and Theoretical Study Explains the Capability of
Carboxylic Acids to Adopt Multiple Binding Modes in the Active Site of Carbonic Anhydrases,
Chemistry. 22 (2016) 97–100. doi:10.1002/chem.201503748.
[22] A. Scozzafava, C.T. Supuran, F. Carta, Polyamines and α-Carbonic Anhydrases, Molecules.
21 (2016). doi:10.3390/molecules21121726.
[23] F. Carta, C. Temperini, A. Innocenti, A. Scozzafava, K. Kaila, C.T. Supuran, Polyamines
inhibit carbonic anhydrases by anchoring to the zinc-coordinated water molecule, J. Med.
Chem. 53 (2010) 5511–5522. doi:10.1021/jm1003667.

[24] F. Carta, A. Maresca, A. Scozzafava, C.T. Supuran, Novel coumarins and 2-thioxo-
coumarins as inhibitors of the tumor-associated carbonic anhydrases IX and XII, Bioorg. Med.
Chem. 20 (2012) 2266–2273. doi:10.1016/j.bmc.2012.02.014.
[25] M. Ferraroni, F. Carta, A. Scozzafava, C.T. Supuran, Thioxocoumarins Show an Alternative
Carbonic Anhydrase Inhibition Mechanism Compared to Coumarins, J. Med. Chem. 59 (2016)
462–473. doi:10.1021/acs.jmedchem.5b01720.
[26] M. Tanc, F. Carta, M. Bozdag, A. Scozzafava, C.T. Supuran, 7-Substituted-sulfocoumarins
are isoform-selective, potent carbonic anhydrase II inhibitors, Bioorg. Med. Chem. 21 (2013)
4502–4510. doi:10.1016/j.bmc.2013.05.032.
[27] K. Tars, D. Vullo, A. Kazaks, J. Leitans, A. Lends, A. Grandane, R. Zalubovskis, A.
Scozzafava, C.T. Supuran, Sulfocoumarins (1,2-benzoxathiine-2,2-dioxides): a class of potent
and isoform-selective inhibitors of tumor-associated carbonic anhydrases, J. Med. Chem. 56
(2013) 293–300. doi:10.1021/jm301625s.
[28] A. Grandane, M. Tanc, L. Di Cesare Mannelli, F. Carta, C. Ghelardini, R. Žalubovskis, C.T.
Supuran, 6-Substituted sulfocoumarins are selective carbonic anhdydrase IX and XII inhibitors
with significant cytotoxicity against colorectal cancer cells, J. Med. Chem. 58 (2015) 3975–
3983. doi:10.1021/acs.jmedchem.5b00523.
[29] A. Grandane, M. Tanc, R. Žalubovskis, C.T. Supuran, Synthesis of 6-aryl-substituted
sulfocoumarins and investigation of their carbonic anhydrase inhibitory action, Bioorg. Med.
Chem. 23 (2015) 1430–1436. doi:10.1016/j.bmc.2015.02.023.
[30] A. Grandane, M. Tanc, R. Zalubovskis, C.T. Supuran, 6-Triazolyl-substituted
sulfocoumarins are potent, selective inhibitors of the tumor-associated carbonic anhydrases IX
and XII, Bioorg. Med. Chem. Lett. 24 (2014) 1256–1260. doi:10.1016/j.bmcl.2014.01.076.
[31] A. Grandane, M. Tanc, R. Zalubovskis, C.T. Supuran, Synthesis of 6-tetrazolyl-substituted
sulfocoumarins acting as highly potent and selective inhibitors of the tumor-associated carbonic
anhydrase isoforms IX and XII, Bioorg. Med. Chem. 22 (2014) 1522–1528.
doi:10.1016/j.bmc.2014.01.043.
[32] A. Nocentini, F. Carta, M. Tanc, S. Selleri, C.T. Supuran, C. Bazzicalupi, P. Gratteri,
Deciphering the Mechanism of Human Carbonic Anhydrases Inhibition with Sulfocoumarins:
Computational and Experimental Studies, Chemistry. 24 (2018) 7840–7844.
doi:10.1002/chem.201800941.
[33] A. Maresca, C. Temperini, H. Vu, N.B. Pham, S.-A. Poulsen, A. Scozzafava, R.J. Quinn,
C.T. Supuran, Non-zinc mediated inhibition of carbonic anhydrases: coumarins are a new class
of suicide inhibitors, J. Am. Chem. Soc. 131 (2009) 3057–3062. doi:10.1021/ja809683v.

[34] A. Maresca, C. Temperini, L. Pochet, B. Masereel, A. Scozzafava, C.T. Supuran,
Deciphering the mechanism of carbonic anhydrase inhibition with coumarins and
thiocoumarins, J. Med. Chem. 53 (2010) 335–344. doi:10.1021/jm901287j.
[35] M. Bozdag, A.M. Alafeefy, A.M. Altamimi, D. Vullo, F. Carta, C.T. Supuran, Coumarins
and other fused bicyclic heterocycles with selective tumor-associated carbonic anhydrase
isoforms inhibitory activity, Bioorganic & Medicinal Chemistry. 25 (2017) 677–683.
doi:10.1016/j.bmc.2016.11.039.
[36] S. Bua, L. Di Cesare Mannelli, D. Vullo, C. Ghelardini, G. Bartolucci, A. Scozzafava, C.T.
Supuran, F. Carta, Design and Synthesis of Novel Nonsteroidal Anti-Inflammatory Drugs and
Carbonic Anhydrase Inhibitors Hybrids (NSAIDs-CAIs) for the Treatment of Rheumatoid
Arthritis, J. Med. Chem. 60 (2017) 1159–1170. doi:10.1021/acs.jmedchem.6b01607.
[37] K. D’Ambrosio, S. Carradori, S.M. Monti, M. Buonanno, D. Secci, D. Vullo, C.T. Supuran,
G. De Simone, Out of the active site binding pocket for carbonic anhydrase inhibitors, Chem.
Commun. (Camb.). 51 (2015) 302–305. doi:10.1039/c4cc07320g.
[38] F. Pacchiano, F. Carta, P.C. McDonald, Y. Lou, D. Vullo, A. Scozzafava, S. Dedhar, C.T.
Supuran, Ureido-substituted benzenesulfonamides potently inhibit carbonic anhydrase IX and
show antimetastatic activity in a model of breast cancer metastasis, J. Med. Chem. 54 (2011)
1896–1902. doi:10.1021/jm101541x.
[39] Y. Lou, P.C. McDonald, A. Oloumi, S. Chia, C. Ostlund, A. Ahmadi, A. Kyle, U. Auf dem
Keller, S. Leung, D. Huntsman, B. Clarke, B.W. Sutherland, D. Waterhouse, M. Bally, C.
Roskelley, C.M. Overall, A. Minchinton, F. Pacchiano, F. Carta, A. Scozzafava, N. Touisni, J.-
Y. Winum, C.T. Supuran, S. Dedhar, Targeting tumor hypoxia: suppression of breast tumor
growth and metastasis by novel carbonic anhydrase IX inhibitors, Cancer Res. 71 (2011) 3364–
3376. doi:10.1158/0008-5472.CAN-10-4261.
[40] F. Pacchiano, M. Aggarwal, B.S. Avvaru, A.H. Robbins, A. Scozzafava, R. McKenna, C.T.
Supuran, Selective hydrophobic pocket binding observed within the carbonic anhydrase II
active site accommodate different 4-substituted-ureido-benzenesulfonamides and correlate to
inhibitor potency, Chem. Commun. (Camb.). 46 (2010) 8371–8373. doi:10.1039/c0cc02707c.
[41] A Study of SLC-0111 and Gemcitabine for Metastatic Pancreatic Ductal Cancer in Subjects
Positive
for
CAIX
-
Full
Text
View
-
ClinicalTrials.gov,
(n.d.).
https://clinicaltrials.gov/ct2/show/NCT03450018 (accessed January 24, 2019).
[42] P.C. McDonald, J.-Y. Winum, C.T. Supuran, S. Dedhar, Recent Developments in Targeting
Carbonic Anhydrase IX for Cancer Therapeutics, Oncotarget. 3 (2012) 84–97.
doi:10.18632/oncotarget.422.

[43] M. Hilvo, Expression of Carbonic Anhydrase IX in Mouse Tissues, Journal of
Histochemistry and Cytochemistry. 52 (2004) 1313–1322. doi:10.1369/jhc.3A6225.2004.
[44] C.T. Supuran, Carbonic Anhydrase Inhibition and the Management of Hypoxic Tumors,
Metabolites. 7 (2017). doi:10.3390/metabo7030048.
[45] J.-Y. Winum, F. Carta, C. Ward, P. Mullen, D. Harrison, S.P. Langdon, A. Cecchi, A.
Scozzafava, I. Kunkler, C.T. Supuran, Ureido-substituted sulfamates show potent carbonic
anhydrase IX inhibitory and antiproliferative activities against breast cancer cell lines, Bioorg.
Med. Chem. Lett. 22 (2012) 4681–4685. doi:10.1016/j.bmcl.2012.05.083.
[46] C. Ward, J. Meehan, P. Mullen, C. Supuran, J.M. Dixon, J.S. Thomas, J.-Y. Winum, P.
Lambin, L. Dubois, N.-K. Pavathaneni, E.J. Jarman, L. Renshaw, I.H. Um, C. Kay, D.J.
Harrison, I.H. Kunkler, S.P. Langdon, Evaluation of carbonic anhydrase IX as a therapeutic
target for inhibition of breast cancer invasion and metastasis using a series of in vitro breast
cancer models, Oncotarget. 6 (2015) 24856–24870. doi:10.18632/oncotarget.4498.
[47] M. Bozdag, F. Carta, M. Ceruso, M. Ferraroni, P.C. McDonald, S. Dedhar, C.T. Supuran,
Discovery of 4-Hydroxy-3-(3-(phenylureido)benzenesulfonamides as SLC-0111 Analogues for
the Treatment of Hypoxic Tumors Overexpressing Carbonic Anhydrase IX, J. Med. Chem. 61
(2018) 6328–6338. doi:10.1021/acs.jmedchem.8b00770.
[48] M. Bozdag, F. Carta, A. Angeli, S.M. Osman, F.A.S. Alasmary, Z. AlOthman, C.T.
Supuran, Synthesis of N’-phenyl-N-hydroxyureas and investigation of their inhibitory activities
on
human
carbonic
anhydrases,
Bioorg.
Chem.
78
(2018)
1–6.
doi:10.1016/j.bioorg.2018.02.029.
[49] M. Bozdag, G. Poli, A. Angeli, E. Lucarini, T. Tuccinardi, L. Di Cesare Mannelli, S. Selleri,
C. Ghelardini, J.-Y. Winum, F. Carta, C.T. Supuran, N-aryl-N’-ureido-O-sulfamates: Potent and
selective inhibitors of the human Carbonic Anhydrase VII isoform with neuropathic pain
relieving
properties,
Bioorganic
Chemistry.
89
(2019)
103033.
doi:10.1016/j.bioorg.2019.103033.
[50] C. Méndez-Blanco, F. Fondevila, A. García-Palomo, J. González-Gallego, J.L. Mauriz,
Sorafenib resistance in hepatocarcinoma: role of hypoxia-inducible factors, Experimental &
Molecular Medicine. 50 (2018) 134. doi:10.1038/s12276-018-0159-1.
[51] G.M. Keating, A. Santoro, Sorafenib: a review of its use in advanced hepatocellular
carcinoma, Drugs. 69 (2009) 223–240. doi:10.2165/00003495-200969020-00006.
[52] F. Bray, J. Ferlay, I. Soerjomataram, R.L. Siegel, L.A. Torre, A. Jemal, Global cancer
statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in

185 countries, CA:
doi:10.3322/caac.21492.
A
Cancer Journal for Clinicians. 68 (2018) 394–424.
[53] R. Dutta, R.I. Mahato, Recent advances in hepatocellular carcinoma therapy, Pharmacol.
Ther. 173 (2017) 106–117. doi:10.1016/j.pharmthera.2017.02.010.
[54] R.G. Gieling, M. Babur, L. Mamnani, N. Burrows, B.A. Telfer, F. Carta, J.-Y. Winum, A.
Scozzafava, C.T. Supuran, K.J. Williams, Antimetastatic effect of sulfamate carbonic anhydrase
IX inhibitors in breast carcinoma xenografts, J. Med. Chem. 55 (2012) 5591–5600.
doi:10.1021/jm300529u.
[55] A. Angeli, D. Tanini, T.S. Peat, L. Di Cesare Mannelli, G. Bartolucci, A. Capperucci, C.
Ghelardini, C.T. Supuran, F. Carta, Discovery of New Selenoureido Analogues of 4-(4-
Fluorophenylureido)benzenesulfonamide as Carbonic Anhydrase Inhibitors, ACS Med Chem
Lett. 8 (2017) 963–968. doi:10.1021/acsmedchemlett.7b00280.
[56] M. Bozdag, M. Pinard, F. Carta, E. Masini, A. Scozzafava, R. McKenna, C.T. Supuran, A
Class of 4-Sulfamoylphenyl-ω-aminoalkyl Ethers with Effective Carbonic Anhydrase Inhibitory
Action and Antiglaucoma Effects, J. Med. Chem. 57 (2014) 9673–9686.
doi:10.1021/jm501497m.
[57] R.G. Khalifah, The carbon dioxide hydration activity of carbonic anhydrase. I. Stop-flow
kinetic studies on the native human isoenzymes B and C, J. Biol. Chem. 246 (1971) 2561–2573.
[58] A. Angeli, F. Carta, G. Bartolucci, C.T. Supuran, Synthesis of novel acyl selenoureido
benzensulfonamides as carbonic anhydrase I, II, VII and IX inhibitors, Bioorg. Med. Chem. 25
(2017) 3567–3573. doi:10.1016/j.bmc.2017.05.014.
[59] E. Bertol, A. Argo, P. Procaccianti, F. Vaiano, M.G. Di Milia, S. Furlanetto, F. Mari,
Detection of gamma-hydroxybutyrate in hair: validation of GC-MS and LC-MS/MS methods
and application to a real case, J Pharm Biomed Anal. 70 (2012) 518–522.
doi:10.1016/j.jpba.2012.07.009.
[60] E. Bertol, F. Mari, G. Orzalesi, I. Volpato, Combustion products from various kinds of
fibers: Toxicological hazards from smoke exposure, Forensic Science International. 22 (1983)
111–116. doi:10.1016/0379-0738(83)90002-6.
[61] A.G.W. Leslie, H.R. Powell, Processing diffraction data with mosflm, in: R.J. Read, J.L.
Sussman (Eds.), Evolving Methods for Macromolecular Crystallography, Springer Netherlands,
2007: pp. 41–51.
[62] G.N. Murshudov, A.A. Vagin, E.J. Dodson, Refinement of Macromolecular Structures by
the
Maximum-Likelihood
Method,
Acta
Cryst
D.
53
(1997)
240–255.
doi:10.1107/S0907444996012255.

[63] A.A. Lebedev, P. Young, M.N. Isupov, O.V. Moroz, A.A. Vagin, G.N. Murshudov,
JLigand: a graphical tool for the CCP4 template-restraint library, Acta Crystallogr. D Biol.
Crystallogr. 68 (2012) 431–440. doi:10.1107/S090744491200251X.
[64] P. Emsley, B. Lohkamp, W.G. Scott, K. Cowtan, Features and development of Coot, Acta
Crystallogr. D Biol. Crystallogr. 66 (2010) 486–501. doi:10.1107/S0907444910007493.
[65] E.M. Westbrook, Crystal-density measurements, in: M.G. Rossmann, E. Arnold (Eds.),
International Tables for Crystallography Volume F: Crystallography Ofbiological
Macromolecules,
Springer
Netherlands,
Dordrecht,
2001:
pp.
117–123.
doi:10.1107/97809553602060000664.
[66] S.C. Lovell, I.W. Davis, W.B. Arendall, P.I.W. de Bakker, J.M. Word, M.G. Prisant, J.S.
Richardson, D.C. Richardson, Structure validation by Calpha geometry: phi,psi and Cbeta
deviation, Proteins. 50 (2003) 437–450. doi:10.1002/prot.10286.
[67] E.F. Pettersen, T.D. Goddard, C.C. Huang, G.S. Couch, D.M. Greenblatt, E.C. Meng, T.E.
Ferrin, UCSF Chimera--a visualization system for exploratory research and analysis, J Comput
Chem. 25 (2004) 1605–1612. doi:10.1002/jcc.20084.

•
•
•
CAIs obtained by merging the multikinase inhibitor sorafenib with the benzene
sulfonamide hCA IX inhibitor SLC-0111 are reported
Sulfamates 23 and 24 decrease proliferation breast cancer cell lines at concentrations
above 10 µM.
The binding modes of compounds 17 and 23 on the hCA I isoform by means of X-
ray crystallographic studies are reported.