Flow synthesis and biological activity of aryl sulfonamides as selective carbonic anhydrase IX and XII inhibitors

Article abstract of DOI:10.1016/j.bmcl.2014.05.086

A series of secondary and tertiary aryl sulfonamides were synthesized under flow conditions and evaluated for their ability to selectively inhibit tumor-associated carbonic anhydrase isoforms IX and XII. The tested compounds revealed to be highly potent CA IX inhibitors in nanomolar range, and to inhibit CA XII activity with different ranks of potencies. Remarkably, 4-methyl-N-phenyl-benzenesulfonamide was a selective nanomolar CA IX inhibitor with an IC₅₀ of 90 nM.

Full text of DOI:10.1016/j.bmcl.2014.05.086

Accepted Manuscript
Flow Synthesis and Biological Activity of Aryl Sulfonamides as Selective Car-
bonic Anhydrase IX and XII Inhibitors
Emiliano Rosatelli, Andrea Carotti, Mariangela Ceruso, Claudiu T. Supuran,
Antimo Gioiello
PII:
S0960-894X(14)00595-2
DOI:
http://dx.doi.org/10.1016/j.bmcl.2014.05.086
BMCL 21700
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
21 March 2014
21 May 2014
22 May 2014
Please cite this article as: Rosatelli, E., Carotti, A., Ceruso, M., Supuran, C.T., Gioiello, A., Flow Synthesis and
Biological Activity of Aryl Sulfonamides as Selective Carbonic Anhydrase IX and XII Inhibitors, Bioorganic &
Medicinal Chemistry Letters (2014), doi: http://dx.doi.org/10.1016/j.bmcl.2014.05.086
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Flow Synthesis and Biological Activity of
Aryl Sulfonamides as Selective Carbonic
Anhydrase IX and XII Inhibitors
Leave this area blank for abstract info.
Emiliano Rosatelli, Andrea Carotti, Mariangela Ceruso, Claudiu T. Supuran, Antimo Gioiello*
SO2Cl
R1
O
O
S
R3
N
R1
1
R2
R2
NH
R3

Bioorganic & Medicinal Chemistry Letters
journal homepage: www.els evier.com
Flow Synthesis and Biological Activity of Aryl Sulfonamides as Selective
Carbonic Anhydrase IX and XII Inhibitors
Emiliano Rosatellia, Andrea Carottia, Mariangela Cerusob, Claudiu T. Supuranc and Antimo Gioielloa,∗
aLaboratory of Medicinal and Advanced Synthetic Chemistry (Lab MASC), Department of Pharmaceutical Sciences, University of Perugia, Via del Liceo 1,
Perugia I-06123, Italy.
bLaboratory of Bioinorganic Chemistry, University of Florence, Via della Lastruccia 3, Sesto Fiorentino (Firenze) I-50019, Italy.
cNeurofarba Dept., Section of Pharmaceutical and Nutriceutical Sciences, University of Florence, Via U. Schiff 6, Sesto Fiorentino (Firenze) I-50019, Italy.
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
A series of secondary and tertiary aryl sulfonamides were synthesized under flow conditions and
evaluated for their ability to selectively inhibit tumor-associated carbonic anhydrase isoforms IX
and XII. The tested compounds revealed to be highly potent CA IX inhibitors in nanomolar
range, and to inhibit CA XII activity with different ranks of potencies. Remarkably, 4-methyl-N-
phenyl-benzenesulfonamide was a selective nanomolar CA IX inhibitor with an IC50 of 90 nM.
2009 Elsevier Ltd. All rights reserved.
Keywords:
Carbonic anhydrases
Flow synthesis
Sulfonamides
Docking
Carbonic anhydrases (CAs; also known as carbonate
dehydratases EC 4.2.1.1) are metalloenzymes expressed in both
prokaryotes and eukaryotes that catalyze the reversible
conversion of carbon dioxide to bicarbonate ions and protons
(CO2 + H2O ꢀ HCO3- + H+).1 CAs are involved in numerous
cellular and physiological processes such as respiration,
electrolytes and pH homoeostasis, and biosynthetic reactions
which require bicarbonate as substrate including lipogenesis,
gluconeogenesis, tumorigenicity and ureagenesis, among others.1
Sixteen human CA isoenzymes belonging to alpha class have
been identified. Twelve of them are characterized by a common
Zn(II) ion at the catalytic site, while they differ in organ and
tissue expression, cellular localization, molecular features,
catalytic activity and responsivity to different classes of
inhibitors.1,2
acidification and to maintain intracellular pH more alkaline; thus,
promoting tumor cell survival in an acidic environment and low
bicarbonate medium. Therefore, considering that in normal
tissues the expression levels of CA IX and XII are extremely low,
the pharmacological targeting of these tumor-associated
isozymes is increasingly asserting as a valuable approach in the
search for novel anticancer treatments with reduced or absent
side effects.
Numerous compounds have been disclosed to inhibit the
diverse CAs.3 They can be generally classified in two main
classes of CA inhibitors (CAi): the metal-complexing anions as
the primary aryl sulfonamides, and the corresponding bioisosters
including sulfamates and sulfamides.1-4,6 These compounds are
able to coordinate the Zn(II) ion of the enzyme catalytic site and
to establish additional interactions promoted by the aryl
substituents in the region nearby the catalytic site.2 However,
there are a limited number of compounds that exhibit high
selectivity toward CA IX and CA XII. Among these, 7,8-
disubstituted coumarins were recently claimed to be nanomolar
CA XII selective inhibitors, while the corresponding 6- or 7-
modified analogues and the parent 2-thioxo derivatives were
described as submicromolar inhibitors of both isoforms (Figure
1).8,9 Much work is however warranted to discover new
compounds with improved specificity and potency.
Among CA isoenzymes, the transmembrane proteins CA IX
and XII have recently emerged as promising therapeutic targets
for the treatment of human cancer.3-5 CO2 is the main byproduct
of all oxidative processes and is produced in large amount in
metabolic active tissues such as tumors, where CA IX and XII
are highly overexpressed in response to hypoxia inducible factor
(HIF) pathway. Since the spontaneous hydration of CO2 is a very
slow process, the catalytic activity of CAs assumes a pivotal role
-
in the maintenance of CO2/HCO3 homeostasis.6,7 Indeed, in
hypoxic tumor cells, both isoforms contribute to extracellular
———
∗ Corresponding author. Tel.: +39-075-585-2318; fax: +39-075-585-5161; e-mail: antimo.gioiello@unipg.it

>100 µM (CA I)
>100 µM (CA II)
70.8 nM (CA IX)
1.0 nM (CA XII)
>100 µM (CA I)
>100 µM (CA II)
56.7 nM (CA IX)
0.98 nM (CA XII)
O
O
O
O
O
O
O
O
O
O
S
Ar-SO2Cl (0.20 mmol)
O
O
O
O
acetone
S
R2
N
HO
R1
72.9 µM (CA I)
> 200 µM (CA II)
0.73 µM (CA IX)
0.64 µM (CA XII)
7.17µM (CA I)
200 µM (CA II)
0.80 µM (CA IX)
0.34 µM (CA XII)
Pump A
R3
1-10
Acetone
25 °C
Reagent
loops
(2 mL)
BPR
Figure 1. Selection of coumarinic derivatives and their inhibitory Ki values
UV
Detector
Pump B
towards carbonic anhydrases.
Reactor Heater
(10 mL)
Fraction
collector
H2O
R1R2NH (0.22 mmol)
NaHCO3 (0.40 mmol)
H2O/PEG400 (1:1, v/v)
In this work, we report the development of a series of aryl
sulfonamides as selective CA IX and XII inhibitors (Figure 2). In
this respect, notwithstanding the wide diversification of the
sulfonamide derivatives in term of structural features, size,
hydrophobic/hydrophilic balance, CA isoform selectivity,3,10-14
the so far reported compounds have the common structural
feature to be primary sulfonamides. In our case, we describe the
synthesis and biological evaluation of secondary aryl
sulfonamides 1-9 characterized by a diverse substitution both at
the amine and aryl sulfonyl portions of the molecule. Moreover,
the tertiary sulfonamide 10 has also been prepared.
Flow rate pump A = Flow rate pump B = 0.25 ml/min
Figure 3. Flow synthesis of sulfonamides 1-10.
The thus obtained aryl sulfonamides 1-10 were tested for their
capability to inhibit the tumor-associated CA IX and XII (Table
1).17 The off-target inhibition of CA isoenzymes I and II was also
assessed. Acetazolamide (AAZ) was used as a reference
standard. As a result, all the tested compounds were found to be
highly potent CA IX inhibitors in nanomolar range, and to inhibit
CA XII activity with different ranks of potencies. Interestingly,
sulfonamides 1-10 were inactive or maintained a moderate
inhibitory profile toward isoforms I and II with compounds 1, 2,
4 and 5 showing a remarkable CA IX selectivity (Table 1).
First Set:
O
O
O
O
O
O
O
O
S
S
S
N
H
N
H
N
H
1
2
3
Table 1. Carbonic anhydrase inhibition data for compounds 1-10.
CO2H
O
O
O
O
IC50 (µM)
S
S
S
N
H
N
Compound
N
H
N
H
CA-I
CA-II
CA-IX
CA-XII
AAZ
0.295
0.015
0.073
0.060
4
5
6
1
2
> 50 (35%)a
> 50 (34%)a
> 50 (40%)a
> 50 (39%)a
> 50 (39%)a
> 50 (38%)a
> 50 (39%)a
> 50 (39%)a
> 50 (36%)a
> 50 (39%)a
> 50 (45%)a
> 50 (44%)a
8.071
0.090
0.076
0.070
0.217
0.094
0.090
0.128
0.079
0.074
0.093
8.442
4.280
0.248
8.570
3.302
0.226
0.174
0.179
0.255
0.266
Second Set:
O
O
O
O
S
O
S
N
H
N
H
3
O2N
O
7
8
4
3.623
O
O
O
O
5
6.672
S
S
N
H
N
6
5.100
10
9
O
7
7.321
8
> 50 (45%)a
> 50 (47%)a
> 50 (45%)a
Figure 2. Small library of secondary and tertiary sulfonamides.
9
10
Products 1-10 were easily generated by an efficient single step
flow protocol using a flow meso-reactor apparatus equipped with
two loop injection systems, two pumps, a 10 mL reactor heater, a
back pressure regulator (BPR), a UV detector and a fraction
collector (Figure 3).15,16 The method consisted in sequentially
injecting/switching through the loops and subsequently mixing
into the flow stream, two solutions constituted by the sulfonyl
chloride (0.20 mmol) in acetone (2 mL) (loop A), and the
appropriate primary or secondary amine (0.22 mmol) and
NaHCO3 (0.40 mmol) in water/PEG-400 (2 mL, 1:1, v/v) (loop
B), respectively (Figure 3). The flow stream was generated by
pumping a reservoir of acetone and water at the total flow rate of
0.5 mL·min-1 through the coil reactor conditioned at 25 °C
(resident time: 15 min.).15,16 The in line UV detector was
instrumental to detect the presence or absence of the product
indicating the end of a reaction and the beginning of the next one.
The final products were isolated by simple extraction with
Et2O/HCl 3 N affording the pure sulfonamides 1-10 in 82-97%
yield, ready for biological evaluation.
aPercent of enzymatic inhibition at 10 µM.
In particular, secondary tolyl sulfonamides 1-6 characterized
by both aromatic and alkylic N-substituents were endowed with a
good inhibitory activity on CA IX enzyme (IC50 ranging between
70 and 217 nM). Interestingly, also the tertiary sulfonamide 10
exhibited a good IC50 value toward CA IX (93 nM). The
biological activity of compounds 1, 7-9 revealed that while
substitution at the aryl nucleus bounded to the sulfamoyl moiety
did not have a significant effect on the efficacy of the compounds
in inhibiting CA IX, it consistently affected the activity on CA
XII. Indeed, 4-methyl-benzenesulfonamido derivative (1)
inhibited CA XII with an IC50 of 8.442 µM, while the 4-nitro-
(7), 3,4-dimethoxy- (8) and 4-acetyl-benzenesulfonamido (9)
analogues exhibited a good inhibitory profile in nanomolar range
(IC50= 174 nM, 179 nM and 255 nM, respectively). The best
compound in terms of selectivity and potency resulted to be 4-

methyl-N-phenyl-benzenesulfonamide (1) that was characterized
by a nanomolar potency on CA IX (IC50= 90 nM), a micromolar
activity on CA XII (IC50 CA IX/XII ratio= 0.01), and devoted by
any activity towards the isoform I and II.
In order to understand the molecular basis responsible for the
selectivity profiles displayed by this series of compounds, a
computational modelling study was performed. Analyzing the
binding sites residues falling within 4 Å from the ligands of the
CA I (pdb code: 1AZM),22 CA II (pdb code: 3K34),23 CA IX
(pdb code: 3IAI)24 and CA XII (pdb code: 2HL4),25 it was
possible to pinpoint interesting differences that could explain the
diverse activities recorded by the tested compounds towards the
CA targets (Figure 4). Remarkably, CA II and CA XII are
identical in the residue typology and only small differences on
the orientation of the side-chains of few residues (e.g. F131 and
V135 with an RMSD of 0.75 and 0.57Å, respectively) can be
observed after the backbone alignment. Conversely, CA I is the
most different isoform being characterized by a unique binding
site composition, which includes residues V62, H67, F91, A121,
L131, A135.
L135
F131
V135
V131
Figure 5. Docking pose of compound 1 in CA IX (green) and CA II (pink).
The main difference in the binding site is in the α-helix at the bottom of the
image, formed by amino acids going from 130 to 137, with sequences
RVDEALGR and DFGKAVQQ for CA IX and CA II, respectively. The
labelled side chains of the aminoacids shown in sticks belongs to the residues,
defining the binding site pocket that are different into the two isoforms.
62
V-HS-H ~ FQ-H-H ~ H-A ~ L---A
N-HA-N ~ IQ-H-H H-V ~ F---V
CA IX N-HS-Q ~ LQ-H-H ~ H-V ~ V---L
CA XII N-HA-N ~ IQ-H-H H-V ~ F---V
91
119
131
141
L-V
L-V
L-V
L-V
CA I
CA II
~
~
~
~
~
~
In summary, we have reported a novel small collection of
secondary and tertiary aryl sulfonamides with potent CA IX and
CA XII inhibitory activity. The compounds were generated by an
efficient, safe, and easily scalable method which can be
instrumental for the rapid preparation of additional, refined
derivatives and for large scale synthesis of specific lead
candidates.15 Further extension of the structure activity
relationships of this class of compounds as well as in vivo
characterization of the most potent and selective CA IX inhibitor
1 will be reported in due course.
Figure 4. Alignment of the binding site residues falling within 4 Å from the
ligands of the four carbonic anhydrases isoforms. Residue numbering is
reported at the first row. Legend: - residue not in the 4 Å range from the
ligand, ~ numbering jump.
Next, we directed our efforts on the analysis of the docking
results performed by Glide v6.0 of the Schrodinger Suite 2013-
226 in simple precision mode (SP), relative to the crystallized
structure of the CA II and CA IX.27 Two factors emerged to be
relevant for the activity: a) the F131 residue in the CA II isoform
is mutated in a valine in the CA IX, and b) the interaction
distance between the Zn(II) at the catalytic site and the closest
oxygen of the sulfonamide group. In particular, in the most
selective 4-methyl-N-phenyl-benzenesulfonamide (1), the tolyl
group displayed a perfect fitting in the V131 region of the CA
IX, while a steric clash was present when the molecule adopted
the same disposition inside the binding pocket of the CA II
(Figure 5). Indeed, the cavity of the CA IX binding site is defined
by a small α-helix (a.a. 130 to 137) that is not perfectly aligned
with the corresponding portion of the CA II. Moreover, the
catalytic site of CA IX may allocate the ligands with a better
fitting because of a wider cavity area. All the other poses
obtained for compound 1 in the CA II gave a different fitting
with even a much higher distance between the interacting oxygen
of the sulfonamide moiety and the Zn metal ion (from 5.72 Å to
6.46 Å). Notably, all the other derivatives of the series displayed
the same binding feature of 1, though compounds 3-5 were likely
to fit better into the CA II binding site probably because of the
higher flexibility of the alkyl groups, while the ortho-phenyl
derivative 6 showed a ‘compact’ disposition into the enzyme
cavity. The nitro derivative 7 displayed a disposition in CA II
comparable to that adopted by 1 within the CA IX with a good
distance from the Zn metal but with a higher exposure of the
nitro group to the solvent.
Supplementary data
Supplementary data associated with this article can be found,
in the online version, at …..
References and notes
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Oncotarget 2012, 3, 84.
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Comb. Sci. 2013, 15, 235.
16. Sulfonyl chloride (0.20 mmol) was dissolved in acetone (2 mL) and the
resulting solution was injected in the reagent loop A. An aqueous
NaHCO3 0.4 M (1 mL) was added to a solution of the amine (0.22 mmol)
dissolved in PEG-400 (1 mL). The H2O/PEG400 solution (2 mL, 1:1,
v/v) was then injected in the reagent loop B. A degassed solution of
acetone and water were connected with pump A and B, respectively and
the flow rate was fixed at 0.5 mL·min−1 (0.25 mL·min−1 + 0.25
mL•min−1). After switching the sample loops, the mixtures exited were
joined in a T-piece, entered in a 10 mL PTFE coil reactor warmed at 25
°C, fitted with the back pressure regulator (100 psi), directed in UV
detector and the output was recovered in a fraction collector. The
reaction mixture was drooped in a tube containing Et2O/HCl 3 N (5 mL,
4:1, v/v). The two phases were separated, and the organic one was
washed with H2O (2 x 1 mL), dried over Na2SO4, and concentrated under
reduced pressure to give the desired pure aryl sulfonamide.
17. An applied photophysics stopped-flow instrument has been used for
assaying the CA catalyzed CO2 hydration activity.18 Phenol red (at a
concentration of 0.2 mM) has been used as an indicator, working at the
absorbance maximum of 557 nm, with 20 mM Hepes (pH 7.4) and 20
mM NaBF4 (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 (10 mM) were prepared in distilled-
deionized water and dilutions up to 0.01 nM were done thereafter with
distilled-deionized water. Inhibitor and enzyme solutions were
preincubated together for 15 min at room temperature prior to assay, in
order to allow for the formation of the E-I complex. The IC50s were
obtained by non-linear least-squares methods using PRISM 3, as reported
earlier19-21
, and represent the mean from at least three different
determinations. All CA isoforms were recombinant and obtained in-
house as reported earlier.19-21
18. Khalifah, R. G. J. Biol. Chem. 1971, 246, 2561.
19. Liu, F.; Martin-Mingot, A.; Lecornué, F.; Jouannetaud, M. P.; Maresca,
A.; Thibaudeau, S.; Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2012;
27, 886.
20. Kazancıoğlu, E. A.; Güney, M.; Sentürk, M.; Supuran, C. T. J. Enzyme
Inhib. Med. Chem. 2012, 27, 880.
21. Cavdar, H.; Ekinci. D. Talaz, O.; Saraçoğlu, N.; Sentürk, M.; Supuran, C.
T. J. Enzyme Inhib. Med. Chem. 2012, 27, 148.
22. Chakravarty, S.; Kannan, K. K. J. Mol. Biol. 1994, 243, 298.
23. Behnke, C. A.; Le Trong, I.; Godden, J. W.; Merritt, E. A.; Teller, D. C.;
Bajorath, J.; Stenkamp, R. E. Acta Crystallogr. D 2010, 66, 616.
24. Alterio, V.; Hilvo, M.; Di Fiore, A.; Supuran, C. T.; Pan, P.; Parkkila, S.;
Scaloni, A.; Pastorek, J.; Pastorekova, S.; Pedone, C.; Scozzafava, A.;
Monti, S. M.; De Simone, G. Proc. Natl. Acad. Sci. USA 2009, 106,
16233.
25. Di Fiore, A.; Scozzafava, A.; Winum, J.-Y.; Montero, J.-L.; Pedone, C.;
Supuran, C.T.; De Simone, G. Bioorg. Med. Chem. Lett. 2007, 17, 1726.
26. Schrodinger Inc., LLC, New York, NY, USA, 2013.
27. Antolin, A. A.; Carotti, A.; Nuti, R.; Hakkaya, A.; Camaioni, E.;
Mestres, J.; Pellicciari, R.; Macchiarulo, A. J. Mol. Graph. Model. 2013,
45, 192.