4-Substituted Benzenesulfonamides Incorporating Bi/Tricyclic Moieties Act as Potent and Isoform-Selective Carbonic Anhydrase II/IX Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.8b00670

As a part of our efforts to expand chemical diversity in the carbonic anhydrases inhibitors (CAIs), three small series of polyheterocyclic compounds (4-6) featuring the primary benzenesulfonamide moiety linked to bi/tricyclic scaffolds were investigated. Highly effective inhibitors against the target tumor-associated hCA IX (low nanomolar/subnanomolar potency levels) showing significant functional selectivity profile toward hCA I, II, and IV isozymes were identified. Molecular docking studies clarified the reasons behind the activity and selectivity of the new compounds.

Full text of DOI:10.1021/acs.jmedchem.8b00670

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Brief Article
4-Substitutedbenzenesulfonamides incorporating bi/tricyclic moieties
act as potent and isoform-selective carbonic anhydrase II/IX inhibitors
Silvia Salerno, Elisabetta Barresi, Giorgio Amendola, Emanuela Berrino, Ciro Milite, Anna Maria Marini,
Federico Da Settimo, Ettore Novellino, Claudiu T Supuran, Sandro Cosconati, and Sabrina Taliani
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00670 • Publication Date (Web): 18 Jun 2018
Downloaded from http://pubs.acs.org on June 18, 2018
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†
∇
†∇
‡
∥
£
Silvia Salerno , Elisabetta Barresi , Giorgio Amendola , Emanuela Berrino , Ciro Milite , Anna
†
†
§
∥
Maria Marini , Federico Da Settimo Ettore Novellino , Claudiu T. Supuran , Sandro Cosco-
,
‡*
†
nati , Sabrina Taliani
†Dipartimento di Farmacia, Università di Pisa, Via Bonanno 6, 56126 Pisa, Italy
‡DiSTABiF, Università della Campania Luigi Vanvitelli, Via Vivaldi 43, 81100 Caserta, Italy
∥NEUROFARBA Department, Sezione di Scienze Farmaceutiche e Nutraceutiche, Università degli Studi di Firenze,
Via Ugo Schiff 6, 50019 Sesto Fiorentino (Florence), Italy
£Dipartimento di Farmacia, Universitꢀ di Salerno, Via Giovanni Paolo II 132, 84084 Fisciano, Salerno, Italy
§Dipartimento di Farmacia, Università di Napoli “Federico II”, Via D. Montesano 49, 80131 Napoli, Italy
ABSTRACT: As a part of our efforts to expand chemical diversity in the Carbonic Anhydrases Inhibitors (CAIs), three small
series of polyheterocyclic compounds (4-6) featuring the primary benzenesulfonamide moiety linked to bi/tricyclic scaf-
folds were investigated. Highly effective inhibitors against the target tumor-associated hCA IX (low nanomolar/subnano-
molar potency levels), showing significant functional selectivity profile towards hCA I, II and IV isozymes were identified.
Molecular docking studies clarified the reasons behind the activity and selectivity of the new compounds.
5
Introduction
(1d−e) systems (Chart 1), designed as geometrically con-
strained analogues of celecoxib (CLX) and valdecoxib
The metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1) is
widespread in organisms all over the phylogenetic tree and
catalyzes a simple physiological reaction, the interconver-
(
VLX), originally launched as cyclooxygenase 2 (COX-2)
specific inhibitors, and later shown also to act as potent
6
CAIs.
sion between CO
a proton. CA inhibition finds pharmacological applications
in several fields including diuretics, antiglaucoma, anti-
2
and bicarbonate, with the generation of
1
Compounds 1a-e behaved as selective CAIs without any in-
hibitory effect on COX-2. In addition, the most interesting
feature was their capability to predominantly exert strong
inhibition of only human (h) isoforms hCA I and II,
whereas their inhibitory activity against hCA III, IV, VA,
VB, VI, VII, IX, XII, XIII, and XIV was up to 2 orders of mag-
nitude lower, potentially resulting in a lower occurrence of
side effects. X-ray crystallographic studies performed on 1e
2
convulsant, and anticancer agents. Sulfonamides and
their bioisosteres (sulfamates, sulfamides), were reported
3
as pharmacologically relevant CA inhibitors (CAIs).
Due to the ubiquity of such enzymes, the selective inhibi-
tion and polypharmacology of inhibitors is an important
3
aspect of all drug design campaigns. Several drug design
(
Chart 1) in complex with hCA II, combined with homology
strategies have been reported ultimately based on the tail
4
modeling, rationalize this inhibition profile: compound 1e
is buried deep into the active site with the sulfonamide ni-
trogen directly bound to the zinc ion; the sulfur atom is out
of the active site and not directly involved in any interac-
tion with the enzyme, only producing a pucker in the ring
geometry; the hydrophobic tricyclic scaffolds protrudes
out of the active site, where it is stabilized by van der Waals
interactions with hydrophobic residues lining the active
site cavity. According to this interaction mode, the differ-
ences in affinity for hCA I and II, with respect to hCA IX
and XII, might be ascribed to point mutations in amino
approach for obtaining sulfonamides/dithiocarbamates,
which exploit more external binding regions within the en-
zyme active site (in addition to coordination to the metal
ion), thus leading to isoform-selective compounds. Thus,
the exploration of novel scaffolds may expand chemical di-
versity aiding the development of novel classes of CAIs
with the desired pharmacologic properties. In this connec-
tion, we recently reported a novel class of tricyclic ben-
zenesulfonamides 1a-e featuring the benzothiopyrano[4,3-
c]pyrazole (1a−c) and pyridothiopyrano[4,3-c]pyrazole
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Chart 1. Structure of previously described (1-3) and newly synthesized (4-6) CAIs.
1
2
acid sequences that produce a reduction in hydrophobicity
in the hydrophobic pockets within the active site of hCA
IX and XII. More recently, class 1 was further investigated
groups (R, R , and R ) to evaluate the effect of these sub-
stituents on the efficacy and selectivity towards different
CA isoenzymes.
5
by moving the benzenesulfonamide function from position
1 to position 2 of the pyrazole system (2a-d) (Chart 1). Con-
Chemistry
7
The key intermediates for the synthesis of the target
compounds 4a-c, 5a-c and 6a-c were the dimethyla-
minomethylene derivatives 7a-c, which were prepared by a
temporaneously, two diverse heteropolycyclic scaffolds
from our in-house database, structurally similar to the py-
razoles of types 1 and 2, that is benzothiopyranopyrimidine
and pyridothiopyrano-pyrimidine, were decorated with a
benzenesulfonamide moiety at the 2-position of the tricy-
previously reported procedure with slight modifications
8,9
(
Scheme 1). Briefly, the commercially available 5-substi-
tuted-1,3-cyclohexandiones were reacted with an excess of
N,N-dimethylformamide dimetylacetal (DMF-DMA) at 100
clic system by an NH linker, yielding compounds 3a–e
7
(
Chart 1) An excellent inhibitory activity against isoforms
°C for one hour to furnish the intermediates 7a-c in quan-
hCA II, IX, and XII was shown by most of the new sulfona-
titative yield.
mides 2 and 3, although without high selectivity profile
7
The 1,3-bielectrophilic reaction of 7a with nucleophilic re-
agents, formamidine, acetamidine or benzamidine hydro-
chloride, in refluxing ethanol for 15-30 hours (TLC analysis)
led to the pyrimidine derivatives 8a-c, that were then re-
acted with an excess of DMF-DMA at 100 °C for one hour
among the various isoforms.
As a part of our efforts to expand chemical diversity in the
CAIs with the aim of identifying potent selective inhibitors,
in the present work, three small series of polyheterocyclic
compounds (4-6) featuring the classical primary
benzenesulfonamide moiety crucial for the binding to the
catalytically metal ion, were synthesized and biologically
evaluated. Specifically, in all the novel classes the sulfur
bridge was replaced with the bioisosteric methylene as our
previous studies highlighted that the sulfur atom was un-
9
10,11
to obtain the intermediates 9a-c in very good yields. De-
rivatives 9a-c were then solubilized in ethanol and reacted
with a slight excess of commercial 4-hydrazinebenzenesul-
fonamide hydrochloride, basically according to described
1
2-14
procedures.
The reaction mixture was heated to 80 °C
for 20 hours and, after cooling, the obtained crude com-
pounds 4a-c were purified by flash chromatography
(Scheme 1). Similarly, the synthesis of the new bicyclic
tetrahydroindazoles 5a-c and tetrahydroquinazoles 6a-c
5
necessary for the interaction of 1e with the enzyme. The
7-substituted-4-(4,5-dihydro-1H-pyrazolo[3,4-
f]quinazolin-1-yl)benzenesulfonamides 4a-c (Chart 1)
maintained a 5,6,6 tricyclic system analogous to derivatives
8
took advantage of the reactivity of derivatives 7a-c
1
-3, but an additional nitrogen atom was introduced in the
(
Scheme 1). When 7a-c were reacted with a slight excess of
pyridine nucleus, converting it into a pyrimidine, possibly
providing an additional or alternative interaction point
with the enzyme.
the commercially available 4-hydrazinebenzenesulfona-
mide hydrochloride in ethanol at 80 °C for 5-20 hours (TLC
analysis), compounds 5a-c were obtained and purified by
recrystallization from ethanol (Scheme 1). Instead, reaction
The bicyclic tetrahydroindazole (5a-c, Chart 1) and
tetrahydroquinazole (6a-c, Chart 1) derivatives were con-
ceived as a structural simplification of compounds 1 and 3,
respectively, in which the phenyl- or pyrido-fused moiety
was removed and a carbonyl group was added in the non-
aromatic ring. The crucial benzenesulfonamide function
was maintained, directly linked at 1-position of the pyra-
zole ring (5a-c, Chart 1) or linked at 2-position of the py-
rimidine ring by an NH spacer (6a-c, Chart 1), in strict sim-
ilarity to derivatives 1 and 3, respectively.
7
of intermediates 7a-c with the guanidine 10 in refluxing n-
butanol, in the presence of sodium hydroxide for 16 hours,
furnished compounds 6a-c, then purified by recrystalliza-
tion from ethanol (Scheme 1).
Results and Discussion
All the newly synthesized compounds 4a-c, 5a-c and 6a-c
were investigated for their enzyme inhibitory capacity
against four physiologically relevant CA isoforms, the hu-
man (h) hCA I, II, IV, and IX (Table 1), by a stopped-flow
Finally, tricyclic compounds 4a-c and bicyclic derivatives
1
5
CO
2
hydrase assay. Acetazolamide (AAZ, 5-acetamido-
5
a-c and 6a-c were decorated with methyl or phenyl
1
,3,4-thiadiazole-2-sulfonamide) was used as standard drug
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Scheme 1.
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Reagents and conditions: (i) DMF-DMA, 100 °C; (ii) formamidine, acetamidine or benzamidine hydrochloride, refluxing etha-
nol; (iii) DMF-DMA, 100 °C; (iv) 4-hydrazinebenzenesulfonamide hydrochloride, ethanol, 80 °C; (v) 4-hydrazinebenzenesulfona-
mide hydrochloride, refluxing ethanol; (vi) concentrated HCl/50% cyanamide water solution, 100 °C; (vii) NaOH, refluxing n-
butanol.
1
5
in the assay. From a general point of view, the data listed
in Table 1 show that the bioisosteric substitution of the sul-
fur bridge with a methylene moiety leads to potent CAIs,
confirming our previous findings of the absence of a direct
involvement of the sulfur in the interaction with the en-
zyme. All the newly synthesized compounds are highly ef-
fective inhibitors against the target tumor-associated hCA
IX, exhibiting potency levels in the low nanomolar/subna-
Sub-nanomolar inhibition against the isoform IX was
achieved by all the compounds featuring a simplified scaf-
fold (5b-c, 6a-c, K
independent by the decoration of the central nucleus, with
the only exception of 5a (K 5.2 nM). The isoforms hCA I
and II were scarcely (hCA I) or effectively (hCA II) inhib-
ited by compounds 5-6 [K values spanning between 272.2
nM to 746.3 nM (the sole exception to this trend is 6a, K
63.9 nM) and 5.2 nM to 8.6 nM (the sole exception to this
trend is 5a, K 78.2 nM), for hCA I and hCA II, respectively),
i
values ranging from 0.55 to 0.89 nM),
i
i
i
nomolar range (K
activity gain of several times compared to the reference
AAZ (K 25.8 nM); the cytosolic isoform hCA I was moder-
i
range from 7.3 nM to 0.55 nM), with an
i
i
while sparing K
bound hCA IV.
i
values were obtained for the membrane-
ately inhibited by compounds 4-6, whereas diverse activi-
ties against isozymes II and IV were observed. All deriva-
tives showed to act as preferential inhibitors of hCA IX,
with selectivity ratios ranging from 61.3 to >1370 against
hCA I, from 7.12 to 577 for hCA II and from 1.8 to 359 for
hCA IV. Specifically, switching the pyridothiopyranopy-
rimidine of 1d-e to the pyrazolo-dihydroquinazoline of 4a-
c affected hCA inhibition depending on the substituent at
7-position: the unsubstituted compound 4a showed a gain
in activity towards all the isoenzyme tested, but particu-
larly for the isoform IX. Insertion of a 7-methyl or 7-phenyl
group on 4a, yielding compounds 4b-c, resulted in a mod-
To clarify the reasons behind the displayed activity and se-
lectivity of the newly designed compounds, molecular
docking studies were undertaken for ligands 4c, 5c, and 6c
as representative compounds of the three series. For this
16
purpose, the latest version of AutoDock4.2 (AD4) was
employed in combination with the available high-
resolution crystal structure of hCA IX bound to an inhibi-
17
tor (PDB code 5FL4, 1.82 Å). Taking into account that a
zinc atom participates to the binding event, the recently
18
released AutoDock4(Zn) force field was employed, which
greatly enhances the accuracy of the pose prediction when
docking zinc-chelating compounds. In particular, a spe-
cialized potential was introduced into the standard Auto-
Dock force field to describe both the geometric and
energetic components of the interactions of zinc-
est decrease in potency hCA IX inhibition (4a K
b K 7.0 nM, 4c K 7.3 nM). However, the installation of
the 7-phenyl substituent conferred a high degree of selec-
tivity to hCA IX when compared the other tested hCAs.
i
0.57 nM,
4
i
i
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Table 1. Inhibition of hCA Isoforms I, II, IV, IX with Sulfonamides 4a-c, 5a-c and 6a-c, and AAZ as reference standard by a
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Stopped-Flow CO Hydrase Assay.
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Ki (nM)a
hCA I
hCA I/hCA IX]b
hCA II
[hCA II/hCA IX]b
hCA IV
[hCA IV/hCA IX]b
cpd
1dc
R
R1
R2
hCA IX
[
H
CH
H
-
-
-
-
193
155
72
328
7500
2340
3250
0.57
7.0
1
ec
3
49
4a
-
-
73.5 [129]
429.0 [61.3]
>10000 [>1370]
746.3 [143.5]
272.2 [309]
295.7 [405]
63.9 [116]
633.4 [712]
686.3 [869]
250
4.6 [8.1]
68.6 [9.8]
4209.2 [577]
78.2 [15]
8.6 [9.8]
5.2 [7.1]
5.2 [9.45]
8.2 [9.2]
6.0 [7.6]
12.1
7.1 [12.5]
581.3 [83]
7228.7 [990]
9.5 [1.8]
4b
CH
3
-
-
4c
C
6
H
5
-
-
7.3
5a
-
H
CH
H
H
CH
H
H
CH
5.2
5b
-
-
-
-
-
3
3
39.7 [45]
213.7 [293]
73.0 [132.7]
319.4 [359]
78.0 [98.7]
74
0.88
0.73
0.55
0.89
0.79
25.8
5c
6 5
C H
6a
H
6b
3
CH
3
6c
6 5
C H
AAZ
a
b
Mean from three different assays. Errors were in the range of ± 5-10% of the reported values (data not shown).
K
i
ratio for the
c
indicated enzyme isoforms. Data from ref 5.
coordinating ligands. To test the predictive power of the
protocol, the co-crystal ligand of the selected X-ray com-
plex, a sulfonamide inhibitor, was re-docked into its cog-
nate receptor structure. Indeed, AD4 yielded a pose which
is virtually superimposable to the experimental one (root
mean squared deviation of 0.72 Å). The encouraging dock-
ing results prompted the employment of the aforemen-
tioned AD4 force field for the docking of the selected hCAs
inhibitors into the hCA IX structure. Analysis of the
achieved results revealed that 4c, 5c, and 6c, chelate the
catalytic zinc in the active site through their negatively
charged nitrogen of the sulfonamide moiety, which is con-
sistent with the pose of all the other sulfonamides com-
plexed to CAs. Furthermore, the sulfonamide in each com-
pound is well positioned to form an H-bond with the back-
bone NH of T200. Apart from these shared interactions,
the different core structure of the three selected CA inhib-
itors induces different interaction patterns with the rest of
the protein structure. Specifically, 4c (Figure 1a) would
preferentially adopt a conformation in which the two ni-
trogens of the pyrimidine ring accept a double H-bond
from the Q71 and Q92 side-chains. Supposedly, the tight
anchoring provided by the double H-bonding interaction
might explain the high affinity of 4c for hCA IX. Interest-
ingly, the pendant phenyl ring of 4c is lodged in a hydro-
phobic gorge lined by L91, L123 and V130 residues. These
residues belong to a so-called “hot spot” region which plays
a key role in the compound selectivity being highly variable
in terms of sequence and, consequently, three-dimensional
1
9
conformation, within each CA isoenzyme. In this respect,
it could be argued that the bulky and lipophilic phenyl sub-
stituent may not be easily allocated in the rather narrow
“hot spot” gorge of hCA I and hCA II, accounting for the
lower inhibitory activity displayed by 4c (Figures 1b, 1c)
against these latter two isoenzymes. On the other hand,
hCA IV features a “hot spot” region lined by rather hydro-
philic residues: N79, K95, and E127, in which the phenyl
20
substituent would not engage favorable contacts. Com-
pounds 5c and 6c, regardless of their own chirality, share a
common binding mode (Figure 1d and Figure S1 in Sup-
porting Information) taking favorable van der Waals con-
tacts with Q92, V130, L199, T201, and P203 residues. These
data would indicate the absence of an enantio-discriminat-
ing binding of these compounds. Interestingly, a similar
binding pose was also experimentally verified for the other
1
7,19
co-crystal ligands of hCA IX.
Moreover, their phenyl
moiety points towards the same hydrophobic “hot spot”
cleft described earlier. Thus, the selectivity of 5c and 6c to
hCA IX could also be ascribed to a difficulty in allocating
bulky and lipophilic substituents in the cleft of other hCAs.
The strong enhancement of potency displayed by most of
the newly synthesized derivatives against hCA IX,
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7
Figure 1. (A) 4c/hCA IX (PDB 5FL4) complex. The protein is depicted as cyan ribbons and sticks while the ligand as orange sticks.
21
(
B) 4c hCA IX docked binding pose within the hCA I (PDB 4WUP) structure. The protein is depicted as green ribbons and its
molecular surface in gray. The ligand is represented as orange sticks and its molecular surface in yellow. (C) 4c hCA IX docked
22
binding pose within the hCA II (PDB 3QYK) structure. The protein is depicted as light blue ribbons and its molecular surface in
gray. The ligand is represented as orange sticks and its molecular surface in yellow. (D) Superimposition of the docked poses of 5c
(
pink sticks) and 6c (blue sticks) in the hCA IX active site. The protein is depicted as cyan ribbons and sticks. In panels A and D
important residues are labeled. H-bonds are shown in red dashed lines while coordination bonds in green dashed lines. The images
were generated using the UCSF Chimera software.
23
combined with their significant functional selectivity pro-
files, could be seen with interest for the design of novel
anti-cancer agents with limited side effects. Indeed, due to
its key role as a survival factor for tumor cells and its in-
volvement in insurgence of resistance to classical anti-
cancer treatments, CA IX represents an excellent drug tar-
get for the development of novel cancer therapeutic ap-
proaches.
1.606 g (7.2 mmol) of 4-hydrazinebenzenesulfonamide hy-
drochloride. The reaction mixture was heated to 80 °C for
5-20 hours (TLC analysis). After cooling, the obtained solid
was purified by recrystallization from ethanol to give the
desired compounds 5a-c (yields, physical, and spectral data
are reported in SI).
General procedure for the synthesis of 7-substituted
4-[(5-oxo-5,6,7,8-tetrahydroquinazolin-2-
Experimental Section
yl)amino]benzenesulfonamides 6a-c. Compounds 7a-
c (1.0 mmol) were solubilized in n-BuOH (3-5 mL) and
8
Chemistry. General directions are in the SI. The purity of
tested compounds is ≥95% (HPLC analysis).
added with 0.300 g (1.0 mmol) of 4-guanidinobenzenesul-
fonamide carbonate 10 and 0.080 g (2.0 mmol) of NaOH.
The reaction mixture was heated up to 120 °C and left to
stir for 16 hours (TLC analysis). After cooling, the obtained
crude solid compounds 6a-c were purified by recrystalliza-
tion from ethanol (yields, physical, and spectral data are
reported in SI).
The following intermediates were obtained according to
methods previously described: 2-[(dimethylamino)meth-
ylene]cyclohexan-1,3-dione 7a, 2-[(dimethylamino)meth-
ylene]-5,5-dimethylcyclohexan-1,3-dione 7b, and 2-[(dime-
thylamino)methylene]-5-phenylcyclohexan-1,3-dione 7c;
2-methyl-7,8-dihydroquinazolin-5(6H)-one 8b, 2-phenyl-
8
9
7,8-Dihydroquinazolin-5(6H)-one 8a. 2-[(Dimethyla-
mino)methylene]cyclohexan-1,3-dione 7a (1.00 g, 6.0
mmol) was solubilized in 10 mL of EtOH and added with
0.480 g (6.0 mmol) of formamidine hydrochloride. The re-
action mixture was heated to 100 °C for 5 hours. After cool-
ing the solid obtained was purified by flash-chromatog-
raphy on silica gel (petroleum ether 40-60/ethyl acetate 1:9
as the eluting system) to furnish 8a as a yellow oil (yield
7,8-dihydroquinazolin-5(6H)-one 8c;
6-[(dimethyla-
mino)methylene]-2-methyl-7,8-dihydroquinazolin-5(6H)-
1
0
one 9b; 6-[(dimethylamino)methylene]-2-phenyl-7,8-di-
1
1
hydroquinazolin-5(6H)-one 9c; N-(4-aminosulfonyl)phe-
nylguanidine carbonate 10.
7
General procedure for the synthesis of 7-substituted
4-(4,5-dihydro-1H-pyrazolo[3,4-f]quinazolin-1-yl)ben-
zenesulfonamides 4a-c. The appropriate derivative 9a-c
(7.o mmol) was solubilized in ethanol (3-5 mL) and added
with 1.851 g (8.3 mmol) of 4-hydrazinebenzenesulfonamide
hydrochloride. The reaction mixture was heated to 80 °C
for 20 hours. After cooling, the obtained solid was purified
by flash chromatography on silica gel column (petroleum
ether 40–60 °C/ethyl acetate 1:9 as the eluting system) to
furnish the desired compounds 4a-c (yields, physical, and
spectral data are reported in SI).
25%; spectral data are reported in SI).
6-((Dimethylamino)methylene)-7,8-dihydro-
quinazolin-5(6H)-one 9a. Compound 8a (0.355 g, 2.4
mmol) was dissolved in 8 mL (3.6 mmol) of DMF-DMA and
heated to 100 °C for about 1 hour. After cooling, the ob-
tained suspension was triturated with ethyl ether to yield
9a as a yellow solid, which was used in the next step with-
out further purification (yield 95%; physical, and spectral
data are reported in SI).
General procedure for the synthesis of 6-substituted
4-(4-oxo-4,5,6,7-tetrahydro-1H-indazol-1-yl)benzene-
sulfonamides 5a-c. The appropriate derivative 7a-c (6.0
8
Supporting Information. General chemistry directions,
yields, physical, and spectral data of compounds 4a-c, 5a-
mmol) was solubilized in ethanol (3-5 mL) and added with
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Journal of Medicinal Chemistry
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c, and 6a-c, spectral data of compounds 8a and 9a, CA in-
8
Schenone, P.; Mosti, L.; Menozzi, G. Reaction of 2-dime-
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thylaminomethylene-1,3-diones with dinucleophiles. I. Synthesis
of 1,5-disubstituted 4-acylpyrazoles. J. Heterocycl. Chem. 1982, 19,
hibition assay and molecular modeling methods. SMILES
molecular formula strings (CSV), coordinates of the pre-
dicted ligand/hCA IX complexes (PDB). This material is
available free of charge via the Internet at
http://pubs.acs.org.
1355–1361.
9
Mosti, L.; Menozzi, G.; Schenone, P. Reaction of 2-dime-
thylaminomethylene-1,3-diones with dinucleophiles. III. Synthe-
sis of 5-acylpyrimidines and 7,8-dihydroquinazolin-5(6H)-ones. J.
Heterocycl. Chem. 1983, 20, 649–654.
10
Mosti, L. Menozzi, G; Schenone, P. Reaction of ketenes
with N,N-disubstituted α-aminomethyleneketones. XX. Synthe-
sis of 2H-pyrano[2,3-f]quinazoline derivatives. J. Heterocycl.
Chem. 1987, 24, 603–608.
*
0
E-mail:
sandro.cosconati@unicampania.it.
Tel:
+39
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2
3
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5
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0
1
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823274789. Fax: +39 0823274585.
11
A.; Petrova, M. V. Chem. Heterocycl. Compd. (N. Y., NY, U. S.)
003, 39, 520-524.
12 Da Settimo, A.; Primofiore, G.; Da Settimo, F.; Marini,
Pyrimido[4,5-f]quinazolines. Tonkikh, N. N.; Strakovs,
2
The manuscript was written through contributions of all au-
thors. All authors have given approval to the final version of
the manuscript. S.S. and E.B. contributed equally to this
A. M.; Taliani, S.; Salerno, S.; Dalla Via, L. Synthesis of pyrim-
ido[1,2-α]benzimidazol-4(10H)-one derivatives and evaluation of
their interactions with DNA. J. Heterocycl. Chem. 2003, 40, 1091–
∇
work.
1
096.
13
Primofiore, G. M., A. M.; Salerno, S.; Da Settimo, F.; Ber-
tini, D.; Dalla Via, L. Synthesis and antiproliferative evaluation of
new aryl substituted pyrido[3',2':5,6]thiopyrano[4,3-c]pyrazoles. J.
Heterocycl. Chem. 2005, 42, 1357-1361.
This work was supported by Progetti di Rilevante Interesse
Nazionale (PRIN) 2015 (grant 2015FCHJ8E_003 to S.C), by Uni-
versity of Pisa (PRA project), and by University of Campania
Luigi Vanvitelli.
14
Marini, A. M.; Da Settimo, F.; Salerno, S.; La Motta, C.;
Simorini, F.; Taliani, S.; Bertini, D.; Gia, O.; Dalla Via, L. Synthesis
and in vitro antiproliferative activity of new substituted
benzo[3',2':5,6]thiopyrano[4,3-d]pyrimidines.
Chem. 2008, 45, 745–749.
J.
Heterocycl.
CA, carbonic anhydrase; CAIs, carbonic anhydrase inhibitors;
CLX, celecoxib; VLX, valdecoxib; COX-2, cyclooxygenase 2;
AAZ, Acetazolamide; AD4, AutoDock4.2.
15
Khalifah, R. G. The carbon dioxide hydration activity of
carbonic anhydrase. I. Stop-flow kinetic studies on the native hu-
man isoenzymes B and C. J. Biol. Chem. 1971, 246, 2561–2573.
1
6
Morris, G. M.; Huey, R.; Lindstrom, W.; Sanner, M. F.;
Belew, R. K.; Goodsell, D. S.; Olson, A. J. AutoDock4 and Auto-
DockTools4: automated docking with selective receptor flexibil-
ity. J. Comp. Chem. 2009, 30, 2785–2791.
1
Supuran, C. T. Structure and function of carbonic anhy-
drases. Biochem. J. 2016, 473, 2023–2032.
Supuran, C. T. Carbonic anhydrases: novel therapeutic
applications for inhibitors and activators. Nat. Rev. Drug Discov.
2
17
Leitans, J.; Kazaks, A.; Balode, A.; Ivanova, J.; Zalubov-
skis, R.; Supuran, C. T.; Tars, K. Efficient expression and crystalli-
zation system of cancer-associated carbonic anhydrase isoform
IX. J. Med. Chem. 2015, 58, 9004–9009.
2
3
008, 7, 168–181.
Supuran, C. T. How many carbonic anhydrase inhibi-
tion mechanisms exist? J. Enzyme Inhib. Med. Chem. 2016, 31, 345–
360.
1
8
Santos-Martins, D.; Forli, S.; Ramos, M. J.; Olson, A. J.,
AutoDock4(Zn): an improved AutoDock force field for small-mol-
4
Hou, Z.; Lin, B.; Bao, Y.; Yan, H. N.; Zhang, M.; Chang,
ecule docking to zinc metalloproteins. J. Chem. Inf. Model. 2014,
X. W.; Zhang, X. X.; Wang, Z. J.; Wei, G. F.; Cheng, M. S.; Liu, Y.;
Guo, C. Dual-tail approach to discovery of novel carbonic anhy-
drase IX inhibitors by simultaneously matching the hydrophobic
and hydrophilic halves of the active site. Eur. J. Med. Chem. 2017,
54, 2371–2379.
19 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. Crystal structure
of the catalytic domain of the tumor-associated human carbonic
anhydrase IX. Proc. Natl. Acad. Sci. USA. 2009, 106, 16233–16238.
1
32, 1–10.
5
Marini, A. M.; Maresca, A.; Aggarwal, M.; Orlandini, E.;
Nencetti, S.; Da Settimo, F.; Salerno, S.; Simorini, F.; La Motta, C.;
Taliani, S.; Nuti, E.; Scozzafava, A.; McKenna, R.; Rossello, A.;
Supuran, C. T. Tricyclic sulfonamides incorporating benzothiopy-
rano[4,3-c]pyrazole and pyridothiopyrano[4,3-c]pyrazole effec-
tively inhibit α- and β-carbonic anhydrase: X-ray crystallography
and solution investigations on 15 Isoforms. J. Med. Chem. 2012, 55,
9619–9629.
20
Zubriene, A.; Smirnoviene, J.; Smirnov, A.; Morkunaite,
V.; Michailoviene, V.; Jachno, J.; Juozapaitiene, V.; Norvaisas, P.;
Manakova, E.; Grazulis, S.; Matulis, D. Intrinsic thermodynamics
of 4-substituted-2,3,5,6-tetrafluorobenzenesulfonamide binding
to carbonic anhydrases by isothermal titration calorimetry. Bio-
phys. Chem. 2015, 205, 51–65.
21
Hen, N.; Bialer, M.; Yagen, B.; Maresca, A.; Aggarwal,
6
Supuran, C. T.; Casini, A.; Mastrolorenzo, A.; Scozzaf-
M.; Robbins, A. H.; McKenna, R.; Scozzafava, A.; Supuran, C. T.
Anticonvulsant 4-aminobenzenesulfonamide derivatives with
branched-alkylamide moieties: X-ray crystallography and inhibi-
tion studies of human carbonic anhydrase isoforms I, II, VII, and
XIV. J. Med. Chem. 2011, 54, 3977–3981.
ava, A. COX-2 selective inhibitors, carbonic anhydrase inhibition
and anticancer properties of sulfonamides belonging to this class
of pharmacological agents. Mini Rev. Med. Chem. 2004, 4, 625–
6
7
Motta, C.; Simorini, F.; Da Settimo, F.; Vullo, D.; Supuran, C. T.,
Sulfonamides incorporating heteropolycyclic scaffolds show po-
tent inhibitory action against carbonic anhydrase isoforms I, II, IX
and XII. Bioorg. Med. Chem. 2016, 24, 921–927.
32.
Barresi, E.; Salerno, S.; Marini, A. M.; Taliani, S.; La
22
10.2210/pdb3qyk/pdb.
Pettersen, E. F.; Goddard, T. D.; Huang, C. C.; Couch, G.
2
3.
S.; Greenblatt, D. M.; Meng, E. C.; Ferrin, T. E., UCSF Chimera--a
visualization system for exploratory research and analysis. J.
Comp. Chem. 2004, 25, 1605–1612.
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